Changes in cultured endothelial cell glycosaminoglycans under hyperglycemic conditions and the effect of insulin and heparin

Background

Diabetic cardiomyopathy (DCM) contributes to cardiac failure in diabetic patients. It is characterized by excessive lipids accumulation, with increased triacylglycerol (TAG) stores, and fibrosis in left ventricle (LV). The mechanisms responsible are incompletely known and no specific treatment is presently defined. We evaluated the possible usefulness of two molecules promoting lipid oxidation, fenofibrate and metformin, in an experimental model of DCM, the Zucker diabetic rat (ZDF).

Methods

ZDF and controls (C) rats were studied at 7, 14 and 21 weeks. After an initial study at 7 weeks, ZDF rats received no treatment, metformin or fenofibrate until final studies (at 14 or 21 weeks). C rats received no treatment. Each study comprised measurements of metabolic parameters (plasma glucose, TAG, insulin levels) and sampling of heart for histology and measurements of TAG content and relevant mRNA concentration.

Results

ZDF rats were insulin-resistant at 7 weeks, type 2 diabetic at 14 weeks and diabetic with insulin deficiency at 21 weeks. Their plasma TAG levels were increased. ZDF rats had at 7 weeks an increased LV TAG content with some fibrosis. LV TAG content increased in untreated ZDF rats at 14 and 21 weeks and was always higher than in C. Fibrosis increased also moderately in untreated ZDF rats. Metformin and fenofibrate decreased plasma TAG concentrations. LV TAG content was decreased by metformin (14 and 21 weeks) and by fenofibrate (14 weeks). Fibrosis was reduced by fenofibrate only and was increased by metformin. Among the mRNA measured, fenofibrate increased Acyl-CoA Oxidase mRNA level, metformin decreased Acyl-CoA Synthase and increased AdipoR1 and pro-inflammatory mRNA levels.

Conclusion

Fenofibrate had favourable actions on DCM. Metformin had beneficial effect on TAG content but not on fibrosis. PPARα agonists could be useful for the prevention and treatment of DCM.     

Metformin Normalizes Type 2 Diabetes-Induced Decrease in Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus

In this study, we observed the effects of metformin, one of the most widely prescribed drugs for the treatment of type 2 diabetes, on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG) in Zucker diabetic fatty (ZDF) rats, which are a model for type 2 diabetes. For this, metformin was administered orally once a day to 14-week-old ZDF rats for 2 weeks and the animals were sacrificed at 16 weeks of age. During this period, blood glucose levels were higher in the vehicle-treated ZDF rats than in the Zucker lean control (ZLC) rats. Metformin treatment significantly decreased the blood glucose levels from 15.5 weeks of age. In the SZDG, Ki67 (a marker for cell proliferation)- and doublecortin (DCX, a marker for differentiated neuroblasts)-immunoreactive cells were much lower in the vehicle-treated ZDF rats than in the ZLC rats. In the metformin-treated ZDF group, Ki67- and DCX-immunoreactive cells were significantly increased in the SZDG compared to those in the vehicle-treated ZDF group. These results suggest that diabetes significantly reduces cell proliferation and neuroblast differentiation in the SZDG and that metformin treatment normalizes the reduction of cell proliferation and neuroblast differentiation in the SZDG in diabetic rats.     

The establishment of a novel non-alcoholic steatohepatitis model accompanied with obesity and insulin resistance in mice

Non-alcoholic steatohepatitis (NASH) is a hepatic manifestation of the metabolic syndrome that can progress to liver cirrhosis. The major aim of this study was to establish a novel NASH mouse model accompanied by obesity and insulin resistance, then explore the molecular mechanisms of NASH and evaluate the effects of both the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist fenofibrate and the PPARgamma agonist rosiglitazone in this established NASH model. The novel model was induced in C57BL/6 mice by 23 weeks of ad libitum feeding of a modified high-fat diet (mHFD), with lower methinione and choline and higher fat content. In comparison to the controls, the model animals developed pronounced obesity, dyslipidemia and insulin resistance. Marked liver lesions characterized by severe steatosis, inflammation, fibrosis, increased hepatic triglyceride content, and elevated serum alanine aminotransferase (ALT) levels were observed in the models. In this novel model, treatment with fenofibrate or rosiglitazone significantly improved insulin sensitivity and corrected dyslipidemia; however, fenofibrate was more effective than rosiglitazone in improving hepatic morphology and ALT levels. Further study showed that long-term feeding of mHFD significantly increased expression of mRNA for hepatic PPARgamma, adipose fatty acid binding protein (ap2) and CD36 and suppressed expression of mRNA for hepatic PPARalpha and carnitine palmitoyl transferase-1a (CPT-1a). These results showed the successful establishment of the combined NASH and obese-insulin resistance mouse model. Additionally, aberrant expressions of hepatic PPARalpha and PPARgamma may play a major role in the pathogenesis of NASH by affecting hepatic lipogenesis and fatty acid oxidation in this novel model.     

Metformin Prevents and Reverses Inflammation in a Non-Diabetic Mouse Model of Nonalcoholic Steatohepatitis

Background

Optimal treatment for nonalcoholic steatohepatitis (NASH) has not yet been established, particularly for individuals without diabetes. We examined the effects of metformin, commonly used to treat patients with type 2 diabetes, on liver pathology in a non-diabetic NASH mouse model.

Methodology/Principal Findings

Eight-week-old C57BL/6 mice were fed a methionine- and choline-deficient plus high fat (MCD+HF) diet with or without 0.1% metformin for 8 weeks. Co-administration of metformin significantly decreased fasting plasma glucose levels, but did not affect glucose tolerance or peripheral insulin sensitivity. Metformin ameliorated MCD+HF diet-induced hepatic steatosis, inflammation, and fibrosis. Furthermore, metformin significantly reversed hepatic steatosis and inflammation when administered after the development of experimental NASH.

Conclusions/Significance

These histological changes were accompanied by reduced hepatic triglyceride content, suppressed hepatic stellate cell activation, and the downregulation of genes involved in fatty acid metabolism, inflammation, and fibrogenesis. Metformin prevented and reversed steatosis and inflammation of NASH in an experimental non-diabetic model without affecting peripheral insulin resistance.     

Hepatic n-3 polyunsaturated fatty acid depletion promotes steatosis and insulin resistance in mice: genomic analysis of cellular targets

Patients with non-alcoholic fatty liver disease are characterised by a decreased n-3/n-6 polyunsaturated fatty acid (PUFA) ratio in hepatic phospholipids. The metabolic consequences of n-3 PUFA depletion in the liver are poorly understood. We have reproduced a drastic drop in n-3 PUFA among hepatic phospholipids by feeding C57Bl/6J mice for 3 months with an n-3 PUFA depleted diet (DEF) versus a control diet (CT), which only differed in the PUFA content. DEF mice exhibited hepatic insulin resistance (assessed by euglycemic-hyperinsulinemic clamp) and steatosis that was associated with a decrease in fatty acid oxidation and occurred despite a higher capacity for triglyceride secretion. Microarray and qPCR analysis of the liver tissue revealed higher expression of all the enzymes involved in lipogenesis in DEF mice compared to CT mice, as well as increased expression and activation of sterol regulatory element binding protein-1c (SREBP-1c). Our data suggest that the activation of the liver X receptor pathway is involved in the overexpression of SREBP-1c, and this phenomenon cannot be attributed to insulin or to endoplasmic reticulum stress responses. In conclusion, n-3 PUFA depletion in liver phospholipids leads to activation of SREBP-1c and lipogenesis, which contributes to hepatic steatosis.     

Effects of fenofibrate and insulin on the biosynthesis of unsaturated fatty acids in streptozotocin diabetic rats

Both insulin and PPAR-alpha up-modulate hepatic Delta9, Delta6 and Delta5 desaturating enzymes involved in the biosynthesis of mono- and polyunsaturated fatty acids. Currently, we have examined for 9 days the independent and simultaneous effects of daily glargine insulin and fenofibrate administration on the insulinemia, glycemia, hepatic acyl-CoA oxidase activity and mRNAs and enzymatic activities of stearoyl-CoA desaturase-1 (SCD-1) and Delta5 desaturase in streptozotocin diabetic rats. Glargine insulin depressed the hyperglycemia of diabetic rats at 4h, but not after 24h of injection. Fenofibrate increased the radioimmunoreactive insulinemia in non-diabetic rats without changing the glycemia. Insulin increased the mRNAs and activities of SCD-1 and Delta5 desaturase depressed in diabetic rats. Fenofibrate increased acyl-CoA oxidase activity, and the mRNAs and activities of both desaturating enzymes in non-diabetic, diabetic and insulin-treated diabetic rats, but was less effective in the mRNAs modification of diabetic animals. Therefore, insulin, and fenofibrate through PPAR-alpha activation, enhance liver mRNAs and activities of SCD-1 and Delta5 desaturases independently and synergistically through different mechanisms. Insulin and fenofibrate independently increased the 18:1/18:0 ratio in liver lipids, increasing the fluidity of the membranes. The 20:4/18:2 ratio was maintained. Fenofibrate increased palmitic acid, but decreased stearic acid percentage in liver lipids.     

12α-Hydroxylated bile acid induces hepatic steatosis with dysbiosis in rats

There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4β-hydroxycholesterol (4βOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4βOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4βOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.     

Metformin Suppresses Diethylnitrosamine-Induced Liver Tumorigenesis in Obese and Diabetic C57BL/KsJ-+Leprdb/+Leprdb Mice

Obesity and related metabolic disorders, such as diabetes mellitus, raise the risk of liver carcinogenesis. Metformin, which is widely used in the treatment of diabetes, ameliorates insulin sensitivity. Metformin is also thought to have antineoplastic activities and to reduce cancer risk. The present study examined the preventive effect of metformin on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in C57BL/KsJ-+Lepr^db/+Lepr^db (db/db) obese and diabetic mice. The mice were given a single injection of DEN at 2 weeks of age and subsequently received drinking water containing metformin for 20 weeks. Metformin administration significantly reduced the multiplicity of hepatic premalignant lesions and inhibited liver cell neoplasms. Metformin also markedly decreased serum levels of insulin and reduced insulin resistance, and inhibited phosphorylation of Akt, mammalian target of rapamycin (mTOR), and p70S6 in the liver. Furthermore, serum levels of leptin were decreased, while those of adiponectin were increased by metformin. These findings suggest that metformin prevents liver tumorigenesis by ameliorating insulin sensitivity, inhibiting the activation of Akt/mTOR/p70S6 signaling, and improving adipokine imbalance. Therefore, metformin may be a potent candidate for chemoprevention of liver tumorigenesis in patients with obesity or diabetes.     

Reduced serum dipeptidyl peptidase-IV after metformin and pioglitazone treatments

Dipeptidyl peptidase-IV (DPP-IV) regulates metabolism by degrading incretins involved in nutritional regulation. Metformin and pioglitazone improve insulin sensitivity whereas glyburide promotes insulin secretion. Zucker diabetic rats were treated with these antidiabetic agents for 2 weeks and DPP-IV activity and expression were determined. Serum DPP-IV activity increased whereas tissue activity decreased as the rats aged. Treatment of rats with metformin, pioglitazone, and glyburide did not alter DPP-IV mRNA expression in liver or kidney. Metformin and pioglitazone significantly (P<0.05) reduced serum DPP-IV activity and glycosylated hemoglobin. Glyburide did not lower DPP-IV activity or glycosylated hemoglobin. Regression analysis showed serum DPP-IV activity correlated with glycosylated hemoglobin (r=0.92) and glucagon-like peptide-1 levels (r=-0.49). Metformin, pioglitazone, and glyburide had no effect on serum DPP-IV activity in vitro, indicating these are not competitive DPP-IV inhibitors. We propose the in vivo inhibitory effects observed with metformin and pioglitazone on serum DPP-IV activity results from reduced DPP-IV secretion.     

Sixteen hours of fasting differentially affects hepatic and muscle insulin sensitivity in mice

Fasting readily induces hepatic steatosis. Hepatic steatosis is associated with hepatic insulin resistance. The purpose of the present study was to document the effects of 16 h of fasting in wild-type mice on insulin sensitivity in liver and skeletal muscle in relation to 1) tissue accumulation of triglycerides (TGs) and 2) changes in mRNA expression of metabolically relevant genes. Sixteen hours of fasting did not show an effect on hepatic insulin sensitivity in terms of glucose production in the presence of increased hepatic TG content. In muscle, however, fasting resulted in increased insulin sensitivity, with increased muscle glucose uptake without changes in muscle TG content. In liver, fasting resulted in increased mRNA expression of genes promoting gluconeogenesis and TG synthesis but in decreased mRNA expression of genes involved in glycogenolysis and fatty acid synthesis. In muscle, increased mRNA expression of genes promoting glucose uptake, as well as lipogenesis and beta-oxidation, was found. In conclusion, 16 h of fasting does not induce hepatic insulin resistance, although it causes liver steatosis, whereas muscle insulin sensitivity increases without changes in muscle TG content. Therefore, fasting induces differential changes in tissue-specific insulin sensitivity, and liver and muscle TG contents are unlikely to be involved in these changes.     

Fructus Xanthii Attenuates Hepatic Steatosis in Rats Fed on High-Fat Diet

Fructus Xanthii (FX) has been widely used as a traditional herbal medicine for rhinitis, headache, cold, etc. Modern pharmacological studies revealed that FX possesses anti-inflammatory, anti-oxidative, and anti-hyperglycemic properties. The present study was designed to investigate the effects of FX on glucose and insulin tolerance, and hepatic lipid metabolism in rats fed on high-fat diet (HFD). Hepatic steatosis was induced by HFD feeding. Aqueous extraction fractions of FX or vehicle were orally administered by gavage for 6 weeks. Body weight and blood glucose were monitored. Glucose and insulin tolerance test were performed. Liver morphology was visualized by hematoxylin and eosin, and oil red O staining. Expression of liver lipogenic and lipolytic genes was measured by real-time PCR. We showed here that FX improved glucose tolerance and insulin sensitivity in HFD rats. FX significantly decreased the expression of lipogenic genes and increased the expression of lipolytic genes, ameliorated lipid accumulation and decreased the total liver triglyceride (TG) content, and thus attenuated HFD-induced hepatic steatosis. In conclusion, FX improves glucose tolerance and insulin sensitivity, decreases lipogenesis and increases lipid oxidation in the liver of HFD rats, implying a potential application in the treatment of non-alcoholic fatty liver disease.     

Metformin reverses fatty liver disease in obese, leptin-deficient mice

There is no known treatment for fatty liver, a ubiquitous cause of chronic liver disease. However, because it is associated with hyperinsulinemia and insulin-resistance, insulin-sensitizing agents might be beneficial. To evaluate this possibility, insulin-resistant ob/ob mice with fatty livers were treated with metformin, an agent that improves hepatic insulin-resistance. Metformin improved fatty liver disease, reversing hepatomegaly, steatosis and aminotransferase abnormalities. The therapeutic mechanism likely involves inhibited hepatic expression of tumor necrosis factor (TNF) alpha and TNF-inducible factors that promote hepatic lipid accumulation and ATP depletion. These findings suggest a mechanism of action for metformin and identify novel therapeutic targets in insulin-resistant states.     

Beneficial Effect of Fenofibrate and Silymarin on Hepatic Steatosis and Gene Expression of Lipogenic and Cytochrome P450 Enzymes in Non-Obese Hereditary Hypertriglyceridemic Rats

The efficacy of fenofibrate in the treatment of hepatic steatosis has not been clearly demonstrated. In this study, we investigated the effects of fenofibrate and silymarin, administered as monotherapy and in combination to existing hepatic steatosis in a unique strain of hereditary hypertriglyceridemic rats (HHTg), a non-obese model of metabolic syndrome. HHTg rats were fed a standard diet without or with fenofibrate (100 mg/kg b.wt./day) or with silymarin (1%) or with a combination of fenofibrate with silymarin for four weeks. Fenofibrate alone and in combination with silymarin decreased serum and liver triglycerides and cholesterol and increased HDL cholesterol. These effects were associated with the decreased gene expression of enzymes involved in lipid synthesis and transport, while enzymes of lipid conversion were upregulated. The combination treatment had a beneficial effect on the gene expression of hepatic cytochrome P450 (CYP) enzymes. The expression of the CYP2E1 enzyme, which is source of hepatic reactive oxygen species, was reduced. In addition, fenofibrate-induced increased CYP4A1 expression was decreased, suggesting a reduction in the pro-inflammatory effects of fenofibrate. These results show high efficacy and mechanisms of action of the combination of fenofibrate with silymarin in treating hepatic steatosis and indicate the possibility of protection against disorders in which oxidative stress and inflammation are involved.     

Postnatal overfeeding promotes early onset and exaggeration of high-fat diet-induced nonalcoholic fatty liver disease through disordered hepatic lipid metabolism in rats

Exposure to overnutrition in critical or sensitive developmental periods may increase the risk of developing obesity and metabolic syndrome in adults. Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome, but the relationship among postnatal nutrition, lipid metabolism, and NAFLD progression during development remains poorly understood. Here we investigated in a rat model whether postnatal overfeeding increases susceptibility to NAFLD in response to a high-fat diet. Litters from Sprague-Dawley dams were culled to three (small litters) or ten (normal litters) pups and then weaned onto a standard or high-fat diet at postnatal day 21 to generate normal-litter, small-litter, normal-litter/high-fat, and small-litter/high-fat groups. At age 16 weeks, the small-litter and both high-fat groups showed obesity, dyslipidemia, and insulin resistance. Hepatic disorders appeared earlier in the small-litter/high-fat rats with greater liver mass gain and higher hepatic triglycerides and steatosis score versus normal-litter/high-fat rats. Hepatic acetyl-CoA carboxylase activity and mRNA expression were increased in small-litter rats and aggravated in small-litter/high-fat rats but not in normal-litter/high-fat rats. The high expression in small-litter/high-fat rats coincided with high sterol regulatory element-binding protein-1c mRNA and protein expression. However, mRNA expression of enzymes involved in hepatic fatty acid oxidation (carnitine palmitoyltransferase 1) and output (microsomal triglyceride transfer protein) was decreased under a high-fat diet regardless of litter size. In conclusion, overfeeding related to small-litter rearing during lactation contributes to the NAFLD phenotype when combined with a high-fat diet, possibly through up-regulated hepatic lipogenesis.     

Pioglitazone reverses down-regulation of cardiac PPARgamma expression in Zucker diabetic fatty rats

Peroxisome proliferator-activated receptor-gamma (PPARgamma) plays a critical role in peripheral glucose homeostasis and energy metabolism, and inhibits cardiac hypertrophy in non-diabetic animal models. The functional role of PPARgamma in the diabetic heart, however, is not fully understood. Therefore, we analyzed cardiac gene expression, metabolic control, and cardiac glucose uptake in male Zucker diabetic fatty rats (ZDF fa/fa) and lean ZDF rats (+/+) treated with the high affinity PPARgamma agonist pioglitazone or placebo from 12 to 24 weeks of age. Hyperglycemia, hyperinsulinemia, and hypertriglyceridemia as well as lower cardiac PPARgamma, glucose transporter-4 and alpha-myosin heavy chain expression levels were detected in diabetic ZDF rats compared to lean animals. Pioglitazone increased body weight and improved metabolic control, cardiac PPARgamma, glut-4, and alpha-MHC expression levels in diabetic ZDF rats. Cardiac [(18)F]fluorodeoxyglucose uptake was not detectable by micro-PET studies in untreated and pioglitazone treated ZDF fa/fa rats but was observed after administration of insulin to pioglitazone treated ZDF fa/fa rats. PPARgamma agonists favorably affect cardiac gene expression in type-2 diabetic rats via activation and up-regulation of cardiac PPARgamma expression whereas improvement of impaired cardiac glucose uptake in advanced type-2 diabetes requires co-administration of insulin.