Metabolic syndrome encompasses cluster of risk factors for cardiovascular disease which includes abdominal obesity, dyslipidemia, hypertension, and hyperglycemia [
Diabetes mellitus co-existing with metabolic syndrome has become a common predicament in society due to change in lifestyle and dietary habits. The number of diabetics with metabolic syndrome is substantial and the prevalence is increasing throughout the world [
The streptozotocin and the Alloxan models of chemically induced diabetes are commonly used to screen antidiabetic drugs [
The present study was designed to develop a unique animal model that will mimic the pathological features seen in a large pool of individuals with long term diabetes and metabolic syndrome, suitable for pharmacological screening of drugs beneficial in this condition. Such a model should replicate the components of metabolic syndrome such as hyperlipidemia, hypertension, and obesity along with type II diabetes mellitus.
Adult male Wistar rats, 10 to 12 weeks old, weighing 150 to 200 gm, were used in the study. The rats were housed in the Central Animal Facility of our own MGM Medical College, Navi Mumbai, India. They were maintained under standard laboratory conditions in the animal house. The study protocol was approved by the Institutional Animal Ethics Committee and conforms to the Committee for the Purpose of Control and Supervision of Experiments on Animals and Indian National Science Academy and Guidelines for the Use and Care of Experimental Animals in Research. Rats were kept in polyacrylic cages (38 × 23 × 15 cm) with not more than four animals per cage, housed in an air-conditioned room, and kept under natural light-dark cycles. The animals were allowed free access to standard diet or High-Fat Diet as the case may be and water
The High-Fat Diet (HFD) was prepared indigenously in our laboratory by using normal pellet diet, raw cholesterol, and mixture of vanaspati ghee and coconut oil (2 : 1). Normal rat pellet diet was powdered by grinding and mixed with 2.5% cholesterol and mixture of vanaspati ghee and coconut oil (5%). The mixture was made into pellet form and put into freezer to solidify. In addition 2% raw cholesterol powder was mixed in coconut oil and administered to the rats by oral route (3 mL/kg).
The HFD along with 2% liquid cholesterol (3 mL/kg) was orally fed to rats for 3 weeks to induce metabolic syndrome. A pilot study was carried out with different doses of STZ (30, 35, and 40 mg/kg) in order to select the appropriate dose of STZ for induction of diabetes. Based on the pilot study results, it was found that 40 mg/kg STZ produced diabetes in experimental rats. Therefore, a single STZ injection (40 mg/kg body wt, i.p., dissolved in 0.01 M citrate buffer, pH 4.5) was standardized to induce diabetes mellitus. Serum glucose estimations (blood sugar >200 mg/dL) were undertaken periodically (days 0, 3, and 7) from the tail vein to confirm the production of diabetes mellitus.
After 3 weeks of dietary manipulation, rats were injected intraperitoneally with STZ (40 mg/kg). The body weight and biochemical parameters (blood glucose, total cholesterol) were estimated 7 days after the vehicle or STZ injection; that is, on 4 weeks of dietary manipulation in rats. The rats with blood glucose (>200 mg/dL), total cholesterol (>110 mg/dL), triglyceride (>150 mg/dL), change in body weight (8% of initial weight), systolic blood pressure (>130 mm Hg), and reduced HDL levels (<35 mg/dL) confirmed presence of metabolic syndrome with diabetes. Thereafter the rats were either fed normal diet or HFD as per the protocol for 10 weeks. Blood samples were collected from the retroorbital plexus under light anesthesia at 0, 4, 7, and 10 weeks for estimation of biochemical parameters. At the ends of experimental period, rats were sacrificed for histopathological evaluation of injury to the heart, aorta, pancreas, liver, and kidney.
In Normal Control group, rats were administered distilled water orally using a feeding cannula for study period of 10 weeks. At the end of 3 weeks, 0.01 M citrate buffer, pH 4.5, was injected intraperitoneally to mimic the STZ injections.
The HFD were fed to rats for 10 weeks to produce metabolic syndrome. At the end of 3 weeks diabetes was induced by a single STZ injection (40 mg/kg body wt, i.p., dissolved in 0.01 M citrate buffer, pH 4.5).
Standardization of HFD and dose of STZ (30, 35, and 40) mg/kg to induce metabolic syndrome co-existing with diabetes mellitus was undertaken in the laboratory. The doses of STZ (30 and 35 mg/kg) did not produce sustained increase in blood glucose (>200 mg/dL). Hence diabetes was not produced by 30 and 35 mg/kg dose of STZ. The 40 mg/kg of STZ produced desired increase in blood glucose levels in HFD fed rats that was maintained through the study period. Hence, 40 mg/kg dose of STZ was selected for the present study.
The HF-DC group showed significant (
Time course of changes in anthropometric parameters in the experimental group.
SN | Variable | Baseline | 4 weeks | 7 weeks | 10 weeks | ||||
---|---|---|---|---|---|---|---|---|---|
NC | HF-DC | NC | HF-DC | NC | HF-DC | NC | HF-DC | ||
1 | Body weight | 157.63 ± 7.11 | 161.14 ± 5.11 | 188.87 ± 6.22 | 235.14 ± 4.59 |
214.12 ± 5.33 | 226.42 ± 4.68 |
237.88 ± 4.99 | 219.14 ± 9.92 |
2 | AC | 14.13 ± 0.49 | 14.28 ± 0.39 | 15.00 ± 0.26 | 17.72 ± 0.48 |
16.31 ± 0.25 | 17.00 ± 0.40 |
17.68 ± 0.70 | 16.58 ± 0.45 |
3 | TC | 13.06 ± 00.40 | 13.14 ± 0.55 | 13.93 ± 0.41 | 16.71 ± 0.48 |
15.25 ± 0.26 | 16.00 ± 0.41 |
16.62 ± 0.74 | 15.57 ± 0.47 |
4 | AC/TC | 1.081 | 1.086 | 1.076 | 1.060 | 1.069 | 1.062 | 1.063 | 1.064 |
NC: Normal Control group (
Rats of the Normal Control and High Fat Diabetic control groups at 10th weeks.
Metabolic parameters in the experimental groups.
SN | Variable | NC | HF-DC |
---|---|---|---|
1 | TG (mg/dL) | 63.75 ± 11.47 | 312.85 ± 62.24 |
2 | HDL (mg/dL) | 32.62 ± 2.56 | 26.57 ± 5.74 |
3 | LDL (mg/dL) | 12.6 ± 2.41 | 62.57 ± 12.44 |
4 | HbA1c (%) | 6.22 ± 0.43 | 12.78 ± 1.50 |
5 | Insulin ( |
6.46 ± 0.65 | 2.94 ± 1.11 |
6 | C-Peptide (ng/mL) | 0.07 ± 0.02 | 0.05 ± 0.035 |
7 | HOMA-IR | 1.57 ± 0.16 | 2.17 ± 0.63 |
8 | HOMA- |
66.6 ± 5.86 | 5.9 ± 1.2 |
9 | Atherogenic index | 1.36 ± 0.20 | 11.34 ± 5.01 |
NC: Normal Control group (
Time course changes of blood glucose level of NC (
Time course changes in total cholesterol among experimental groups of NC (
Time course changes in triglyceride of NC (
Study variables in the experimental groups.
SN | Variables | NC | HF-DC |
---|---|---|---|
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1 | Hs-CRP (mg/dL) | 0.86 ± 0.11 | 2.2 ± 0.52 |
2 | Systolic blood pressure (mm Hg) | 101.7 ± 1.52 | 149.6 ± 4.04 |
|
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1 | SGPT | 62.77 ± 11.58 | 98.50 ± 10.35 |
|
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1 | Creatinine | 0.35 ± 0.07 | 1.36 ± 0.45 |
NC: Normal Control group (
Time course changes in CPK-MB of NC (
The systolic blood pressure was raised significantly (
Histopathological changes of the myocardium and aorta. (a) The NC group rat heart revealed the noninfarcted architecture of the myocardium. (b) The HF-DC group rats showed myofibril damage and demonstrated marked edema, confluent areas of myonecrosis separation of myofibers, congested blood vessels, and mild inflammation. (c) NC group rat aorta showed the normal architecture. (d) Histopathology of HF-DC aorta showed fat cell deposition in vessel wall (atherosclerosis).
Histopathological changes of the Pancreas. (a) The NC group of rats pancreas were characterized by an organized pattern and showed normal architecture of beta cell mass. (b) The HF-DC group of rat pancreas damaged islets of Langerhans and the atrophy of beta cells showed reduced beta cell mass. The arrow showed beta cell mass.
Histopathology of the liver. (a) The liver of the NC group rats shows normal architecture of central vein, peripheral vein, and hepatocytes. (b) The HF-DC group showed degeneration of hepatocyte and congestion in liver.
Histopathology of the kidney. (a) Histopathology of NC group kidney showed normal structure of the kidney. (b) The HF-DC group showed congestion of glomerulus, damaged tubules, inflammation, and cloudy degeneration in tubules.
Immunohistochemistry of NC group pancreas showed increased localization of insulin in the NC as compared to HF-DC (Figure
Immunohistochemical localization of insulin. (a) Immunohistochemistry of NC group pancreas showed increased localization of insulin. (b) The HF-DC group showed decreased insulin localization and hence loss of beta cell functions.
Metabolic syndrome includes central obesity, insulin resistance, elevated blood pressure, impaired glucose tolerance, and dyslipidaemia. The number of adults with metabolic syndrome is substantial and the prevalence is increasing throughout the world. In the Indian subcontinent, 45% of males and 38% of females are diagnosed with metabolic syndrome. Majority of individuals diagnosed as metabolic syndrome are also diabetics. With the increase in proportion of such individuals with both the disease conditions co-existing, diabetes and metabolic syndrome need to be addressed not as independent diseases or separate entities but as a unique disease combination that requires urgent attention.
There are several animal models of diabetes as well as metabolic syndrome. However, there is no experimental model where both diabetes and metabolic syndrome co-exist. Hyperglycemia, hypertension, hyperlipidemia, and low grade inflammation confer combined architecture of metabolic derangements which may initiate changes in heart, pancreas, liver, and kidney. It is therefore essential to establish models to target all these risk factors for the treatment and reduction of clustering factors of diabetes with metabolic syndrome as such unique pathogenesis cannot be adequately studied in either the diabetes or metabolic syndrome animal models alone. In the search to combat these risk factors together, efforts were directed to develop a suitable animal model that would mimic all the symptoms of human metabolic syndrome as well as diabetes to screen the potential target compounds [
Such a unique experimental model would closely reflect the natural history and characteristic of metabolic syndrome along with human type II diabetes as well as respond to the pharmacological treatment. Further, it was kept in mind while developing the model that it should be less expensive, easily available, taking relatively short periods for development, reproducible, and displaying the various components of metabolic syndrome and diabetes mellitus. In the absence of such a unique experimental model where both the disease conditions co-exist, development of an animal model is of paramount importance and utility.
Metabolic syndrome includes central obesity, insulin resistance, elevated blood pressure, impaired glucose tolerance, and dyslipidaemia. Patients of metabolic syndrome may not have overt diabetes. However the objective of the present study was to develop an animal model which was essentially diabetic and in addition should possess the other components of metabolic syndrome such as dyslipidemia, central obesity, and hypertension.
The present study standardized different doses of STZ (30, 35, and 40 mg/kg) to be used for induction of diabetes after HFD was fed to the experimental rats. Diabetes co-existing with metabolic syndrome was successfully established with 40 mg/kg dose of STZ in the present study.
Various anthropometric parameters such as body weight, thoracic circumferences (TC), abdominal circumference (AC), and their ratios (AC/TC) were evaluated in the healthy normal control (NC) and High Fat Diabetic control (HF-DC) groups. The HF-DC group showed significant (
The present study evaluated several metabolic parameters such as blood glucose, glycosylated hemoglobin (HbA1c), serum insulin, and C-Peptide. The blood glucose levels in the HF-DC group rats were significantly higher as compared to NC group rats at 4th, 7th, and 10th week. Results do not coincide with the observations made in various animal models of metabolic syndrome as overt diabetes is not an essential requirement for this syndrome. Kuate et al. [
In addition to hyperglycemia, the present study results also found poor glycemic control as indicated by increased HbA1c levels in HF-DC group as compared with NC. Kehkashan and Waseem [
Thus, the biochemical results showed increase in blood glucose with concomitant decrease in insulin and C-peptide levels in conformity with histopathological and immunohistochemical findings. The pancreas HF-DC group of rats demonstrated damaged islets of Langerhans, atrophy of beta cells, and reduced beta cell mass as compared to NC. Immunohistochemical localization of insulin showed increased secretion of insulin in the NC as compared to HF-DC group. This suggests that those beta cells are functional in the NC group, secreting insulin as compared to HF-DC group. HF-DC caused beta cell dysfunction and loss of beta cell mass resulting in decreased insulin localization as seen in the slide.
The triglycerides and cholesterol levels in the HF-DC group rats were significantly higher as compared to NC group rats at 4th, 7th, and 10th week. Dyslipidemia is a hallmark of metabolic syndrome. These results concur with studies undertaken by Kuate et al. [
The present study also determined total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) at the end of the 10th week (end parameter). Abnormal lipid profile as reflected by raised LDL, TC, and reduced HDL levels was observed in the HF-DC group as compared with NC group. The previous studies by Munshi et al. [
Individuals with metabolic syndrome and diabetes have a twofold elevated risk of having a heart attack or stroke. Thus, it is critical that the cardiovascular complication of metabolic syndrome and diabetes are also replicated in the experimental models. Atherogenic index of plasma which assesses the risk of developing atherosclerosis log (TG/HDL-C) was also calculated. Universally, atherogenic index of plasma has been used by researchers as a significant predictor of atherosclerosis and as an independent cardiovascular risk factor. In the present study increase in the atherogenic index was observed in the HF-DC group as compared with NC group. Atherogenic dyslipidemia as evidenced by elevated serum triglyceride levels, increased levels of small dense low-density lipoprotein (sd LDL) particles, and decreased levels of HDL-C was observed in the HF-DC suggesting that the HF-DC rats are more prone to developing coronary artery diseases.
CPK-MB, an enzyme found primarily in the myocardium, is widely used to evaluate the existence and extent of myocyte injury [
The myocardial injury induced by High-Fat Diet and STZ shown by biochemical marker was also confirmed by histopathological assessment. The present study showed that the HF-DC group rats subjected to HFD and STZ injury demonstrated marked edema, confluent areas of necrosis and separation of myofibers, congested blood vessels, and mild inflammation as compared to the NC group. The histology of the aorta of HF-DC group rats also showed atherosclerotic deposition in the vessel wall. Munshi et al. [
The other cardiac markers hs-CRP and systolic blood pressure were measured at 10th week of study and were found to be significantly raised in HF-DC group as compared with NC. Hypertension, one of the important components of metabolic syndrome, was mimicked successfully in the experimental model of diabetes and metabolic syndrome. Raised blood pressure confirmed presence of hypertension, one of the important components of metabolic syndrome in present experimental model induced by High-Fat Diet and STZ. The study by Kuate et al. [
Metabolic syndrome is also associated with an increased risk of nonalcoholic fatty liver disease and kidney dysfunction. The present study also confirmed a decline in hepatic and renal function as shown by biochemical findings and histopathological assessment. Hepatic cells of the HF-DC group showed degeneration, scattered necrotic cells, congestion in the central vein, and fat deposition as compared to NC. The HF-DC group rats also demonstrated congestion of glomerular blood vessels, tubular necrosis, inflammation, and cloudy degeneration as compared to NC group. Therefore, this model may also be used to study hepatic steatosis and nephropathy.
There are no reported experimental models using High-Fat Diet and low dose streptozotocin where pathogenesis of diabetes with metabolic syndrome has been mimicked. However, recently, Kuate et al. [
Absolute insulin deficiency (significant fall in serum insulin levels) in the HFD-diabetic control group rats as compared to normal control group rats was observed in the present model, in contrast to the model reported by Kuate et al. [
Thus, the present study has attempted to develop a unique rodent model of metabolic syndrome in the setting of diabetes mellitus. Although presently there is no perfect animal model of these comorbidities, the present study for the first time has successfully developed an experimental model with specific attributes (dyslipidemia, hypertension, and diabetes) that makes them useful for studying the mechanisms and potential therapies of metabolic syndrome in the setting of diabetes.
The present study has developed a unique rodent model of metabolic syndrome, with diabetes as an essential component. The developed model will be helpful in screening of different pharmacological compounds.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The study is funded by Indian Council of Medical Research vide grants received, no. 58/2/2014-BMS.