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.     

Cloning and expression of stearoyl-CoA desaturase 1 (SCD-1) in the liver of the Sichuan white goose and landes goose responding to overfeeding

The EST sequence of goose (Anser cygnoides) Stearoyl-CoA desaturase 1(SCD-1) was obtained from a subtractive cDNA library. To further investigate the role of SCD-1 in lipid metabolism in geese, 5′-RACE and 3′-RACE were carried out in this study to obtain the complete cDNA sequence of goose SCD-1, which contained a 29-bp 5′ UTR, a 1074-bp open reading frame (ORF) encoding 357 amino acids, and a 125-bp 3′ UTR. The expression of SCD-1 was measured in several tissues, and the effects of overfeeding on the expression of SCD-1 were studied. The results of real time RT-PCR demonstrated that, compared to the brain, goose SCD-1 mRNA was more abundant in the liver. Overfeeding markedly increased the mRNA expression of SCD-1 in the liver of Sichuan White and Landes geese, and gene expression was markedly higher in the Sichuan White goose than in the landes goose. The mRNA abundance of SCD-1 in the liver had significant positive correlations with triacylglycerol (TG) content in liver lipids and in the levels of plasma insulin and very low-density lipoproteins (VLDL) levels in Sichuan white geese. However, the mRNA abundance of SCD-1 in the livers of Landes geese had only significant positive correlations with the TG content in liver lipids. In conclusion, SCD-1 is not only critical for hepatic steatosis in geese but is also important for the difference in lipid deposition in the livers of the two breeds.     

A1 adenosine receptor partial agonist lowers plasma FFA and improves insulin resistance induced by high-fat diet in rodents

There is substantial evidence in the literature that elevated plasma free fatty acids (FFA) play a role in the pathogenesis of type 2 diabetes. CVT-3619 is a selective partial A(1) adenosine receptor agonist that inhibits lipolysis and lowers circulating FFA. The present study was undertaken to determine the effect of CVT-3619 on insulin resistance induced by high-fat (HF) diet in rodents. HF diet feeding to rats for 2 wk caused a significant increase in insulin, FFA, and triglyceride (TG) concentrations compared with rats fed chow. CVT-3619 (1 mg/kg) caused a time-dependent decrease in fasting insulin, FFA, and TG concentrations. Acute administration of CVT-3619 significantly lowered the insulin response, whereas glucose response was not different with an oral glucose tolerance test. Treatment with CVT-3619 for 2 wk resulted in significant lowering of FFA, TG, and insulin concentrations in rats on HF diet. To determine the effect of CVT-3619 on insulin sensitivity, hyperinsulinemic euglycemic clamp studies were performed in C57BL/J6 mice fed HF diet for 12 wk. Glucose infusion rate was decreased significantly in HF mice compared with chow-fed mice. CVT-3619 treatment 15 min prior to the clamp study significantly (P < 0.01) increased glucose infusion rate to values similar to that for chow-fed mice. In conclusion, CVT-3619 treatment lowers FFA and TG concentrations and improves insulin sensitivity in rodent models of insulin resistance.     

A defect in the activity of Delta6 and Delta5 desaturases may be a factor predisposing to the development of insulin resistance syndrome

GLUT-4 (glucose transporter) receptor, tumor necrosis factor-alpha (TNF-alpha), interleukins-6 (IL-6), daf-genes and PPARs (peroxisomal proliferation activator receptors) play a role in the development of insulin resistance syndrome and associated conditions. But, the exact interaction between these molecules/factors and the mechanism(s) by which they produce insulin resistance syndrome is not clear. I propose that a defect in the activity of the enzymes Delta6 and Delta5 desaturases that are essential for the formation of long chain metabolites of essential fatty acids, linoleic acid and alpha-linolenic acid, is a factor in the development of insulin resistance syndrome. Long chain polyunsaturated fatty acids (LCPUFAs) increase cell membrane fluidity and enhance the number of insulin receptors and the affinity of insulin to its receptors; suppress TNF-alpha, IL-6, macrophage migration inhibitory factor (MIF) and leptin synthesis; increase the number of GLUT-4 receptors, serve as endogenous ligands of PPARs, modify lipolysis, and regulate the balance between pro- and anti-oxidants, and thus, play a critical role in the pathogenesis of insulin resistance. In the nematode, Caenorhabditis elegans, the protein encoded by daf-2 is 35% identical to the human insulin receptor; daf-7 codes a transforming growth factor-beta (TGF-beta) type signal and daf-16 enhances superoxide dismutase (SOD) expression. Melatonin has anti-oxidant actions similar to daf-16, TGF-beta and SOD. Calorie restriction enhances the activity of Delta6 and Delta5 desaturases, melatonin production, decreases daf-2 signaling, free radical generation, and augments anti-oxidant defenses that may explain the beneficial effect of diet control in the management of obesity, insulin resistance, and type II diabetes mellitus. These evidences suggest that the activities of Delta6 and Delta5 enzymes play a critical role in the expression and regulation of GLUT-4, TNF-alpha, IL-6, MIF, daf-genes, melatonin, and leptin by modulating the synthesis and tissue concentrations of LCPUFAs. Caloric restriction delays ageing by activating Sir 2 deacetylase in yeast, and expression of Sir 2 (SIRT1) in human cells. Both insulin and insulin-like growth factor-1 (IGF-1) attenuated this response. SIRT1 sequesters the proapoptotic factor Bax, prevents stress-induced apoptosis of cells, and thus, prolongs survival. In addition, SIRT1 repressed PPAR-gamma, and overexpression of SIRT1 attenuated adipogenesis, and upregulation of SIRT in differentiated fat cells triggered lipolysis and loss of fat, events that are known to attenuate insulin resistance and prolong life span. It remains to be seen whether LCPUFAs have a regulatory role in SIRT1 expression and control Sir 2 deacetylase activity. Thus, calorie restriction or reduced food intake has a role not only in the pathobiology of insulin resistance, but also in other associated conditions such as obesity, type II diabetes mellitus, ageing, and longevity.     

Effects of diabetes and insulin on hepatic delta6 desaturase gene expression

It has been recognized that rat liver microsomal Delta6 desaturation activity is defective in experimental diabetes, a fact that may be reverted by means of insulin treatment. In the present study, we used streptozotocin-induced diabetic rats in order to determine the regulatory role of insulin on the expression of hepatic Delta6 desaturase gene. The abundance of hepatic Delta6 desaturase mRNA in the diabetic rats is sevenfold lower than in the control. Insulin administration to diabetic rats induces Delta6 desaturase mRNA eightfold within 24 h. The effect of insulin on the Delta6 desaturase mRNA was inhibited 70% with dibutyryl-cAMP and theophylline administration and 90% by cycloheximide administration. Therefore, our data demonstrate that the activity of hepatic Delta6 desaturase in response to insulin is, at least in part, regulated by pretranslational events that require the synthesis of an unknown protein(s).     

Inhibition of intestinal bile acid transporter Slc10a2 improves triglyceride metabolism and normalizes elevated plasma glucose levels in mice

Interruption of the enterohepatic circulation of bile acids increases cholesterol catabolism, thereby stimulating hepatic cholesterol synthesis from acetate. We hypothesized that such treatment should lower the hepatic acetate pool which may alter triglyceride and glucose metabolism. We explored this using mice deficient of the ileal sodium-dependent BA transporter (Slc10a2) and ob/ob mice treated with a specific inhibitor of Slc10a2. Plasma TG levels were reduced in Slc10a2-deficient mice, and when challenged with a sucrose-rich diet, they displayed a reduced response in hepatic TG production as observed from the mRNA levels of several key enzymes in fatty acid synthesis. This effect was paralleled by a diminished induction of mature sterol regulatory element-binding protein 1c (Srebp1c). Unexpectedly, the SR-diet induced intestinal fibroblast growth factor (FGF) 15 mRNA and normalized bile acid synthesis in Slc10a2-/- mice. Pharmacologic inhibition of Slc10a2 in diabetic ob/ob mice reduced serum glucose, insulin and TGs, as well as hepatic mRNA levels of Srebp1c and its target genes. These responses are contrary to those reported following treatment of mice with a bile acid binding resin. Moreover, when key metabolic signal transduction pathways in the liver were investigated, those of Mek1/2-Erk1/2 and Akt were blunted after treatment of ob/ob mice with the Slc10a2 inhibitor. It is concluded that abrogation of Slc10a2 reduces hepatic Srebp1c activity and serum TGs, and in the diabetic ob/ob model it also reduces glucose and insulin levels. Hence, targeting of Slc10a2 may be a promising strategy to treat hypertriglyceridemia and diabetes.     

Roles of plasma interleukin-6 and tumor necrosis factor-alpha and FFA and TG in the development of insulin resistance induced by high-fat diet

The role of adipokines in development of insulin resistance still remains controversial. The purpose of the present study was to examine the dynamic changes of fasting plasma levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), free fatty acids (FFA) and insulin in a Sprague-Dawley rat insulin resistant model induced by high-fat diet. Heterotopic deposition of triglycerides (TG) in liver, skeletal muscles and pancreatic islet was also investigated. The fasting plasma level of insulin in rats in the high-fat diet group was significantly higher than that in the normal diet group on day 21 (P<0.01), suggesting that an increased insulin resistance developed in the high-fat diet group. However, no significant difference in the plasma IL-6 level was observed between the two groups (P>0.05), although in both groups, the plasma IL-6 level was significantly higher on day 21 than that of the day 0 (P<0.05). The plasma FFA level in the high-fat diet group began to increase significantly on day 21 (P<0.05), and elevated markedly on day 28, was positively correlated to the fasting plasma insulin level. Histological study revealed a more abundant TG deposition in liver and skeletal muscles (from quadriceps femoris) in the high-fat diet group than in the normal diet group on day 21, and the liver deposition was even higher on day 28. However, no deposition was observed in pancreatic islets. The plasma TNF-alpha level remained unchanged throughout the duration of the experiment. These results indicate that the progression of insulin resistance in high-fat diet rats is closely related to the plasma FFA elevation and the heterotopic deposition of TG in liver and skeletal muscles, but is unrelated to the plasma TNF-alpha and IL-6 levels.     

Effect of Octreotide on Hepatic Steatosis in Diet-Induced Obesity in Rats

Background

Non-alcoholic fatty liver disease (NAFLD) caused by liver lipid dysregulation is linked to obesity. Somatostatin (SST) and its analogs have been used to treat pediatric hypothalamic obesity. However, the application of such drugs for the treatment of NAFLD has not been evaluated.

Objective

This study aimed to investigate the expression levels of important regulators of hepatic lipid metabolism and the possible effect of the SST analog octreotide on these regulators.

Methods

SD rats were assigned to a control group and a high-fat diet group. Obese rats from the high-fat diet group were further divided into the obese and octreotide-treated groups. The body weight, plasma SST, fasting plasma glucose (FPG), insulin, triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and free fatty acid (FFA) levels were measured. Hepatic steatosis was evaluated based on the liver TG content, HE staining and oil red O staining. The SREBP-1c, ACC1, FAS, MTP, apoB and ADRP expression levels in the liver were also determined by RT-PCR, qRT-PCR, western blot or ELISA.

Results

The obese rats induced by high-fat diet expressed more SREBP-1c, FAS and ADRP but less MTP protein in the liver than those of control rats, whereas octreotide intervention reversed these changes and increased the level of apoB protein. Compared to the control group, obese rats showed increased liver ACC1, SREBP-1c and apoB mRNA levels, whereas octreotide-treated rats showed decreased mRNA levels of apoB and SREBP-1c. This was accompanied by increased body weight, liver TG contents, FPG, TG, TC, LDL-C, FFA, insulin and derived homeostatic model assessment (HOMA) values. Octreotide intervention significantly decreased these parameters. Compared to the control group, the obese group showed a decreasing trend on plasma SST levels, which were significantly increased by the octreotide intervention.

Conclusion

Octreotide can ameliorate hepatic steatosis in obese rats, possibly by decreasing hepatic lipogenesis and increasing TG export from hepatocytes.     

Suppression of acute ethanol-induced hepatic steatosis by docosahexaenoic acid is associated with downregulation of stearoyl-CoA desaturase 1 and inflammatory cytokines

Excessive alcohol consumption can lead to hepatic steatosis. Omega-3 (n-3) polyunsaturated fatty acids (PUFA) have been shown to be effective in reducing hepatic accumulation of triglycerides (TG) by downregulation of TG biosynthesis in the liver. The aim of this study was to examine whether supplementation with the n-3 PUFA, docosahexaenoic acid (DHA), can effectively reduce acute alcohol-induced hepatic steatosis. Acute alcohol-induced hepatic steatosis was generated in 9-week-old male mice (C57BL/6J) by oral gavage of ethanol (4.7 g/kg BW) diluted in water (60%, v/v), with or without DHA (250 mg/kg BW), every 12 h for 3 administrations. Compared to the control (ethanol-alone) group, animals supplemented with DHA were protected against ethanol-induced TG accumulation in the liver. Accordingly, hepatic stearoyl-CoA desaturase-1 (SCD-1) expression, serum alanine aminotransferase (ALT) activity, and the levels of inflammatory cytokines (such as IL-6 and TNF-α) in the liver were significantly reduced, whereas the expression of heme oxygenase-1 (HO-1), an enzyme that can improve cell survival in liver tissue, was markedly increased in DHA-supplemented mice compared to the control animals. There were no differences in serum TG level and hepatic production of reactive oxygen species (ROS) between the two groups. Our findings demonstrate that DHA supplementation protects against acute ethanol-induced hepatic steatosis, which may be associated with reduced expression of SCD-1 and inflammatory cytokines.     

Effects of gender and GH secretory pattern on sterol regulatory element-binding protein-1c and its target genes in rat liver

We investigated whether the sexually dimorphic secretory pattern of growth hormone (GH) in the rat regulates hepatic gene expression of sterol regulatory element-binding protein-1c (SREBP-1c) and its target genes. SREBP-1c, fatty acid synthase (FAS), and glycerol-3-phosphate acyltransferase (GPAT) mRNA were more abundant in female than in male livers, whereas acetyl-CoA carboxylase-1 (ACC1) and stearoyl-CoA desaturase-1 (SCD-1) were similarly expressed in both sexes. Hypophysectomized female rats were given GH as a continuous infusion or as two daily injections for 7 days to mimic the female- and male-specific GH secretory patterns, respectively. The female pattern of GH administration increased the expression of SREBP-1c, ACC1, FAS, SCD-1, and GPAT mRNA, whereas the male pattern of GH administration increased only SCD-1 mRNA. FAS and SCD-1 protein levels were regulated in a similar manner by GH. Incubation of primary rat hepatocytes with GH increased SCD-1 mRNA levels and decreased FAS and GPAT mRNA levels but had no effect on SREBP-1c mRNA. GH decreased hepatic liver X receptor-alpha (LXRalpha) mRNA levels both in vivo and in vitro. Feminization of the GH plasma pattern in male rats by administration of GH as a continuous infusion decreased insulin sensitivity and increased expression of FAS and GPAT mRNA but had no effect on SREBP-1c, ACC1, SCD-1, or LXRalpha mRNA. In conclusion, FAS and GPAT are specifically upregulated by the female secretory pattern of GH. This regulation is not a direct effect of GH on hepatocytes and does not involve changed expression of SREBP-1c or LXRalpha mRNA but is associated with decreased insulin sensitivity.     

Effects of dietary mulberry, Korean red ginseng, and banaba on glucose homeostasis in relation to PPAR-alpha, PPAR-gamma, and LPL mRNA expressions

Despite lack of scientific evidences to support its therapeutic efficacy, the use of herbal supplements has significantly increased. The purpose of this study was to evaluate the effects of traditional anti-diabetic herbs on the progress of diabetes in db/db mice, a typical non-insulin-dependent model. Five different experimental diets were as follows: control diet, 0.5% mulberry leaf water extract diet, 0.5% Korean red ginseng diet, 0.5% banaba leaf water extract diet, and 0.5% combination diet (mulberry leaf water extract/Korean red ginseng/banaba leaf water extract, 1:1:1). Blood levels of glucose, insulin, HbA1c, and triglyceride were measured every 2 weeks. At 12 weeks of age, animals were sacrificed, and tissue mRNA levels of PPAR-alpha, PPAR-gamma, and LPL were determined. Results indicated that mulberry leaf water extract, Korean red ginseng, banaba leaf water extract, and the combination of above herbs effectively reduced blood glucose, insulin, TG, and percent HbA1c in study animals (p<0.05). We also observed that the increased expressions of liver PPAR-alpha mRNA and adipose tissue PPAR-gamma mRNA in animals fed diets supplemented with test herbs. The expression of liver LPL mRNA was also increased with experimental diets containing herbs. The efficacy was highest in animals fed the combination diet for all of the markers used. These results suggest that mulberry leaf water extract, Korean red ginseng, banaba leaf water extract, and the combination of these herbs fed at the level of 0.5% of the diet significantly increase insulin sensitivity, and improve hyperglycemia possibly through regulating PPAR-mediated lipid metabolism.     

Phosphoenolpyruvate carboxykinase overexpression selectively attenuates insulin signaling and hepatic insulin sensitivity in transgenic mice

The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steady-state levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.     

Overexpression of APOC1 in obob mice leads to hepatic steatosis and severe hepatic insulin resistance

Obese obob mice with strong overexpression of the human apolipoprotein C1 (APOC1) exhibit excessive free fatty acid (FFA) and triglyceride (TG) levels and severely reduced body weight (due to the absence of subcutaneous adipose tissue) and skin abnormalities. To evaluate the effects of APOC1 overexpression on hepatic and peripheral insulin sensitivity in a less-extreme model, we generated obob mice with mild overexpression of APOC1 (obob/APOC1(+/-)) and performed hyperinsulinemic clamp analysis. Compared with obob littermates, obob/APOC1(+/-) mice showed reduced body weight (-25%) and increased plasma levels of TG (+632%), total cholesterol (+134%), FFA (+65%), glucose (+73%), and insulin (+49%). Hyperinsulinemic clamp analysis revealed severe whole-body and hepatic insulin resistance in obob/APOC1(+/-) mice and, in addition, increased hepatic uptake of FFA and hepatic TG content. Treatment of obob/APOC1(+/-) mice with rosiglitazone strongly improved whole-body insulin sensitivity as well as hepatic insulin sensitivity, despite a further increase of hepatic fatty acid (FA) uptake and a panlobular increase of hepatic TG accumulation. We conclude that overexpression of APOC1 prevents rosiglitazone-induced peripheral FA uptake leading to severe hepatic steatosis. Interestingly, despite rosiglitazone-induced hepatic steatosis, hepatic insulin sensitivity improves dramatically. We hypothesize that the different hepatic fat accumulation and/or decrease in FA intermediates has a major effect on the insulin sensitivity of the liver.     

Effects of leptin,troglitazone, and dietary fat on stearoyl CoA desaturase

Leptin, troglitazone, and high fat feeding profoundly influence the lipid content of various tissues. To determine if they affect the expression of stearoyl CoA desaturase (SCD)-1 and -2, their mRNA was measured in livers of normal, hyperleptinemic, troglitazone-treated, and fat-fed rats. Hyperleptinemia, which reduces tissue TG by downregulating lipogenic enzymes and upregulating fatty acid oxidation, lowered SCD-1 96% below controls and reduced SCD-2 slightly. By contrast, hepatic SCD-1 mRNA of leptin-resistant fa/fa rats was five times wild-type controls, but SCD-2 mRNA was 66% lower. High fat feeding lowered SCD-1 by 80%, possibly by inducing hyperleptinemia. Troglitazone treatment, which reduces nonadipose tissue TG of fa/fa rats without downregulating lipogenic enzymes, raised SCD-2 13-fold but lowered SCD-1 by 25%. The findings suggest that leptin controls SCD-1 expression and that troglitazone's antilipotoxic action may involve SCD-2 upregulation.     

Interaction between growth hormone and insulin in the regulation of lipoprotein metabolism in the rat

The importance of insulin for the in vivo effects of growth hormone (GH) on lipid and lipoprotein metabolism was investigated by examining the effects of GH treatment of hypophysectomized (Hx) female rats with and without concomitant insulin treatment. Hypophysectomy-induced changes of HDL, apolipoprotein (apo)E, LDL, and apoB levels were normalized by GH treatment but not affected by insulin treatment. The hepatic triglyceride secretion rate was lower in Hx rats than in normal rats and increased by GH treatment. This effect of GH was blunted by insulin treatment. The triglyceride content in the liver changed in parallel with the changes in triglyceride secretion rate, indicating that the effect of the hormones on triglyceride secretion was dependent on changed availability of triglycerides for VLDL assembly. GH and insulin independently increased editing of apoB mRNA, but the effects were not additive. The expression of fatty-acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and sterol regulatory element-binding protein-1c (SREBP-1c) was increased by GH treatment. Insulin and GH had no additive effects on these genes; instead, insulin blunted the effect of GH on SREBP-1c mRNA. In contrast to the liver, adipose tissue expression of SREBP-1c, FAS, or SCD-1 mRNA was not influenced by GH. In conclusion, the increased hepatic expression of lipogenic enzymes after GH treatment may be explained by increased expression of SREBP-1c. Insulin does not mediate the effects of GH but inhibits the stimulatory effect of GH on hepatic SREBP-1c expression and triglyceride secretion rate.