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The metabolic effects of prolonged starvation and refeeding in sturgeon and

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Article  in  Journal of Comparative Physiology B · June 2011

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J Comp Physiol B (2012) 182:63–76
DOI 10.1007/s00360-011-0596-9
ORIGINAL PAPER
The metabolic effects of prolonged starvation and refeeding
in sturgeon and rainbow trout
Miriam Furné • Amalia E. Morales • Cristina E. Trenzado
Manuel Garcı́a-Gallego • M. Carmen Hidalgo •
Alberto Domezain • Ana Sanz Rus
Received: 18 November 2010 / Revised: 2 June 2011 / Accepted: 7 June 2011 / Published online: 24 June 2011
 Springer-Verlag 2011
Abstract The present study examines the particular
metabolic strategies of the sturgeon Acipenser naccarii in
facing a period of prolonged starvation (72 days) and
subsequent refeeding (60 days) compared to the trout On-
corhynchus mykiss response under similar conditions.
Plasma metabolites, endogenous reserves, and the activity
of intermediate enzymes in liver and white muscle were
evaluated. This study shows the mobilization of tissue
reserves during a starvation period in both species with an
associated enzymatic response. The sturgeon displayed an
early increase in hepatic glycolysis during starvation. The
trout preferentially used lactate for gluconeogenesis in liver
and white muscle. The sturgeon had higher lipid-degrada-
tion capacity and greater synthesis of hepatic ketone bodies
than the trout, although this latter species also showed
strong synthesis of ketone bodies during starvation. During
refeeding, the metabolic activity present before starvation
was recovered in both fish, with a reestablishment of tissue
reserves, plasmatic parameters (glucemia and cholesterol),
and enzymatic activities in the liver and muscle. A com-
pensatory effect in enzymes regarding lipids, ketone bod-
ies, and oxidative metabolism was displayed in the liver of
both species. There are metabolic differences between
sturgeon and trout that support the contention that the
Communicated by G. Heldmaier.
M. Furné  A. E. Morales  C. E. Trenzado 
M. Garcı́a-Gallego  M. Carmen Hidalgo  A. Sanz Rus (&)
Dpt. Biologı́a Animal, Facultad de Ciencias,
Campus Fuentenueva, Universidad de Granada,
18071 Granada, Spain
e-mail: anasanz@ugr.es
A. Domezain
Bioteco, S.L., Granada, Spain
•
sturgeon has common characteristics with elasmobranchs
and teleosts.
Keywords Metabolism  Starvation  Sturgeon  Trout 
Liver  Muscle
Introduction
Starvation is undergone and tolerated by many fish species,
both in their natural environment during migrations and
reproduction (Hinch et al. 2005; Miller et al. 2009) as well
as in fish farming. To survive these periods of unfavorable
feeding conditions, fish reduce their energy expenditures,
which are derived largely from protein turnover (Salem
et al. 2007), and mobilize their endogenous reserves to
obtain the energy required to maintain vital processes
(brain function, respiration, regulation of mineral balance,
etc.). This response entails metabolic changes that are
species dependent (Wang et al. 2006). Moreover, intra-
specific adjustments to these conditions depend on differ-
ent factors such as fish age or nutritional state (Navarro and
Gutiérrez 1995).
The feeding habits of the species also influence their
metabolic response to food deprivation and, in this sense,
carnivorous fish are better adapted because, under natural
conditions, they feed less frequently and undergo periods
of inanition, whereas omnivorous and herbivorous fish
continually ingest food (Bond 1996).
In some fish species, the first energy reserve mobilized
to face food deprivation is liver glycogen (Hung et al.
1997; Figueiredo-Garutti et al. 2002; Metón et al. 2003).
Also, glycogen hydrolysis helps to maintain the homeo-
stasis of blood glucose during the first stages of starvation.
In parallel to glycogen mobilization, lipid reserves are used
123
64
to obtain energy. In these species, the muscle protein would
be the last reserve mobilized (Navarro and Gutiérrez 1995).
In contrast, some species try to preserve liver glycogen
stores, degrading protein for gluconeogenesis and lipid
and/or protein being mobilized as energy substrates
(Sheridan and Mommsen 1991; Navarro and Gutiérrez
1995; Gillis and Ballantyne 1996).
On the other hand, some authors suggest that ketone bodies
are not an important energy source during fish starvation
(Zammit and Newsholme 1979; Black and Love 1986).
However, this assumption is still under debate, since other
authors have shown evidence of ketone bodies supporting the
activity of vital organs under such circumstances (Singer et al.
1990; de Roos 1994; Soengas et al. 1996).
Refeeding after a starvation period may trigger a vari-
able response that depends on such factors as fish species,
age, environmental conditions, food-deprivation period,
and the feeding history prior to starvation (Navarro and
Gutiérrez 1995). In general, most refed fish show a fast
weight recovery known as compensatory growth, mainly
supported by the rapid restoration of its metabolic profile
(Black and Love 1986; Lim and Ip 1989; Méndez and
Wieser 1993; Böhm et al. 1994; Soengas et al. 1996; Metón
et al. 2003; Morales et al. 2004).
In the search for new fish farming species, some Euro-
pean countries such as Italy and Spain have introduced the
culture of the sturgeon Acipenser naccarii, also known as
the Adriatic sturgeon. This species migrates seasonally
from the Po, Ticino, and Adige rivers to the Adriatic Sea
(Clementi et al. 1999). Recent studies have shown that the
historic distribution of this species included some Spanish
rivers (Garrido-Ramos et al. 1997; Hernando et al. 1999;
De la Herrán et al. 2004).
The sturgeon deserves special attention from the
standpoint of science and economics. This species of fish
lives from the Devonian, and is now an endangered spe-
cies. Moreover it constitutes a valued fish, with all of its
parts used commercially (skin, cartilage, meat, and of
course the eggs). The decline in the stocks of most aci-
penserids has increased the efforts to improve the hus-
bandry for restocking and aquaculture. Among the
available studies on Acipenser naccarii, the morphological
and physiological adjustments to changes in environmental
salinity (Cataldi et al. 1995, 1999; McKenzie et al. 1999,
2001a, b; Martı́nez-Álvarez et al. 2002, 2005; Carmona
et al. 2004), ontogenesis of several organs (Cataldi et al.
2002; Vázquez et al. 2002; Grandi and Chicca 2004; Icardo
et al. 2004), aspects related to feeding and nutrition (Agradi
et al. 1993; Soriguer et al. 2002), and digestive (Furné et al.
2005, 2008) and antioxidant (Trenzado et al. 2006; Furné
et al. 2008) capacities have been evaluated. Also, the
metabolic organization of this sturgeon species has been
evaluated in a recent study reporting marked differences
123
J Comp Physiol B (2012) 182:63–76
with respect to more advanced teleosts (Furné et al. 2009b).
Despite these studies, there is still much to be learned about
the physiology of this fish so interesting from a phyloge-
netic viewpoint.
The aim of this study was to assess the metabolic
strategies of Acipenser naccarii in facing a period of pro-
longed starvation and subsequent refeeding. In this study,
we compare this response to that of rainbow trout, one of
the first farmed species in which the physiology has been
widely studied. Knowledge of how fish respond to starva-
tion periods will improve the fish farmer’s ability to pro-
vide nutrition during feeding periods and thereby help to
optimize Acipenser naccarii culture. With this purpose,
plasma metabolite levels, liver and white-muscle compo-
sition, and the activity of key intermediary metabolic
enzymes in liver and white muscle were evaluated in
sturgeon and trout.
Materials and methods
Experimental animals
A total of 40 Adriatic sturgeon (Acipenser naccarii),
1? year old, 552.12 ± 22.03 g mean weight, and 40
rainbow trout (Oncorhynchus mykiss), 1? year old,
299.74 ± 26.65 g mean weight, were used. Both species
were obtained from the fish farm, Sierra Nevada S.L.
(Riofrio, Granada, Spain). The maintenance and feeding
conditions were those of the fish farm. The tank size was
7.35 m3 and the water flow was 1 L per second. During the
refeeding period, both species, were fed the same artificial
diet supplied before the starvation period (Le Gouesant,
France), with an approximate chemical composition of
480 g kg-1 protein, 140 g kg-1 lipid, 114 g kg-1 ash, and
150 g kg-1 carbohydrates. The water temperature was
maintained at 14 ± 1C with a 12-:12-h photoperiod.
The fish were starved for a total period of 72 days. Five
fish per species were sampled on days 1, 2, 5, 10, 40, and
72 of the food-deprivation period. Later, the fish were refed
for 2 months and five fish of each species were sampled on
days 10 and 60 of the refeeding period. Fish sampled on
day 1 were used as a control.
Sampling
The animals were quickly killed by cerebral percussion.
Blood samples were taken from the caudal vessels using
heparinized syringes in \1 min, transferred to heparinized
tubes, and kept on ice until centrifugation. Following blood
collection, the liver and a sample of white muscle from the
dorsal anterior region were quickly removed; liver and
white-muscle samples were divided into two subsamples
J Comp Physiol B (2012) 182:63–76
for enzyme assays and glycogen determinations. Tissue
samples were frozen in liquid nitrogen and stored in the
laboratory at -80C until analysis.
Blood and tissue processing
Blood samples were centrifuged immediately after
extraction (650g, 10 min) and plasma was separated and
stored at -80C until metabolite determinations were
made. Tissues were rapidly thawed and weighed samples
were manually homogenized using a Potter homogenizer
with a glass pestle. All procedures were performed on ice.
For the glycogen assays, samples of liver and white muscle
were thawed on ice and quickly homogenized in ice-cold
distilled water at a ratio of 1:5 (w:v) with no centrifugation
and stored at -80C until analyzed. Tissue samples for
enzyme assays were homogenized in nine volumes of ice-
cold 100-mM Tris–HCl buffer containing 0.1 mM EDTA
and 0.1% (v:v) Triton X-100, pH 7.8. Homogenates were
centrifuged at 30,000g for 30 min at 4C (Centrikon H-401
centrifuge) and the resulting supernatants were aliquoted
and stored at -80C for later enzyme assays.
Plasma and tissue metabolite determinations
Plasma glucose was assayed by a standard enzymatic-col-
orimetric test, based on the glucose oxidase method
(Labkit, 30236, Barcelona, Spain). Total plasma lipid, tri-
glycerides, and total HDL and LDL cholesterol were ana-
lyzed using standard colorimetric tests (Labkit 30345,
30360, 30184, 30189, 30181). The protein concentration in
plasma and tissue homogenates was analyzed using the
method of Bradford (1976), with bovine-serum albumin
used as a standard. Extraction of total lipids from liver and
muscle samples was performed by the method of Folch
et al. (1957). Glycogen in liver and muscle homogenates
was assayed as glucose after treatment with amylogluco-
sidase (Roehrig and Allred 1974).
Enzyme assays
The activity of the enzymes hexokinase (HK; EC 2.7.1.1),
pyruvate kinase (PK; EC 2.7.1.40), lactate dehydrogenase
(LDH; EC 1.1.1.27), fructose 1,6-bisphosphatase (FBPase;
EC 3.1.3.11), citrate synthase (CS; EC 4.1.3.7), glucose
6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49), malic
enzyme (ME; EC 1.1.1.40), b-hydroxyacyl CoA dehydro-
genase (HOAD; EC 1.1.1.35), glycerol kinase (GK; EC
2.7.1.30), alanine aminotransferase (GPT; EC 2.6.1.2),
aspartate aminotransferase (GOT; EC 2.6.1.1), glutamate
dehydrogenase (GDH; EC 1.4.1.2), acetoacetyl CoA thio-
lase (ACoAT; EC 2.3.1.9), and b-hydroxybutyrate dehy-
drogenase (b-OHBDH; EC 1.1.1.30) was assayed in liver
65
and white muscle. The assay conditions, in a final volume of
200 ll, were as described previously (Furné et al. 2009b).
All enzyme assays were performed at 25C using a
PowerWaveX microplate scanning spectrophotometer (Bio-
Tek Instruments, Inc., USA) and run in duplicate in 96-well
microplates (UVStar, Greiner Bio-One, Germany). The
optimal substrate and protein concentrations for the mea-
surement of maximal activity for each enzyme in each
tissue were established by preliminary assays. The enzy-
matic reactions were started by adding the tissue extract.
The change in absorbance of NADH or NADPH at
340 nm was monitored to determine the activity of most
enzymes (millimolar extinction coefficient, 6.22). Citrate
synthase activity was determined by monitoring the
reduction of DTNB [5,50 -dithiobis(2-nitrobenzoic acid)] at
412 nm (millimolar extinction coefficient, 13.6). Acetoac-
etyl CoA thiolase activity was determined at 313 nm
(millimolar extinction coefficient, 20.5). Enzyme activities
are reported in milliunits (nmol of substrate transformed
per min) per mg of protein.
Statistical analysis
All results are expressed as mean ± SEM. The data were
analyzed by one-way analysis of variance (ANOVA) using
an SPSS version 13.0 for Windows software package.
Whenever a significant effect was indicated (P \ 0.05),
Duncan’s multiple-range test was used to compare treat-
ment means (Duncan 1955).
Results
Liver and muscle reserves
Liver glycogen declined significantly at 2 and 5 days of
food deprivation in the sturgeon and trout, respectively,
these levels being maintained for the rest of the starvation
period. Refeeding led to the restoration of the liver gly-
cogen in both species. Hepatic proteins declined signifi-
cantly at 5 days without food in both species, remaining
stable throughout the food-deprivation period and not
recovered after refeeding. Liver lipid reserves fell drasti-
cally in the sturgeon and became statistically significant
after 2 days of starvation, while in the trout levels did not
vary. After refeeding, liver lipid reserves increased sig-
nificantly in the sturgeon (Table 1).
Glycogen, protein, and lipid content in white muscle
significantly decreased at 5 days of food deprivation in
both species, with the levels remaining stable for the rest of
the starvation period. After refeeding, only the protein
content in the muscle of the sturgeon and the lipid content
in both species were restored (Table 2).
123
66 J Comp Physiol B (2012) 182:63–76

Table 1 Changes of glycogen, protein and lipid content in liver (% wet weight) during starvation (S) and refeeding (R) in sturgeon A. naccarii
(St) and trout O. mykiss (T)
Days Glycogen Protein Lipid
St T St T St T

1S 5.22 ± 0.58a 7.23 ± 0.21a 10.76 ± 0.66a* 12.49 ± 1.88a 55.32 ± 2.31b* 5.25 ± 0.23
b a a a
2S 3.69 ± 0.12 * 8.80 ± 0.84 11.60 ± 0.67 13.06 ± 2.29 30.22 ± 1.32c,* 4.95 ± 0.39
5S 2.78 ± 0.19b,* 4.46 ± 0.24b 6.90 ± 0.42c 7.78 ± 0.56c 25.15 ± 1.21d,* 4.37 ± 0.46
10S 3.71 ± 0.32b 3.17 ± 0.53b 7.70 ± 0.26bc,* 9.70 ± 0.45b 17.32 ± 0.23e,* 4.13 ± 0.35
40S 2.38 ± 0.11b 2.46 ± 0.27b 9.34 ± 0.77b 9.28 ± 0.40b 4.73 ± 0.21e 4.03 ± 0.12
b b b, ab e
72S 2.59 ± 0.25 2.64 ± 0.23 9.69 ± 0.61 * 10.98 ± 0.72 5.45 ± 0.35 4.97 ± 0.21
10R 4.63 ± 1.04a 3.81 ± 0.73b 9.03 ± 0.55b 8.71 ± 0.35b 21.63 ± 0.99c,* 4.32 ± 0.20
60R 4.35 ± 0.72a,* 8.40 ± 1.16a 9.27 ± 0.70b 8.45 ± 0.26b 60.02 ± 2.50a,* 6.91 ± 0.24
Values are mean ± SEM (n = 5; number of fish sampled per sampled points)
Different superscript letters in the same column indicate significant differences (P B 0.05) among sampled points within each species
* Significant differences (P B 0.05) between species at each sampled point

Table 2 Changes of glycogen, protein, and lipid content in white muscle (% wet weight) during starvation (S) and refeeding (R) in sturgeon A.
naccarii (St) and trout O. mykiss (T)
Days Glycogen Protein Lipid
St T St T St T

1S – – 5.20 ± 0.51cd,* 7.22 ± 0.45c 25.54 ± 3.22c,* 19.52 ± 2.09c

c a d c b
2S 0.355 ± 0.129 0.944 ± 0.017 5.61 ± 0.74 6.83 ± 0.58 17.32 ± 1.03 15.12 ± 0.49b
5S 0.125 ± 0.017a 0.110 ± 0.006b 3.57 ± 0.14ab 3.15 ± 0.17a 8.24 ± 1.21a 9.65 ± 2.83ab
10S 0.141 ± 0.025a 0.146 ± 0.019b 2.96 ± 0.17a 3.14 ± 0.20a 6.86 ± 1.46ad,* 12.69 ± 2.74bc
40S 0.126 ± 0.017a 0.122 ± 0.023b 2.85 ± 0.43a 3.28 ± 0.43a 4.00 ± 0.81d 9.32 ± 2.26ab
a b ab a d,
72S 0.113 ± 0.010 0.152 ± 0.013 3.29 ± 0.08 3.58 ± 0.15 4.22 ± 0.81 * 7.31 ± 0.22a
a b ab a b,
10R 0.105 ± 0.006 0.169 ± 0.043 3.46 ± 0.25 3.83 ± 0.36 15.40 ± 1.52 * 10.12 ± 0.04ab
b b bc b c
60R 0.204 ± 0.036 0.245 ± 0.050 4.19 ± 0.32 4.79 ± 0.32 22.86 ± 4.62 17.37 ± 3.28c
Values are mean ± SEM (n = 5; number of fish sampled per sampled points)
Different superscript letters in the same column indicate significant differences (P B 0.05) among sampled points within each species
* Significant differences (P B 0.05) between species at each sampled point

Plasma metabolites The total plasma cholesterol in the sturgeon declined at

The plasma glucose levels fell from the first day of star-
vation in the sturgeon. The trout underwent significant
hypoglycemia at 5 days of starvation, and the glucose levels
remained low until the end of the food-deprivation period.
After refeeding, the plasma glucose levels significantly
recovered in both fish. In relation to plasma protein levels, no
conclusive changes were found throughout the experimental
time in either species. Trout displayed a higher plasma protein
concentration than sturgeon over the experimental period.
A summary for plasma metabolites throughout starvation and
refeeding can be found in Table 3.
In the sturgeon and trout, total lipids and triglycerides in
plasma showed no conclusive change during starvation, but
values significantly increased after 2 months of refeeding
(Table 3).
40 days of starvation, whereas in trout the concentration
fell at 5 days and stabilized throughout the food-depriva-
tion period. During refeeding, the total cholesterol values
in the sturgeon recovered, while in the trout these values
increased significantly, reaching levels higher than the
initial ones. The HDL and LDL cholesterol significantly
fell in both species at 5 days of food deprivation. In trout,
the HDL values increased after refeeding (Table 3).
Liver metabolism
The glycolytic enzyme HK peaked in activity at 5 days of
starvation in the sturgeon. From this time onward, activity
significantly declined until 10 and 40 days of starvation in
the trout and sturgeon, respectively. After the experimental
time, the values of HK recovered in both species.

123
 Table 3 Changes of plasma metabolite levels during starvation (S) and refeeding (R) in sturgeon A. naccarii (St) and trout O. mykiss (T) expressed as mg 100 ml-1
Specie Days
1S 2S 5S 10S 40S 72S 10R 60R

Glucose
J Comp Physiol B (2012) 182:63–76

St 90.82 ± 2.14ab,* 48.67 ± 7.44bc,* 55.82 ± 5.31c 41.94 ± 3.61ab,* 30.11 ± 1.80a,* 38.62 ± 4.33ab,* 95.82 ± 5.79d 91.43 ± 5.63d
c c b ab a b c
T 103.47 ± 11.30 88.56 ± 6.69 66.15 ± 4.99 56.37 ± 2.18 47.86 ± 2.29 68.57 ± 3.96 87.14 ± 4.75 96.61 ± 8.90c
Proteins
St 1,804.4 ± 284.5a,* 1,848.1 ± 160.0a,* 2,906.7 ± 503.7bc,* 1,805.0 ± 64.4a,* 2,104.6 ± 677.3ab 2,122.0 ± 154.7ab,* 2,447.5 ± 294.7ab 3,456.4 ± 128.7c,*
bc c abc bc a ab a
T 4,747.2 ± 285.0 5,106.0 ± 187.6 4,202.4 ± 215.9 4,502.4 ± 401.0 3,297.2 ± 491.6 3,874.9 ± 197.2 3,166.5 ± 646.8 5,233.4 ± 412.8c
Total lipids
St 517.7 ± 44.0b,* 624.0 ± 56.2b,* 458.2 ± 28.5ab,* 561.2 ± 1.4b,* 163.9 ± 33.4a,* 548.0 ± 77.4b,* 706.9 ± 57.8b,* 1,154.6 ± 287.3c,*
a a a a a a a
T 1,434.2 ± 119.8 1,342.7 ± 140.9 1,421.8 ± 192.9 1,391.9 ± 233.7 1,101.5 ± 100.6 1,319.1 ± 126.3 1,181.6 ± 142.2 3,645.4 ± 397.5b
Triglycerides
St 342.36 ± 31.44abc 431.11 ± 58.39bc 381.01 ± 84.23abc,* 259.13 ± 16.66ab,* 139.81 ± 11.08a 344.96 ± 13.02ab 540.25 ± 37.10b,* 1,208.42 ± 208.42c
T 440.9 ± 66.5b 336.7 ± 41.4ab 161.5 ± 6.0ab 143.3 ± 14.0a 134.2 ± 16.6a 324.4 ± 76.3ab 369.5 ± 66.1ab 1,725.8 ± 257.3c
Total cholesterol
St 73.57 ± 5.24bc,* 94.54 ± 6.38d,* 54.55 ± 4.67ab,* 65.34 ± 2.41b,* 39.32 ± 3.60a,* 36.43 ± 6.20cd,* 68.59 ± 6.58bc,* 66.48 ± 8.47bc,*
b b a a a a a
T 314.83 ± 22.77 312.12 ± 18.78 200.00 ± 10.38 205.34 ± 26.50 155.57 ± 14.73 196.34 ± 8.49 196.34 ± 13.11 409.09 ± 46.05c
HDL cholesterol
St 9.10 ± 0.88b,* 13.69 ± 3.79c,* 5.03 ± 1.05a,* 3.34 ± 0.97a,* 4.38 ± 0.57b,* 5.96 ± 1.27bc,* 8.04 ± 1.03a,* 9.02 ± 1.00a,*
a a b b a b a
T 116.41 ± 7.47 101.71 ± 9.21 85.53 ± 7.61 85.22 ± 7.90 86.56 ± 5.68 84.83 ± 6.80 101.56 ± 1.61 175.31 ± 9.68b
LDL cholesterol
St 34.27 ± 2.09c,* 36.95 ± 5.52c,* 6.89 ± 3.13a 5.77 ± 2.27a 7.78 ± 2.22ab 15.00 ± 3.76b 9.75 ± 1.65ab 9.62 ± 1.48ab,*
T 199.64 ± 8.05b 210.19 ± 14.14b 12.00 ± 3.17a 14.54 ± 5.31a 12.53 ± 2.84a 15.69 ± 2.72a 10.82 ± 2.75a 21.71 ± 5.12a
Values are mean ± SEM (n = 5; number of fish sampled per sampled point)
Different superscript letters in the same line indicate significant differences (P B 0.05) among sampled points within each species
* Significant differences (P B 0.05) between species at each sampled point
67

123
68
PK activity in the sturgeon increased significantly at
5 days of food deprivation, with a subsequent decrease in
activity and reached values similar to control, which
remained stable throughout the starvation and refeeding
period. In trout, the PK activity significantly fell at 2 days
of starvation, followed by a further decline in values
throughout the starvation period. During refeeding, trout
displayed activity that surpassed the control values
(Fig. 1).
In the sturgeon, no significant variations in LDH activity
during the starvation period were found. In contrast, in
trout the activity significantly rose at 5 days of food
Fig. 1 Changes of hexokinase (HK), pyruvate kinase (PK), fructose
1,6-bisphosphatase (FBPase), and lactate dehydrogenase (LDH)
activities in liver during starvation (solid line) and refeeding (broken
line) in the Adriatic sturgeon (dark line) and rainbow trout (faint line).
Fig. 2 Changes of glucose 6-phosphate dehydrogenase (G6PDH),
malic enzyme (ME), b-hydroxyacyl CoA dehydrogenase (HOAD),
and glycerol kinase (GK) activities in liver during starvation (solid
line) and refeeding (broken line) in the Adriatic sturgeon (dark line)
123
J Comp Physiol B (2012) 182:63–76
deprivation and this level remained constant throughout the
starvation period. Refeeding induced the recovery of the
initial values (Fig. 1). The FBPase activity did not signif-
icantly change during starvation and refeeding in either
species. The values of FBPase activity were higher in the
trout than in the sturgeon (Fig. 1).
G6PDH activity was not affected by starvation in either
species, but increased significantly over the 60 days of
refeeding in both species. This activity proved higher in
trout than in sturgeon (Fig. 2).
ME activity was significantly higher in the trout com-
pared to the sturgeon. In the sturgeon, this activity
Significant differences within each species for the different experi-
mental days are indicated with different letters. The asterisk indicates
significant differences between species at the same sampling point
(P B 0.05)
and rainbow trout (weak line). Significant differences within each
species for the different experimental days are indicated with different
letters. The asterisk indicates significant differences between species
at the same sampling point (P B 0.05)
J Comp Physiol B (2012) 182:63–76
significantly increased at 5 days of food deprivation, fol-
lowed by a decline for the rest of the starvation period. In
the trout, ME activity decreased steadily for the first few
days of starvation, the reduction becoming significant from
10 days of food deprivation onwards. After refeeding, the
ME activity increased in both species (Fig. 2).
HOAD activity did not significantly change either with
starvation or refeeding in the sturgeon and trout, with
values being higher in the former than the latter (Fig. 2).
GK activity significantly rose at 5 days of starvation and
remained higher than control for the entire deprivation
period in both species. Refeeding did not prompt signifi-
cant changes in this activity in the sturgeon, whereas trout
registered significant increases, reaching the highest values
of the entire experimental period (Fig. 2).
Hepatic GOT, GPT, and GDH displayed higher activity
in the trout than the sturgeon, and increased GPT activity
was displayed in the trout during the first few days of the
starvation period. Refeeding induced the recovery of initial
enzyme activity in both species (Fig. 3).
CS remained without abrupt variations during the star-
vation period in both species. Refeeding in the sturgeon
and trout significantly boosted CS activity, which reached
values higher than control (Fig. 3). Activity values were
significantly higher in the trout than in the sturgeon.
The enzyme ACoAT at 5 and 10 days of starvation
displayed a significant increase of activity in the trout
followed by a drop to control values that was sustained
throughout the starvation period. During refeeding, activity
values in trout were higher than control. In general, the
trout displayed higher activity compared to the sturgeon. In
the sturgeon this enzyme activity followed a downward
Fig. 3 Changes of glutamate oxalacetate transaminase (GOT), glu-
tamate pyruvate transaminase (GPT), glutamate dehydrogenase
(GDH), and citrate synthase (CS) activities in liver during starvation
(solid line) and refeeding (broken line) in the Adriatic sturgeon (dark
69
trend during the starvation period, which was significant at
2 days of starvation. Refed sturgeon showed an ACoAT
activity slightly lower than the control, whereas in refed
trout this activity significantly increased. In general,
b-OHBDH remained unchanged in the trout for the entire
experimental period, while the sturgeon displayed a sig-
nificant increase at 10 days of food deprivation. At 60 days
of refeeding activity in both species, b-OHBDH signifi-
cantly increased in relation to control values. Activity
values were significantly higher in sturgeon than in trout
until 40 days of starvation (Fig. 4).
White-muscle metabolism
HK activity significantly decreased at 40 days and increased
at 72 days of inanition in the sturgeon. In the trout, values did
not reflect remarkable changes during the starvation period
(Fig. 5). Refeeding prompted the recovery to control activity
in sturgeon, but no changes were shown in trout. PK activity
significantly increased at 5 days of starvation in both species,
diminishing afterward to reach control values toward the end
of this period in sturgeon, while in trout the levels were not
recovered. Refeeding did not cause any variations in the
sturgeon, whereas this activity was significantly higher in trout
than in control (Fig. 5). Throughout the experimental time,
trout displayed higher PK values than sturgeon.
FBPase activity dropped significantly at 2 days of star-
vation in the sturgeon, the activity being sustained for this
period. In contrast, the trout showed an initial significant
increase at 5 days without food. During starvation, trout
showed higher activity values than the sturgeon. After the
refeeding, the FBPase activity did not totally recover in the
line) and rainbow trout (weak line). Significant differences within
each species for the different experimental days are indicated with
different letters. The asterisk indicates significant differences between
species at the same sampling point (P B 0.05)
123
70
Fig. 4 Changes of acetoacetyl CoA thiolase (ACoAT) and b-
hydroxybutyrate dehydrogenase (bOHBDH) activities in liver during
starvation (solid line) and refeeding (broken line) in the Adriatic
sturgeon (dark line) and rainbow trout (weak line). Significant
Fig. 5 Changes of hexokinase (HK), pyruvate kinase (PK), fructose
1,6-bisphosphatase (FBPase), lactate dehydrogenase (LDH), b-
hydroxyacyl CoA dehydrogenase (HOAD), and glycerol kinase (GK)
activities in white muscle during starvation (solid line) and refeeding
(broken line) in the Adriatic sturgeon (dark line) and rainbow trout
sturgeon, while in the trout values remained similar to
control (Fig. 5).
LDH activity showed a significant decline at 5 days of
starvation in the sturgeon that was maintained throughout this
period. Refeeding led to a recuperation of LDH activity in the
sturgeon to control values. Trout did not show significant
variations during the experimental period, with the activity
values being significantly higher than those of sturgeon (Fig. 5).
GK activity significantly increased at 5 days of starva-
tion, reaching the highest activity value at day 10 in both
species. In the sturgeon, control values recovered for the
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J Comp Physiol B (2012) 182:63–76
differences within each species for the different experimental days are
indicated with different letters. The asterisk indicates significant
differences between species at the same sampling point (P B 0.05)
(weak line). Significant differences within each species for the
different experimental days are indicated with different letters. The
asterisk indicates significant differences between species at the same
sampling point (P B 0.05)
remaining experimental period, but in trout remained high
after refeeding. In general, activity values were more
marked in the sturgeon compared to the trout (Fig. 5). At
5 days of starvation, HOAD activity in both species
showed a downward trend, which was clearly significant in
sturgeon; from this time on, the values rose significantly to
control levels at the end of starvation in both species.
Refeeding depressed HOAD activity in both species, and
the values fell to levels lower than control. HOAD activity
was significantly higher in the trout compared to the stur-
geon (Fig. 5).
J Comp Physiol B (2012) 182:63–76
CS activity significantly peaked during starvation at
5 days in trout and 10 days in the sturgeon, followed by a
significant decline in both species. Refeeding increased
activity in both fish (Fig. 6).
ME activity (Fig. 6) significantly peaked at 5 and 10 days
of starvation in the trout and sturgeon, respectively, followed
by a significant dip. Refeeding produced a recovery of initial
values in the trout, while values surpassed initial levels in the
sturgeon. In general, trout displayed higher activity values
than did the sturgeon. G6PDH activity peaked at 5 days of
starvation in both species and was followed by a significant
fall that in sturgeon reached control values. Refeeding did not
cause significant variations (Fig. 6).
GOT activity significantly peaked in the starvation
period at 5 and 10 days in the trout and sturgeon, respec-
tively, followed by a decrease in activity to control values
in both species. Refeeding did not trigger activity changes
in either species (Fig. 6).
GPT activity significantly diminished in sturgeon at
40 days of starvation, and this fall persisted until the end of
the experimental time. GPT activity in the trout peaked at
5 days without food and was followed by a drop to control
values. In trout, refeeding resulted in a recovery of initial
values. GDH activity did not significantly change during
starvation or the refeeding period (Fig. 6).
Fig. 6 Changes of citrate synthase (CS), glucose 6-phosphate
dehydrogenase (G6PDH), malic enzyme (ME), glutamate oxalacetate
transaminase (GOT), glutamate pyruvate transaminase (GPT), gluta-
mate dehydrogenase (GDH), and citrate synthase (CS) activities in
white muscle during starvation (solid line) and refeeding (broken line)
71
Discussion
There is a plethora of studies regarding somatic and
enzymatic parameters, and changes in plasmatic metabo-
lites during the starvation period in many fish species.
Detailed knowledge of these aspects may be necessary to
develop better approaches to fish physiology (Navarro and
Gutiérrez 1995; Guderley et al. 2003; Chatzifotis et al.
2011). In a previous report, we studied the effect of star-
vation and refeeding period on digestive enzymes (Furné
et al. 2008) and oxidative stress parameters (Furné et al.
2009a) in sturgeon and trout. The current work intends to
complete the previous through study of the effects of
starvation and refeeding on key intermediary metabolism
enzymes in both fish species. Furthermore, we determi-
nated the metabolic organization of both fish species in fish
farming conditions after 1 day of starvation, which is
considered as a control condition (Furné et al. 2009b).
In the present experiment, the first few days of starva-
tion triggered a mobilization of liver glycogen to cover
energy demands both in the sturgeon as well as the trout,
with this response being earlier in the sturgeon. Observed
changes are consistent with the situation found in most
species, in which hepatic glycogen is the main substrate
mobilized in fish during starvation (Machado et al. 1988;
in the Adriatic sturgeon (dark line) and rainbow trout (weak line).
Significant differences within each species for the different experi-
mental days are indicated with different letters. The asterisk indicates
significant differences between species at the same sampling point
(P B 0.05)
123
72
Navarro and Gutiérrez 1995; Pérez-Jiménez et al. 2007;
Barcellos et al. 2010). Although glycogenolysis occurs in
the sturgeon, glucose demands and a minor content of
hepatic glycogen in this species would explain the notable
decrease of glycemia at 2 days of starvation, unlike trout,
which showed a more gradual response over time. Also,
this marked glucose mobilization in sturgeon was sup-
ported by the peak in HK and PK activities at the beginning
of the food-deprivation period. However, this enzymatic
profile was not displayed in trout. A similar trend was
found in both species for PK and HK, displaying lower
activity values from day 10 without food. Lower PK and
HK activities agree with the stabilization of hypoglycemia
and of the low hepatic glycogen levels. In this study, GPT
and LDH gluconeogenic activity was more marked in the
trout than in the sturgeon, suggesting that gluconeogenic
processes in the liver appear to be more relevant in the
former species (Furné et al. 2009b). Induction of glu-
coneogenic processes (increase of GPT and LDH activities)
and a decrease of glycolytic enzymes (HK and PK) in the
trout liver during the starvation period would explain the
more gradual decline of glycemia in this species. Lower
activity of glycolytic enzymes has also been reported for
other species during starvation (Shimeno et al. 1993; Kir-
chner et al. 2005). Furthermore, hepatic gluconeogenesis
during prolonged starvation has been reported by other
authors in the rainbow trout (Moon et al. 1989) and yellow
perch (Perca flavescens; Foster and Moon 1991). In
mammals, the conservation of the glycogenic reserves
during prolonged food restriction appears to be vital for the
maintenance of tissue integrity (Phan et al. 1974) In par-
allel, French et al. (1981) reported that gluconeogenesis
from non-carbohydrate substrates such as amino acids and
lactate was supported by the GPT and LDH activities,
which remained high in the trout from 5 days of the star-
vation period onwards.
A greater utilization of hepatic lipids for energy pur-
poses in the sturgeon compared to the trout is supported by
a higher hepatic lipid content in this species as well as a
reduction in these reserves throughout the starvation per-
iod, as opposed to trout, in which the hepatic lipid content
did not change (Table 1). It is known that the use of hepatic
lipids as an energy source during starvation depends on the
species, the lipid-reserve tissue, and the strategy followed
to mobilize other reserves such as carbohydrates. In ref-
erence to fatty-acid mobilization to obtain energy, HOAD
showed a relatively high activity during starvation in
sturgeon. This fact would indicate that the sturgeon liver
preferentially utilizes fatty acids as an energy resource,
while trout does not. GK activity was similar in both spe-
cies, increasing at 5 days of inanition. The enzymatic
pattern observed would indicate a greater incorporation
of glycerol, from the degradation of triglycerides, to the
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J Comp Physiol B (2012) 182:63–76
glycolytic pathway in both species, but relatively accen-
tuated in the sturgeon (Fig. 2).
The maintenance of triglyceride catabolism could
encourage the use of acetylCoA to the synthesis of ketone
bodies in the sturgeon, supported by higher b-OHBDH
activity. The higher b-OHBDH activity reveals a higher
capacity to synthesize ketone bodies in the sturgeon liver
compared with trout (Furné et al. 2009b), which is sus-
tained throughout the starvation period. This finding is
supported by control values before starvation in ACoAT
activity being higher in the sturgeon than in trout. Con-
versely, an increase of ACoAT during starvation in the
liver of the trout rather than sturgeon, would indicate that
throughout the starvation period the trout synthesizes
ketone bodies in response to the Krebs cycle collapse by
acetyl CoA excess from catabolic activity.
On the other hand, ME activity clearly declined in the
trout from the first few days of food deprivation and dis-
played a steady decline in values in both species during the
remaining starvation period. The decreased ME activity
could indicate lower lipid synthesis in both species
(Fig. 2). In fact, the negative influence of starvation on
lipid synthesis in salmonids has been reported by other
authors (Lin et al. 1977; Jürss et al. 1986). In this sense, the
marked decrease in plasma HDL and LDL cholesterol in
both species during the starvation period would explain a
low lipid mobilization from liver and perivisceral tissue in
the sturgeon and trout, respectively. In the trout, the
decrease in HDL and LDL during starvation was supported
by the previously reported decreased digestive somatic
index (DSI) during starvation, unlike in the sturgeon (Furné
et al. 2008).
The enzyme CS peaked only at the beginning of star-
vation period in both species, coinciding with a higher
reserve mobilization. Oxidative metabolism was sustained
during this experimental period (Fig. 3).
In the white muscle of both species, glycogen levels
significantly fell at 5 days without food, coinciding with
the peak of PK glucolytic activity, which would be indic-
ative of a mobilization of carbohydrate stores as an energy
source at the beginning of starvation. The mobilization of
muscle glycogen in the first stages of food deprivation has
been previously reported by Black and Love (1986) in
Atlantic cod. However, in other species submitted to star-
vation, no variations were found in muscle glycogen
reserves (Dave et al. 1975; Gutiérrez et al. 1991; Blasco
et al. 1992; Barcellos et al. 2010). In the present study,
starvation lowered muscle lipid levels from the second day
in both species. The decrease of muscle lipid levels and the
increase in HOAD and GK activities could be indicative of
a mobilization of muscle lipid reserves for energy pur-
poses. In contrast to the sturgeon, which showed a decrease
in LDH and FBPase activity during the starvation period,
J Comp Physiol B (2012) 182:63–76
the trout had a steady LDH activity and increased FBPase
activity in the muscle, indicating an activation of the glu-
coneogenic pathways (Fig. 5), as previously reported in
other fish species (Ferguson and Storey 1991). This
response, added to higher hepatic LDH activity, shows that
trout preferentially utilize lactate as a gluconeogenic sub-
strate in both tissues under starvation conditions. In addi-
tion, the increase and/or steadiness in GPT activity during
the first few days of starvation together with the decrease in
muscle protein during the starvation period appears to
indicate the use of this substrate as an energy source in both
species. The muscular GOT and GDH also remained active
during food deprivation, corroborating the relevance of
protein catabolism for energy in fish under this condition.
The proteins are the main energy source in muscle under
starvation, given that, in absolute terms, it is the main
muscle component (Machado et al. 1988). Some authors,
such as Lowery and Somero (1990) or Beaulieu and
Guderley (1998), hold that the adaptive response of muscle
to food deprivation involves more the glucolytic activity of
white muscle than the oxidative activity of red muscle. The
initial activity peak followed by the maintenance of control
CS activity in both species corroborates the oxidative uti-
lization of different substrates with energy purposes for the
maintenance of the muscular function. The undetected
activity of ACoAT and b-OHBDH in the present study
confirms that in both species (sturgeon and trout), the
ketone bodies played a minor role in white-muscle
metabolism, in agreement with previous studies (Moyes
et al. 1989).
Our results are consistent with previous findings (Mén-
dez and Wieser 1993), which postulate the use of the
energy reserves in the muscle of fish subjected to starvation
by utilizing glycogen, lipid reserves, and structural protein
as an energy source.
During the refeeding period, metabolic activity prior to
starvation period was recovered in the liver of both spe-
cies, with a reestablishment of tissue reserves and plas-
matic parameters (glucose and cholesterol), which
decreased during the starvation period. This was pro-
moted by a recovery of enzymatic activities altered dur-
ing the period without food. In fact, some of these
activities, linked to the metabolism of lipids and ketone
bodies, such as G6PDH, ME, GK, ACoAT and bOHBDH
displayed a higher activity than control values, which
would be related to a compensatory metabolic effect
during refeeding in response to prolonged starvation (Ali
et al. 2003). Liver glycogen restoration during refeeding
agrees with previous studies in Salmo salar (Soengas
et al. 1996). Thus, some authors have reported an increase
in hepatic glycogen after refeeding substantially sur-
passing initial values (Machado et al. 1988; Shimeno
et al. 1990). Conversely, in the sturgeon, lower lipid
73
levels were restored during refeeding, in agreement with
the importance of the liver as a reserve tissue in the
sturgeon (Trenzado et al. 2006; Furné et al. 2009b) and as
opposed to trout, which, as mentioned above, would
export lipids from the liver to the perivisceral tissue. In
white muscle, refeeding reestablished the enzymatic
profile prior to starvation in both species, in agreement
with previous reports (Guderley et al. 2003). Contrary to
the situation in the liver, white muscle showed no com-
pensatory effect in enzyme activity in either species.
Lipid stores recovered with refeeding, but muscle gly-
cogen and proteins were not completely restored after this
experimental period.
During the starvation period, the liver displayed marked
metabolic differences between species, while in white
muscle these differences were less pronounced and in
cardiac muscle were not found. Metabolic response in the
heart was previously not observed when we studied the
oxidative status in both species during starvation and re-
feeding conditions (Furné et al. 2009a).
Our previous studies on the trout and sturgeon under
starvation have shown that digestive enzymatic activity
was altered in both species alike, and the capacity to digest
proteins and lipids was recovered after 60 days of refeed-
ing (Furné et al. 2008). During starvation, both species
showed an alteration in oxidative status in tissues also
(Furné et al. 2009a).
In a previous study (Furné et al. 2009b), it was con-
cluded that the metabolism of the sturgeon Acipenser
naccarii was characteristic of an animal with sedentary
behavior. Also, taking into account the relatively greater
capacity to mobilize carbohydrates, it can be considered a
fish of dietary habits far closer to an omnivore rather than a
strict carnivore such as the trout. In this species of stur-
geon, as in elasmobranchs, the liver is the main organ for
the deposit for lipids, and it has a lower level of circulating
lipids in the plasma. Nevertheless, it can also oxidize fatty
acids in extrahepatic tissues, as in teleosts. A greater
capacity to synthesize ketone bodies was revealed in the
liver of sturgeon, giving it the ability to utilize them as fuel
in the peripheral tissues, offsetting the low capacity to
transport free fatty acids in the plasma, as in elasmo-
branchs. Together, our findings are supported by studies on
other sturgeon species, as well as the theory that sturgeons
are in an intermediate evolutionary position between
elasmobranchs and teleosts.
In summary, the present study confirms our findings of a
previous study (Furné et al. 2009b) and, in addition, it
suggests that both species have survival mechanisms for
prolonged starvation and subsequent recovery. In the liver,
the response during a starvation period is more heteroge-
neous between species than in the muscle. It was found
that:
123
74
• Tissue-reserve mobilization during the starvation per-
iod in both species (glycogen, lipids, and proteins) had
an associated enzymatic response.
• Sturgeon displayed an early increased hepatic glycol-
ysis during the starvation period.
• Trout preferentially utilized lactate for gluconeogenic
purposes in the liver and white muscle.
• The sturgeon had a higher lipid-degradation capacity
and synthesis of hepatic ketone bodies than the trout,
although the latter species also revealed a marked
synthesis of ketone bodies during starvation.
• During refeeding, the metabolic-activity level before
starvation was recovered in the trout and sturgeon,
with a reestablishment of tissue reserves, plasmatic
parameters (glucemia and cholesterol), and liver and
muscle enzymatic activities. A compensatory effect in
enzymes regarding lipids, ketone bodies, and oxida-
tive metabolism was displayed in the liver of both
species.
This study shows that both species, sturgeon and trout,
showed a significant metabolic adaptive response to pro-
longed starvation. There are metabolic differences between
the sturgeon and trout that support the contention that the
sturgeon has characters in common with elasmobranches
and teleosts. Finally, the results of the present study could
be of great interest for fish farming when under certain
circumstances fish could undergo starvation periods.
Acknowledgments This work was supported by the Spanish Min-
isterio de Educación y Ciencia, under the research project CGL2006/
12193.
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