Type 2 diabetes, characterized by peripheral insulin resistance, is a major lifestyle disorder of the 21st Century. Raw garlic homogenate has been reported to reduce plasma glucose levels in animal models of Type-1 diabetes. However, no elaborative studies have been conducted to evaluate the effect of raw garlic on insulin resistance. This study was designed to investigate the effect of raw garlic on fructose induced insulin resistance and associated metabolic syndrome in diabetic rats. Male Sprague Dawley rats (n=7 per group) weighing 200-250 gm body weight were fed diet containing 65% cornstarch (control group) and 65% fructose with (Dia+Garl group) and without (diabetic group) raw garlic homogenate (250 mg/kg/day) for 8 weeks. Whole garlic cloves were homogenized with water to make a fresh paste for this study. At the end of 8 weeks, serum glucose, insulin, triglyceride and uric acid levels, as well as insulin resistance, as measured by glucose tolerance test, were significantly (p<0.05) increased in fructose fed rats when compared to the cornstarch fed (control) rats. Administration of raw garlic to fructose fed rats (Dia+Garl group) significantly (p<0.05) reduced serum glucose, insulin, triglyceride and uric acid levels, as well as insulin resistance when compared with fructose fed rats. Garlic also normalised the increased serum level of nitric oxide (NO) and decreased level of H2S after fructose feeding. Although body weight gain and serum glycated haemoglobin levels of fructose fed rats (Diabetic group) were not significantly different from control rats, significant reduction of those parameters was observed in fructose fed rats after garlic administration (Dia+Garl group). Our study demonstrates that raw garlic homogenate is effective in improving insulin sensitivity while attenuating metabolic syndrome in fructose-fed rats.
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Key Words: Allium sativum L., Fructose, Diabetes, Metabolic syndrome, Nitric oxide, Hydrogen sulphide.
Type-2 diabetes is the most common form of diabetes constituting 80% of all diabetic population. The World Health Organization has predicted that developing countries would have to bear the major burden of this disease. It has been estimated that there will be a 42% increase from 51 to 72 million affected individuals in the developed countries. In developing countries these figures are much higher and are expected to show 170% increase from 84 to 228 million . The countries with the largest number of diabetic people are, and will be in the year 2025, India, China and United States . The long term consequences of type-2 diabetes make it imperative to focus on the development of novel treatment strategies for the management of insulin resistance and metabolic syndrome.
It is well known that dietary factors play a key role in the prevention of diabetes and other metabolic disorders [3-5]. Among all such agents, garlic has attracted the attention of modern medical science because of its widespread over the counter use. The salutary effects of garlic in type 1 diabetes have been well established. Several studies document the efficacy of garlic in reducing blood glucose in various animal models of type 1 diabetes mellitus. [6-10].The hypoglycemic effect of garlic has been attributed to the presence of allicin and sulfur compounds . However, the effect of raw garlic homogenate which is enriched with allicin has not been reported in the insulin resistance diabetic model. Thus present study was designed to investigate the effect of raw garlic on insulin resistance and associated metabolic syndrome in established model of type 2 diabetes mellitus characterized by insulin resistance and metabolic syndrome [11-13].
2. Materials and Methods
2.1 Preparation of garlic Homogenate
Fresh garlic (Allium Sativum L.) was purchased from local market in Hyderabad, India. Individual bulbs were put in a grinder to form a juicy paste as described [6,14]. The garlic homogenate was prepared freshly each day.
2.2 Animals and Treatment
All animal experiments were undertaken with the approval of Institutional Animal Ethical Committee of IICT, Hyderabad. Male Sprague-Dawley rats (200-250gms) were purchased from the National Institute of Nutrition (NIN), Hyderabad, India. The animals were housed in BIOSAFE, an animal quarantine facility of the Indian Institute of Chemical Technology (IICT), Hyderabad, India. The animal house is maintained at temperature 22 Â± 2Â°C with relative humidity 50 Â± 15 % and 12 hour dark/light cycle throughout the study. Animals were randomly divided into three groups (n=7). control group was fed 65% corn starch diet (Research diet, USA), whereas diabetic group was fed 65% fructose diet (Research diet, USA), for the induction of diabetes and associated metabolic disorders, [15,16] while the third group (Dia+Garl) was fed 65% fructose diet along with raw garlic homogenate (250mg/kg) for a period of 8 weeks.
2.3 Intraperitoneal glucose tolerance test
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In a separate experiment, rats from all three groups were injected intraperitoneally with a glucose load of a freshly prepared 2 gm/kg of body weight. Blood was collected from the retro-orbital plexus at different intervals, 5, 30, 60 and 120 min as well as just before injecting the glucose load (0 min) for the measurement of blood glucose. Blood glucose was measured using glucometer (One Touch Horizon, Singapore).
2.4 Biochemical assay
The rats in all groups were analysed for different biochemical parameters at different time intervals to confirm the induction of diabetes and metabolic syndrome. Serum glucose and triglyceride levels were determined after 3 and 8 weeks of feeding, while insulin, glycated haemoglobin, uric acid, total cholesterol, nitric oxide and H2S were determined after 8 weeks of feeding. The biochemical parameters i.e., glucose, triglyceride, uric acid and total cholesterol were determined by an auto blood analyser (Bayer Corp. USA), and H2S concentration was measured as described by Cai et. al, 2007(17). Commercial kits were used for the measurement of serum nitric oxide (Assay Design, USA.), serum insulin (Mercodia, Sweden) and blood glycated haemoglobin (Biosystem, Spain). Blood was collected from retro-orbital plexus using small capillary tubes, centrifuged at 4000 rpm for 10min. at 4o C, and serum was collected for all biochemical assays.
2.4.1 Serum glucose, uric acid, total cholesterol and triglyceride
Serum samples were analysed for estimation of glucose, triglyceride, uric acid, and total cholesterol using an auto blood analyser (Bayer Corp. USA). Glucose, triglyceride, uric acid, and total cholesterol kits were obtained from Siemens, India.
2.4.2 Serum nitric oxide
Nitric oxide was determined by a commercially available kit (Assay design, USA). Assay is based on reduction of NO3- into NO2- using nitrate reductase. The azo dye is produced by diazotization of sulfanilic acid (Griss Reagent-1) with NO2- and then subsequent coupling with N-(1-napthyl)-ethylene diamine (Griss Reagent-2). The azo dye was measured calorimetrically at 540nm. Serum NO level was expressed as nmol/L.
2.4.3 Serum hydrogen sulphide (H2S)
Serum H2S concentration was measured as described by Cai et. al, 2007(17) after some modifications. Briefly, 0.1 ml serum was added into a test tube containing 0.125 ml 1% zinc acetate and 0.15 ml distilled water. Then 0.067 ml 20mM N, N-dimethyl-phenylene diamine dihydrochloride in 7.2M HCL was added. This was followed by addition of 0.067 ml 30mM FeCl3 in 1.2M HCL. The absorbance of resulting solution was measured with a spectrophotometer at a wavelength of 670 nm. The H2S concentration in a solution was calculated according to the calibration curve of sodium hydrogen sulphide (NaHS: 3.12-400Âµmol) and data were expressed as H2S concentration in Âµmol/L.
2.4.4 Estimation of glycated haemoglobin
Glycated haemoglobin was estimated by using ion exchange microcoloumns (Biosystem Ltd, Spain). After preparing the hemolysate, where the labile fraction is eliminated, haemoglobin's were retained by a cationic exchange resin. Haemoglobin A1c (HbA1c) was specifically eluted after washing away the haemoglobin A1a and A1b fractions, and was quantified by direct spectrophotometric reading at 415 nm.
2.4.6 Estimation of serum insulin levels
Quantitative estimation of serum insulin was done by rat insulin ELISA kits (Mercodia, USA). It is a solid phase two-site enzyme Immunoassay. It is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. During incubation, insulin in the sample reacts with peroxidase-conjugated anti-insulin antibodies which are then bound to microtitration well. A simple washing step removes unbound enzyme loaded antibody. The bound conjugate detected by reaction 3, 3', 5, 5'-tetramethylbenzidine. The reaction was stopped by adding acid and read using a spectrophotometer at 450 nm.
2.5 Statistical analysis
All values are expressed as mean Â± SEM. Data were statistically analysed using one way ANOVA for multiple group comparison, followed by student unpaired 't' test for group wise comparison. Significance was set at Pâ‰¤0.05. Data were computed for statistical analysis by using Graph Pad Prism Software.
3.1 Body weight gain
There was no significant difference in body weight gain between control and diabetic groups after 8 weeks of feeding. However, a significant (p<0.05) decrease in body weight gain was observed in Dia+Garl group when compared to both control and diabetic groups (Table-1).
3.2 Glucose levels
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After 3 weeks of feeding, no significant change in blood glucose levels was observed in fructose fed rats (diabetic group) compared to rats from control group (Fig. 1A). But after 8 weeks of feeding, rats from the diabetic group showed a significant (p<0.05) increase in blood glucose levels compared to control rats. However this increased serum glucose levels in fructose feeding rats was significantly (p<0.05) decreased after chronic administration of garlic (Dia+Garl group) (Fig. 1B).
3.3 Triglyceride levels
Serum triglyceride levels were measured at different time intervals during the study. A significant increase in serum triglyceride levels was observed after 3 and 8 weeks of fructose feeding in rats from diabetic group. However this increased serum triglyceride level in fructose feeding rats was significantly (p<0.05) decreased after chronic administration of garlic (Dia+Garl group) (Fig. 1C & 1D).
3.4 Serum insulin levels
After 8 weeks, serum insulin levels were significantly (pË‚0.01) higher in the diabetic group when compared to the control group. Chronic administration of garlic (Dia+Garl group) significantly (p<0.05) reduced serum uric acid levels when compared to diabetic group (Fig.2A).
3.5 Glycated Haemoglobin
After 8 weeks, no significant increase in serum glycated haemoglobin levels was observed in diabetic group as compared to control. However, a significant (p<0.05) decrease in serum glycated haemoglobin levels was observed in Dia+Garl group when compared to diabetic group (Fig.2B).
3.6 Total cholesterol levels
After 8 weeks, no significant change in serum cholesterol level was observed between control and diabetic group. Similarly no change in cholesterol level was observed after chronic administration of garlic (Fig.2C).
3.7 Uric acid levels
After 8 weeks, serum uric acid levels were significantly (p<0.05) increased in the diabetic group as compared to the control group. Chronic administration of garlic (Dia+Garl group) significantly (p<0.05) reduced serum uric acid level as compared to diabetic group (Fig.2D).
3.8 Nitric oxide levels
Serum nitric oxide levels were significantly (p<0.05) increased in the diabetic group after 8 weeks as compared to the control group. Chronic administration of garlic (Dia+Garl group) significantly (p<0.05) reduced serum nitric oxide levels in fructose fed rats when compared to the diabetic group (Fig.3A).
3.9 Hydrogen sulphide levels
Serum hydrogen sulphide levels were significantly decreased (p<0.05) in the diabetic group after 8 weeks as compared to control group. Chronic administration of garlic (Dia+Garl group) significantly (p<0.05) increased serum hydrogen sulphide levels in fructose fed rats when compared to diabetic group (Fig.3B).
3.10 Intraperitoneal glucose tolerance test
An intraperitoneal glucose load led to marked increase in blood glucose levels in diabetic group, at 5 and 30 min, compared to the control group. Chronic administration of garlic (Dia+Garl group) prevented this rise in serum glucose levels and was observed to be lower than the control group (Fig. 3C).
High fructose corn syrup (HFCS) - a corn-based sweetener that has been on the market since approximately 1970, is a popular food sweetener. Consumption of fructose in the form of HCFS is high in many countries including USA. Between 1970 and 1990, the consumption of HFCS increased over 1,000 percent . High fructose intake over long periods is known to be hazardous for human beings as well as animals [18-20]. In the present study, a fructose rich diet has been used for the induction of diabetes, which is characterized by insulin resistance and metabolic syndrome very much similar to that human type-2 diabetes. Previous studies have shown that long-term fructose feeding induces diabetes associated with insulin resistance and metabolic syndrome in experimental animals such as rats and mice [16,21-23]
In the present study, rats were fed with a 65% fructose diet for a period of eight weeks in order to induce diabetes associated with insulin resistance and metabolic syndrome. Although blood triglycerides level was increased after 3 weeks of high fructose feeding, we observed an increase in blood glucose level only after 8 weeks. Along with triglycerides, increase in other biochemical parameters associated with the metabolic syndrome such as uric acid and plasma insulin levels However, blood cholesterol and glycated haemoglobin were not significantly affected in this rat model. Most importantly, insulin resistance, an important pathogenic mechanism in human Type-2 diabetes and cause of all metabolic complications, was present in this model of diabetes, as evidenced by the altered glucose tolerance test. Current medical research focuses on correcting insulin resistance the primary underlying disorder in type 2 diabetes.
Naturally occurring compounds represent a valuable source of such therapeutic agents of which garlic (Allium sativum) holds a unique position in history and is well recognized for its therapeutic potential for diabetes and metabolic complications. Being the most common form of garlic intake, fresh garlic homogenate or raw garlic has been subjected to intense scientific study [24,25]. We have previously shown that garlic homogenate at 250 mg/kg dose in rat is more effective against heart disease without any adverse effect [14,25]. Several studies have reported the hypoglycaemic effect of garlic, which has been attributed primarily to the presence of allicin-type compounds [16,26,27]. The exact mechanism/s of garlic in diabetes is still not clear, however, some reports suggest that increased secretion of insulin or its release from bound insulin after garlic treatment might be responsible for this effect .
Although the antidiabetic effect of raw garlic has been well established in the type-I experimental diabetic model [6-10] no experimental study has yet been conducted to evaluate its effect on insulin resistance as well as other metabolic complications. In the present study we evaluated whether oral administration of raw garlic homogenate improves insulin sensitivity and associated metabolic syndromes in fructose fed rats. Oral administration of raw garlic for a period of eight weeks showed beneficial effect on fructose fed diabetic rats. There was significant reduction of blood glucose and improvement of insulin sensitivity in garlic treated rats. Other metabolic complications like increased serum triglyceride, insulin and uric acid levels observed in diabetic rats were also normalised after garlic administration. There is evidence that fructose-induced insulin resistance is mediated by fructose-induced hyperuricemia or hypertriglyceridemia [21,22]. Lowering serum uric acid and triglyceride after garlic administration might be responsible for improving insulin resistance in fructose fed rats. An interesting observation of the present study was that chronic administration of garlic reduced body weight gain significantly compared to both control and diabetic rats. Previously Elkayam et al (2003) also reported that allicin, one of the components of raw garlic paste, reduced weight gain in fructose fed rats . Reduction of body weight gain could also be responsible for improving insulin sensitivity in fructose fed rats.
NO and H2S are key players in disease progression [30-32]. Similar to NO, H2S is considered to be an important vasodilator, inducing endothelium-dependent and K+-ATP channel-dependent vasorelaxation in vivo and in vitro . Increased serum level of NO  and decreased level of H2S  has been reported in diabetic patients. In the present study we measured both serum NO and H2S levels in diabetic rats. Serum NO level was significantly higher while H2S level was significantly lower in diabetic rats compared to control. Importantly, chronic administration of garlic normalised both gaseous molecules in fructose fed rats.
We concluded that high fructose feeding for 8 weeks induced diabetes along with insulin resistance and metabolic disorder. Oral administration of raw garlic homogenate increased insulin sensitivity and reduced metabolic complications in diabetic rats. Further human studies are essential to establish the role of garlic in controlling type 2 diabetes and its complications.