Anti-Hyperlipidemic and Myocardial Enhancing Effects of Berberine in High Fat Diet/Streptozotocin-Induced Diabetic Rats; Possible Role of Adiponectin

Aim: Berberine (BBR) is an isoquinoline alkaloid with promising anti-diabetic effect. The current study aimed to investigate the antihyperlipidemic and myocardial protective effect of BBR in high fat diet (HFD)/streptozotocin(STZ)-induced type 2 diabetic rats, highlighting the role of adiponectin. Materials and Methods: Rats were fed a HFD for 4 weeks followed by a single intraperitoneal injection STZ (35mg/kg body weight). Diabetic rats were treated either with 50mg/kg or 100mg/kg BBR for 4 weeks. Results: The present results showed a significant increase in fasting and post-prandial glucose levels with concomitant declined serum insulin and liver glycogen levels in diabetic rats. Serum levels of total cholesterol, triglycerides and free fatty acids (FFAs) showed a significant increase in the diabetic rats. Treatment with BBR significantly ameliorated serum glucose, insulin and lipids, and liver glycogen content. In addition, diabetic rats exhibited significantly increased serum AST, CK-MB and LDH, and cardiovascular risk indices, an effect that was markedly reversed following treatment with BBR. Oral supplementation of BBR significantly increased serum levels of adiponectin. Conclusion: The ameliorative effect of BBR on carbohydrate and lipid variables in diabetes may be attributed to its insulin releasing capacity, lipid lowering effect, and increasing adiponectin levels.


Introduction
Type 2 diabetes mellitus (T2DM), the frequent form of diabetes, represents more than 90% of all diabetic patients [1]. Insulin resistance and impaired insulin secretion are two main characteristics of type 2 diabetes [2]. Insulin resistance in T2DM could be provoked by glucotoxicity, oxidative stress, lipotoxicity and inflammation [3]. Recently, the International Diabetes Federation (IDF) reported that the number of diabetic patients was 415 million in 2015 and is expected to rise to 642 million by 2040 [4]. If ineffectively controlled in diabetic patients, chronic hyperglycemia can cause serious complications in different organs [4]. Diet, exercise and several pharmacological agents are treatment approaches for type 2 DM. However, the use of currently used pharmacological agents is associated with adverse side effects such as weight gain, hypoglycemia and edema [5]. Therefore, search for effective alternatives with minimal or no side effects became a necessity. Berberine (BBR), an isoquinoline alkaloid originally isolated from Coptischinensis (Huanglian), showed several pharmacological effects including, antioxidant, anti-inflammatory, anticancer, antimicrobial and hepato protective properties [6][7][8][9][10]. Reducing hyperglycemia was also among the beneficial effects of BBR [11,12] however, the exact anti-diabetic mechanism is not completely understood. Insulin resistance and T2DM have been associated with dysfuncton in adipocytes [13]. Studies have demonstrated the role of adipose tissue in energy expenditure and insulin sensitivity [14] which is mediated through the secretion of diverse adipocytokines such as adiponectin [13,15]. Reduced adiponectin levels can provoke insulin resistance [13,15]. We hypothesized that the increase in circulating adiponectin levels may mediate, at least in part, BBR's anti-diabetic activity. Therefore, this study was undertaken to investigate the modulatory role of BBR on adiponectinand hyperlipidemia in high fat diet (HFD)/

Induction of type 2 diabetes mellitus
The rats received HFD (58% fat, 25% protein and 17% carbohydrate, as a percentage of total kcal) ad libitum. The composition and preparation of HFD were described by Reed et al [16]. After 4 weeks, rats were injected intraperitoneally (i.p.) with 35mg/kg streptozotocin (STZ; Sigma, USA) dissolved in cold citrate buffer (pH 4.5) in a dose volume of 1ml/kg [17]. Seven days after STZ injection, rats were screened for blood glucose levels. Rats having glucose levels more than 200mg/dl, after 2 h of glucose (3g/kg) intake were considered diabetic.

Experimental design
Eighteen diabetic and 6 normal rats were divided into 4 groups, each consisting of six (N = 6) animals as following: Group 1 (Control): normal rats received normal diet and the vehicle 1% carboxy methyl cellulose (CMC) by gastric intubation daily for 4 weeks and served as control rats.
The rats were allowed to continue to feed on their diets until the end of the study. Twenty-four hr after the last treatment, the rats were fasted overnight, sacrificed and blood samples were collected for analysis.

Biochemical study
Glucose levels were determined using reagent kit purchased from Spin react (Spain) according to Trinder [19] method. Glycogen content in the liver was assayed following the method of Seifter, et al. [20]. Total cholesterol and triglycerides were estimated using reagent kit purchased fromVitro Scientific (Egypt) according to the method of Allain, et al. [21] and Fossati and Prencipe [22], respectively. HDL-cholesterol concentration was measured according to the method of Allain, et al. [21] using kits purchased from Spinreact (Spain).Serum free fatty acids (FFAs) were estimated according to the method of Harris [23].
Serum LDL-and vLDL cholesterol were calculated using the equations:

Determination of serum insulin and adiponectin and Homeostasis Model of Insulin Resistance (HOMA-IR)
Serum insulin and adiponectin were determined using specific ELISA kits (R&D systems, USA) following the manufacturer's instructions. The insulin resistance was evaluated by homeostasis model assessment estimate of insulin resistance (HOMA-IR) as follows [29]:

Statistical analysis
The obtained data were analyzed by the one-way ANOVA followed by Tukey's test post hoc analysis using Graphpad Prism 5 (San Diego, CA, USA). The results were presented as mean ± standard error (SEM) and a P value <0.05 was considered significant.

BBR attenuates hyperglycemia in HFD/STZ-diabetic rats
The HFD/STZ-induced diabetic rats showed significant (P<0.001) increase in fasting and post-prandial serum glucose levels when compared with the control group, as depicted in Figure 1A. Treatment with BBR at either 50 or 100mg/kg for 4 weeks significantly (P<0.001) improved both fasting and postprandial glucose levels in diabetic rats. Liver glycogen content showed a significant (P<0.01) decrease in HFD/STZinduced diabetic rats when compared with the control rats Nutrition & Food Science International Journal ( Figure 1B). Oral supplementation of 50mg/kg BBR produced a significant (P<0.05) amelioration in liver glycogen content. Similarly, treatment of the diabetic with 100mg/kg BBR significantly (P<0.05) increased liver glycogen content when compared to the diabetic group

BBR alleviates insulin sensitivity in HFD/STZ-diabetic rats
In comparison with the control group, the HFD/STZ-induced Diabetic rats showed a significant (P<0.001) decrease in serum insulin levels, as depicted in Table 1. Treatment of the diabetic rats with 50 as well as 100 mg/kg BBR significantly (P<0.001) ameliorated serum insulin levels when compared with the diabetic control rats. The higher BBR dose markedly (P<0.01) increased serum insulin levels as compared to the lower dose. In the same context, diabetic rats showed a significant (P<0.001) increase in HOMA-IR when compared with the control rats. Supplementation of BBR significantly decreased HOMA-IR index at either 50 mg (P < 0.05) or 100 mg/kg (P<0.01) dose (Table 1).

Nutrition & Food Science International Journal
The present data showed significant (P<0.001) increase in serum total cholesterol (Figure 2A), triglycerides ( Figure 2B), LDL-cholesterol ( Figure 2C), vLDL-cholesterol ( Figure 2D) and FFA ( Figure 2F) levels in HFD/STZ induced diabetic rats when compared to the normal control rats. Treatment of HFD/STZadministered rats with 50mg/kg BBR significantly (P<0.001) improved serum cholesterol, triglycerides and FFA levels when compared to diabetic control rats. The higher BBR dose (100mg/ kg) ameliorated all parameters of the lipid profile and as well as FFA levels. Serumlevels of HDL-cholesterol showed nonsignificant (P>0.05) variation between all studied groups as represented in (Figure 2E). To investigate the cardio-protective effect of BBR in diabetic rats, we determined its effect on cardiovascular risk and atherogenic indices, and serum activities of AST, CK-MB and LDH. As depicted in Figure 3, HDF/STZ-induced diabetic rats exhibited a significant (P<0.001) increase in T. cholesterol/ HDL-cholesterol ratio when compared to their respective control group ( Figure 3A). Similarly, the LDL-cholesterol/HDLcholesterol ratio showed a significant increase in HDF/STZinduced diabetic rats when compared with the control group ( Figure 3B). On the other hand, HDF/STZ-induced diabetic rats exhibited a significantly (P<0.001) decreased antiatherogenic index value when compared with the control rats ( Figure 3C).

BBR increase serum levels of adiponectinin HFD/STZdiabetic rats
The Data illustrated in Figure 5 showed a significant (P<0.001) decrease in serum adiponectin levels of the diabetic rats received HFD and STZ when compared with their corresponding control rats. Oral administration of either 50 or 100mg/kg BBR markedly (P<0.001) alleviated serum adiponectin levels in HFD/ STZ-induced diabetic rats.

Discussion
In HFD/STZ-induced diabetic animals, our data showed significant increase in fasting and post-prandial glucose levels. Similar findings have been demonstrated in our previous studies Mahmoud et al. [17,30]. STZ is known to induce rapid destruction of pancreatic β-cells leading to impaired glucose stimulated insulin release and insulin resistance, both of which are marked features of type 2 diabetes [31]. The elevated blood glucose is a result of reduced glucose uptake in muscle and adipose tissue [32], and increased gluconeogenesis, hepatic glucose production [33] and glycogen breakdown [34].
The anti-hyperglycemic effect of BBR could be connected to its ability to increase insulin secretion and glucose storage as glycogen in the liver of HFD/STZ-induced type 2 diabetic rats. Previous data have demonstrated the ability of BBR to stimulate insulin secretion in HFD-fed rat [40] and repair destructed or exhausted islets [41]. In addition, liver glycogen may be considered a reliable marker for assessing anti-hyperglycemic potential of any drug [42]. Generally, decreased hepatic glycogen synthesis and glycolysis, and increased hepatic glucose production are the major symptoms in type 2 diabetes [43]. Our results revealed an enormous depletion in hepatic glycogen content which were in accordance to those of Lavoie and Van de Werve [44], and Ahmed et al. [45,46].
In the present study, the increased liver glycogen content after treatment with berberine could be attributed to amelioration of the glycogenolytic and gluconeogenic pathways secondary to the increase of insulin levels in the blood. In diabetic patients, changes in concentrations of plasma lipids are complications frequently observed and certainly contribute to the development of coronary heart disease (CHD) [47]. Abnormalities in lipid metabolism play a key role in the occurrence of atherosclerosis in patients with diabetes [48]. The present data showed significant hyperlipidemia in HFD/STZ-induced diabetic rats as previously reported [45,49,50]. Treatment with of the diabetic rats with BBR significantly improved all parameters of the lipid profile, demonstrating anti-hyperlipidemic effect.
In the same context, BBR has been reported to decrease plasma lipids in high-fat diet induced obese rats and in diabetic rats fed with high-cholesterol diet [12,51,52]. The lipid lowering effect of BBR may be due to increasing insulin secretion or stabilization of the hepatic LDL receptor (LDLR) by stimulated extracellular signal-regulated kinase-dependent pathway [53]. Moreover, BBR reduced circulating FFA in HFD/STZ-induced rats. Decreasing blood FFA has been proposed as a potent strategy for prevention and/or treatment of insulin resistance [54]. Therefore, the decreased level of serum FFA by BBR may participate in its insulin sensitizing effects.
CHD is a life threatening diabetes complication and its risk increases two-or more folds in diabetes [29]. Dyslipidemia in diabetes has been reported to be strongly associated with CHD [55]. Increased triglycerides and total cholesterol levels and decreased HDL-cholesterol represent atherogeniclipid profile, which leads to the development of CHD [56]. As a favorable effect on lipid profile was observed following treatment with BBR, this indicated that it might help prevent the progression of CHD. In addition, several atherogenic indices such as total cholesterol/ HDL-cholesterol and LDL-cholesterol/HDL-cholesterol have been used to predict CHD risk [57]. Reduction of these indices in BBR supplemented diabetic rats strongly supported the notion that BBRsupplementation may lead to reduction in the risk of developing CHD.
The cardioprotective effect of BBR was further confirmed through assessment of AST, CK-MB and LDH which were significantly ameliorated in the serum of diabetic rats. This myocardial protective effect of BBR appears to be mediated not only through alleviating glucose homeostasis and lipid metabolism.
Studies on ischemia/reperfusion-induced myocardial infarction [58] and type 2 diabetic rats [59] demonstrated that BBR exerts cardioprotective effect through modulation of the sympathetic activity of myocardium. Moreover, we hypothesized that the increased adiponectin levels may have a role in the beneficial effect of BBR in diabetic rats. In the present study, HFD/STZ diabetic rats exhibited diminished serum adiponectin levels as we previously reported [46,60], an effect that was reversed following administration of BBR.
Reduced levels of serum adiponectin have been reported to be associated with the etiology of obesity and type 2 diabetes [61]. Studies have demonstrated that adiponectin stimulates AMPK and regulates glucose metabolism [62], enhances glucose transport and fat oxidation in muscle [63], reduces hepatic glucose production [62], and activates peroxisome proliferator activated receptor-α with subsequently decreased liver and muscle TG content [64]. In conclusion, the ameliorative effect of BBR on carbohydrate and lipid variables in diabetes may be attributed to its insulin releasing capacity, lipid lowering effect, and increasing adiponectin levels.

Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.