Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149

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Comparative Biochemistry and Physiology, Part B

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b

Review

Glucosensing and glucose homeostasis: From fish to mammals

Sergio Polakof a, b,⁎, Thomas P. Mommsen c, José L. Soengas d
a
INRA, UMR 1019, UNH, CRNH Auvergne, Clermont-Ferrand, France
b
Clermont Université, Université d'Auvergne, Unité de Nutrition Humaine, Clermont-Ferrand, France
c
Department of Biology, University of Victoria, Victoria, BC, Canada V8P 3N5
d
Laboratorio de Fisioloxía Animal, Departamento de Bioloxía Funcional e Ciencias da Saúde, Facultade de Bioloxía, Universidade de Vigo, 36310 Vigo, Spain

a r t i c l e i n f o a b s t r a c t

Article history: This review is focused on two topics related to glucose in vertebrates. In a first section devoted to glucose
Received 13 June 2011 homeostasis we describe how glucose levels fluctuate and are regulated in different classes of vertebrates. The
Received in revised form 20 July 2011 detection of these fluctuations is essential for homeostasis and for other physiological processes such as
Accepted 22 July 2011
regulation of food intake. The capacity of that detection is known as glucosensing, and the different
Available online 17 August 2011
mechanisms through which it occurs are known as glucosensors. Different glucosensor mechanisms have
Keywords:
been demonstrated in different tissues and organs of rodents and humans whereas the information obtained
Glucose homeostasis for other vertebrates is scarce. In the second section of the review we describe the present knowledge
Glucosensing regarding glucosensor mechanisms in different groups of vertebrates, with special emphasis in fish.
Fish © 2011 Elsevier Inc. All rights reserved.
Food intake
Insulin secretion

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1240
2. Glucose homeostasis: a comparative view . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1240
2.1. Elasmobranch and teleostean fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1240
2.2. Amphibians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1260
2.3. Reptiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1260
2.4. Birds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1270
2.5. Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1270
3. Glucosensing: the mammalian model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1280
3.1. Peripheral glucosensing: pancreatic β-cell glucose detection as a general mechanism . . . . . . . . . . . . . . . . . . . . . . . . . 1280
3.2. Central glucosensing: glucose-excited and glucose-inhibited neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1290
3.2.1. Glucose excited neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1290
3.2.2. Glucose inhibited neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300
3.3. Functions involving glucosensing cells in mammals (Table 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300
3.3.1. Food intake. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300
3.3.2. Counterregulation to hypoglycemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300
3.3.3. Insulin secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1310

Abbreviations: 2-DG, 2-deoxyglucose; AgRP, agouti related peptide; AMPK, AMP-activated kinase; ANLS, astrocyte-neuron lactate shuttle; AP, area postrema; Arc, arcuatus
hypothalamic nucleus; cAMP, cyclic adenosine monophosphate; CART, cocaine–amphetamine-related transcript; Cav1.2/1.3 or VDCC, Calcium channel, voltage-dependent, L type;
CCK, cholecystokinin; CFTR, Cystic fibrosis transmembrane conductance regulator; CRF, corticotropin releasing factor; DMH, dorsomedial hypothalamus; DMNX, dorsal motor
nucleus of the vagus; GE neuron, glucoase-excited neuron; GI neuron, glucose-inhibited neuron; GK, glucokinase; GKA, glucokinase activator; GLP-1, glucagon-like peptide 1; GLUT,
glucose facilitative transporter; GSIS, glucose-stimulated insulin secretion; GTT, glucose tolerance test; HGE, high glucose excited; HGI, high glucose inhibited; HK, hexokinase; IRS-PI
(3)K, insulin receptor substrate 1/phosphoinositide 3-kinase; JAK/STAT, janus kinase/signal transducer and activator of transcription; KATP, ATP-sensitive potassium channels; Kir6.2,
inward rectifier K+ channel pore type 6.2; LH, lateral hypothalamus; LXR, liver receptor X; NPY, neuropeptide Y; NTS, nucleus of the tractus solitaries; POMC, pro-opiomelanocortin;
PVN, paraventricular nucleus; ROS, reactive oxygen species; SGLT, Na+-coupled glucose transporters; SSTR, somatostatin receptor; SUR1, sulfonylurea receptor type-1; T1R2/T1R3,
taste receptors; VMH, ventromedial hypothalamus.
⁎ Corresponding author at: INRA, Human Nutrition Unit-UMR 1019, Clermont-Ferrand, France. Tel.: + 33 473624895; fax: + 33 473624638.
E-mail address: sergio.polakof@clermont.inra.fr (S. Polakof).

1096-4959/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpb.2011.07.006
124 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149

3.4. Glucosensing neurons as metabolic sensors: interactions with other nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
0
3.5. Hormonal interaction with the glucosensor system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
131
3.6. Pathological intolerance: diabetes. Is glucose intolerance in mammals related to glucosensing? . . . . . . . . . . . . . . . . . . . . 131
0
4. Glucosensing: the emerging picture in fishes (Fig. 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
133
4.1. Why glucoregulate? Why is glucose important for carnivorous fish species? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
0
4.2. First indirect evidence of glucose detection in fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
0
4.3. Central glucose detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
0
4.3.1. Fish brain as a glucose consumer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
0
4.3.2. Components of the glucosensor system (see Table 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
0
4.3.3. Possible functions related to the glucosensing system (see Table 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
0
4.4. Peripheral glucosensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
0
4.4.1. Components of the glucosensing system (see Table 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
0
4.4.2. Brockmann bodies: involvement of the glucosensor system in insulin secretion (see Table 1) . . . . . . . . . . . . . . . . . 139
0
4.4.3. Glucosensing in intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
140
4.5. Other glucose sensing mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
0
5. Glucosensing in other vertebrate groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
0
6. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
0
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
0

1. Introduction vertebrates though more studies are needed to characterize the

Blood glucose concentration is the net result of the difference
between rates of glucose entry and removal from the circulation.
Glucose homeostasis is maintained by a feedback mechanism
designed to keep blood glucose levels close to a set point
characteristic for each species. In vertebrates blood glucose levels
are positively correlated to metabolic rate (Umminger, 1977) and
display an allometric relationship with body weight (Kjeld and
Ólafsson, 2008). However, these correlations do not allow extending
physiological conclusions among vertebrates. In the first part of the
present review we will deal with the different mechanisms employed
to maintain glucose levels in different vertebrates, and the uses and
dependence on this metabolite within each group.
Key to the homeostatic control of glucose is the existence of
sensors located in different parts of the body that continuously
monitor blood glucose variations. The molecular basis of glucodetec-
tion is relatively well understood in mammalian pancreatic β-cells
(Marty et al., 2007) and neurons excited by glucose (GE) found in
areas of the brain such as the septum, amygdale, striatum, motor
cortex, hindbrain and hypothalamus (Levin et al., 2004a,b; Moran,
2010). The mechanism is common to both cell types requiring glucose
uptake by the low-affinity glucose transporter type 2 (GLUT-2),
glucose phosphorylation by glucokinase (GK), and the subsequent
metabolism of glucose through glycolysis to increase the intracellular
ATP/ADP ratio. This leads to the closure of KATP channels, membrane
depolarization, the entry of Ca 2+, which triggers increased neuronal
activity and neurotransmitter secretion in brain regions and insulin
release in pancreatic β-cells. This mechanism can be denoted as the
classical glucosensor system. Other glucosensor systems have been
found in peripheral locations in mammals such as the L-cell of the
intestine (Reimann et al., 2008), glucose-inhibited α-cells (Rorsman
et al., 2008), hepatoportal vein (Donovan, 2002), liver (Magnuson and
Matschinsky, 2004) and carotid body (Pardal and López-Barneo,
2002). Also in mammals, glucosensing systems independent on GK
such as those based on SGLT1, LXR or sweet taste receptors have been
described in recent years (Mitro et al., 2007; González et al., 2009;
Nakagawa et al., 2009). Detecting changes in glucose levels must be
also necessary in vertebrates other than mammals that maintain
constant glucose levels and face changes in glucose availability due to
dietary habits or seasonal changes. The few studies available suggest
that common mechanisms for glucose sensing are present in
presence of these systems and to evaluate the functions which they
are involved within each particular phyla. In amphibians, reptiles and
birds the studies carried out to date only provided partial information
regarding the presence of individual components of putative
glucosensor systems. In fish, several studies carried out in recent
years in rainbow trout (Polakof et al., 2006, Polakof et al., 2007a,b,c,
2008a,b,c, 2009, 2010, 2011) despite being considered for many years
as a model of glucose intolerant species, have demonstrated the
existence of glucosensing mechanisms in central and peripheral
locations, which are similar to those described in mammals.
Therefore, we will describe from a comparative point of view what
we know at present about glucosensor mechanisms in vertebrates.
2. Glucose homeostasis: a comparative view
Blood glucose concentration depends on a wide variety of factors
and its level at any time is the net result of the difference between
rates of glucose entry into and removal from the circulation.
Furthermore, when the renal reabsorptive capacity for glucose is
exceeded (renal threshold), urinary loss of glucose becomes an
additional factor influencing the maintenance of the blood glucose
concentration. From a comparative point of view, Umminger (1977)
showed that blood glucose levels are positively correlated with
metabolic rate across the vertebrate subphylum. Moreover, glycemia
also has an allometric relationship with the body mass and, in general,
the larger the vertebrate the lower the circulating glucose levels
(Kjeld and Ólafsson, 2008). However useful this correlation may be in
theory it cannot be extrapolated to physiological conclusions among
vertebrates. In the following paragraphs we will discuss the blood
glucose levels in the different groups of vertebrates and the uses and
dependence on this metabolite for each group.
2.1. Elasmobranch and teleostean fish
The unusual energy metabolism of elasmobranchs compared to
teleost fish is characterized by limited or absence of fatty acid
oxidation in cardiac and skeletal muscles and a significant reliance on
the ketone bodies and amino acids as oxidative fuels in these tissues
(Speers-Roesch and Treberg, 2010). Carbohydrates are utilized as a
fuel source in elasmobranch tissues, with plasma glucose levels often
lower (around 3–4 mM) than in comparatively sized teleosts with
 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149 125

similar metabolic activities and at comparative temperatures. In some
species, values around 10 mM are not uncommon and following
stress, levels can double (Hoffmayer et al., in press), though these
increases are transient. While the overall contribution of glucose to
energy metabolism is unclear (Ballantyne and Robinson, 2010;
Speers-Roesch and Treberg, 2010) it should be mentioned that the
rate of hepatic gluconeogenesis is low compared with teleosts
(Mommsen and Moon, 1987). A number of findings support the
idea that, conceptually, glucose and insulin in elasmobranchs show
interactions similar to other vertebrates, but responses tend to be
minor compared with other vertebrates, including teleosts: 1)
exogenous heterologous insulin exerts a slow hypoglycemic effect
(Anderson et al., 2002); 2) severe hypoglycemia induced by insulin
treatment was tolerated for several days in dogfish (Squalus
acanthias) (DeRoos and DeRoos, 1979); and 3) levels of circulating
glucose are maintained relatively constant during fasting (DeRoos et
al., 1985) and feeding (Walsh et al., 2006), although Kajimura et al.
(2008) reported an increase in plasma glucose concentration after
several weeks of fasting in dogfish.
In contrast to the limited information available in elasmobranchs, a
plethora of information is available for teleost fish. In teleost fish
glucose homeostasis has been discussed in numerous studies (Wilson,
1994; Moon, 2001; Stone, 2003), although some key issues remain
under debate. While temperature alone cannot explain the differences
observed in glycemia with respect to other vertebrate groups (Cowey
et al., 1975), several hypotheses arose including that fish glucose
metabolism (unlike birds and mammals) was adapted to long-term
food deprivation conditions. In addition to having generally lower
values than other vertebrate groups, averaging 3.55 mM (Fig. 1), fish
blood glucose levels are very variable intraspecies, especially among
elasmobranchs (Patent, 1973; DeRoos and DeRoos, 1979) (see Fig. 1).
In lampreys (Larsen, 1976) and Scorpaeniformes (MacCormack and
Driedzic, 2007) glycemia values can be as low as 0.08 mM or even
undetectable (Scott, 1921), which put glucose in the category of the
non-preferred energy substrate for elasmobranchs. This led to the
idea that ectotherm vertebrates in general would rely on the most
abundant metabolites, which is not always necessarily glucose. This is
contrasted by the fact that in many fish species blood glucose levels
are very stable during food deprivation periods (Navarro and
Gutiérrez, 1995) and suggests that fish have the mechanisms to
maintain this metabolite stable over long periods, which implies that
glucose must be important for some physiological functions. Howev-
er, this is not the case for all the species studied, since in carps (and
other warm-water species), and American eel (Anguilla rostrata)
(Cornish and Moon, 1986) glycemia can be stable for weeks or
months, while in other species, such as sea bass (Dicentrarchus
labrax), brown trout (Salmo trutta) and dogfish (Scyliorhinus canicula)
blood glucose levels are more sensitive to food deprivation, and lower
glycemia can be found after a few days of fasting (Navarro and
Gutiérrez, 1995). With the exception of eel (Cornish and Moon, 1986)
and highly active skipjack tuna (Katsuwonus pelamis) (Weber et al.,
1986), glucose turnover in fish has been reported to be lower than in
mammals and birds (Lin et al., 1978; Bever et al., 1981; Garin et al.,
1987). However, some of the data from the latter studies must be
interpreted carefully, as no rapid mixing and equilibration of glucose
pools as well as missing out on the initial phase of glucose dilution
could result in underestimation of true values. Machado et al. (1989)
found that food deprivation induced a progressive reduction in the
rate of glucose utilization, probably linked to depressed glucose
metabolism under such circumstances. While carbohydrate – largely
glucose 6-phosphate derived from endogenous glycogen – is the
almost exclusive driving force for white muscle during short-term
anaerobic activity in fish, glucose makes only a minor contribution as
oxidative fuel during sustained swimming (Weber and Haman, 1996)
with West and colleagues showing that the largest tissue in fish
(white muscle) was only responsible of 10–15% of the glucose
utilization (West et al., 1993; 1994). While the white muscle was a
poor glucose consumer, other smaller organs in fish were “strictly
dependent”, including brain, kidney and gills, using 60–90% of the
glucose taken up (Blasco et al., 2001). The fact that glucose stimulates
insulin secretion (Thorpe and Ince, 1976; Emdin, 1982; Ronner and
Scarpa, 1987) and that streptozotocin-injected salmon lose control of
glycemia (Plisetskaya and Duan, 1994) certainly indicates that fish do
have a glucose homeostasis system. However, in some studies the
possibility of a poor system controlling glycemia has been proposed,
as in some occasions the insulin secretion does not seem to be enough

Fig. 1. Individual (dots) and median (bars) glycemia values of different species (N ranging from 19 in amphibians to 511 in birds) representing 5 vertebrate groups, which were
obtained from references available in literature. Different letters indicant significant differences among groups. The boundary of the box closest to zero indicates the 25th percentile,
a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the
90th and 10th percentiles. Data are presented as means ± SE. Comparisons among groups were performed using one-way ANOVA (SigmaStat; SPSS). Post hoc comparisons were
made using a Student–Newman–Keuls test, and differences were considered statistically significant at P b 0.05.
126 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
to control circulating glucose levels, like after a glucose load in
chinook salmon (Oncorhynchus tshawytscha) (Mazur et al., 1992).
Glucose increased the expression of the somatostatin receptor (SSTR)
2 mRNA in a dose-dependent manner in rainbow trout Brockmann
bodies and liver (Kittilson et al., 2011). If this effect on expression is
reflected in functional protein, increases in the Brockmann bodies
SSTRs would affect the feedback loop between somatostatins and
insulin (Sheridan and Kittilson, 2004). Specifically, this would result
in a weakening of glucose effects on insulin release, likely preventing
sharp bursts in insulin release and also decreasing the kinetics of
glucose removal from plasma. Glucose-dependent increases in the
effectiveness of circulating somatostatins on peripheral tissues would
be somewhat counterintuitive, since this would lead to increased
glycogenolysis, and this provide a feed-forward loop for plasma
glucose. This scenario is somewhat reminiscent of that found in
diabetic mice showing increased expression of SSTR2 in their
endocrine pancreas (Strowski and Blake, 2008). Surprisingly, the
effects of glucose on the endocrine pancreas are not restricted to
regulating the synthesis and release of insulin in beta cells, but other
cells and hormones are also affected. This co-release of opposing
hormones, if tightly timed, can dampen the effects of insulin and thus
provide very tight maintenance of normoglycemia, and this appears to
be key when mammalian and fish systems diverge. Two key
observations set the mammals apart from the fishes and birds, and
neither of these observations seems to be related to the thermal
condition of the model system. Across all species, mammals are
characterized by relatively uniform plasma glucose concentrations
about 7 mM and by a very tight control of normoglycemia. Fish
systems subscribe to neither. Plasma glucose varies substantially
between species and within species and under different physiological
conditions. Conditions that would be considered pathological for a
mammal, like extended periods of hyperglycemia or severe hypogly-
cemia, hardly seem to alter fish glycemia at all. Moreover, Harmon
et al. (1991) showed that glucose stimulates somatostatin secretion,
which in turn increases glycemia through the striking inhibition of
insulin secretion and the stimulation of glucagon release, a common
phenomenon in amphibians and human type 2 diabetics. In this
sense, although it has been shown that fish can largely tolerate
hyperglycemia without displaying the classical symptoms of diabetic
mammals, increased glycosylated hemoglobin was reported in
hyperglycemic/hyperinsulinemic Indian perch (Anabas testudineus)
(Barma et al., 2006). Finally, while GTTs (glucose tolerance tests) were
traditionally performed in fish (reviewed in Moon, 2001), the results
were highly variable, although it seems that at least two ideas can be
highlighted: first, hyperglycemia in fish after a GTT is more persistent
than in mammals, and second, omnivorous fish return to basal blood
glucose levels more rapidly than carnivorous fish. However, in the last
case, it is also worth mentioning that in general the omnivorous fish
have lower basal glucose levels than the carnivorous species.
2.2. Amphibians
Glucose homeostasis in amphibians has not received much
attention. The first data of blood sugar reported levels in fasted
frogs (Rana pipiens) ranging from 1.9 to 3.6 mM (Lesser, 1913). These
early results were confirmed later by Bosman and Zwarenstein (1930)
in Xenopus laevis, who also offered the first available data on
amphibian glucose tolerance. The authors concluded that when
subjected to a GTT (~ 700 mg/kg) at the same environmental
temperature (20 °C) both human and frog reach the same maximum
circulating blood glucose levels (about 10 mM) 30 min after admin-
istration. However, blood glucose levels in frog returned to basal
levels 4 times slower than in human, and this delay was – not
surprisingly – increased when the temperature was reduced to 5 °C.
GTTs were also performed more recently in the salamander (Necturus
maculosus), resulting in a much higher increase in glycemia (7-fold
vs. control) even when the amount of glucose used was lower
(250 mg/kg) than in the frog experiment (Copeland and DeRoos,
1971). Despite these differences between GTTs, amphibians in general
contain the lowest blood circulating glucose levels among vertebrates
(Umminger, 1977). Thus, published values average 2.7 mM (19
species) (Fig. 1), with some extreme values probably influenced by
nutritional conditions (0.55 mM in Rana catesbeiana; now Lithobates
catesbeianus) or stress (7 mM in Calotriton asper). Other studies on
amphibians also showed that these animals respond to heterologous
insulin with a significant hypoglycemia (Copeland and DeRoos, 1971;
DeRoos and Parker, 1982; Branco, 1997). Other hormones do not seem
to affect blood glucose levels in amphibians, like glucagon (Wurster
and Miller, 1960) — although in vitro, glucagon lead to the expected
activation of glycogen phosphorylase and glucose production in
isolated wood frog hepatocytes (Mommsen and Storey, 1992), while
other hormones exert similar effects to those described in mammals,
such as somatostatins (Vethamany Globus et al., 1977). Actually,
instead of causing immediate coma and eventual death, insulin-
induced hypoglycemia results in hypometabolism and behavioral
hypothermia in Rana catesbeiana (Rocha and Branco, 1998). More-
over, insulin is only able to cause hypoglycemia at normal or high
temperature, while at low temperature the effect disappears
(Rocha and Branco, 1998). However, it has to be kept in mind that
heterologous insulins – mostly bovine – have been used in studies
with amphibians and homologous insulin may lead to different
results. Overall, the changes observed in circulating blood glucose in
amphibians may be more related to environmental adjustments
rather than to glucose homeostasis. This is also supported by the
hypoxia-induced hyperglycemia (D'Eon et al., 1978; De Castro e Silva
et al., 1992) and the use of glucose as an anti-freeze molecule in
the wood frog (Rana sylvatica), the gray tree frog (Hyla versicolor),
spring peeper (Pseudacris crucifer), and forest frogs (H. chrysoscelis,
P. triseriata) (Mommsen and Storey, 1994; Storey and Storey, 2004).
In these animals glucose is not used as a primary energetic substrate,
but serves as the sole cryoprotectant produced by R. sylvatica,
Pseudacris crucifer and P. triseriata, whose plasma glucose levels can
typically rise to 150–300 mM in freeze-exposed winter animals,
compared with 1–5 mM in unfrozen controls (Storey and Storey,
2004). Glucose production by wood frogs is stimulated only when
frogs begin to freeze and the sugar is cleared again post-thaw (Storey
and Storey, 2004), probably to limit any metabolic damage that could
accrue from a long-term elevation of glucose over the winter, similar
to the damage occurring in tissues of diabetic patients exposed to
sustained high glucose levels in the 10–45 mM range (Chan et al.,
2006; Aronson, 2008; Negre-Salvayre et al., 2009).
2.3. Reptiles
Glucose homeostasis in reptiles, as in amphibians, is largely
unchartered territory. The average blood glucose levels in 176 species
is around 5.4 mM (Fig. 1), which means 2-fold higher than in
amphibians. One of the first reports of reptile blood sugar levels
came from Luck and Keeler (1929), who determined that glucose
levels in Arizona captured Crotalus atrox and Crotalus oregonus were
similar to the samples taken from humans. However, the variability in
the reptile group is very high, and not all the studied species show
such similarity with mammals. Insulin treatment results in persistent
hypoglycemia in alligators (Penhos et al., 1967; Lance et al., 1993) and
snakes (Sidorkiewicz and Skoczylas, 1974), while glucagon treatment
induces a hyperglycemic effect also in alligators (Penhos et al., 1967).
In this sense, although pancreatectomy results in loss of glucose
homeostasis control and persistent hyperglycemia (up to 30 mM)
(Penhos et al., 1967), diabetes has been rarely documented in reptiles
(Frye, 1991), even if hyperglycemia can appear after a period of stress
(Britton and Kline, 1939). The role played by glucose on reptile
metabolism remains uncertain. In lizards, glucose turnover is 10-fold
 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
lower than in mammals, which was explained as an advantage against
low temperatures in these animals (Guppy et al., 1987). Similarly, in
the desert iguana (Dipsosaurus dorsalis) glucose does not seem to play
a major role in metabolism, since changes in levels during exercise are
minor (Donovan and Gleeson, 2006). However, glucose does play a
more important role in the steady state recovery period in which
glucose oxidation maintains ATP production for basal functions and is
stored as glycogen when present in excess. Moreover, some changes
in glucose turnover in reptiles could be related to its use as substrate
by glucose-dependent tissues, like the brain and the heart. While
these studies suggest a minor role for glucose, other studies in reptiles
showed that circulating glucose levels are sensitive to fasting and
migration (Gist, 1972; Martin and Bagby, 1973; Stamper et al., 2005;
McCue, 2007), dropping in the same proportion as in mammals and
much more than in fish (Navarro and Gutiérrez, 1995). In those
studies the use of glucose during fasting was not assessed. However
for some species it seems that ketone bodies are responsible to
maintain basal metabolism under such conditions (McCue, 2007). The
importance of glucose was also reported in cases of stress, during
which reptiles experience rapid hyperglycemia, most likely stimulat-
ed by catecholamine release, either after handling (Jessop et al., 2003)
or exposure to high temperatures (Ray and Maiti, 2001). Other data
that could shed some light about the importance of glucose for
reptiles can be found in winter reptiles, including box turtles
(Terrapene carolina, T. ornata), hatchling painted turtles (Chrysemys
picta), the Siberian salamander (Salamandrella keyserlingi) and the
European common lizard (Lacerta vivipara), which use (as many
amphibian species) glucose as cryoprotectant (Grenot et al., 2000;
Storey and Storey, 2004). However, unlike winter frogs, some lizards
only tolerate up to 30 mM of glucose, 10-fold less than the
amphibians. The reason for this difference in blood glucose levels is
probably the time of exposure, as the lizards become hyperglycemic
long before and after the freezing period, while frogs remain super-
hyperglycemic only minutes before and after freezing.
2.4. Birds
The first studies of avian glucose homeostasis were reported in the
1870–90s by Langendorff (1879) and Minkowski (1893), using
pancreatectomized pigeons and ducks, indicating an absence of
glycosuria (even following external glucose loads) and operated birds
usually died in less than a week after complete pancreas removal. Later,
Koppanyi et al. (1926) were able to generate both hyperglycemia and
glycosuria, but using huge amounts of glucose (6–13 g/kg) (Koppanyi et
al., 1926). Surprisingly, in pancreatectomized chickens, hyperglycemia
was only transient — similar to what had been noted by Weintraud
(1894) and Kausch (1896) for other granivores, and the birds returned
to basal levels in a week, suggesting a change in the metabolism of the
liver or another organ in birds producing insulin when the pancreas was
removed. Moreover, the fact that the drop in glycemia was initially
unrelated to insulin titers cannot be ruled out. Even more surprising
were the results in some granivorous species that became hypoglycemic
after total pancreatectomy (Sitbon et al., 1980). During these studies
researchers starting with Sprague and Ivy (1936) came to the realization
that birds have the highest blood glucose levels among vertebrates: of
the 511 species analyzed in the present review, birds averaged 15.6 mM
(Fig. 1), almost 2-fold higher than mammals, and 7-fold higher than
amphibians (lowest values among vertebrates). However, data also
show a very high inter-species variability, as glucose can reach 28 mM in
some species in healthy status, and because birds can also experience
hyperglycemia during stress situations or at the beginning of fasting
periods (Krautwald-Junghanns, 2007). While such high values can be
assigned to diabetic mammals, ‘normal’ values in mammals (about
7 mM) would be considered as pathological hypoglycemia in birds (only
4 of the 501 species were below 7 mM). Glucose tolerance tests in birds
showed that some species are more tolerant than others. While in the
127
American kestrel 1 g/kg of oral glucose caused a peak of glycemia only
5 min after administration (Minick, 1978), 4 g/kg of glucose produced
maximum glucose levels in chickens and owls at 30 and 90 min,
respectively (Myers and Klasing, 1999). Comparing tests on the same
conditions, Myers concluded that omnivorous species (chicken) are
significantly more tolerant to glucose than carnivorous ones (owls),
which are also supported by higher gluconeogenic rates in the latter
group. Although diabetes mellitus has been recorded in some
domesticated birds (Hochleithner, 1994), with blood glucose levels
reaching 111 mM, this is a rare disease for this group (Oglesbee et al.,
1997; Lumeij, 1999; Gancz et al., 2007). The relatively high glucose
levels in birds are certainly related to a different glucose homeostasis
regulation in comparison with other vertebrate groups rather than to a
difference in body temperature (birds have the highest temperature
among vertebrates), since in the Malachite sunbirds (Nectarinia famosa)
low temperatures (5 °C) are associated with higher blood circulating
glucose levels (Downs et al., 2010). Thus, the insulin/glucagon ‘couple’
does not seem to operate in the same way in birds than in mammals,
since circulating glucagon levels exceed those of insulin (~2-fold)
(Hazelwood, 1984; Minick et al., 1996), resulting in low insulin/
glucagon ratios and suggesting that birds are normally in a catabolic
state like diabetic, starving, or exercising mammals (Hazelwood, 1984).
Moreover, Minick (1986) has shown that insulin levels in birds
(especially those of carnivorous species) are low and do not correlate
with those of glucose and unlike mammals, insulin does not contribute
to fasting glucose levels (Minick, 1986; Minick and Duke, 1991; Minick
et al., 1996). Thus, it is probable that the low insulin/glucagon ratio is
responsible for high blood glucose levels and that metabolism is largely
‘glucagon-driven’. As such, the elevated rates of gluconeogenesis
reported in the avian liver (Hazelwood and Lorenz, 1959), being
especially marked in carnivorous species, where high protein intake
allows them to produce two-fold more glucose than other species
(Migliorini et al., 1973). This ratio decreases during periods of food
deprivation inducing increased glycemia and gluconeogenic rates
concomitantly (Hazelwood and Lorenz, 1959; Brady et al., 1978; Cherel
et al., 1988; Sartori et al., 1995). However, when the fasting period is too
long, glycemia finally falls, probably as a consequence of a reduced
gluconeogenic rate. The importance of glucose for bird metabolism is
clearly demonstrated by the fact that during fasting periods glucose
(instead of lipid) availability is the determinant variable that drives
energetic metabolism (Bernard et al., 2003). Similarly, except for the
first minutes of flight (Rothe et al., 1987), there are no differences in
blood glucose levels between resting and flying birds, showing that
these animals will defend stable blood glucose levels even during
periods of high energy turnover (Brackenbury and El-Sayed, 1984;
Bordel and Haase, 1993; Jenni-Eiermann et al., 2002). High glucose
levels are known to generate an increase in levels of reactive oxygen
species and to increase the levels of glycosylated hemoglobin levels.
However, when compared with mammals, the production of reactive
oxygen species in birds is low, probably due to the higher levels of
antioxidants, including enzymes and uric acid (Ku and Sohal, 1993;
Klandorf et al., 1999). In spite of high core temperature and high plasma
glucose concentrations, the levels of glycosylated hemoglobin in birds
range from 0.5 to 1.5% (Miksik and Hodný, 1992; Holmes et al., 2001),
while in mammals they could reach 6%. How can birds tolerate such high
constant blood glucose circulating levels? Some of the possible
explanations to this protection against high glucose levels include a
low permeability to glucose of (nucleated) avian erythrocytes as well as
shorter half-life, which would limit the formation and accumulation of
glycosylated hemoglobin (Holmes et al., 2001).
2.5. Mammals
All mammalian cells use glucose as a fuel for their basic functions.
Interestingly, despite the fluctuations in food consumption and activity
level throughout the course of a day, most mammals maintain stable
128 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
blood glucose levels. Blood glucose levels are determined by relative
rates of glucose entry into the blood and uptake into the tissues. Hence,
stable glucose levels are the result of whole-body glucose production
that matches whole-body glucose use (Cherrington, 1999). In order to
maintain stable glucose levels, an intricate system of neural, hormonal
and direct nutrient responses are initiated after stress (exercise or
hypoglycemia) or a meal. The liver is the predominant source of glucose
production, with small contributions by the kidney and the gut while
peripheral tissues (skeletal muscle and fat) and the central nervous
system remove glucose from the plasma especially. While an insulin-
sensitive glucose transporter (GLUT-4) is rate limiting in several
peripheral tissues (muscle, fat), in the liver, however, glucose moves
freely across the plasma membrane in either direction. At blood glucose
levels of approximately 8.33 mM the liver does not take up or supply
glucose to the circulation. This level is termed the “steady state” or the
“glucostatic level” at which the mechanisms of normal supply and
removal of glucose are operating at equal rates. At glucose levels above
8.33 mM glucose removal is greater than supply whereas at levels below
8.33 mM glucose supply is greater than removal, but the fasting blood
glucose level in most mammals is about 5 mM (Cherrington, 1999).
When administered orally to a normal mammal, typical changes in
blood glucose concentration include (Kaneko, 2008): 1) During the
absorptive phase the rate of entry of glucose into the circulation exceeds
that of removal and the blood glucose rises. 2) As the blood glucose rises,
hepatic glucose output is inhibited and the release of insulin from the
pancreas is stimulated by the rising blood glucose. 3) In 30 to 60 min, the
peak level of blood glucose is reached, after which it begins to fall.
During this phase of falling blood glucose, the rates of removal now
exceed those of entry and the regulatory mechanisms directed toward
removal of glucose are operating maximally. At the same time, hepatic
glucose output decreases and the blood glucose falls rapidly. 4) When
the blood glucose reaches its baseline level, it continues to fall below the
original level for a short time and then returns to its baseline level. This
hypoglycemic phase is due to the inertia of the regulatory mechanisms
because, in general, the higher the glycemia the greater the subsequent
hypoglycemia. Although diabetes mellitus has been reported in virtually
all laboratory mammals (gerbils, guinea pigs, hamsters, mice, rats,
nonhuman primates) and in horses, cattle, sheep, and pigs, it is most
frequently found in dogs and cats. A persistent fasting hyperglycemia is
the single most important diagnostic criterion of diabetes mellitus
(American Diabetes Association, 2010). In the diabetic mammal, the
hyperglycemia itself tends to compensate in part for the decrease in
peripheral utilization. This occurs as a mass action effect that promotes
the uptake of glucose into the peripheral tissues. In this way, the diabetic
can continue to use some glucose when insulin is decreased, but only at
the expense of increased glucose production and hyperglycemia. As the
deficiency of insulin progressively becomes more severe, the equilib-
rium level of blood glucose is established at higher levels, and ultimately
the equilibrium is never established without therapeutic intervention.
However, Delaere et al. (2010) recently postulated that the
importance of glucose may not be only based on its participation in
the energy production in the mammalian cells. While the brain, kidneys
and gut are often cited as being among the most glucose-utilizing
organs, it is today known that all of them can utilize efficiently other
substrates especially ketone bodies (Delaere et al., 2010). Thus, it is
probable that the glucose requirement of biological tissues is the
consequence of ‘qualitative’ reasons (to enable, for example, specific
metabolic pathways) rather than ‘quantitative’ purposes (such as for
energy supply). This probably explains why the regular supply of
glucose to living cells is so important, and also why sensing of plasma
glucose concentrations is so essential for the whole body.
3. Glucosensing: the mammalian model
The body continuously adjusts its metabolism to keep blood
glucose concentrations at a constant value. Glucose homeostasis in
humans and most other studied mammals is maintained by a
feedback mechanism designed to keep the blood glucose close to a
set point characteristic for each species (see above). Key to this
homeostatic control is the existence of sensors located in different
parts of the body that continuously monitor blood glucose variations.
They respond to changes in glycemia by triggering hormonal
secretion or activation of the autonomic nervous system to control
glucose uptake, utilization or production and also to control energy
expenditure and food intake. The molecular basis of glucodetection is
relatively well understood for insulin secretion by pancreatic β-cells.
Apart from the classical glucosensor in pancreatic β-cells, glucosen-
sing cells have been found in peripheral locations such as the L-cell of
the intestine (Reimann et al., 2008), glucose-inhibited α-cells (Rorsman
et al., 2008), hepatoportal vein (Donovan, 2002), liver (Magnuson and
Matschinsky, 2004) and carotid body (Pardal and López-Barneo, 2002),
as well as in central locations within the brain where glucosensing
neurons have been located to areas such as the septum, amygdala,
striatum, motor cortex, hindbrain and hypothalamus (Levin et al.,
2004c; Moran, 2010).
3.1. Peripheral glucosensing: pancreatic β-cell glucose detection as a
general mechanism
The pancreatic β-cell glucosensor links variations in extracellular
glucose concentrations to insulin secretion (Matschinsky, 1996).
Glucose-stimulated insulin secretion (GSIS) is initiated by glucose
uptake by GLUT2 and phosphorylation by glucokinase (hexokinase IV,
GK). Glucose 6-phosphate is then metabolized through the glycolytic
pathway and activates mitochondrial metabolism to generate cou-
pling factors. ATP is the best described of these factors, and a rise in
cytoplasmic ATP/ADP concentration leads to closure of KATP channels
(composed by Kir6.2 and SUR1 subunits), depolarization of the
plasma membrane and opening of voltage-sensitive L-type Ca 2+
channels [composed of a pore-forming α1-subunit and regularly
associated β and disulfide-linked α2δ subunits (Reuter, 1996)]. The
entry of Ca 2+ then triggers exocytosis of the insulin granules (Schuit
et al., 2001; Navarro-Tableros et al., 2007). A KATP channel-
independent pathway also has been described, in which glucose
stimulates insulin secretion, even when the KATP channel is main-
tained open (diazoxide treatment) and the cells are depolarized by
extracellular K + (Henquin, 2000). Both the KATP channel-dependent
and -independent pathways require energy production from glucose.
In both pathways, glucose entry is usually not limiting, since the
expression level of GLUT2 allows a rate of glucose uptake in excess of
the rate of glucose phosphorylation by GK (Heimberg et al., 1993).
Glucose phosphorylation represents the rate-controlling step in GSIS,
as indicated by the similarity in the dose-dependent increase in
glucose phosphorylation, utilization, oxidation and GSIS
(Matschinsky, 1996). GK gene transcription is affected by a variety
of factors, including insulin, cAMP and biotin. GK activity is also
regulated at the post-translational level mainly by glucose (Magnuson
and Matschinsky, 2004) (Fig. 2).
The liver has a complex response to changes in the glucose
concentration, being able to alternate between glucose utilization and
production. GK is essential for induction by glucose of key glycolytic
and lipogenic enzymes, such as liver pyruvate kinase, acetyl-CoA
carboxylase, and fatty acid synthase, and repression of genes involved
in gluconeogenesis such as phosphoenolpyruvate carboxykinase
(Vaulont et al., 2000). Thus, GK can be considered as a glucosensor
for the regulation of these glucose-responsive genes (Magnuson and
Matschinsky, 2004).
Recently, a combination of in vitro, in situ, molecular biology and
clinical studies has formed the basis of the knowledge about taste
receptor proteins in the glucose-sensing enteroendocrine cells and
the secretion of incretins by these cells (Renwick and Molinary, 2010).
Margolskee et al. (2007) showed that the sweet-taste receptor
 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
Glucose
GLUT2
R 1R 1
.
R6
S US U
KI
GLUCOKINASE
K+
Glycolysis
Ca2+
(ATP/ADP)
-cell / GE neuron
Fig. 2. General admitted glucosensing mechanism in mammalian β-cells and glucose excited (GE) neurons. Glucose enters the cell through GLUT 2, and is phosphorylated by GK,
acting as the gatekeeper, and regulating the production of cytosolic ATP in a subcellular compartment. The ATP closes KATP channels in the plasma membrane, causing depolarization.
In turn, this leads to Ca2+ influx through L-type Ca2+ channels, stimulating the release of insulin (β-cells) or the action potential frequency (GE neurons). Glucokinase (GK), GLUT2
(glucose facilitative transporter type 2), KATP (ATP-sensitive inward rectified K+ channel), Kir6.2 (pore-forming subunit of KATP), SUR (sulfonylurea receptor).
subunit T1R3 and the G-protein α-gustducin are expressed in the
enteroendocrine cells and are responsible for intestinal glucose
sensing (Dyer et al., 2003). Other mechanisms independent of glucose
metabolism have been demonstrated in recent years. It was proposed
that hormone secretion was somehow linked to the sugar-uptake
mechanism, supported by the finding that incretin hormone secretion
required the concomitant presence of luminal sodium and was
inhibited by the glucose uptake blocker phloridzin (Sykes et al.,
1980). Subsequent studies in GLUTag (tumoral cell line expressing the
proglucagon gene and secreting immunoreactive GLP-1) and primary
crypt cells identified the sodium-coupled glucose transporter SGLT1
as the likely mediator of the direct responsiveness of K- and L-cells to
luminal sugars (Gribble et al., 2003; Parker et al., 2009).
3.2. Central glucosensing: glucose-excited and glucose-inhibited neurons
The reliance of the brain on glucose to meet its energy demands
suggests that, within the context of glucose homeostasis, brain
glucosensing may be of vital importance. Specialized neurons able
to modulate their firing activity in response to changes in extracellular
glucose levels were first demonstrated in the 1960s (Anand et al.,
1964; Oomura et al., 1969). These are glucose-excited (GE; previously
called glucose-responsive) neurons that increase their firing rate
when extracellular glucose concentrations are elevated, or glucose-
inhibited (GI; previously called glucose-sensitive) neurons that are
activated by a decrease in extracellular glucose concentration or by
cellular glucoprivation (Routh, 2002; Yang et al., 2004). Both types of
neurons are highly represented in regions involved in the control of
energy homeostasis and food intake, suggesting that they are able to
receive inputs from blood nutrient and hormone levels, as well as
from peripheral and central sensory systems, and use this information
to generate the appropriate physiological responses (Marty et al.,
2007; McCrimmon, 2008). Glucosensing neurons are found in certain
distinct hypothalamic regions, namely the ventromedial hypothala-
mus (VMH) (which includes the arcuatus nucleus or Arc and the
ventromedial nucleus or VMN), the paraventricular nucleus (PVN)
and the dorsomedial hypothalamus (DMH) (Levin et al., 2004c), as
129
K+
2
KATPchannel
R 1R 1
S US U
Vm
Ca2+ L-type Ca
channel
2+
Ca2+
Increase of
firing rate
Ca2+
Insulin
secretion
well as the hindbrain, including the area postrema (AP) and dorsal
motor nucleus of the vagus (DMNX) (Marty et al., 2007). In the Arc,
the presence of GE and GI neurons responsive to glucose over either a
low range (0–5 mM) or a high range (5–20 mM) of glucose
concentrations has been described, the latter are referred to as HGE
(high glucose excited) or HGI (high glucose inhibited) neurons,
respectively (Fioramonti et al., 2004).
While it may be argued that GE neurons are simply a more
sensitive version of non-glucose-sensing neurons, which would also
be stimulated by glucose (especially if they are energy depleted), GI
neurons respond in the opposite way to that expected from the
general stimulatory fuel effects of glucose, and their operation is thus
clearly different from a general energy-related effect (Karnani and
Burdakov, 2011).
Glucosensing neurons of both types often co-exist in the same
locations. Thus, the Arc contains neuropeptide Y/agouti related pep-
tide (NPY/AgRP)-GI (Muroya et al., 1999) and pro-opiomelanocortin/
cocaine–amphetamine-related transcript (POMC/CART)-GE neurons
(Ibrahim et al., 2003), the LH contains orexin GI neurons (Burdakov
et al., 2005) and the nucleus of the tractus solitarius (NTS) con-
tains both GE and GI neurons (Dallaporta et al., 1999). Up to 20–
30% of neurons in those areas have glucosensing properties (Levin,
2006).
3.2.1. Glucose excited neurons
There is considerable evidence that the glucosensing mechanism
used by GE neurons has many similarities with the pancreatic β-cell
described above (Yang et al., 1999; Schuit et al., 2001). The presence of
GLUT2 in hypothalamic nuclei where GE neurons are present has indeed
been reported (Arluison et al., 2004; Kang et al., 2004) as has the
presence of GK (Dunn-Meynell et al., 2002; Levin et al., 2004c). In these
neurons, the increase in extracellular glucose leads to an augmentation
of the ATP-to-ADP ratio and the closure of KATP channels (Levin et al.,
2001; Miki et al., 2001), which leads to plasma membrane depolariza-
tion and Ca2+ entry through voltage-gated channels, thereby increasing
neuronal activity and neurotransmitter secretion (Moriyama et al.,
2004). However, there is also indication that neuronal gluco-detection
130 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
could be independent of KATP channels (Fioramonti et al., 2004), GK
(Song and Routh, 2005) or GLUT2 (Kang et al., 2004). For instance, the
activation of an Arc GE neuron population by glucose seems to depend
on the glucose-regulated activity of transient response potential (TRP)
channels (Fioramonti et al., 2004). Moreover, a recent study identified
the heterodimeric G-protein-coupled sweet receptor T1R2/T1R3 as a
candidate membrane-bound brain glucose sensor that allows neurons
to change their firing rates independently of intracellular glucose
metabolism (Ren et al., 2009).
3.2.2. Glucose inhibited neurons
Since GI neurons show a decrease in activity as glucose levels rise,
it is easy to think that these neurons should use signaling mechanisms
close to those of pancreatic α-cells. Although the mechanism is less
clear than in GE neurons, suppression of firing activity may be
controlled by the increase in the ATP-to-ADP ratio, which leads to an
increase in Na +, K +-ATPase activity (Oomura et al., 1974; Silver and
Erecinska, 1998) and/or opening of ATP-regulated chloride channels
possibly belonging to the CFTR family (Song et al., 2001; Routh, 2002;
Fioramonti et al., 2007), which hyperpolarize the plasma membrane.
For GI orexin neurons, it has been shown that tandem-pore K +
channels could mediate their inhibition by glucose (Burdakov et al.,
2006). In addition, GK mRNA is expressed in ~ 40% of GI neurons, and
may also pose a regulatory role in those neurons (Kang et al., 2006).
More recently, evidence has also emerged that the AMP-kinase
(AMPK) plays a key role in the sensing pathway used by GI neurons
(McCrimmon, 2008). Although initially it was shown that orexin-A/
hypocretin-1 was not present in rat GI neurons (Liu et al., 2001;
Parton et al., 2007), other studies have demonstrated an acute
inhibition of orexin/hypocretin neurons by glucose in the mouse brain
(Muroya et al., 2001; Yamanaka et al., 2003), suggesting that the
glucosensing sensitivity can be also species-dependent in mammals.
Interestingly, the glucose responses of orexin/hypocretin cells
displaying a unique sugar selectivity, are also insensitive to GK
inhibitors and cannot be mimicked by the intracellular ATP, suggest-
ing that they do not require conventional glucose-metabolizing
machinery (González et al., 2008). In this sense, it was suggested
that although GK is expressed in about 40% of GI neurons it may also
hold a regulatory role in those neurons (see above), and therefore
AMPK activity would be the most important molecule involved in GI
neuronal glucose sensing (Jordan et al., 2010). Mechanistically, it is
possible that AMPK activation in GI neurons leads to the phosphor-
ylation and thereby inactivation of plasma membrane Cl − and/or
other ion channels (Song and Routh, 2005), leading to depolarization,
and activation of the neuron. Consistently, studies of Murphy et al.
(2009) suggest that in VMH GI neurons activated AMPK phosphor-
ylates neuronal nitric oxide synthase (nNOS) leading to nitric oxide
production, which in turn amplifies AMPK activation and thereby
closure of the Cl − channel.
The data reported by Glowik et al. (1997) are of special interest
since they discovered and characterized glucose-induced inhibition
neurons present in the X organ of the crab (Cancer borealis). Similar to
the orexin neurons described above, glucose induced large-amplitude
membrane hyperpolarization which was due to activation of K +-
selective currents with leak-like (voltage-independent) properties.
The authors concluded that glucose-stimulated K + channels probably
form a component of the current in the crab cells, and speculated that
they are activated by glucose binding to a plasma membrane site that
shows different sensitivity and specificity for sugars than known
passive glucose-transport systems.
3.3. Functions involving glucosensing cells in mammals (Table 1)
3.3.1. Food intake
The theory of glucostatic regulation of food intake proposed by
Mayer (Mayer, 1953) is still under debate. Accordingly, reductions in
glucose availability during hypoglycemia produce hunger in humans
(Heller and Cryer, 1991) and stimulate food intake in rats (Bernardis
et al., 1981). In addition, glucoprivation at both systemic (Smith and
Epstein, 1969) and central (Miselis and Epstein, 1975) levels also
enhances food intake in rodents. However, data supporting a satiating
effect of raised blood or brain glucose levels are conflicting (Levin
et al., 2004a,b). Recent findings also question in mammals the
physiological relevance of hypothalamic glucose-sensing in the
regulation of feeding (Blouet and Schwartz, 2010). Thus, although
very low or high levels of glucose availability may alter feeding in
mammals, it is unclear whether acute changes of glucose within the
physiological range may have any primary role in regulating normal
meal initiation or termination. In contrast, the levels of basal glucose
may participate in the state-dependent regulation of food intake as
one of many signals integrated by metabolic sensing neurons at the
level of their membrane potential (Levin et al., 2004a,b). Glucose
infusion into the hypothalamus leads to a decrease in food intake and
body weight in rodents (Davis et al., 1981; Kurata et al., 1986),
suggesting that one of the functions of hypothalamic glucosensing is
appetite control. Furthermore, NPY and POMC hypothalamic neurons
(Clark et al., 1984; Marx, 2003) have been shown to respond to
changes in glucose concentration (Muroya et al., 1999; Ibrahim et al.,
2003; Burdakov et al., 2005). On the contrary, glucoprivic feeding was
recently suggested to involve glucose-sensing neurons in the VMH,
since it was reduced by blockade of GK in VMH (Dunn-Meynell et al.,
2009). Moreover, at least some of the orexin/hypocretin neurons
control key peripheral mediators of glucose homeostasis via the
sympathetic nerves promoting shuttling of glucose from liver to
muscle (Yamanaka et al., 2003). Thus, it is possible that the glucose
inhibition of orexin/hypocretin cells provides negative feedback in a
circuit that consists of orexin/hypocretin cells, sympathetic nerves,
the pancreas, and the liver (Karnani and Burdakov, 2011). A role for
central GLUT2, GK and KATP in feeding regulation has been suggested
in several studies. Thus, food intake is inhibited by blocking or
suppressing GLUT2 (null mice and antisense oligonucleotides) (Wan
et al., 1998), GK (pharmacological inhibition) (Sanders et al., 2004)
and KATP (inactivation of the Kir6.2 gene) (Miki et al., 2001). Although
the role of glucose in the regulation of feeding was evaluated in many
studies (Mayer, 1953; Mobbs et al., 2005), changes in blood glucose
may not be a critical primary regulator of ingestive behavior, and it is
likely that glucose levels play a role in altering the overall activity of
metabolic sensing neurons involved in ingestive behavior and energy
homeostasis (Levin, 2007). In glucosensing neurons glucose may act
as one of several metabolic and hormonal regulators of their
membrane potential and final activity (Levin, 2006).
As it receives blood from the whole of the gut, the portal vein
appears to be a key site in the initial sensing of plasma glucose
concentrations. Several studies have established that infusions of
glucose into the portal vein can induce a decrease in spontaneous food
intake or acquisition of food preferences (Tordoff and Friedman,
1986). Therefore, it has been hypothesized that activation of the
portal glucose signal plays a key role in the decrease of hunger that
takes place under postprandial conditions. However, various other
studies have shown that portal delivery of glucose does not determine
termination of an ongoing meal but, instead, determines the size of
the next meal (Strubbe and Steffens, 1977). This strongly suggests
that the portal glucose signal is related to satiety phenomena, but not
satiation phenomena (Delaere et al., 2010).
3.3.2. Counterregulation to hypoglycemia
A fall in blood glucose levels induces a rapid counterregulatory
response to restore normoglycemia that involves the activation of
glucagon secretion from pancreatic α-cells and catecholamines from
the adrenal glands, as well as the activation by the autonomic nervous
system of hepatic glucose production (Marty et al., 2007). The VMH is
especially important for the counterregulatory response, as local
 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
glucoprivation results in pancreatic glucagon secretion (Borg et al.,
1995) and glucose infusion into the VMH suppresses glucagon
secretion in response to falling blood glucose (Borg et al., 1997).
However, the hindbrain also participates in the coordination of the
counterregulatory response, since glucoprivation in the brain stem
produces sympathoadrenal activation in decerebrated rats (DiRocco
and Grill, 1979). In humans, several studies also have been conducted
on this subject, showing that hypoglycemia activates glucosensing
brain areas including the hypothalamus, brainstem, anterior cingu-
lated cortex, uncus, putamen, medial frontal gyrus, and posterior
cingulated (Tesfaye and Seaquist, 2010).
To link particular glucosensing cells with the physiological responses
that control, molecular markers of these cells are required. The
involvement of central GLUT2-dependent glucosensors in the counter-
regulatory response was demonstrated in null mice for GLUT2, which
present an exaggerated glucagon secretion (Marty et al., 2005).
Hypothalamic GK participates in glucagon secretion, since its local
inhibition depresses the hyperglycemic response induced by glucopriva-
tion, and is restored when GK expression is normalized (Sanders et al.,
2004). The involvement of KATP channel in central glucose counter-
regulation is apparent since channel inactivation (Miki et al., 2001) or
inhibition (Evans et al., 2004) impairs while activation amplifies
(McCrimmon et al., 2005) the counterregulatory hormone responses to
hypoglycemia in rats.
3.3.3. Insulin secretion
Pancreatic β-cells act as glucosensors in mammals by adjusting
insulin output to the prevailing blood glucose level. Glucose uptake and
further metabolism are essential events for β-cells to secrete insulin in
response to increased plasma glucose (Newgard and McGarry, 1995;
Prentki, 1996). Due to the high Km of both GLUT2 and GK, the rate of
glucose utilization by β-cells increases across a wide spectrum of
physiological glucose levels and because of that the insulin secretory
response in β-cells is proportional to glucose concentrations within that
range (Nolan and Prentki, 2008). Thus, the glucosensing mechanism of
the β-cell is essential for the maintenance of glucose homeostasis
(MacDonald et al., 2005). This is exemplified by the role that β-cell plays
in the pathogenesis of type 2 diabetes mellitus, where a relative defect in
the insulin response to glucose is (at least) as important as peripheral
insulin resistance in the development of the disease (Ashcroft and
Rorsman, 2004). In stark contrast to all other mammals, murine rodents
express two insulin genes (Shiao et al., 2008) — a situation commonly
found in teleosts (Mommsen et al., 2002; Irwin, 2004). Surprisingly, the
interactions between glucose sensors and such multiple insulin genes
(expression, synthesis, release, etc.) remain to be studied.
3.4. Glucosensing neurons as metabolic sensors: interactions with other
nutrients
Unlike most neurons that utilize glucose and other metabolites to
fuel the metabolic demands associated with their activity, glucosen-
sing neurons use glucose as a signal to alter their activity (see
Section 3.3.). However, many of these neurons also respond to a
variety of metabolites from the periphery which relay to them the
metabolic status of the body (Minami et al., 1990; Song and Routh,
2005), and for this reason, they have been called “metabolic sensing
neurons” (Levin, 2002). In such neurons glucose acts as one of several
metabolic regulators of their membrane potential and final activity,
some of them responding to changes in ambient fatty acids (Wang
et al., 2006), lactate (Song and Routh, 2005) or ketone body (Minami
et al., 1990) levels by altering their activity. Indeed, it was previously
shown based on the metabolic coupling between astrocytes and
neurons (Magistretti et al., 1999) that the astrocyte-neuron lactate
shuttle (ANLS) plays a critical role for glucosensing function (Lam
et al., 2005), since lactate is sensed as a metabolic signal to regulate
the activation of glucosensing neurons (Marty et al., 2007).
131
The notion of interactions between glucose and lipid sensing in the
pancreatic tissue is clearly represented by the fact that elevated fatty
acid concentrations are able to modify the secretory response to
glucose (Sako and Grill, 1990; Zhou and Grill, 1994). Moreover, free
fatty acids have been demonstrated to decrease the expression of key
components of the glucose-signaling pathway such as GLUT2 and GK
(Gremlich et al., 1997). At the central level, recent data revealed an
interaction between glucose and fatty acids to regulate oleic acid
sensing in neurons of these hypothalamic nuclei, implying that
subtypes of lipid-sensing neurons are activated or inhibited, depend-
ing on the hypo-, normo- or hyperglycemic status (Wang et al., 2006;
Le Foll et al., 2009). The role played by the reactive oxygen species
(ROS) signaling in glucose and lipid sensing by AgRP/NPY and POMC
neurons is of special interest. Thus, Andrews et al. (2008) observed
that during positive energy balance, GE-POMC neurons accumulate
ROS as a result of increased glucose utilization. In a contrary manner,
during negative energy balance, when low glucose levels activate
AgRP/NPY neurons, elevated fatty acid oxidation in these cells does
not increase intracellular ROS levels. Thus, persistent high ROS levels
in active POMC neurons seem to favor satiety during energy surplus,
whereas in active AgRP/NPY neurons ROS production needs to be
buffered to allow for the appropriate orexigenic responses to energy
deficiency (Andrews et al., 2008; Horvath et al., 2009).
Although mammalian β-cells are most responsive to glucose as a
nutrient secretagogue (Prentki, 1996), they can be markedly affected by
the presence or absence of other nutrients, like amino acids and lipids
(Nolan et al., 2006; Newsholme et al., 2007). Thus, certain combinations
of amino acids are able to actively promote insulin secretion and augment
GSIS (Newsholme et al., 2007), promote glucagon secretion from α-cells,
increase ATP production (Sener and Malaisse, 2002) or depolarize plasma
membrane (Nenquin et al., 2004). The interaction between amino acid
metabolism with glucose and lipid metabolism is likely to be of
importance in regulating appropriate insulin secretion after a mixed
carbohydrate/lipid/protein meal (Nolan and Prentki, 2008). Although
free fatty acids do not stimulate insulin secretion in the absence of
glucose, there is substantial evidence that they are essential for GSIS to
occur (Nolan et al., 2006).
3.5. Hormonal interaction with the glucosensor system
Both GE and GI neurons are responsive to key peripheral feeding
hormones. Inhibition (Spanswick et al., 1997) and activation (Muroya et
al., 2004) of GE neurons were described in mammals after leptin
administration, suggesting that different subpopulations of these
neurons present opposite physiological responses and functions. In
addition, leptin has been shown to inhibit the majority of GI neurons
(Mountjoy and Rutter, 2007). In contrast, ghrelin, in common with the
action of low glucose concentrations, activates Arc GI neurons (Kohno et
al., 2003), while insulin inhibits VMN and Arc GE neurons via activation
of KATP channels (Spanswick et al., 2000). Thus, KATP channel activation
might represent a mechanism allowing for integration of hormonal and
nutrient (glucose)-dependent modulation of glucosensing in the
hypothalamus. This notion is supported by the finding that insulin
attenuates the ability of VMN GE neurons to sense decreased glucose via
the activation of PI3 kinase and KATP channels (Cotero and Routh, 2009).
Neuropeptides involved in controlling feeding have also direct effects on
glucosensing neurons: both orexins (Muroya et al., 2004) and NPY
(Wang et al., 2004) have been shown to inhibit GE neurons, while the
POMC peptide product, α-melanocyte stimulating hormone, activates
them (Wang et al., 2004).
3.6. Pathological intolerance: diabetes. Is glucose intolerance in
mammals related to glucosensing?
Deterioration of β-cell function contributes to the progression of
type 2 diabetes (Kahn, 2000). As well, several monogenic forms of
 132
Table 1
Summary of physiological functions in vertebrates related to glucosensing systems.

S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
Insulin secretion Food intake Glucose homeostasis

Fish –Glucose stimulates insulin secretion in several species –Hyperglycemia reduced food –Hypoglycemia strongly activates
–Main glucosensing markers found in some species intake related to changes in glucosensing markers glucosensing markers
–Insulin is stimulated by glycolytic intermediates –Glucosensing markers are –Central glucose administration
–Indirect evidence suggest the involvement of KATP, distributed in brain areas related to food intake control modifies liver glucose metabolism
though distal markers of the glucosensing markers are unknown –No glucosensing neurons were
electrophysiologically characterized
Amphibians –Glucose acts as insulin secretagogue No data available No data available
–Non-glucokinase dependent model
–Insulin secretion is dependent on glucose oxidation
–Weak response of KATP
–Involvement of calcium channels
Reptiles –Glucose stimulates biphasic insulin secretion No data available No data available
–Possible involvement of glucose transport
–Very high threshold when compared to mammals
Birds –Important increases in glucose result only in transient insulin secretion –Glucose transport in the brain No data available
–GLUT8 may have substitute GLUT2 in the system does not involve GLUT2
–GK present in the pancreatic tissue but unlikely involved in insulin secretion –Pharmacological activation of
–Lack of response to glycolytic intermediates (except for D-glyceraldehyde) central GK putatively reduces food intake
–Possible conservation of distal steps in the glucosensing system
–Very high threshold when compared to mammals
Mammals –Glucose-stimulated insulin secretion is initiated by glucose uptake –Glucosensing neurons are divided in glucose-excited –Glucosensing neurons involved in the
through GLUT2 and phosphorylation by glucokinase. Glucose 6-phosphate is and glucose-inhibited neurons depending on their response to glucose. counterregulation to hypoglycemia —
then metabolized through the glycolytic pathway and activates mitochondrial –Glucosensing neurons are found in brain areas involved in food Glucosensing neurons participate in the
metabolism to generate coupling factors. ATP is the best described of those intake, where orexigenic and anorexigenic neuropeptides are co-expressed activation of the nervous and endocrine
factors, and a rise in cytoplasmic ATP/ADP concentration leads to closure of KATP pathways involved in restoring normoglycemia
channels, depolarization of the plasma membrane and opening of voltage-sensitive after hypoglycemic episodes.
L-type Ca2+ channels. A KATP-independent pathway has been also described.
 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
diabetes are directly linked to the presence of defects in the
glucosensing machinery of the β-cell (Porter and Barrett, 2005) and
type 2 diabetes has been linked to polymorphisms in the glucose
signal transduction machinery (Ashcroft and Rorsman, 2004). Thus,
heterozygous inactivating mutations of GK cause a subtype of
maturity onset diabetes of the young, for which there are more than
190 published mutations (Porter and Barrett, 2005). The defect in GK
leads to a rise in the plasma level of glucose at which insulin is
triggered, and patients experience raised fasting glucose levels
throughout life (Gloyn, 2003).
On the other hand, Cranston et al. (2001) studying type 1 diabetes
patients shown that impairment in central glucosensing and
neurotransmission may constitute the primary contributor to hypo-
glycemia unawareness.
4. Glucosensing: the emerging picture in fishes (Fig. 3)
4.1. Why glucoregulate? Why is glucose important for carnivorous fish
species?
In elasmobranchs the tolerance to hypoglycemia (DeRoos and
DeRoos, 1979) questions the necessity of central glucose sensing
mechanisms involved in counterregulatory responses though the
responses in insulin release to changes in glucose levels (Anderson
et al., 2002) pointed to the existence of glucosensing-dependent
mechanisms involved in insulin release in the pancreas. In any case,
there are no studies available regarding these topics in elasmobranchs
yet and certainly they deserve attention in the future. Therefore the
next sections are mostly focused on teleost fish.
Teleost fish in general are unable to rapidly lower circulating
glucose levels following a glucose load or a high carbohydrate meal,
thus leading to the interpretation that fish are glucose intolerant
(Moon, 2001) (see Section 2). Even considering that fish carbohydrate
metabolism (especially in carnivorous species) seems to play only a
subordinate role to lipids and proteins (Hemre et al., 2002),
carbohydrates are key to the metabolism of all fish species. Then,
why not regulate protein metabolism or lipid instead of glucose
metabolism? The answer is not known in fish yet. In mammals,
glucose is the fuel substance used by most tissues under normal
circumstances to release needed energy, and when necessary, glucose
is readily synthesized from non-carbohydrate sources. Thus, glucose is
the preferred (if not compulsory) substrate employed by certain
tissues for energy purposes, like brain, retina, red blood cells, and
adrenal medulla. In teleosts, some evidence also pointed to the use of
glucose as a main substrate in several tissues such as red cells in the
sea raven (Hemitripterus americanus) (Sephton et al., 1991), or brain
in rainbow trout (Oncorhynchus mykiss) (DiAngelo and Heath, 1987).
Glucose is also critical for the maintenance of several tissues in
cyclostomes like lamprey (Petromyzon marinus) (Foster et al., 1993)
and Atlantic hagfish (Myxine glutinosa) (Sidell et al., 1984). Moreover,
glucose levels fluctuate in response to environmental challenges
which together with its necessity for the function of different tissues
including brain (Soengas and Aldegunde, 2002), suggest that glucose
may function as a metabolic signal coordinating physiological
responses to maintain homeostasis within the animal.
4.2. First indirect evidence of glucose detection in fish
Several studies in fish suggest the possible existence of brain
glucosensors (see Table 2). These studies found that: 1) experimentally
induced hyperglycemia led to decreased food intake, whereas increased
food intake occurred after intracerebroventricular treatment of rainbow
trout with 2-deoxyglucose (2-DG, a non-metabolizable carbohydrate)
(Soengas and Aldegunde, 2004); 2) parallel changes exist between brain
glycogen levels and GK activity and expression, and changes in plasma
glucose levels in rainbow trout (Polakof et al., 2007a); 3) rainbow trout
133
refed for 7 days after 14 days of food deprivation displayed increased GK
activity and expression in both hypothalamus and hindbrain and
increased glycogen levels at the same time as plasma glucose levels
increased (Soengas et al., 2006); 4) glucose use in Myoxocephalus
scorpius brain increased under hypoglycemic conditions (MacCormack
and Driedzic, 2007); and, 5) changes in GK activity/expression as well as
in glycogen levels occurred in hypothalamus and hindbrain of rainbow
trout when subjected to changes in circulating glucose levels (Polakof et
al., 2008a,b,c). Each of these results is consistent with the response of the
mammalian glucosensor, at least in GE neurons (see Section 3.2.1.).
Several lines of evidence also suggest the existence of a
glucosensor system linked to insulin secretion in the teleost
pancreatic cells (see Table 2). Although amino acids are the most
potent insulin secretagogues in fish (Navarro et al., 2002; Andoh,
2007), in almost all in vivo and in vitro studies carried out to date,
glucose was able to stimulate the release of insulin (Epple et al., 1987;
Mommsen and Plisetskaya, 1991). Besides the ability of glucose to
stimulate insulin secretion in fish, other indirect evidence supports
the existence of a glucosensor system in the fish pancreas (most
studies were carried out in Brockmann bodies, BB). These include:
1) the presence of GK in Atlantic halibut (Hippoglossus hippoglossus)
BB (Tranulis et al., 1997); 2) the findings that 2-DG and mannose
stimulate insulin secretion in channel catfish (Ronner and Scarpa,
1987; Ronner, 1991); 3) the finding by Ronner and Scarpa (1987) that
K + (despite the high concentrations used in their experiments)
induces a pronounced insulin secretion in channel catfish (Ronner
and Scarpa, 1987); and, 4) changes in GK activity/expression, Glut2
expression, and glycogen levels in rainbow trout BB when subjected
to changes in glucose (Polakof et al., 2007b).
Taken into account these findings, several studies carried out in
recent years provided for the first time direct evidence regarding
glucosensing capacity in fish (see below).
4.3. Central glucose detection
4.3.1. Fish brain as a glucose consumer
Although fish brain can utilize other fuels besides glucose,
including ketone bodies, lactate, fatty acids or amino acids (Soengas
and Aldegunde, 2002), it has the highest glucose utilization rates per
unit weight of all tissues examined in rainbow trout, with rates
exceeding those reported in the rat (Washburn et al., 1992).
The main distinction between piscine systems and the mammalian
situation is an enormous tolerance of fish to hypoglycemia. Some
teleosts survive after the administration of pharmacological doses of
insulin reaching blood glucose concentrations close to undetectable
without any observable symptoms of neural disorders (Mommsen
and Plisetskaya, 1991). However, when active fish that contain
relatively low levels of brain glycogen are made hypoglycemic,
convulsions and death, similar to the mammalian situation do occur
(Leibson, 1973). To explain these contrary reports, it was pointed out
by some authors that this lack of ill effects of extreme hypoglycemia in
fish is based on their primary dependence on protein and lipid
metabolism, and that glucose requirements of tissues like the brain
are exceptionally small. However, a reduced importance of glucose
metabolism in brain conflicts with the findings in several studies
addressing important amounts of glycogen in brain tissue not only in
teleosts but also in cyclostomes (Schmidt and Wegener, 1988; Foster
et al., 1993; Plisetskaya et al., 1993), which are certainly much higher
than those reported in other vertebrates.
In addition, glucose-6-phosphatase activities were reported in
brain of several fish species (Rovainen, 1970; Plisetskaya et al., 1985;
Polakof and Songas, personal communication). Thus, it is possible that
fish brain endowed with endogenous carbohydrate stores is to a
certain extent autonomous from glucose supplied through the blood,
especially if the metabolic challenge is of limited duration.
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S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
Table 2
Evidence regarding presence of glucosensing components n different tissues (brain, liver or pancreas) of vertebrates with emphasis on differing nutritional habits.

Glucosensing components

GLUT2 Glucokinase Glycolytic pathway KATP channel Calcium channel

Mammals Herbivorous GK is present in sheep (Ovies aries) (liver) (1), and central (but not peripheral), glucosensors regulates luteinizing hormone secretion (40)
β cells in rabbit Rabbit (liver and β cells) (8, 54) Insulin release is accelerated Tolbutamide induces (71) and Channel blockers do not affect
(Oryctolagus cuniculus) are Cururo (Spalacopus cyanus) by GLU and MAN, but not by GAL, diazoxide abolishes (37) insulin insulin secretion in rabbit (41)
permeable to glucose (8) Coypu (Myocastor coypus) 3-OMG, FRU, ribose, 2-DG, secretion in rabbits
(liver) (7) NAGA and MH in rabbits (8)
Omnivorous Rodents, man: Rodents, man Rodents, man: Rodents, man: Rodents, man:
β cells, liver (64), and brain (63) (brain, β cells, liver) (34) β cells and brain β cells, liver and brain (35) β cells (33) and brain (30)
Hamster (55)
(Mesocricetus auratus),
Akodon (Akodon olivaceus),
Pig (Sus scrofa) (7) and
Guinea pig (Cavia porcelus) (59) (liver)
Carnivorous – Cat (60) and dog (liver) (32) 3-OMG stimulates and MH inhibits Kir6.2 and SUR1 are expressed in Channel blockers inhibit insulin
insulin secretion in dogs (31) canine β cells (12) secretion in dogs
Diazoxide inhibits (29) while (21)
sulfamides and K+ stimulate
insulin secretion in dogs (27, 46)
Birds Herbivorous – Goose (Anser anser) liver (67) – – –
Omnivorous GLUT2 is expressed in liver Chicken (liver, pancreas) (3) MH produces hyperglycemia in Tolbutamide and K+ induce insulin Calcium elevates
(not in brain) (70), while GLUT8 Duck (Cairina moschata x Anas chicken (58) secretion and hypoglycemia in insulin secretion
was found in plathyrhynchos) (liver) (2) GA induces small insulin secretion chicken (10, 50) in chicken
pancreas and brain of chicken (56) Japanese quail (Coturnix coturnix while MAN does not initiate insulin Sulfonylureas do not stimulate (28, 39)
japonica) (liver) (69) release in chicken (49) insulin secretion in ducks (27)
Pigeon (Columba livia) and
Mallard (Anas platyrhynchos) (59)
GKA inhibits food intake in chicken
(brain GK?) without altering insulin
secretion (non-functional pancreatic
GK?) (74)
Carnivorous – Barn owl (liver) (38) – Sulfonylureas do not induce insulin –
secretion in penguins (9)
Reptiles Herbivorous – Chilean turtle (liver) – Poor effect of sulfonylureas on KATP –
(Geochelone chilensis) (7) in turtle CNS (25)
Carnivorous GLU, arginine and leucine stimulate insulin secretion in the green anole (Anolis carolinensis) (47, 48)
Table 2 (continued)
Glucosensing components

GLUT2 Glucokinase Glycolytic pathway KATP channel Calcium channel

Amphibians Carnivorous – Toad (Bufo arenarum)
(β cells) (16)
Chilean frog
(Calyptocephalella caudiverbera)
Clawed frog (Xenopus laevis)
Horned frog (Ranita cornuda) Axolotl
(Ambistoma mexicanum) (liver) (7)
Glucose stimulates insulin secretion in the grass frog (Litoria raniformis) and A. means (19, 63)
Teleost Herbivorous Tilapia (GLUT1 brain) (72) Tilapia (pancreas) (26)
Omnivorous – Carp and
Killifish (Fundulus heteroclitus) (liver)
(5, 42)
D-GLU, GA, MAN, FRU and DHA
stimulate insulin secretion, while
GAL, 2-DG, iodoacetate,
dinitrophenol or L-glucose do not
(14–17)
GLU stimulates insulin secretion in
tilapia (23)
GLU, 2-DG and MAN, but not GAL
increase insulin secretion in channel
catfish (52, 53)
GLU induces insulin secretion in
carp (18)
K+ and tolbutamide increase, but Nifedipine blocks insulin
diazoxide and BPDZ44 inhibit secretion in toad (15)
insulin secretion in toad (15, 17) Calcium stimulates insulin
Tolbutamide increases insulin secretion in A. means (19)
secretion in Amphiuma means (19)
– –
K+ induces a pronounced insulin –
secretion in channel catfish
(53)

Carnivorous Trout (liver, pancreas, brain)
(43, 61)
Cod (liver) (22)
Sea bass (brain) (75)
and zebrafish (liver, brain) (76)
GLUT2 blockade decreases
the glucosensing response
in rainbow trout brain
but not in pancreas (44)
Invertebrates Cancer borealis
Trout (liver, pancreas, brain) (5, 43)
Seabream, Atlantic salmon, zebrafish,
perch, Senegalese sole
(Solea senegalensis),
European sea bass, rainbow smelt
(Osmerus mordax) (liver)
(5, 6, 11, 13, 66)
Atlantic halibut (pancreas) (65)
Zebrafish (brain, liver) (20, 51, 78)
GLU induces insulin secretion in
European eel (24),
anglerfish (Lophius americanus) (36),
brown trout (4),
rainbow trout (68), yellowtail (18)
and red seabream (18)
MAN and GLY increase the
glucosensing response rainbow trout
brain but not in pancreas (44)
KATP is expressed in brain of rainbow
trout and zebrafish (45, 73),
in rainbow trout pancreas (45)
and in Atlantic salmon (*)
The KATP channel is regulated as in
mammals in rainbow trout brain
but it does not in pancreas (44)
Presence of glucose-stimulated K+
channels in neurons of the X-organ
S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
VDCC is expressed in zebrafish
pancreas and brain (57)
Nifedipine does not inhibit the
glucosensing system in trout
brain but it does in pancreas (44)

References: (1) Ballard et al., 1972; (2) Berradi et al., 2004; (3) Berradi et al., 2005; (4) Blasco et al., 2001; (5) Blin et al., 1999; (6) Borrebaek and Christophersen, 2001; (7) Cárdenas et al., 1979; (8) Coore and Randle, 1964; (9) Chieri et al.,
1972; (10) Datar et al., 2006; (11) Dias et al., 2004; (12) Donley et al., 2005; (13) Enes et al., 2006; (14) Francini and Gagliardino, 1998; (15) Francini and Gagliardino, 1995; (16) Francini et al., 2000; (17) Francini et al., 1997; (18) Furuichi and
Yone, 1981; (19) Gater and Balls, 1977; (20) Gonzalez-Alvarez et al., 2009; (21) Gristwood et al., 1992; (22) Hall et al., 2006; (23) Hrytsenko et al., 2008; (24) Ince, 1979; (25) Jiang et al., 1992; (26) Joy, 2002; (27) Kajinuma et al., 1974; (28)
King and Hazelwood, 1976; (29) Kuzuya et al., 1974; (30) Levin et al., 2004a,b; (31) Lindskog et al., 1998; (32) Maccioni and Babul, 1980; (32) MacDonald et al., 2005; (34) Magnuson and Matschinsky, 2004; (35) Miki and Seino, 2005; (36)
Milgram et al., 1991; (37) Milner et al., 1975; (38) Myers and Klasing, 1999; (39) Naber, 1974; (40) Ohkura et al., 2000; (41) Patel et al., 1987; (42) Picard and Schulte, 2004; (43) Polakof et al., 2007b; (44) Polakof et al., 2007c; (45) Polakof et
al., 2008c; (46) Pørksen et al., 1996; (47) Rhoten, 1973; (48) Rhoten, 1974a,b; (49) Rideau, 1998; (50) Rideau and Simon, 1989; (51) Robison et al., 2008; (52) Ronner, 1991; (53) Ronner and Scarpa, 1987; (54) Salas et al., 1965; (55) Schuit et
al., 2001; (56) Seki et al., 2003; (57) Sidi et al., 2004; (58) Smith and Baranowski-Kish, 1976; (59) Stanley et al., 1984; (60) Tanaka et al., 2005; (61) Teerijoki et al., 2000; (62) Telib, 1968; (63) Thorens, 2001; (64) Thorens et al., 1988; (65)
Tranulis et al., 1997; (66) Tranulis et al., 1996; (67) Ureta et al., 1973; (68) Vega-Rubín de Celis et al., 2004; (69) Wals and Katz, 1981; (70) Wang et al., 1994; (71) Watanabe et al., 2002; (72) Wright JR, Jr. et al., 1998; (73) Zhang et al., 2006;
(74) Rideau et al., 2010; (75) Terova et al., 2009; (76) Castillo et al., 2009; (77) González-Álvarez et al., 2009; (78) Glowik et al., 1997; (*) Leong et al., 2010. GenBank accession number: NM_001140360; Diaz and Planas, 2006. GenBank
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In fish, food deprivation generally leads to decreased plasma
glucose levels in many species (Navarro and Gutiérrez, 1995). In the
mammalian brain, hypoglycemia associated with food deprivation
enhances glucose uptake the first days of fasting whereas a depression
in transport rates occurs over longer periods (Davson and Segal,
1996). However, in fish, induced hyperglycemia does not alter glucose
uptake into brain (Blasco et al., 1996), suggesting that fish brain could
be metabolically independent on the systemic glucose supply. In fact,
the capacity of brain to use exogenous glucose decreases during food
deprivation in salmonids (Soengas et al., 1996; Soengas et al., 1998).
In addition, in hypoglycemic salmonids (induced by insulin treatment
or food deprivation) glycogen levels decrease in brain, supported by
appropriate changes in glycogen synthase/glycogen phosphorylase
activity ratios (Soengas et al., 1996; Soengas et al., 1998; Polakof et al.,
2007b; Polakof et al., 2008a), which demonstrate that local glycogen
metabolism can sustain glucose demand in neural tissue indepen-
dently on changes in circulating glucose levels. Then, under conditions
of severe hypoglycemia or long periods of food deprivation, brain
glycogen stores are mobilized providing glucose to support brain
metabolism. This phenomenon has also been described in mammals,
where brain glycogen levels provide fuel for extended periods when
glucose supply is inadequate (Choi et al., 2003). In addition, trout and
human brains share other common metabolic behaviors, like the
ability to replenish glycogen stores after hypoglycemic episodes in a
“super-compensated” manner (Choi et al., 2003; Soengas et al., 2006).
Then, although it is possible that the brain adjust to alterations in
glycemia through more than one mechanism in fish as in mammals
(Choi et al., 2003), one of them could include changes in brain
glycogen metabolism. In this sense, even when fish brain shows a
relative metabolic independency on systemic blood supply under
normal conditions, brain glycogen may play a significant neuropro-
tective role in vivo. Among the possible mechanisms involved in the
initiation of the depletion of brain glycogen stores we may postulate
that the ability to detect both local and systemic changes in glucose
levels could play a significant role. In this context, several results in
rainbow trout suggest that changes in glycemia are detected in
discrete brain areas where changes in local glycogen levels also
occurred (Soengas et al., 2006; Polakof et al., 2007b). Accordingly, in
rainbow trout hypoglycemia has a stronger effect than hyperglycemia
on glycogen levels in brain (Soengas et al., 2006; Polakof et al., 2007b),
in agreement with the lack of changes in glucose uptake by the brain
in hyperglycemic brown trout (Blasco et al., 1996).
4.3.2. Components of the glucosensor system (see Table 2)
Brain glucose sensors are specialized neurones that respond to
fluctuations in local extracellular glucose concentration with changes
in their firing rate. Neuronal glucosensing requires glucose uptake by
the low-affinity glucose transporter GLUT2, glucose phosphorylation
by GK, and the subsequent metabolism of glucose to increase the
intracellular ATP/ADP ratio, this in turn leads to the closure of KATP
channels, membrane depolarization, calcium entry, and increasing
(GE neurons) neuronal activity and neurotransmitter secretion
(Marty et al., 2007). To date there are no evidences regarding the
presence of GE or GI neurons in fish brain and the results obtained
regarding changes in the expression/activity of putative components
of the glucosensor system described in fish brain (see below) are the
result of a mixed response of many neurones present in brain areas
like the hypothalamus or hindbrain. Another handicap of the present
studies in fish is the lack of electrophysiological studies characterizing
the response of neurones in these areas to changes in glucose
concentration to fully characterize changes in firing rate of neurons in
response to changes in glucose concentration. Moreover, in mammals
hypothalamic astrocytes can also detect changes in glucose availabil-
ity (Blouet and Schwartz, 2010) by interacting with glucosensing
neurons function through lactate production allowing the possibility
of glial cells also present in fish brain to be involved in glucosensing
capacity, a possibility that has not been assessed in piscine systems
yet.
Although several authors suggested that glucodetection could be
GLUT2-independent (Kang et al., 2004), the GLUT2 isoform is the
most probable carrier involved in neuronal glucosensing in mammals
(Thorens, 2001). In fish, GLUT2 gene expression was found in brain of
several species including zebrafish (Castillo et al., 2009), rainbow
trout (Soengas et al., 2006; Polakof et al., 2007b) and sea bass
(Dicentrarchus labrax) (Terova et al., 2009). GLUT2 expression
changed under different glycemic conditions in brain of rainbow
trout (Polakof et al., 2007b), but not in cod (Gadus morhua) (Hall et al.,
2006). Besides these studies in the whole brain, expression of GLUT2
has been characterized in several discrete brain regions of rainbow
trout (Polakof et al., 2007b). Additional evidence regarding the
presence of members of the GLUT carrier family in fish brain was
obtained from pharmacological blockade of glucose transport in vitro,
although this approach did not allow discrimination between
different isoforms (Polakof et al., 2007c). The expression of GLUT1
has been also detected in brain of several fish species including
zebrafish (Danio rerio) (Jensen et al., 2006), cod (Hall et al., 2005), sea
bass (Terova et al., 2009) and tilapia (Oreochromis niloticus)
(Hrytsenko et al., 2010), though in contrast to the situation of
GLUT2 (see above), there is no evidence regarding its possible
involvement in glucosensing. Thus, in the only study in which GLUT1
expression was assessed in tilapia brain in response to changes in
glucose levels no changes were observed (Hrytsenko et al., 2010).
GK is the key molecule for glucosensing and is considered the
major glucosensing marker in mammals (Magnuson and Matschinsky,
2004; Kang et al., 2006). GK gene expression and activity are found in
all vertebrate groups (see Table 2), and in mammals two alternative
promoters are present for GK which are responsible for transcription
initiation in a mutually exclusive manner in distinct tissues (Iynedjian,
2009). The upstream promoters drive the synthesis in non-hepatic
tissues including endocrine pancreas and brain (Iynedjian, 2009).
In fish, the studies carried out to date regarding this issue addressed
the presence of one promoter in liver of gilthead sea bream (Sparus
auratus) (Egea et al., 2007).
GK gene expression in fish whole brain was only studied in two
species: rainbow trout (Panserat et al., 2000) and zebrafish (González-
Álvarez et al., 2009). GK gene expression and activity were reported in
five discrete brain areas: telencephalon, hindbrain, midbrain, hypo-
thalamus (Soengas et al., 2006), and pituitary (Polakof et al.,
unpublished). Moreover, GK immunoreactivity was found in several
brain areas of rainbow trout (Polakof et al., 2009), including lateral
hypothalamus, anterior, posterior and lateral tuberal nucleus, that are
homologous to areas in mammals where glucosensing neurons are
found (Lynch et al., 2000). In addition, GK immunoreactivity was also
observed in other areas where the glucosensor system is probably
functional, such as the preoptic area and the oculomotor nucleus
(Polakof et al., 2009). GK was observed not only in neurons but also in
tanacytes (Polakof et al., 2009), whose potential glucosensing
capacity was also proposed in rodent brain (Rodríguez et al., 2005).
In contrast to data obtained in a carnivorous fish species like rainbow
trout, GK gene expression was not detected in the brain of an
omnivorous fish species like common carp (Cyprinus carpio) (Panserat
et al., 2000).
The activity and expression of GK was observed to increase in
hypothalamus and hindbrain of rainbow trout when subjected to
increased glucose concentration either in vivo or in vitro (Polakof et al.,
2007b,c). In zebrafish an increase in the amount of carbohydrates in
the diet did not result in changes in GK expression in the whole brain
(González-Álvarez et al., 2009) but this is not surprising considering
the diluting effect of sampling the whole tissue in that study thus
masking the possible effects located in specific brain areas.
The KATP channel plays a fundamental role in glucosensing linking
changes in glucose metabolism to cell membrane electrical activity
 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
(Burdakov, 2007; Hibino et al., 2010). There are limited studies
indicating the presence of KATP channel in the brain of non-rodent
species. In fish, the two components of the KATP channel, namely SUR
and Kir, are expressed in the brain of several teleosts, with sequences
highly similar to mammalian Kir6.2 and SUR1, including Kir6.-like,
and SUR-like in rainbow trout (Polakof et al., 2008c), Kir8 in Atlantic
salmon (Salmo salar) (Leong et al., 2010. GenBank accession number:
NM_001140360), and Kir6.3 and SUR1 in zebrafish (Zhang et al.,
2006). Moreover, indirect pharmacological evidence of the existence
of KATP channels in the brain of non-mammalian vertebrates was
obtained in turtle (Jiang et al., 1992) and rainbow trout (Polakof et al.,
2007c). In mammals, the genes encoding L-type calcium channels in
neurons are Cav1.2 and Cav1.3 (Namkung et al., 2001). In fish, the
presence of this type of calcium channel has not been fully studied,
and with gene expression of both Cav1.2 and Cav1.3 found only in
zebrafish brain (Sidi et al., 2004). Also, indirect evidence based on
pharmacological tests carried out with the L-type channel inhibitor
nifedipine suggests that this isoform of the Ca 2+ channel is not active
in trout brain (Polakof et al., 2007c).
4.3.3. Possible functions related to the glucosensing system (see Table 1)
4.3.3.1. Regulation of food intake. The theory of glucostatic regulation
of food intake proposed by Mayer (Mayer, 1953) is still under debate
(see Section 3.3.1.). In fish, as in other vertebrates, the main regulator
of food intake is the energy content of the meal, since fish eat almost
exclusively until acquiring an optimal energetic status (Boujard and
Médale, 1994; Morales et al., 1994). Among dietary components, high
levels of protein (Gurure et al., 1995) or lipids (Gélineau et al., 2001)
negatively affect food intake in fish. Dietary carbohydrates should
participate weakly in the regulation of food intake in fish. However,
several studies report decreases in food intake in fish fed with
carbohydrate-enriched diets, including European sea bass (Peres and
Oliva-Teles, 2002; Moreira et al., 2008; Enes et al., 2010), rainbow
trout (Kaushik et al., 1989), Atlantic salmon (Hemre et al., 1995),
catfish (Clarias batrachus) (Erfanullah and Jafri, 1998), and European
eel (Anguilla anguilla) (Suárez et al., 2002).
Several studies carried out in the last few years using rainbow
trout suggest that induced changes in glycemia, either through
pharmacological approaches or through feeding fish with diets
containing different amounts of carbohydrates, may modulate the
ingestive response in fish (Table 1). In a first study, decreased food
intake was observed after experimentally induced hyperglycemia,
whereas increased food intake was noticed in glucodeprived rainbow
trout (Soengas and Aldegunde, 2004). When trout were fed with
high-carbohydrate diets or were injected IP with glucose they showed
sustained hyperglycemia accompanied by decreased food intake,
whereas in trout fed a diet without carbohydrates or injected with
insulin a clear hypoglycemia accompanied by increased food intake
were observed (Polakof et al., 2008a, b). In these long-term studies, as
well as in previous shorter-term studies (Soengas et al., 2006; Polakof
et al., 2007b), hyperglycemia in trout was expressed in glucosensing
central areas (hypothalamus and hindbrain) by increased GK activity
and transcript levels, glycogen accumulation, and in some cases
augmented glycolytic potential, whereas converse changes occurred
during hypoglycemic episodes. These results reinforce the suggestion
that in response to high glucose levels the glucosensing system is
activated in fish brain, and such activation probably modulates the
decrease in food intake reported in these animals (Table 1), a response
similar to that found in birds (Denbow, 1999) and mammals (Seino
and Miki, 2003; Marty et al., 2007). In rainbow trout, gene expression
of the KATP channel is unaffected by high glucose levels (Polakof et al.,
2008c), although in vitro evidence suggests that pharmacologically it
works as in mammals (Polakof et al., 2007c). In addition, although the
glucosensing function is yet to be explored in other fish species,
137
Kir6.3/SUR1-containing channels exists in zebrafish (Zhang et al.,
2006).
How are changes in glucosensing parameters related to changes in
food intake? It is well known in mammals that neurons in
glucosensing areas produce peptides involved in the control of food
intake (Schwartz et al., 2000; Blouet and Schwartz, 2010). Thus, for
instance neurons from the mammalian arcuate nucleus in the
hypothalamus producing NPY/AgRP appear to be GI whereas neurons
producing POMC/CART appear to be GE (Dunn-Meynell et al., 2002)
resulting in increased expression of POMC and CART and decreased
expression of NPY and AgRP when glucose levels rise (Mobbs et al.,
2005). The mRNA of these genes has been detected in the brain of
different fish species in areas analogous to those characterized in
mammals (Cerdá-Reverter and Canosa, 2009), and GK protein is
present in specific brain areas related to food intake regulation and
energy homeostasis, some of them homologous to those known to
contain glucosensing neurons in mammals such as VMN, Arc, PVN and
LH in hypothalamus (Polakof et al., 2009). Therefore, the areas in
which GK and peptides involved in the control of food intake are
present are generally coincident, suggesting a functional relationship
between them. In this way, a recent study (Conde-Sieira et al., 2010)
demonstrated that the expression of several neuropeptides involved
in the regulation of food intake in glucosensing central areas
(hypothalamus and hindbrain) of rainbow trout is regulated by
changes in glycemia, in a way compatible with the effects of changes
in glucose levels on food intake already reported in the same species
(Polakof et al., 2008a, b). The most important changes observed were
the decreased mRNA levels of NPY and increased levels of CART and
POMC in the hypothalamus of hyperglycemic trout whereas in
hindbrain increased mRNA levels of CART and corticotropin releasing
factor (CRF) were also noticed. Moreover, stress induced by high
stocking density in the same species (Conde-Sieira et al., 2010)
induced marked changes in the expression of all peptides assessed in
the glucosensing areas of fish under normoglycemic conditions such
as increased mRNA levels of NPY and CRF as well as decreased mRNA
levels of POMC and CART in hypothalamus. In hindbrain, the
expression of the four neuropeptides NPY, POMC, CART, and CRF
decreased in stressed fish. All these changes in peptide expression
suggest that the decrease in food intake observed in fish under stress
(Wendelaar Bonga, 1997) may be related to the activation of
glucosensor systems through changes in the expression of these
peptides. The mechanisms through which stress is altering the food
intake response induced by the activation of the glucosensor systems
are not previously known but probably some of the factors involved in
the activation of the hypothalamic–pituitary–interrenal axis like CRF
are likely involved.
Evidence obtained in recent years in fish provided evidence for a
modulatory role of different peptides involved in food intake
regulation in the activity of glucosensor areas. Thus, leptin interacts
with the glucosensing system in rainbow trout both in vivo (Aguilar et
al., 2010) and in vitro (Aguilar et al., 2011). Leptin treatment in
rainbow trout induced in hypothalamus and hindbrain dose-dependent
changes in parameters related to glucosensing, through the JAK/STAT
and IRS-PI(3)K pathways. Central leptin administration reduces NPY
mRNA levels in the hypothalamus and telencephalon of goldfish
(Volkoff et al., 2003) or in the whole brain of grass carp (Li et al., 2010)
whereas in rainbow trout IP injection of leptin induced in hypothalamus
a transient reduction and elevation of NPY and POMC mRNA levels,
respectively (Murashita et al., 2008). Moreover, in a recent in vitro
study (Aguilar et al., 2011) significant dose-dependent effects of
leptin treatment in reducing NPY mRNA levels in hypothalamus of
rainbow trout were demonstrated which were apparently specific
for leptin using JAK/STAT and IRS-PI(3)K pathways. Considering the
orexigenic action of NPY in fish it seems that the anorectic effect of
leptin can be mainly mediated by the reduced expression of NPY
occurring in hypothalamus, and this expression can be related to the
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activation of the glucosensing system occurring simultaneously in
the same area.
Another food intake-regulatory peptide that could be involved in
the modulation of glucosensing systems is ghrelin. In fish ghrelin is
involved in hormone release (growth hormone, prolactin, insulin-like
growth factor-1), reproduction, gastrointestinal motility, and food
intake regulation (Kaiya et al., 2008). The effect of ghrelin treatment
on central glucosensing was recently assessed in rainbow trout
(Polakof et al., in press) where ghrelin stimulated the glucosensing
system in trout brain probably as a helper signal upon glucose arrival
in the postprandial state. This is strongly supported by the fact that
glucose stimulated both the brain glucosensing system (Polakof et al.,
2007b) and ghrelin secretion (Riley et al., 2009) in fish, and that
ghrelin also acts as a glucosensing activator in rainbow trout.
It is important to emphasize that all studies described to date in
fish either in vivo or in vitro were carried out using whole brain or
selected areas of the brain. As far as we are aware, there are no studies
in non-mammalian species carried out with isolated neurons
responding to changes in glucose levels. However, the results
obtained in studies to date point to the fact that certain aspects of
glucosensing are conserved across species, which may indicate that
these steps are essential for function. However, nature may have
derived more than one mechanism for sensing (Sweet and
Matschinsky, 1997) and this has to be addressed in future studies.
4.3.3.2. Counterregulation to changes in glycemia. In mammals, a fall in
plasma glucose levels is rapidly detected and triggers a sequence of
counterregulatory responses that mainly involve: (1) suppression of
insulin secretion; (2) release of counterregulatory hormones, which
rapidly promotes endogenous glucose production and limits periph-
eral glucose utilization; and, (3) subjective awareness of hypoglyce-
mia (McCrimmon, 2008). Both vagal and sympathetic outflow to the
pancreas and the liver are implicated in this regulation (Blouet and
Schwartz, 2010). Central glucose detection is implicated in the
pancreatic counterregulatory response to hyperglycemia. In fish,
many studies report that plasma insulin levels decrease and plasma
glucagon levels increase when fish are subjected to natural or
experimental deprivation of food (reviewed in Navarro and Gutiérrez,
1995). Moreover, insulin expression was also apparently enhanced
when zebrafish were exposed to high glucose levels (Jurczyk et al.,
2011). In rainbow trout, we demonstrated for the first time in fish,
that brain hyperglycemia (caused by intracerebroventricular injection
of glucose) elicits changes in hepatic carbohydrate metabolism to
counterregulate the high glucose levels detected in the CNS (Table 1)
(Polakof and Soengas, 2008). Although changes in insulin and
glucagon as well as central glucoprivation tested in this kind of
approach have to be carried out to elucidate the involvement of the
glucosensing system in this counterregulation process, evidence
obtained to date suggest that the detection of high glucose levels in
the CNS of fish is needed to modulate the hepatic response to changes
in glycemia, in a way similar to the mammalian model (Marty et al.,
2007). Furthermore, rainbow trout intracerebroventricular treated
with leptin (Aguilar et al., 2010), glucagon-like peptide-1 (GLP-1)
(Polakof et al., 2011a), and cholecystokinin (CCK) (Polakof et al.,
2011b) elicited changes in liver metabolism that could be involved in
the counterregulatory response to changes in glycemia and be
mediated through activation of the sympathetic system. The possible
involvement of the interrenal axis cannot be ignored since in a recent
study in rainbow trout hyperglycemia per se induced an increase in
plasma cortisol levels (Conde-Sieira et al., 2010).
Finally, recent studies carried out in rainbow trout provided
evidence for a hyperglycemic action of GLP-1 (Polakof et al., 2011a)
and CCK (Polakof et al., unpublished), which are at least in part is
related to the presence of an ancestral gut–brain axis in which GLP-1
and CCK are involved. Besides a global impact on glucose homeostasis,
both peptides also induced an activation of central glucosensing
systems. Thus, CCK seems to activate glucosensing system in response
to the increased glycemia and/or vagal/splanchnic afferents in
hypothalamus whereas in hindbrain a possible action through specific
CCK-1 receptors cannot be excluded. Furthermore, GLP-1 also
activates glucosensing systems either indirectly caused by hypergly-
cemia (hypothalamus) or likely through GLP-1R and/or vagal/
splanchnic afferences (hindbrain). All these processes result in
changes in metabolic parameters related to energy homeostasis
control, probably mediating the GLP-1- and CCK-induced inhibition of
food intake.
4.4. Peripheral glucosensing
4.4.1. Components of the glucosensing system (see Table 2)
Although many studies have been conducted regarding insulin
effects and secretion in teleosts, its mechanism of secretion is not fully
known. In the last years several lines of evidence helped to elucidate
partially the components involved in the mechanism of insulin
secretion. Thus, Hrytsenko et al. (2008) assessed in tilapia BB the
effects of glucose and arginine on transcription, translation and
secretion of insulin demonstrating that both glucose and arginine can
induce insulin secretion in a dose-dependent manner. Possibly, the
basal level of the insulin gene transcription in tilapia β-cells is high
and could be further stimulated by supraphysiological concentrations
of stimulators. In rainbow trout two different insulin-encoding
mRNAs named INS1 and INS 2 have been observed in BB and brain
(Caruso et al., 2008).
The first evidence of glucose transport into piscine pancreatic
tissue was reported in the toadfish Opsanus tau, where transport of D-
but not L-glucose suggested the presence of a stereospecific carrier
system for the transport of sugars across the β-cell membrane
(Cooperstein and Lazarow, 1969). Gene expression of a GLUT2 carrier
was described for the BB of rainbow trout (Polakof et al., 2007b),
which changed in response to variations in glucose levels (Polakof et
al., 2007b), indicative of a possible role of this carrier in glucosensing
capacity. Also in rainbow trout BB, cytochalasin B (a glucose carrier
blocker) decreased sugar transport across the cell membrane (Polakof
et al., 2007c). In contrast, GLUT1 expression did not change in BB of
tilapia in response to changes in glucose levels (Hrytsenko et al.,
2010) supporting the specificity of the GLUT2 response.
The presence of an inducible GK enzyme in pancreatic tissue of fish
was described initially in the Atlantic halibut (Tranulis et al., 1997),
and more recently in rainbow trout (Polakof et al., 2007b) and tilapia
(Joy, 2002). Strikingly, while trout GK activity is regulated by glucose
administration and dietary carbohydrates (Polakof et al., 2007b;
2008b,c), gene expression does not respond directly to glucose, but it
does to high carbohydrate diets (Polakof et al., 2008b,c), suggesting a
differential regulation for this enzyme at transcriptional and post-
transcriptional levels. The presence of GK in BB reinforces the central
role in glucose regulation of this tissue in fish, since in amphibians
(Francini et al., 2000), birds (Berradi et al., 2005) and mammals
(Schuit et al., 2001) GK is only expressed in glucose-sensitive tissues.
Although GK constitutes the flux determining step for glycolysis in
mammals (see above) where glucose-dependent ATP production acts
on the KATP channel and final insulin secretion, several glycolytic and
ATP-production intermediates may also regulate this glucosensing
system (MacDonald et al., 2005). In fish, just a few studies have
elucidated the involvement of the glycolytic pathway as part of the
glucosensor system in pancreatic tissues. In general, in a way similar
to that observed in amphibians (Francini and Gagliardino, 1998), birds
(Rideau, 1998), and mammals (Yang et al., 1999), the glycolytic
pathway is involved in the proximal events that transduce the
glucosensing signal from glucose phosphorylation to ATP production
in fish pancreatic tissue (see below) (Ronner and Scarpa, 1987;
Ronner, 1991; Polakof et al., 2007c).
 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
Although the first evidence of the presence of KATP channels in fish
BB was based on indirect tests, such as incubations of pancreatic tissue
with channel effectors (Ronner and Scarpa, 1987; Polakof et al.,
2007c), more recently gene expression of both Kir6.-like and SUR-like
components was reported in trout BB (Polakof et al., 2008c). Indeed,
gene expression of a KATP-like channel in trout BB together with its
recent description in dog (Canis familiaris) pancreas (Donley et al.,
2005), supports the presence of this glucosensor component in β-cells
of carnivorous vertebrates, since up to date, the channel had been only
described in omnivorous mammals (Miki and Seino, 2005).
Voltage-dependent calcium channels (VDCC) have to date been
only characterized in the pancreas of rodents (MacDonald et al.,
2005), where the isotypes involved in glucosensing are Cav1.2 and
Cav1.3, with Cav1.2 being the most important (Namkung et al., 2001;
Nitert et al., 2008). In fish, both Cav1.2 (Rottbauer et al., 2001) and
Cav1.3 (Sidi et al., 2004) gene expression was found in pancreas of
zebrafish. Moreover, the presence of VDCC in BB of rainbow trout has
been also suggested by pharmacological characterization (Polakof
et al., 2007c), with responses being similar to those obtained in
mammals (Gristwood et al., 1992), birds (King and Hazelwood, 1976)
and amphibians (Francini and Gagliardino, 1995).
4.4.2. Brockmann bodies: involvement of the glucosensor system in
insulin secretion (see Table 1)
In mammals, pancreatic β-cells secrete insulin in response to a
sensing mechanism triggered by an increase in extracellular glucose
above basal levels. In fish, the first hypotheses suggesting the existence
of a glucosensor system linked to insulin secretion were postulated by
Ronner and co-workers utilizing isolated perfused BB as a model for
studying pancreatic endocrine secretion (Ronner and Scarpa, 1982).
Although it is possible that other glucose carriers may exist in fish
BB, data available up to date strongly suggest the involvement of
GLUT2 carrier isoform in the glucosensor system that lead to insulin
secretion in fish, as occurs in mammals (Schuit et al., 2001). This
statement is also supported by the fact that GLUT2 is the only glucose
transporter normally detected in rodent β-cells (Thorens et al., 1988).
In rainbow trout, GLUT2 gene expression parallels in general changes
in glycemia either induced by dietary carbohydrates (Polakof et al.,
2008b; Polakof et al., 2008c) or glucose (Polakof et al., 2007b). Indeed,
the time necessary to achieve this induction is similar in fish and
mammals (Ferrer et al., 1993), in agreement with the role of glucose
as major regulator of GLUT2 gene expression in rodent β cells (Tiedge
and Lenzen, 1995). Although decreased expression (Unger, 1991)
occurs simultaneously with the loss of GSIS in mammalian models of
type 2 diabetes, this does not seem to be the case in rainbow trout
(diabetic-like metabolic behavior) that displays an efficient adapta-
tion to dietary carbohydrates (Polakof et al., 2008c).
The role of GK as a signal molecule for insulin secretion in fish was
initially hypothesized in channel catfish (Ronner, 1991). However,
incubation of rainbow trout BB with 2-DG showed that this molecule
was unable to induce GK and to activate the glucosensor system
(Polakof et al., 2007c) thus resembling the mammalian model, where
glucose cannot be replaced by 2-DG in glucosensing function
(Grodsky et al., 1963). Moreover, the key role of GK as a metabolic
limiting step for insulin secretion is evidenced in fish through several
in vitro approaches. Thus, sugars other than glucose that can be also
metabolized by GK (and therefore mimic glucosensing response in
mammalian β-cells) like mannose (Gasa et al., 2000), also elicited in
pancreatic tissue an increased glucosensing response in trout (Polakof
et al., 2007c), and insulin secretion in channel catfish (Ronner and
Scarpa, 1987; Polakof et al., 2007c). In contrast, non- or poorly-GK-
metabolized substrates, like galactose or fructose (Grodsky et al.,
1963) failed to stimulate insulin release in fish BB (Ronner and Scarpa,
1987). Similar to mammals (Tiedge and Lenzen, 1995), GK activity
and gene expression in trout seem to be under glucose control, with
both parameters changing in parallel with glycemia. Glucose alone
139
increases GK activity in trout BB both in vivo (Polakof et al., 2007b)
and in vitro (Polakof et al., 2007c), or after fish were fed with
carbohydrate-enriched diet (Polakof et al., 2008b; Polakof et al.,
2008c). Interestingly, in these studies changes in GK gene expression
were always less robust than those in GK activity, and GK gene
expression was only induced by dietary carbohydrates, and not by
glucose alone, which agrees with the predominant posttranscriptional
regulation of this enzyme in mammals (Chen et al., 1994). The
positive response of GK activity and expression to changes in plasma
glycemia in trout strongly suggests that this enzyme acts as a
glucosensor in pancreatic tissue (Fig. 3). However, the involvement of
GK on insulin secretion needs to be elucidated, since no studies are
currently available regarding changes in this enzyme and hormone
secretion in fish.
As mentioned above, GK is the flux determining step for glycolysis.
Early metabolic products of glycolysis in mammals mimic effects of
glucose to excite β cells leading to insulin secretion (Dean et al., 1975).
This characteristic of glycolytic metabolites is demonstrated in fish
(Ronner and Scarpa, 1987) However, not all glycolytic intermediates
are able to induce insulin secretion. Distal glycolytic metabolites have
no effect on insulin secretion in mammals (Newgard and McGarry,
1995; Noel et al., 1997; Lenzen et al., 2000). Similarly, glycerol, lactate
and pyruvate failed to activate the glucosensor system in fish (Polakof
et al., 2007c). The involvement of glycolysis generating metabolic
coupling factors to trigger insulin secretion was also apparent
employing proximal and distal glycolytic inhibitors. Compounds like
iodoacetate, dinitrophenol and 2-DG clearly inhibit insulin secretion
in amphibians (Francini and Gagliardino, 1998) and mammals
(Motoyoshi et al., 1998). Strikingly, in a study by Ronner (1991) 2-
DG acted as an insulin secretagogue in channel catfish, which is not
consistent with recent results obtained in rainbow trout (see above).
A tight coupling between glycolysis and mitochondrial oxidation
has been considered to be crucial for more distal steps in the signal
generation in β-cells, and thus increases in extracellular glucose
concentration induced a dose-dependent increase in the ATP/ADP
ratio closing KATP channels (Schuit et al., 2001). In fish, several lines of
evidence suggest that the involvement of the KATP channel in the
glucosensor system is linked to insulin secretion. Thus, K + is able to
induce insulin secretion in fish, although at high concentrations
(Ronner and Scarpa, 1987). In addition, pharmacological effectors of
the KATP channel in all vertebrates, like diazoxide and tolbutamide
(Chieri et al., 1972; Kuzuya et al., 1974; Francini et al., 1997), also
modulate the glucosensing system in trout BB (Polakof et al., 2007c).
However, the changes observed in that study were not conclusive,
since although diazoxide was able to inhibit the glucosensing
response, tolbutamide failed to elicit any change (Polakof et al.,
2007c). Gene expression of both components of the KATP channel in
trout, Kir6.-like and SUR-like, seems to be under glucose control, since
trout fed with a high-carbohydrate diet displayed an up-regulated
KATP channel gene expression (Polakof et al., 2008c). However, in
mammals glucose reduces (Moritz et al., 2001) and glucose
deprivation increases (Smith et al., 2006) gene expression and
synthesis of the channel, respectively adjusting them to maintain a
sustained response in insulin secretion. Although the basis for these
striking differences in both pharmacological and transcriptional
regulation of the KATP channel in trout with respect to mammals
remains unknown, these discrepancies could contribute to the
impaired insulin secretion in trout fed a high carbohydrate diet
(Capilla et al., 2003; Polakof et al., 2008c).
Finally, despite the key role of VDCC in insulin secretion in
mammals (MacDonald et al., 2005), no studies have been performed
in fish that clearly correlate calcium currents with insulin secretion.
However, preliminary evidence of this possible relationship was
found in rainbow trout after BB incubation with nifedipine, a calcium
channel blocker, which increased the glucosensing response (Polakof
et al., 2007c). This blocker completely counteracts insulin secretion
140 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
induced by calcium in amphibians (Francini and Gagliardino, 1995),
birds (King and Hazelwood, 1976), and mammals (Gristwood et al.,
1992), suggesting a similar role for L-type calcium channel in piscine
pancreatic tissue.
From the studies conducted to date, we can hypothesize that the
glucose intolerance experienced by carnivorous fish species may be
related, at least in part, to the inability to detect glucose in the
pancreatic tissues where insulin is normally secreted.
4.4.3. Glucosensing in intestine
Glucosensing capacities have been reported in mammals in
gastrointestinal tract cells, including enterocytes, enteroendocrine
cells (K and L), and enteric neurons (Gribble et al., 2003). Although
the mechanisms remain to be fully elucidated, some of the actors
involved in this function are likely the same as those present in the
glucosensing mechanism of pancreatic β-cells (Schuit et al., 2001),
including GLUT2, GK or KATP (Reimann and Gribble, 2002), while
others seem to be more gastrointestinal-specific, such as SGLTs (Díez-
Sampedro et al., 2003; Moriya et al., 2009).
There is evidence for the presence of putative glucosensors in the fish
intestine, including 1) an increase in glucose uptake presumably
through SGLT-1 in enterocytes of black bullhead (Ictalurus melas) fed
a diet rich in carbohydrates (Soengas and Moon, 1998); 2) an increase in
SGLT-1 expression in the intestine of rainbow trout fed a diet rich in
carbohydrates (Kirchner et al., 2008); and 3) changes in GLUT2
expression in intestine of zebrafish in response to changes in glucose
levels (Castillo et al., 2009). A recent study (Polakof et al., 2010)
provided evidence for the presence in the rainbow trout intestine of
glucosensing components, including GLUT2, SGLT-1, GK, SUR-like and
Kir-like transcripts. GLUT2 and SGLT-1 transcripts, and also SGLT-1
immunoreactivity were detected in enterocytes and occasionally in
enteroendocrine cells, which together with the fact that GK gene
expression and activity were enhanced in midgut of hyperglycemic
trout and that GK was localized in enteroendocrine cells, constitutes the
first evidence for a glucosensing function in putative enteroendocrine
cells of trout gut. Further support comes from the presence of KATP
components (SUR1 and Kir6.2) in a way similar to that found in both L-
and K-cells in mammals (Reimann et al., 2008; Parker et al., 2009).
Fig. 3. Glucosensing sites and components of the glucosensor systems characterized in rainbow trout to date, including those in brain regions, intestine and Brockmann bodies. The
different glucosensing markers found in each case are also indicated: GK (Glucokinase) GLUT2 (glucose facilitative transporter type 2), KATP (ATP-sensitive inward rectified K+
channel), Kir6.2 (pore-forming subunit of KATP), SUR (sulfonylurea receptor), SGLT (Na+-coupled glucose transporters).
Although the involvement of enteric neurons in the reception
of luminal glucose might be indirect (Pfannkuche and Gäbel,
2009), glucose is also reported to directly influence the activity of
enteric neurons in mammals though the mechanism underlying
this ability remains under debate. The fact that in trout gut GK
protein was also localized in equivalent cell types strongly sup-
ports the hypothesis of glucosensing neurons also present in trout
gut.
Finally, the sweet taste receptors are expressed in the luminal
membrane of gut enteroendocrine cells (Nakagawa et al., 2009) and
respond to changes in glucose levels using a glucose sensing
mechanism that was en recently evaluated in the trout intestine
(Polakof and Soengas, unpublished).
4.5. Other glucose sensing mechanisms
The glucosensing model consisting of GLUT2, GK, and K +-ATP
channels has been challenged in recent years in mammals by several
observations that in the hypothalamus 1) intracellular ATP levels do
not always increase in response to glucose in hypothalamic neurons,
2) KATP channels are not always required for glucose sensing in
hypothalamic nuclei, and 3) some, but not all, glucosensing
hypothalamic neurons express the three components. This has lead
to alternative mechanisms including the response of glucosensing
systems to other substrates such as lactate or the presence of
alternative mechanisms such as electrogenic entry of glucose through
SGLT-1 or non-transporting glucose sensing through the sweet-taste
receptor or LXR receptor (Blouet and Schwartz, 2010). It is quite
probable that the different other models co-exist in a tissue like the
hypothalamus, but the precise contribution of these mechanisms in
particular cells needs to be demonstrated (Blouet and Schwartz,
2010). In fish, the main focus has been to describe the presence of
glucosensors based on GLUT2, GK, and K +ATP channels (studies by
Polakof et al.) but there is preliminary evidence for the possible
presence of alternative glucosensing mechanisms as well.
In mammals it seems advantageous to uncouple glucosensing from
the energy status of the cell since ATP levels can be also monitored by
lactate according to the astrocyte-neuron lactate shuttle (ANLSH)
mechanism (Pellerin and Magistretti, 2004). In fish, studies in
 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
rainbow trout demonstrated that lactate is used in brain at rates
similar to those of glucose (Soengas et al., 1998), and that lactate
could mimic glucose effects on the glucosensing system (Polakof and
Soengas, 2008), which suggest that an ANLSH similar to the
mammalian model may exist in fish brain allowing the possibility of
the central glucosensing system based on GK being also mediated by
substrates other than glucose like lactate.
In mammals, LXR mediates a glucosensing pathway responding to
changes in glucose levels by altering the expression of glycolytic and
gluconeogenic enzymes (Mitro et al., 2007). In fish there is recent
evidence regarding the presence of LXR in liver and brain of trout and
salmon (Cruz-García et al., 2009) as well as in gilthead sea bream
adipocytes (Cruz-Garcia et al., 2011). However, no studies are
available in which the expression of LXR is assessed in response to
changes in glucose levels in any putative area presumably related to
glucosensing such as brain or pancreas. However, in preliminary
studies carried out in rainbow trout (Polakof and Soengas, unpub-
lished), a change in LXR expression was observed in intestine in
response to changes in glucose levels.
Recent evidence in mammalian hypothalamus indicate the
presence of a glucosensing mechanism independent of metabolism
based on the sodium-dependent glucose carrier SGLT-1 (González et
al., 2009). In this model, the generation of ATP is not an essential
prerequisite for sugar sensing since neurons can be metabolically
excited by glucose transported through SGLT-1. Since SGLT-1 is
expressed in fish tissues in which the classical metabolic GLUT2-GK
mechanism is present like brain and liver (and possibly others like
intestine), the existence of a non-metabolic glucosensor mechanism
in fish based on SGLT-1 cannot be excluded.
Finally, in mammals other glucosensor systems are based on sweet
taste receptors present in the tongue, and in pancreatic β-cells and
enterocytes (Nakagawa et al., 2009). Moreover, the signal transduc-
tion mechanism based on sweet-taste receptors is also found in
several regions of mammalian brain like hypothalamus, forebrain,
hippocampus (Ren et al., 2009) and the expression of the genes
involved changed in the hypothalamus in response to changes in
blood glucose levels. In fish, there are no studies characterizing the
presence of these receptors in any tissue proposed to be involved in
glucosensing. However, in preliminary studies carried out with
rainbow trout intestine the expression of components of the sweet-
taste receptor was observed to change in response to changes in
glucose concentration (Polakof and Soengas, unpublished).
5. Glucosensing in other vertebrate groups
There is evidence of a glucosensor system in pancreatic tissue of
amphibians, but central glucosensors have yet to be assessed. Among
the first indirect evidence of this phenomenon is one study in the
salamander Taricha torosa by Wurster and Miller (1960) who noted
that injection of glucose produced degranulation of the β-cells, while
the administration of alloxan (a β-cell toxic drug) did not affect
degranulation. Although some authors have stated that insulin is
produced by the endocrine pancreas in amphibians and is regulated
by plasma glucose levels in a manner similar to mammalian insulin
(Penhos and Ramey, 1973), later studies on this issue do not fully
support this contention. The laboratory of Francini and co-workers
developed a culture system of pancreatic tissue of the toad Bufo
arenarum and demonstrated that a glucosensing mechanism could be
involved in mediating the insulin secretion in this toad, and that the
insulin secretion process is not completely equivalent to that reported
in mammals (Francini and Gagliardino, 1995, 1998; Francini et al.,
1997; 2000). In their earlier studies they demonstrated indirectly the
existence of KATP channels mediating the GSIS in the toad pancreatic
tissue, based on the increased insulin secretion when the tissue was
incubated with KCl or tolbutamide (a potassium channel blocker)
(Francini and Gagliardino, 1995; Francini et al., 1997). However, the
141
dose used to block the channel was much higher than that reported in
mammals, suggesting a lower affinity of these channels to sulfonyl-
ureas, which therefore could imply differences in the glucosensing
system. In order to support these data, they also activated the KATP
channel using diazoxide, pyridothiadiazine or clonidine, which
reduced the insulin secretion (Francini et al., 1997), confirming the
presence of KATP channels in the amphibian pancreatic tissue,
probably integrated into a glucosensing system. In these studies
they also provided indirect evidence for the presence of calcium
channels since reduced insulin secretion was observed when the
tissue was incubated with calcium channel blockers like nifedipine
(Francini and Gagliardino, 1995) or tetraethylammonium (Francini et
al., 1997). Insulin secretion could also be increased with glucose
concentration, and this could be mimicked by glycolytic intermediates
like glyceraldehyde (Francini et al., 1997). Further support was found
after the incubation of the pancreatic tissue in the presence of
glycolytic inhibitors (iodoacetate) or blockers (dinitrophenol) which
also reduced the GSIS (Francini et al., 1997). Using non-metabolized
glucose analogs they arrived at the conclusion that, as in mammals
(see above), the GSIS in toads was relative to the potency of the sugar
used and to the oxidation of such substrate. Thus, in amphibians as in
mammals, the GSIS shows stereospecific properties and is highly
dependent on the ATP/ADP ratio after the oxidation of glucose
through glycolysis (Francini and Gagliardino, 1998). In turn, the
addition of dinitropehnol (which reduce the ATP/ADP ratio) resulted
in decreased insulin secretion (Francini et al., 1997). Finally, they
reported that the GSIS in amphibians differs from mammals in the
glucose phosphorylation step, in which the glucosensing marker GK is
involved. Although they did not measure phosphorylation activities,
using non-metabolizable glucose analogs like 2-DG (for HK) and
mannoheptulose (for GK) they showed that the GSIS decreased in the
toad pancreatic tissue (Francini et al., 2000). Moreover, the toad
presented higher HK/GK activity ratios, which does not agree with the
mammalian and piscine models, but would allow the production of
insulin at the low glucose circulating levels present in this animal that
average 2.7 mM. As a whole, the data obtained in amphibians, though
mostly obtained from a single species, strongly suggest that a
glucosensing system, although adapted to the physiological glucose
circulating levels of the group (the lowest among vertebrates), could
be involved in the GSIS.
Little is known regarding the existence of glucosensors in reptilian
tissues. All results available to date were published by Rothen in the
1970s using Anolis carolinensis. Thus, Anolis pancreatic incubations
secreted insulin when stimulated by glucose and they do in a biphasic
way, as reported in mammals (Rhoten, 1973). Indirect evidence also
comes from the presence of some of the glucosensing markers, such as
those involved in glucose transport (GLUT2) since cytochalasin B
treatment nearly doubled insulin secretion in a manner similar to that
described in mammals (Rhoten, 1974a). One of the main conclusions
drawn from Rhoten's studies is that the threshold for in vitro insulin
secretion in saurians, which was established at 11 mM, was two-fold
higher than that reported in mammals (Rhoten, 1974b). Thus, while
glucose circulating levels in Anolis are normally above 10 mM, the
response to glucose is the same as that of mammals, whose glycemia
is about 7 mM. Although this lower sensitivity to glucose can be
compared to that of diabetic mammals, it is also possible that this
could constitute an adaptation to the high circulating glucose levels
reported in reptiles, whereas amphibians was adapted to low levels.
While avian glucose circulating levels are much higher than those
of the rest of vertebrates (see Fig. 1 and above) supraphysiological
glucose concentrations are needed (30–40 mM) in vitro to stimulate
pancreatic insulin secretion (Basabe et al., 1975; Naber and Hazelwood,
1977; Honey et al., 1980; Colca and Hazelwood, 1981). In vivo, the
elevation of blood glucose levels in duck and chicken produced only
transient insulin responses (Langslow et al., 1970; Colca and Hazelwood,
1976). Moreover, this glucose insensitivity of the avian pancreas seems
142 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
to be also related to genetic background (Rideau et al., 1986).
However, this higher insulin release threshold for glucose in birds
when compared with mammals (2–3 times higher) may also reflect
an adjustment to the normal blood glucose levels found in birds. This
lack of sensitivity to glucose of the avian pancreas could certainly
suggest that either no glucosensing mechanism is present in this
vertebrate group or if present major differences exist in comparison
with the model described in mammals. For instance, GLUT2 was not
reported in chicken pancreas, where its function could possibly be
assumed by another isoform (GLUT8), although its involvement in
insulin secretion has to be proven (Seki et al., 2003). The differences
observed between mammalian and avian insulin secretion mecha-
nism could also be due to a rate-limited step on glycolysis. For
instance, although the main glucosensing marker (GK) was found in
the pancreatic tissue of the chicken (Berradi et al., 2005), a recent
study reported that a GK activator RO0281675 (see (Grimsby et al.,
2003)) caused hypoglycemia without altering significantly insulin
levels (Rideau et al., 2010). These findings are of major relevance for
the glucosensing model, as they strongly suggest that although GK is
present, its activation at the pancreatic level may not be related to
insulin secretion as in mammals. This hypothesis is further supported
by the fact that chickens are resistant to streptozotocin (Simon
and Dubois, 1980; Danby et al., 1982) that selectively inhibits GK
in mammalian pancreatic tissue (Lenzen, 2008). An alternative for
the GK function in the GSIS can be the other hexokinases. Smith
and Baranowski-Kish reported that the administration of manno-
heptulose (a selective HK inhibitor that does not inhibit GK) result-
ed in hyperglycemia in chickens (Smith and Baranowski-Kish,
1976). Moreover, while a very weak insulin response was found at
physiological concentrations of the glycolytic intermediate D-
glyceraldehyde, no effect of mannose alone was observed (Rideau
and Simon, 1989; 1992). Therefore, the glucose insensitivity of the
avian β-cells is not attributable to a rate limiting-step of glycolysis
before the triose phosphate step, but it does suggest major
differences in pancreatic avian metabolism. Thus, the metabolic
threshold that permits the switching of the β-cell from the resting to
the active state is much higher in chickens than in mammals (Rideau,
1998). On the contrary, while the initiation of insulin secretion
by fuel nutrient appears to be different in birds and mammals,
the membrane depolarization events seem to be conserved. Thus,
tolbutamide (a KATP channel blocker) (King and Hazelwood, 1976;
Naber and Hazelwood, 1977) and K + in the medium (Rideau and
Simon, 1989) increased rapidly insulin secretion in the chicken
pancreas in vitro, demonstrating that the KATP channel is involved in
the GSIS process in birds as in mammals. Strong support for this role
was found in the tolbutamide-induced hypoglycemia found in
chickens fed with this drug (Seki et al., 2001). The distal event taking
place in the GSIS also seems to be present in the avian pancreas,
includes the calcium channel. Early studies on the chicken pancreas
reported that the lack of magnesium in the media (which normally
would compete with calcium) increased dramatically insulin
secretion in vitro (King and Hazelwood, 1976). On the contrary, the
absence of calcium in the perfusion medium abolished the insulin
secretion (Naber and Hazelwood, 1977). Similar alterations in the
glucosensing system linked to the GSIS are reported in hyperglyce-
mic mammalian models (Portha et al., 1974; Bonner-Weir et al.,
1983), and it has been suggested that avian abnormal glucosensing
system could be related to such high glycemic condition (Rideau,
1998). However, the fact that birds have higher blood glucose levels
than mammals does not make them hyperglycemic by definition, and
as stated before, it is possible that the glucosensing system (if any)
could be adjusted to these relatively high glucose levels. At this point,
one study published by Datar et al. (2006) clearly showed that blood
glucose in the chicken increased gradually from embryo (5.3 mM) to
adulthood (22.1 mM). More interestingly, they showed that 5–6 days
old chickens are indeed responsive to glucose, in contrast with the
results obtained in most of the studies discussed above that used 4–
6 week old chickens. These ontogenic changes in the sensibility to
glucose could certainly be due to changes in the chicken glucosensing
elements in the pancreatic tissue. However, no further studies were
reported on this interesting hypothesis.
Few data are available in the literature concerning the presence of
central glucosensor markers in birds. Glucose transport in the avian
brain seems to be unrelated to the glucosensing marker GLUT2
(Wang et al., 1994), in opposition to the mammalian model.
However, other transporters were described, although their involve-
ment in a putative glucosensing machinery has not been explored
(Braun and Sweazea, 2008). Concerning the main glucosensing
maker, GK, no direct evidence has been found about its presence in
bird brain. However, it has been reported that the GKA administra-
tion to chicken reduced food intake (Rideau et al., 2010), which could
be related to a putative activation of the brain GK, as noted in fish
(Polakof et al., 2008b) and mammals (Matschinsky et al., 2006); this
needs to be addressed.
As a whole, the studies carried out in other vertebrate groups
addressing the presence of glucosensor systems in pancreatic tissue
responding to specific changes in circulating glucose levels (depen-
dent on the vertebrate group) with changes in insulin secretion. The
components of these glucosensor systems are only partially elucidat-
ed, and in some cases are similar to those of mammalian or fish
models but not in others. In contrast, there is no evidence regarding
the presence of putative glucosensor systems in brains of amphibians,
reptiles and birds, which certainly needs to be addressed to fill the gap
existing now in our comparative knowledge from fish to mammals.
6. Conclusions and perspectives
While control and regulation of glucose homeostasis is currently
well known in representative mammalians species (especially in
rodents and humans) some of the dogmas created around these
species do not seem to be extended to the rest of the vertebrates.
Although blood glucose in mammals is about 7 mM, there is an
important inter-species variability suggesting that further studies are
needed in other mammalian species to contrast some of the findings
reported in rodents and humans, two highly studied species. Blood
glucose levels in fish and amphibians are definitely lower than in
mammals, based mostly on its ectothermic nature, but there is also an
evolutionary component as the other group of ectotherms (reptiles)
have higher glycemia than these two groups. While glucose levels in
amphibians are very homogenous across species, this is not the case in
fish. In amphibians, glucose does not seem to be of major importance
and only a few species rely on glucose exclusively possibly related to
the anti-freezing characteristics of this carbohydrate. Thanks to the
importance of fish in aquaculture, a significant number of studies are
reported in numerous fish species regarding the control of glucose
homeostasis. While carnivorous species appear to be less glucose
tolerant than omnivorous, important differences in glycemia control
are described ranging (under normal conditions) from undetectable
glucose levels (dogfish) to 17 mM glucose (Glyptoperichthys gibbceps),
a freshwater omnivorous species (MacCormack et al., 2003). The wide
spectrum of species, diets and varieties make difficult to generalize to
a common glucose homeostasis pattern in fish. However, the fact that
some species have low blood glucose levels does not seem to be due to
the lack of a regulatory system, but rather to environmental
conditions and nutritional requirements. In some cases, glycemia
seems also to be related to the simplest way to obtain energy. Finally,
reptiles and birds seem to be separated through evolution from their
closer relatives (fish and amphibians as ectotherms vs. reptiles, and
mammals as endotherms vs. birds). These species present the highest
glycemia values detected among the ectotherms (reptiles) and
endotherms (birds). While the origin of such high glucose levels in
reptiles remains a mystery, in birds the high glycemia values were
 S. Polakof et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 123–149
suggested to be related to an extremely low insulin/glucagon ratio
and its subsequent metabolic consequences (mainly abnormally high
endogenous glucose production, see Section 2). Moreover, some
recent studies also demonstrated that increased blood glucose levels
are observed during the development of these animals, suggesting a
type of programmed hyperglycemia.
As stated above, the ability to accurately detect circulating
glucose levels and its fluctuations is vital to guarantee the correct
adjustment of the animal to environmental challenges and changes
in glucose availability and/or demand. However, the presence of
putative glucosensing system might be also related to the reliance of
the different species on glucose. While a more or less established
central glucosensing system is known in mammals, the fact that this
system has been only described in rodents (and partially in humans)
does not allow one to extend this model to the rest of mammals. In
this sense, further studies in other species, especially carnivores are
vitally important to build further conclusions about glucose
regulation in this group. In opposition to this, in fish a glucosensing
system has been proposed in a carnivorous species like the rainbow
trout. Since no studies have been carried out in omnivorous or
herbivorous fish, it remains to be established whether or not the
glucosensing mechanism observed in rainbow trout is a common
feature in fish. In the remaining vertebrate groups (amphibians,
reptiles, and birds) no studies are available regarding central
glucosensing capacities, and thus important evolutionary gap needs
to be filled.
More information is available regarding peripheral glucosensors.
As in mammals, in many other vertebrates a glucosensor system is
responsible of controlling insulin secretion. While this seems to be the
case in some fish species (mostly omnivorous) and amphibians, the
pancreatic cells model however needs to be fully explored in reptiles,
where the last studies were carried out 40 years ago. As for glycemia,
the avian glucosensing model does not seem to fit the mammalian
dogma. Thus, while some of the distal steps seem to be operative, a
clear different pattern in the proximal events controlling seems to
exist in birds.
There are several key challenges there were propose to researchers
dealing with glucose homeostasis control and glucosensing. Thus,
while data in mammals needs to be clearly extended to species other
than rodents and humans, experiments in other vertebrate groups
need to go deeper in the mechanisms and especially in the endocrine
system controlling glucose homeostasis. Concerning glucosensing, the
lack of information regarding central glucosensors in amphibians,
reptiles and birds is the most important objective to be accomplished.
Moreover, in order to complement and make more robust the current
knowledge on glucosensing in fish some key advances are needed. For
instance, the mix of cells in which central glucosensing studies have
been carried out in rainbow trout needs to be solved by neuronal
culture models and electrophysiological experiments that confirm the
activation of GE- and GI-like neurons. Similar studies are also needed
for peripheral glucosensors, including the dissociation of α- and β-
cells as well as the characterization of calcium channels, completely
ignored to this point. Finally, the isolation of the enteric nervous
system also represents an important challenge in non-mammalian
species in order to establish the presence (if any) of gastrointestinal
glucosensors.
Acknowledgments
The authors' research has been supported in recent years by grants
from Ministerio de Ciencia e Innovación and FEDER (AGL2004-08137-
c04-03/ACU, AGL2007-65744-C03-01/ACU, and AGL2010-22247-
C03-03), Xunta de Galicia (Consolidación en estructuración de
unidades de investigación competitivas), and Universidade de Vigo
(Contrato-Programa grupos de investigación consolidados).
143
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