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Insulin resistance precedes glucose intolerance and hyperleptinaemia in high-fat simple carbohydrate-fed C57BL/6J mice
Insulinooporność poprzedza nietolerancję glukozy i hiperleptynemię u myszy C57BL/6J otrzymujących karmę wysokotłuszczową i zawierającą cukry proste
1Father George Albuquerque Pai Cell and Molecular Biology Laboratory, Department of Postgraduate Studies and Research in Biotechnology, St Aloysius College (Autonomous), Mangalore, Karnataka, India
2PG Department of Biochemistry, St Aloysius College (Autonomous), Mangalore, Karnataka, India
3Protein Engineering Laboratory, Department of Chemical Engineering, Indian Institute of Technology, Bombay (IIT B), Powai, India
Introduction: Very few systematic studies are done during the onset and progression of metabolic syndrome in suitable animal models. In this paper we present the effect of High-Fat Simple Carbohydrate (HFSC) feed on the metabolic hormones in C57BL/6J mice to understand the sequence of events leading to impairment of glucose homeostasis.
Material and methods: One-month-old male C57BL/6J mice were fed with control (C group) and HFSC (T group) feed (n = 30 each) respectively for five months. The glucose tolerance was studied by Oral Glucose Tolerance Test (OGTT) whereas serum insulin and leptin were quantified using ELISA kits, and serum cortisol was quantified using CLIA kits.
Results: Insulin resistance index and HOMA-IR levels were higher in the mice of group T as compared to age-matched mice of group C within one month and significantly higher after and five months of feeding. The total area under the glucose tolerance test curve (AUC) and the insulin curve (AUC ins) was found to significantly increase in the mice of T group as compared to the mice of C group as early as two months of feeding and was elevated after 5 months post feeding. Comparison of the Matsuda index revealed that pancreatic beta cell function was significantly lower in mice of T group as compared to mice of C group by five months of feeding. Feptin levels fluctuated during the lst-4th month and by the 5th month significant hyperleptinaemia was detected. There was no significant change in cortisol levels in mice of group T as compared to mice of group C after five months of feeding.
Conclusions: HFSC feed induces insulin resistance by the first month and progressively impairs glucose tolerance, resulting in hyperleptinaemia by the fifth month in male C57BF/6J mice.
(Endokrynol Pol 2016; 67 (6): 592–598)
Key words: insulin resistance; oral glucose tolerance; cortisol; leptin; HFSC diet; C57BL/6J mice; metabolic syndrome; Matsuda index; pancreatic islet function
Wstęp: Dostępnych jest bardzo niewiele badań systematycznych oceniających wystąpienie i progresję zespołu metabolicznego na odpowiednich modelach zwierzęcych. W niniejszej pracy przedstawiono wpływ podawania myszom C57BF/6J karmy wysokotłuszczowej i zawierającej cukry proste (HFSC, High Fat Simple Carbohydrate) na sekwencje zdarzeń prowadzących do zaburzeń homeostazy glukozy.
Materiał i metody: Jednomiesięcznym samcom myszy C57BF/6J podawano przez 5 miesięcy karmę kontrolną (grupa C) lub HFSC (grupa T) (n = 30 w każdej grupie). Tolerancję glukozy oceniono na podstawie doustnego testu tolerancji glukozy (OGTT, oral glucose tolerance test), natomiast stężenia insulin i leptyny w surowicy oznaczono, używając metody ELISA, a do oznaczenia stężenia kortyzolu w surowicy użyto metody CLIA.
Wyniki: Wskaźnik insulinooporności HOMA-IR był wyższy u myszy z grupy T niż u dobranej pod względem wieku myszy z grupy C już w ciągu pierwszego miesiąca, a po 3 i 5 miesiącach diety HFSC różnice były istotne statystycznie. Całkowite pole pod krzywą (AUC, area under the curve) w teście tolerancji glukozy oraz pole pod krzywą insuliny (AUC ins) zwiększyło się istotnie u myszy z grupy T w porównaniu z myszami z grupy C, co było widoczne już po 2 miesiącach podawania karmy HFSC i było podwyższone przez 5 miesięcy od zakończenia podawania tej karmy. Porównanie wskaźnika Matsudy wykazało, że po 5 miesiącach czynność komórek beta trzustki była istotnie upośledzono u myszy z grupy T w porównaniu z myszami z grupy C. Stężenia leptyny wahały się w okresie 1.–4. miesiąca, a po 5 miesiącach wykryto istotną hiperleptynemię. Po 3 miesiącach nie stwierdzono istotnych zmian stężeń kortyzolu u myszy z grupy T w porównaniu z grupą C.
Wnioski: U samców myszy C57BF/6J dieta HFSC wywołała insulinooporność już po pierwszym miesiącu, a następnie powodowała stopniowe pogarszanie tolerancji glukozy, co po pięciu miesiącach doprowadziło do hiperleptynemii.
(Endokrynol Pol 2016; 67 (6): 592–598)
Słowa kluczowe: insulinooporność; doustny test tolerancji glukozy; kortyzol; leptyna; dieta HFSC; myszy C57BL/6J; zespól metaboliczny; wskaźnik Matsudy; czynność wysp trzustkowych
This work was funded by a UGC Minor Research Project awarded to Swarnalatha BN (MRP(S)113-12/13-KAMA002/UGC-SWRO), a CSIR SRF fellowship awarded to D’Souza Serena Stephen (08/439(0004)/2010-EMR-1), and a UGC Major Research Project awarded to Dr Asha Abraham (F. No. 39-251/2010(SR) dated 1st February 2011).
Asha Abraham M.D., M.SC, Ph.D. Father George Albuquerque Pai Cell and Molecular Biology Laboratory, Department of Postgraduate Studies and Research in Biotechnology, St Aloysius College (Autonomous), Mangalore-575003, Karnataka, India,. phone: +91 944 9555 802, fax: 0824 2449 705, e-mail: email@example.com
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. AH the experiments were carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India with approval from the Institutional Animal Ethics Committee (Sanction No. SAC/IAEC/110/2011, 30th March 2011).
The rampant mushrooming of fast food outlets has led to a change in dietary patterns across the globe. Most of the food items available are categorically rich in fat and simple carbohydrates. Food influences hormones such as insulin [1, 2] and leptin [3, 4]. Insulin and leptin, both anorexic hormones, are known to suppress appetite . Among the animal models used for nutritional studies, C57BL/6J mice seem to respond better to hormonal changes caused by diet. High-fat diet-fed C57BL/6J mice develop obesity, hyperglycaemia, hyperinsulinaemia, and impaired glucose-stimulated insulin secretion , as well as peripheral and central leptin insensitivity . Paradoxical observations exist with respect to the role of leptin and insulin resistance. Leptin appears to act in both insulin sensitisation and resistance phenotype . Most of the studies are based on feeding a high-fat diet that does not mimic the present day fast foods. Recognising the importance of simple carbohydrates in weight gain and other associated complications of glucose homeostasis, we specially formulated and prepared High-Fat Simple Carbohydrate Diet (HFSC) feed in our laboratory.
HFSC-fed C57BL/6J male mice progressively gained body weight, developed hyperglycaemia, obesity, hypercholesterolaemia, and hypertriglyceridemia during a five-month study . Given the observed increase in body weight, we wanted to study the leptin profile in the developed animal model due to its anorectic mode of action in body weight regulation. Also, the glucose levels that were studied by us primarily focused on fasting blood glucose. This did not provide us with an insight into tolerance of the animal model to the glucose flux that could be obtained by a glucose tolerance test. Along with insulin, cortisol also plays an important role in maintaining the glucose levels of the body by stimulating gluconeogenesis as well as in fat, carbohydrate, and lipid metabolism . To get a better understanding of the observed disrupted biochemical profile, in the present investigation we have tried to assess the timeframe of disruption of glucose homeostasis in C57BL/6J mice in response to the HFSC feed. In this study we evaluated the glucose tolerance as well as insulin, cortisol, leptin, and pancreatic insulin function in experimental C57BL/6J mice during a five-month period.
Material and methods
Pathogen-free, one-month-old, male C57BL/6J mice obtained by breeding (breeder stock procured from the National Institute of Nutrition, Hyderabad, India) were used for the experimental study. Male mice were chosen for the study because the energy balance-related pathways are affected due to hormonal variations associated with oestrous cycle [11–13]. Also, the body composition and body fat distribution differs in males and females wherein females have higher percentage of body fat and less abdominal fat compared to males . The mice were divided into control and test groups (n = 30 each) and were fed with control feed and high-fat simple carbohydrate feed respectively at the rate of 5 g/day according to Indian National Science Academy regulations .
The control feed consisted of wheat flour (79% w/w), infant milk spray (Amul, India) (16% w/w) (used as the source of proteins and vitamins) and soya bean oil (4.67% v/w), whereas the HFSC feed contained porcine fat (13% w/w), sucrose (10% w/w), corn starch (7% w/w), and infant milk spray (Amul, India) (70% w/w). Total energy content from control feed was 3582.369 Kcal/kg and from HFSC feed was 5064.581 Kcal/kg . The mice were provided with fresh food and water, both ad libitum, daily. All the experiments were carried out according to CPCSEA India guidelines with approval from the Institutional Animal Ethics Committee (sanction No. SAC/IAEC/110/2011, 30th March 2011). These guidelines are in accordance with the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Testing, and Education issued by the New York Academy of Sciences Ad Hoc Committee on Animal Research.
Oral glucose tolerance test (OGTT)
Oral glucose tolerance test was performed every month in accordance with Buetner et al. (2006) . The mice were starved for 16 hours prior to the procedure. A glucose load (1 g/kg body weight) was orally administered to each mouse. The blood was collected by tail vein bleeding, and blood glucose levels were then measured at 0 (before glucose administration) 30, 60, 90, and 120 minutes post glucose load using a glucometer (One touch Horizon, India). The serum obtained was used for insulin analysis.
The serum insulin analysis was performed using an Ultra-Sensitive Mouse Insulin ELISA kit according to the manufacturer’s instruction (Crystal Chem Inc., USA).
Homeostatic Model of Assessment-Insulin Resistance (HOMA-IR) was calculated according to the given formula :
HOMA-IR = Glucose [mmol/L] * Insulin [m/U/mL]
The Matsuda index of insulin sensitivity was calculated as follows, with glucose and insulin values in mmol/L and pmol/L, respectively :
10,000/(G0 × I0 × Gm × Im)0.5
where G0 and I0 are pre-meal values for insulin and glucose, respectively, and Gm and Im are mean postmeal values during the first 120 minutes of the glucose tolerance test.
Total area under the curve of both glucose and insulin was calculated during 0-120 minutes following oral liquid glucose load .
The ratio of AUC (ins/glc) and Matsuda index provides an insight into the β cell function .
Leptin was analysed from the serum obtained by tail vein bleeding using Mouse Leptin ELISA kit according to the manufacturer’s instructions (RayBio®, USA).
Serum cortisol was analysed by Chemiluminescence Immuno assay (CLIA) technique by Bioscan Diagnostics and Health Care Centre (Kadri, Shivabaugh, Karnataka, India).
The obtained data was statistically analysed using Graph Pad InStat Version 3.1. Specifically, the OGTT data (post Students t-test) was further subjected to Bonferroni correction in order to calculate the correct error because after each month of OGTT the mice were sacrificed for further analysis in the study. For example, for the fifth month, only those values below 0.01 were considered significant (p < 0.05/5 = 0.01). Accordingly, the corrections were applied for the other months as well. The statistical approach was confirmed by a statistician, Dr Mariamma Philip (Assistant Professor, NIMHANS, Bangalore, India).
HFSC feed significantly increased fasting blood glucose in the mice of group T as compared to the mice of group C (p < 0.05) after one month of feeding. However, oral glucose tolerance was unaffected (i.e. the ability of mice of group T to bring the blood glucose levels to normal levels was not affected) at the end of one month of feeding with HFSC diet (Table I). Glucose tolerance was found to be significantly impaired after two months of feeding the HFSC diet. The blood glucose levels were significantly (p < 0.025) higher in the mice of group T after 0 minutes and 30 minutes of glucose load as compared to the mice of group C (Table I). This impairment only worsened in the mice of group T throughout the feeding regimen as compared to the mice of group C, shown by significantly higher levels of the blood glucose levels of mice of group T at 0 minutes (p < 0.001), 30 minutes (p < 0.01), 60 minutes (p < 0.01), and 120 minutes (p < 0.01) post glucose load after three months of feeding (Table I). After four and five months of feeding, significantly (p < 0.01 and p < 0.001) higher impaired glucose tolerance was observed until 120 minutes of feeding with the blood glucose levels rapidly becoming very high in the mice of group T as compared to the mice of group C (Table I). The total area under the glucose curve (AUC glc) (Fig. 1) and total area under the insulin curve (AUC ins) (Fig. 2) were found to significantly increase in the mice of group T as compared to the mice of group C as early as within two months of feeding and was elevated until five months of feeding. Comparison of the Matsuda index revealed that pancreatic beta-cell function was significantly lower in mice of group T as compared to mice of group C from one month, and worsened by five months of feeding (Fig. 3). The ratio of AUC (ins/glc) and Matsuda index (Fig. 4) further confirms that the pancreatic beta-cell function was affected from the first month and deteriorated by fifth month.
Table I. Blood glucose levels in male C57BL/6J MICE following oral glucose load during five-month study
Tabela I. Stężenia glukozy we krwi samców myszy C57BL/6J po doustnym obciążeniu glukozą w trakcie 5-miesięcznego badania
|Month||0 minutes||30 minutes||60 minutes||90 minutes||120 minutes|
|1 mo||94.25 ± 7.36||158.6 ± 9.02||136.8 ± 7.42||112.8 ± 3.23||95.8 ± 1.02|
|2 mo||97.25 ± 1.03||173 ± 22.35||165.8 ± 13.38||106.8 ± 2.35||95.4 ± 0.97|
|3 mo||89.5 ± 2.15||133.83 ± 5.79||120.83 ± 2.53||103 ± 4.86||89 ± 2.28|
|4 mo||91.83 ± 4.23||137.16 ± 9.39||110.33 ± 2.67||93.66 ± 3.27||88.83 ± 2.93|
|5 mo||98.25 ± 0.86||185.8 ± 4.65||169.8 ± 2.39||127.25 ± 3.32||97.6 ± 0.92|
|Month||0 minutes||30 minutes||60 minutes||90 minutes||120 minutes|
|1 mo||161.33 ± 18.49*||169.6 ± 5.17||152 ± 10.75||136.8 ± 10.42||104 ± 4.50|
|2 mo||165 ± 12.46#||228 ± 9.97**||187.8 ± 7.79||118.4 ± 4.94||117.6 ± 8.19|
|3 mo||131.83 ± 5.3***||171.86 ± 10.86**||159 ± 9.82**||133.6 ± 4.55||125.66 ± 1.62**|
|4 mo||141.16 ± 7.78**||196.83 ± 9.1***||178.33 ± 7.72***||174 ± 14.28***||154.16 ± 13.42***|
|5 mo||164.6 ± 3.20***||255.4 ± 13.94**||282.8 ± 22.59***||225.6 ± 11.47***||207.6 ± 12.33***|
The HOMA-IR levels of the mice of group T were higher than the mice of group C within one month and significantly higher after three months (p < 0.001) and five months (p < 0.001) of feeding (Table II). An interesting observation was that the HOMA-IR of mice of group T was lower after five months of feeding as compared to HOMA-IR after three months of feeding of the same group. Corresponding analysis of the insulin levels using the serum samples obtained during OGTT showed that the insulin levels of the mice of group T were significantly higher than the mice of group C (p < 0.01) as early as within one month of feeding but only 30 minutes post glucose load (Table III). Significantly higher levels of insulin were observed in the mice of group T at all time points (0 minutes [p < 0.001], 30 minutes [p < 0.01], and 120 minutes [p < 0.025]) after five months of feeding as compared to the mice of group C (Table III).
Table II. Homeostatic model of insulin resistance analysis (HOMA-IR) of male C57BL/6J mice during the five-month study
Tabela II. Analiza danych modelu homeostatycznego oceny insulinooporności (HOMA-IR) u samców myszy C57BL/6J w trakcie 5-miesięcznego badania
|0 Day||1 mo||3 mo||5 mo|
|Control||1.86 ± 0.009||2.35 ± 0.37||2.61 ± 0.31||3.007 ± 0.46|
|Test||1.86 ± 0.03||4.13 ± 0.84||33.92 ± 4.12***||18.22 ± 1.39***|
Table III. Insulin levels of male C57BL/6J mice following oral glucose load during the five-month study
Tabela III. Stężenia insulin u samców myszy C57BL/6J po doustnym obciążeniu glukozą w trakcie 5-miesięcznego badania
|Control [μIU/mL]||Test [μIU/mL]|
|0 minutes||30 minutes||120 minutes||0 minutes||30 minutes||120 minutes|
|1 mo||10.29 ± 1.72||8.58 ± 0.70||11.07 ± 1.60||16.95 ± 3.44||17.42 ± 1.85**||16.06 ± 1.95|
|5 mo||12.41 ± 1.92||17.59 ± 2.62||16.88 ± 1.78||44.91 ± 3.32***||60.37 ± 6.77**||76.01 ± 18.88*|
In our study the leptin levels were lower in mice of group T, although not significantly after a month of feeding as compared to mice of group C (Fig. 5), indicating that the body’s sensitivity to leptin was not lost yet. However, leptin levels in the mice of group T fluctuated during the second to the fourth month period, reaching significantly (p < 0.05) higher concentration than the mice of group C after feeding for five months (Fig. 5). Also, the cortisol levels did not show any significant change in mice of group T as compared to mice of group C after five months of feeding (Table IV).
Table IV. Cortisol levels of male C57BL/6J after five months of feeding HFSC diet
Tabela IV. Stężenia kortyzolu u samców myszy C57BL/6J po 5 miesiącach stosowania karmy HFSC
|Cortisol [μg/dL]||8.58 ± 0.62||9.76 ± 0.49|
A previous study from our laboratory has shown that HFSC feed could induce features of metabolic syndrome like obesity, hyperglycaemia, hypercholesterolaemia, and hypertriglyceridaemia in male C57BL/6J mice . The increased levels of fasting blood glucose that were noted in our study could indicate hepatic insulin resistance because this glucose is derived from glycogenolysis from the liver [20, 21]. Glucose tolerance was found to be significantly impaired after two months of feeding with the HFSC diet because there was a failure to clear the high blood glucose levels post glucose load. This impairment only worsened in mice of group T throughout the feeding regimen as compared to mice of group C. Our results are in agreement with a previous study ; however, glucose intolerance was much more severe in the current study. This could be due to hepatic insulin resistance, muscle insulin resistance, and impaired insulin secretion . High Insulin levels observed in our study further confirms it.
The total area under the glucose tolerance test curve (AUC) and the insulin curve (AUC ins) was found to significantly increase in the mice of group T as compared to the mice of group C as early as after two months of feeding and was elevated until five months of feeding implying impaired glucose tolerance. This could be due to insulin resistance triggered by HFSC feed. Several studies  show that insulin sensitivity is reduced in human subjects with reduced glucose tolerance. HOMA-IR further confirmed insulin resistance in our animal model. The increased levels of insulin in the mice of group T after five months of feeding as compared to mice of group C indicate that the mice of group T had OGTT-stimulated hyperinsulinaemia as compared to mice of group C. A study reported that 54% of obese children and adolescents have higher fasting or OGTT-stimulated hyperinsulinaemia . This supports our study. Insulin resistance was also observed in prediabetic patients, which increased in parallel to waist circumference . Pancreatic beta cell function as revealed by Matsuda index was significantly lower in mice of group T as compared to mice of group C by one month and worsened by five months of feeding. The ratio of AUC (ins/glc) is an insulin secretion index whereas the Matsuda index is an OGTT-stimulated index and provides an insight into the insulin sensitivity . The product of these two indices is a measure of pancreatic β-cell function. This combination is distinct for different degrees of glucose tolerance. It is found to decrease from Normal Glucose Tolerance to Impaired Glucose Tolerance to Diabetes. In our study, the insert insulin of group T had reduced values as compared to mice of group C, indicating decreased pancreatic β-cell function. This is further supported by histopathological observations in the pancreas of HFSC-fed C57BL/6J mice in our previous study .
Leptin is associated with insulin resistance in patients with metabolic syndrome . Insulin is known to stimulate leptin release . In our study, leptin levels were found to be lower in mice of group T after one month of feeding, but not significantly as compared to mice of group C, indicating that sensitivity to leptin has not been lost. A study reported no difference in the leptin levels of pre-diabetic patients as compared to normal patients . This supports our finding. However, leptin levels were significantly higher in mice of group T than mice of group C after feeding for five months. A contrasting report was found in a human study wherein no difference was observed in the leptin levels of metabolic syndrome and non-metabolic syndrome patients . This could also be due to differences in diets consumed. Diets were found to have varied effects on leptin, as discussed subsequently. Long-term intake of a high-fat diet was found to positively correlate with plasma leptin levels in male rats  whereas a short-term, moderately high-fat diet was found to reduce circulating leptin levels . Also, the type of dietary fats seems to affect plasma leptin levels . In our study, insulin secretion was not found to be modulated by hyperleptinaemia even though leptin is known to inhibit insulin secretion in the pancreas via leptin receptors on the pancreatic β-cells . This could be due to polymorphisms in the leptin receptors or leptin resistance in the pancreas, which needs to be evaluated.
There was no significant change in cortisol levels in mice of group T than mice of group C after five months of feeding. Higher cortisol levels were reported to reduce insulin secretion . However, in our study insulin secretion, as stated earlier, was higher in mice of group T than mice of group C, indicating loss of regulation.
High incidence of metabolic syndrome worldwide warrants more focussed research for elucidating the causative factors. Among other factors, fast foods, which are energy dense, could also play a role in the aetiology of the syndrome. In the present investigation we have tried to systematically evaluate the time frame of disruption of glucose homeostasis by feeding HFSC feed (which mimics calorie-rich fast foods) to male C57BL/6J mice. Our study shows that HFSC feed induces glucose intolerance within two months of feeding probably by inducing insulin resistance and pancreatic beta cell dysfunction. Hyperleptinaemia was also noted in HFSC-fed C57BL/6J mice, which further shows that our animal model displays essential features of metabolic syndrome. Further studies on the hypothalamus adrenal axis in this model might shed more light onto the cell signalling mechanisms contributing to this syndrome. These observations may have clinical significance, especially with changing nutritional patterns globally.
Swarnalatha BN would like to thank the University Grants Commission, South Western Regional Office Bangalore, Karnataka, India for providing the Minor Research Project. D’Souza Serena Stephen thanks Council of Scientific and Industrial Research, New Delhi, India for providing the Senior Research fellowship, and Dr Asha Abraham would like to thank the University Grants Commission, New Delhi, India for providing the Major Research Project. All authors thank the Principal and Management of St Aloysius College for providing the facilities, Rev. Dr Leo D’Souza S.J. (Director, Laboratory of Applied Biology, St Aloysius College), his lab members, and Ms Sushma Patrao (Lecturer, St Aloysius College) for all their help and support
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