DIETETIC MANAGEMENT OF TYPE 2 DIABETES MELLITUS: A
Variations arose from
TRIAL OF LOW CARBOHYDRATE DIET IN TYPE 2 DIABETIC
RATS
Herry Herman1, 2, Endri Septiadi3, Dinar Mutiara3, Maria Theresia1, Shellita
melanie Astuti setiawan1,4, Regina Chintya Fani1, Dewi Marhaeni Diah Herawati5
1
Magister Program in Basic Medical Science
2
Dept. of Orthopaedic Surgery and Traumatology, Faculty of Medicine
Universitas Padjadjaran
3
Department of Medical Nutrition, Faculty of Medicine, Universitas Jenderal
Achmad Yani
4
Faculty of Medicine, Universitas Pasundan
5
Division of Nutrition, Department of Basic Medicine, Faculty of Medicine
Universitas Padjadjaran
ABSTRACT
A conventional dietetic Management of type 2
Diabetes Mellitus (DM) is prescribed by the Indonesian
endocrine association (Perkeni) as the consensus for non-
pharmacological management of type 2 Diabetes Mellitus in
Indonesia. This diet comprises of 40-65% calorie contribution
from carbohydrate. In this study we seek to determine in rat
model of type II diabetes mellitus, if an alternative lower
carbohydrate diet, where the contribution of calorie from
carbohydrate is below 25%, may result in a better control of
fasting blood sugar level, insulin level, and weight gain
Following positive induction of type II DM utilizing a
combination of high fat diet and streptozotocin, but prior to
dietetic intervention, fasting blood glucose level and body
weight were uniformly increased among test rats. Insulin level
is uniformly increased too, confirming the induction of type II
DM. Succeeding the dietetic intervention, to compare the effect
of conventional diet, low carbohydrate diet, and control lab
chow diet, significant differences were found in the variances of
body weight (p<0.01), fasting blood glucose level (p <0.01) and
blood insulin level (p <0.05). Post Hoc Duncan analysis with t-
test, identified significant differences in body weight (p<0.01),
fasting blood glucose level (p<0.01) and blood insulin level
(p<0.01) only between low carbohydrate diet group with the
other two groups.
We concluded that low carbohydrate dietetic
intervention in our T2DM rat model is sufficient for the control of
fasting blood glucose level, body weight gain, and blood insulin
level.
Keywords: Diabetes Mellitus type II, dietetic control of
Diabetes, type II diabetic rat model
INTRODUCTION
Different from type I Diabetes, Type II diabetes is
amenable to non-insulin managements. This includes diet and
exercise; that were able to reverse impaired glucose tolerance1-
5. The use of diet and exercise in established type II DM is
more controversial, as results vary6-9.
multiple factors; genetics and non-genetics.
As an Outbred population, homogenization of human
trial subjects’ genetic background, for the study of
pathogenesis and intervention of type II DM, proved hard to do
and full of limitations. Rats as an inbred population, is in unison
in their genetics background, that transplantation among
individuals was readily tolerated without any rejection. Non-
genetics factors too, are troublesome to control in human trial
subjects, among them are the non-genetic predisposition,
nutritional state prior and during the development of diabetes,
duration of diabetes, and history of intervention1, 10, 11. Hence,
rat offer an attractive alternative as trial subjects.
In Human, multiple mechanisms have been
associated with the advent of DM11-18, attributing the
contribution of specific mechanism toward DM pathogenesis is
always plagued with clause. In mice, a single mechanism may
be used on all trial subjects, and any ensuing differences may
be singled out to the intervention used to generate the
differences.
Both the uniform genetic background and the ability
to use one common inducing mechanism, offer an ideal
combination for the study of DM.
Type II DM is characterize by insulin resistance,
where increased level of blood insulin is needed to maintain an
acceptable, yet elevated, blood glucose level. Multiple
mechanisms exist those were associated with the advent of
type II Diabetes, not a single one deemed unique to type II
DM1, 10-13, 16-18. One of the mechanism available to mimic the
increased level of insulin and blood glucose in rat and mice,
was the streptozotocin induction of DM19, where both type I and
type II DM may ensues, depending on the adjunct method used
in combination. The making of type II DM requires the salvage
of beta islets by high fat diet with streptozotocin induction19, 20,
otherwise type I DM shall ensue. Streptozotocin is a well know
antibiotics toxic to glut2 utilizing organs, including pancreas,
and used for the control of metastatic beta islet carcinoma 19.
Limitation of blood volume in mice limits the number
of diagnostic test that may be investigate, experimentation in
rats offer ample blood samples for multiple assays, and also
longer life span. We established a protocol to induced type II
DM in rats, with the used of customized high fat diet, and
streptozotocin induction. Rats with consistent elevation of blood
glucose level above 140 mg/dl upon 3 separate tests, were
classify as t2DM, and used in the experiments1, 19-22.
The consensus of the Indonesian endocrine
association on the dietetic management of type II DM was a
diet comprising of 40-65% calorie originating from
carbohydrate, less than 30 % from fat, and 10-20% from
protein (Soelistijo et al. 2015). A low carbohydrate diet is a diet
with less than 25% calorie contribution from carbohydrate7, 23,
24. We reason that with low carbohydrate content, the resulting
blood glucose will also be low, hence requires less insulin to
maintain normal blood glucose level2, 25. To test this idea, we
compare the result of 8-weeks dietetic intervention in t2DM significance of statistics p-value between groups were added to
rats, between low carbohydrate diet, conventional diet (ala clarify differences.
Perkeni), and a control regular lab chow diet.
RESULTS AND DISCUSSION
MATERIALS AND METHODS
Fasting Blood Glucose Level
Induction of type II Diabetes Mellitus Rat Model
30 male26 wistar rat with average age of 11 weeks
(70-90 days) with average body weight of 197 gr (150-300 gr)
and fasting blood glucose level of 97.5 gr/dl (70-110 gr/dl) were
given 1 month high fat diet (53% fat, 25% Carbohydrate, 22%
protein) followed by a single dose intra peritoneal injection of
40 mg/kg body weight Streptozotocin (MP-Bio, USA) according
to Reed et al19, 21, 22 Rat with fasting blood Glucose more than
140 gr/DL upon 3 separate times of measurements is taken as
t2DM1, 19-22.
Figure 1. Fasting Blood Glucose Level in pre intervention
Diet Intervention and post intervention Diabetic Rat groups as compared to
pre-induction Non Diabetic group
Two types of diet were prepared for the experiments;
ns: non significant **: p<0.01 convention: Conventional Diet
a Conventional Diet, which was composed of 42%
Group, Low Carb : Low Carbohydrate Diet Group
carbohydrate, and 16% fat, in accordance to Perkeni
consensus, and a Low Carbohydrate diet, which is composed After streptozotocin induction of type II diabetes
of 60% fat and 24% carbohydrate. Dry analysis of the diet used mellitus fasting blood glucose level were uniformly increased
were conducted to validate the composition of carbohydrate, fat among all test rats. From average of 97.55 mg/dL in the pre-
and protein. induction state to 310-335 mg/DL post induction. The increase
of fasting blood glucose level and the increase insulin level
Duration of Intervention (see below), is consistent with type II DM instead of type I DM
Intervention were conducted for 8 weeks, each where insulin level is low.
animal was maintained in its own cage, and were given 40 gr of Following 8 weeks of nutritional intervention,
food pellet daily, the remains of food were collected and significant differences between groups in fasting blood glucose
replace with new pellet daily. No significant variation of level was identified by analysis of variance (p anova <0.01).
remaining food observed. Further analysis wit Post hoc Duncan t-test identified a
significant difference only between low carbohydrate diet group
Blood withdrawal and blood examination with the other two groups (p<0.01), meanwhile conventional
Blood was withdrawn with capillary method from the diet group and lab chow diet, did not differ.
tail of the rat. Validation of the induction of diabetes was by Table 1. Pre and Post Intervention Fasting Blood
fasting blood glucose level, upon 3 separate times exam Glucose level variance analysis by One- way Anova
utilizing glucometer from easytouch®. Blood insulin level was
measured with ELISA kit (Bioassay Technology, Shanghai,
China). Single calibrated scale was used to measure body
weight.
Ethical Clearance
Ethical clearance was obtained from the Internal
Review Board of Faculty of Medicine, Universitas Padjadjaran.
Table 2. Post Hoc Duncan t-test Analysis between
post intervention groups
Statistical Analysis
Analysis tool pack for Microsoft office 2013 ANOVA
test were used to analyzed variance differences among the 3
groups; low carbohydrate diet, convention diet and control lab
chow diet, , and. Significant anova analysis, were continued
with, post hoc Duncan t-test among groups, assuming equal or Our hypothesis that the low carbohydrate content in
unequal variance based on inter-group anova statistics. Means the low carbohydrate diet associate with lower level of fasting
were represented as histogram with 1 standard errors, blood glucose, seemed justified.
 Of note here, the mean fasting blood sugar level of
125.5 mg/dL in the low carbohydrate group, was 50% or more
than that of the pre intervention level of 310.50 mg/dL. Upon
comparison with the fasting blood glucose level in the pre
induction state in non-diabetic mice (average 97.55 mg/dL), the
level in post intervention low carbohydrate group, though, was
still statistically higher (p<0.01), the level was lower than 140
mg/dL threshold we used to classify as diabetic in the first
place.
We share the same reduction in blood glucose level,
seen by Part et al in rats27, and Yancy et. al in human28 upon
ketogenic/low carbohydrate intervention.
Dietary composition of conventional diet, consensus
of Perkeni, was inadequate in lowering fasting blood glucose
level in T2DM rat model. It seems the carbohydrate content is
still too high for desirable fasting blood glucose level control.
Body Weight
Figure 2. Average Body Weight in pre intervention and
post intervention Diabetic Rat groups as compared to pre-
induction Non Diabetic group.
ns: non significant **: p<0.01 convention: Conventional Diet
Group, Low Carb : Low Carbohydrate Diet Group
With the induction of type 2 diabetes, body weight
increased uniformly in all test rats, from average of 196.65 gr.
to 286-299 gr, without significant variance differences among
assigned group (p=0.82). Following 8 weeks of diet
intervention, a significant variance difference of body weight
was identified (p anova <0.01). Post-hoc Duncan t-test analysis
between group, only identify significant differences, between
the low carbohydrate diet group with the control (p<0.01) and
with the conventional diet group (p<0.01).
Table 3. Pre and Post Intervention Body Weight
variance analysis by One- way Anova
Table 4. Post Hoc Duncan t-test Analysis between
post intervention groups
T2DM Rats treated with Low Carb Diet has
significantly lower body weight compared to Rats from the
conventional diet group (p<0.01), and importantly, reverses the
gain of weight following T2DM induction (p>0.05). On the other
hand, Post nutritional intervention mean body weight of Rats
belonging to the conventional diets group, did not differ from
that of the control group (p>0.05).
We share the same reduction in body weight as seen
in rat by Park et. al.27 , or in mice by Srivastava et. al.29 , or in
human by Yancy et. al.28 . With low Carbohydrate content,
and high fat content, metabolism is switch to utilizing ketone
bodies as source of energy. One of this ketone bodies, the beta
hydroxybutirate was a known appetite suppressor. We also
proposed the reduction in reassembly of triglycerides due to
low production of glycerol from low glucose level30. Circulating
free fatty acid, will not be deposited to fat tissue, and remain in
circulation as ready to use fuel. Srivastava et. al.29 proposed a
stimulation of sympathetic nerve activity resulting in further
lipolysis by the increase of c-amp byproduct during ketogenic
or low carbohydrate diet. Further, C-amp rise increases AMP
kinase activity, which suppressed the activity of lypogenic gene
SREBP131 .
Blood Insulin Level
Figure 3. Blood insulin level in pre intervention and post
intervention Diabetic Rat groups.
ns: non significant **: p<0.01 convention: Conventional Diet
Group, Low Carb : Low Carbohydrate Diet Group
Before nutritional intervention, the induction of
diabetes, resulted in blood insulin level of 3.66 to 3.9 mCU/mL
that did not vary between group (p=0.97). Following 8 weeks of
dietary intervention, significant variance difference was
identified (p anova <0.05). Post hoc Duncan t-test analysis
identified means differences only between the low
carbohydrate diet with the control group (p<0.05) with the
conventional diet (p<0.05)
Table 5. Pre and Post Intervention Blood Insulin
level variance analysis by One- way Anova

Table 4. Post Hoc Duncan t-test Analysis between
post intervention groups
We did not measure pre-induction insulin level and
hence not included in the analysis. Significant differences of
blood insulin level were found in the post intervention group
between that of the control diet and low carbohydrate diet
(p<0.05). Differences in insulin level between rats in the
conventional diet and low carbohydrate diet groups, shied away
from reaching statistics significance (p=0.05).
Low Carb Diet decreased blood insulin level following
8 week of nutritional intervention, not seen in conventional diet
intervention. Conventional diet intervention was unable to
achieve desirable control of blood insulin level in T2DM rat
model.
Decrease level of blood insulin represent direct
response to low carbohydrate content in Low Carbohydrate
Diet. May also represent positive feedback to increase insulin
sensitivity as proposed by Yancy28 . Increase insulin sensitivity
is proposed to be the result of increased AMP kinase activity,
due to stimulation by increased c-amp production during low
carbohydrate/ketogenic diet32
Limitation to translating this animal result to human
trial is numerous, the strength here though, is the ability to
singled out one specific mechanisms of induction and a specific
intervention on a similar disease shared by human and rats.
CONCLUSION
We concluded that Low Carbohydrate Diet
intervention, but not the Conventional Diet (ala Perkeni), was
sufficient for the control of fasting blood glucose level, blood
insulin level and body weight in Type II Diabetes Mellitus rat
model.
ACKNOWLEDGEMENTS
The funding for this research was provided by the
Internal Grant Award from Universitas Padjadjaran to HH. We
thank the animal research laboratory of department of clinical
pharmacology for housing our animal and experiments. We are
indebted to the animal facility of Brawijaya University, for
technical training in developing T2DM rat model.
REFERENCES
1. Soelistijo SA, Novida H, Rudijanto A, Soewondo P,
Suastika K, Manaf A, et al. Konsensus Pengelolaan dan
Pencegahan Diabetes Mellitus tipe II di Indonesia: PB
Perkeni; 2015. 82 p.
2. Chang CR, Francois ME, Little JP. Restricting
carbohydrates at breakfast is sufficient to reduce 24-hour
exposure to postprandial hyperglycemia and improve
glycemic variability. Am J Clin Nutr. 2019;109(5):1302-9.
3. Maja Cigrovski Berkovic MC, Bilic-Curcic I, Gradiser M,
Herman-Mahecic D, Cigrovski V, Ivandic M. Are We
Compensating for the Lack of Physical Activity in Our
Diabetic Patients with Treatment Intensification? Sports
(Basel, Switzerland). 2017;5(3):58.
4. Kanat M, DeFronzo RA, Abdul-Ghani MA. Treatment of
prediabetes. World J Diabetes. 2015;6(12):1207-22.
5. Francois ME, Gilbertson NM, Eichner NZM, Heiston EM,
Fabris C, Breton M, et al. Combining Short-Term Interval
Training with Caloric Restriction Improves ß-Cell Function
in Obese Adults. Nutrients. 2018;10(6):717.
6. Lee SH, Cho MH, Kim YH, Chung S. Two Cases of
Successful Type 2 Diabetes Control with Lifestyle
Modification in Children and Adolescents. J Obes Metab
Syndr. 2017;26(1):71-5.
7. Hari A, Fealy C, Solomon TPJ, Haus JM, Kelly KR,
Barkoukis H, et al. Exercise-induced improvements in
glucose effectiveness are blunted by a high glycemic diet in
adults with prediabetes. Acta diabetologica.
2019;56(2):211-7.
8. Heiskanen MA, Motiani KK, Mari A, Saunavaara V,
Eskelinen J-J, Virtanen KA, et al. Exercise training
decreases pancreatic fat content and improves beta cell
function regardless of baseline glucose tolerance: a
randomised controlled trial. Diabetologia. 2018;61(8):1817-
28.
9. Madsen SM, Thorup AC, Overgaard K, Jeppesen PB. High
Intensity Interval Training Improves Glycaemic Control and
Pancreatic β Cell Function of Type 2 Diabetes Patients.
PLoS One. 2015;10(8):e0133286-e.
10. Abdul-Ghani M, DeFronzo RA. Is It Time to Change the
Type 2 Diabetes Treatment Paradigm? Yes! GLP-1 RAs
Should Replace Metformin in the Type 2 Diabetes
Algorithm. Diabetes Care. 2017;40(8):1121-7.
11. Parrillo L, Spinelli R, Nicolò A, Longo M, Mirra P, Raciti GA,
et al. Nutritional Factors, DNA Methylation, and Risk of
Type 2 Diabetes and Obesity: Perspectives and
Challenges. Int J Mol Sci. 2019;20(12):2983.
12. Walaszczyk E, Luijten M, Spijkerman AMW, Bonder MJ,
Lutgers HL, Snieder H, et al. DNA methylation markers
associated with type 2 diabetes, fasting glucose and
HbA(1c) levels: a systematic review and replication in a
case-control sample of the Lifelines study. Diabetologia.
2018;61(2):354-68.
13. Sikhayeva N, Iskakova A, Saigi-Morgui N, Zholdybaeva E, signaling in a rat model of non-obese type 2 diabetes. Exp
Eap C-B, Ramanculov E. Association between 28 single Biol Med (Maywood). 2011;236(2):194-204.
nucleotide polymorphisms and type 2 diabetes mellitus in 28. Yancy WS, Jr., Foy M, Chalecki AM, Vernon MC, Westman
the Kazakh population: a case-control study. BMC Med EC. A low-carbohydrate, ketogenic diet to treat type 2
Genet. 2017;18(1):76-. diabetes. Nutr Metab (Lond). 2005;2:34.
14. Vaishya S, Sarwade RD, Seshadri V. MicroRNA, Proteins, 29. Srivastava S, Baxa U, Niu G, Chen X, Veech RL. A
and Metabolites as Novel Biomarkers for Prediabetes, ketogenic diet increases brown adipose tissue
Diabetes, and Related Complications. Front Endocrinol mitochondrial proteins and UCP1 levels in mice. IUBMB
(Lausanne). 2018;9:180-. Life. 2013;65(1):58-66.
15. Blaslov K, Naranđa FS, Kruljac I, Renar IP. Treatment 30. Ho-Palma AC, Toro P, Rotondo F, Romero MDM, Alemany
approach to type 2 diabetes: Past, present and future. M, Remesar X, et al. Insulin Controls Triacylglycerol
World J Diabetes. 2018;9(12):209-19. Synthesis through Control of Glycerol Metabolism and
16. Portha B, Grandjean V, Movassat J. Mother or Father: Who Despite Increased Lipogenesis. Nutrients. 2019;11(3):513.
Is in the Front Line? Mechanisms Underlying the Non- 31. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J,
Genomic Transmission of Obesity/Diabetes via the et al. Role of AMP-activated protein kinase in mechanism
Maternal or the Paternal Line. Nutrients. 2019;11(2):233. of metformin action. J Clin Invest. 2001;108(8):1167-74.
17. Udler MS. Type 2 Diabetes: Multiple Genes, Multiple 32. Omar B, Zmuda-Trzebiatowska E, Manganiello V,
Diseases. Curr Diab Rep. 2019;19(8):55-. Goransson O, Degerman E. Regulation of AMP-activated
18. Chung ST, Hsia DS, Chacko SK, Rodriguez LM, Haymond protein kinase by cAMP in adipocytes: roles for
MW. Increased gluconeogenesis in youth with newly phosphodiesterases, protein kinase B, protein kinase A,
diagnosed type 2 diabetes. Diabetologia. 2015;58(3):596- Epac and lipolysis. Cell Signal. 2009;21(5):760-6.
603.
19. Deeds MC, Anderson JM, Armstrong AS, Gastineau DA,
Hiddinga HJ, Jahangir A, et al. Single dose streptozotocin-
induced diabetes: considerations for study design in islet
transplantation models. Lab Anim. 2011;45(3):131-40.
20. Glastras SJ, Chen H, Teh R, McGrath RT, Chen J, Pollock
CA, et al. Mouse Models of Diabetes, Obesity and Related
Kidney Disease. PLoS One. 2016;11(8):e0162131-e.
21. Reed MJ, Meszaros K, Entes LJ, Claypool MD, Pinkett JG,
Gadbois TM, et al. A new rat model of type 2 diabetes: the
fat-fed, streptozotocin-treated rat. Metabolism.
2000;49(11):1390-4.
22. Skovso S. Modeling type 2 diabetes in rats using high fat
diet and streptozotocin. J Diabetes Investig. 2014;5(4):349-
58.
23. Agriculture USDoHaHSaUSDo. 2015 – 2020 Dietary
Guidelines for Americans. 8th Edition. 2015.
24. Agriculture DGACUCfNPaPRotDGACotDGfADo. Dietary
Guidelines for Americans, 2010. 2010.
25. Maki KC, Phillips-Eakley AK, Smith KN. The Effects of
Breakfast Consumption and Composition on Metabolic
Wellness with a Focus on Carbohydrate Metabolism. Adv
Nutr. 2016;7(3):613S-21S.
26. Blesson CS, Schutt A, Chacko S, Marini JC, Mathew PR,
Tanchico D, et al. Sex Dependent Dysregulation of Hepatic
Glucose Production in Lean Type 2 Diabetic Rats. Front
Endocrinol (Lausanne). 2019;10:538-.
27. Park S, Kim DS, Kang S, Daily JW, 3rd. A ketogenic diet
impairs energy and glucose homeostasis by the attenuation
of hypothalamic leptin signaling and hepatic insulin