Adult rats prenatally exposed to ethanol have increased gluconeogenesis and impaired insulin response of hepatic gluconeogenic genes.

Rat offspring exposed to ethanol (EtOH rats) during pregnancy are insulin resistant, but it is unknown whether they have increased gluconeogenesis. To address this issue, we determined blood glucose and liver gluconeogenic genes, proteins, and enzyme activities before and after insulin administration in juvenile and adult EtOH rats and submitted adult EtOH rats to a pyruvate challenge. In juvenile rats, basal glucose; peroxisome proliferator-activated receptor-coactivator-1alpha protein and mRNA; and phosphoenolpyruvate carboxykinase enzyme activity, protein, and mRNA were similar between groups. After insulin injection, these parameters failed to decrease in EtOH rats, but glucose decreased by 30% and gluconeogenic enzymes, proteins, and mRNAs decreased by 50-70% in control rats. In adult offspring, basal peroxisome proliferator-activated receptor-coactivator-1alpha protein and mRNA levels were 40-80% higher in EtOH rats than in controls. Similarly, basal phosphoenolpyruvate carboxykinase activity, protein, and mRNA were approximately 1.8-fold greater in EtOH rats than in controls. These parameters decreased by approximately 50% after insulin injection in control rats, but they remained unchanged in EtOH rats. After insulin injection in the adult rats, glucose decreased by 60% in controls but did not decrease significantly in EtOH rats. A subset of adult EtOH rats had fasting hyperglycemia and an exaggerated glycemic response to pyruvate compared with controls. The data indicate that, after prenatal EtOH exposure, the expression of gluconeogenic genes is exaggerated in adult rat offspring and is insulin resistant in both juvenile and adult rats, explaining increased gluconeogenesis. These alterations persist through adulthood and may contribute to the pathogenesis of Type 2 diabetes after exposure to EtOH in utero.     

Effect of dichlorvos on hepatic and pancreatic glucokinase activity and gene expression, and on insulin mRNA levels

Several studies have shown that organophosphate pesticides affect carbohydrate metabolism and produce hyperglycemia. It has been reported that exposure to the organophosphate pesticide dichlorvos affects glucose homeostasis and decreases liver glycogen content. Glucokinase (EC 2.7.1.1) is a tissue-specific enzyme expressed in liver and in pancreatic beta cells that plays a crucial role in glycogen synthesis and glucose homeostasis. In the present study we analyzed the effect of one or three days of dichlorvos administration [20 mg/kg body weight] on the activity and mRNA levels of hepatic and pancreatic glucokinase as well as on insulin mRNA abundance in the rat. We found that the pesticide affects pancreatic and hepatic glucokinase activity and expression differently. In the liver the pesticide decreased the enzyme activity; on the contrary glucokinase mRNA levels were increased. In contrast, pancreatic glucokinase activity as well as mRNA levels were not affected by the treatment. Insulin mRNA levels were not modified by dichlorvos administration. Our results suggest that the decreased activity of hepatic glucokinase may account for the adverse effects of dichlorvos on glucose metabolism.     

Hepatic 11 beta-hydroxysteroid dehydrogenase 1 involvement in alterations of glucose metabolism produced by acidotic stress in rat

11 beta-hydroxysteroid dehydrogenase (HSDs) enzymes regulate the activity of glucocorticoids in target organs. HSD1, one of the two existing isoforms, locates mainly in CNS, liver and adipose tissue. HSD1 is involved in the pathogenesis of diseases such as obesity, insulin resistance, arterial hypertension and the Metabolic Syndrome. The stress produced by HCl overload triggers metabolic acidosis and increases liver HSD1 activity associated with increased phosphoenolpyruvate carboxykinase, a regulatory enzyme of gluconeogenesis that is activated by glucocorticoids, with increased glycaemia and glycogen breakdown. The aim of this study was to analyze whether the metabolic modifications triggered by HCl stress are due to increased liver HSD1 activity. Glycyrrhetinic acid, a potent HDS inhibitor, was administered subcutaneously (20 mg/ml) to stressed and unstressed four months old maleSprague Dawley rats to investigate changes in liver HSD1, phosphoenolpyruvate carboxykinase (PECPK) and glycogen phosphorylase activities and plasma glucose levels. It was observed that all these parameters increased in stressed animals, but that treatment with glycyrrhetinic acid significantly reduced their levels. In conclusion, our results demonstrate the involvement of HSD1 in stress induced carbohydrate disturbances and could contribute to the impact of HSD1 inhibitors on carbohydrate metabolism and its relevance in the study of Metabolic Syndrome Disorder and non insulin-dependent diabetes mellitus.     

Stimulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) activity by low concentrations of circulating glucose in perfused rat liver.

1. After nicotinic acid treatment, rat liver glycogen is depleted and phosphoenolpyruvate carboxykinase activity increased, to about twice the initial value. 2. The increase in phosphoenolpyruvate carboxykinase activity promoted by nicotinic acid is prevented by cycloheximide or actinomycin D, suggesting that this effect is produced by synthesis of the enzyme de novo. 3. Despite the enhancement of phosphoenolpyruvate carboxykinase activity and glycogen depletion, which occurs 5h after the injection of nicotinic acid, the gluconeogenic capacity of liver is low and considerably less than the values found in rats starved for 48h. 4. When the livers of well-fed rats are perfused in the presence of low concentrations of glucose, the activity of phosphoenolpyruvate carboxykinase significantly increases compared with the control. 5. This increase is not related to the glycogen content, but seems to be also the result of synthesis of the enzyme de novo, since this effect is counteracted by previous treatment with cycloheximide or actinomycin D. 6. Phosphoenolpyruvate carboxykinase activity is not increased in the presence of low concentrations of circulating glucose when 40 mM-imidazole (an activator of phosphodiesterase) is added to the perfusion medium. 7. Addition of dibutyryl cyclic AMP to the perfusion medium results in an increase in phosphoenolpyruvate carboxykinase activity, in spite of the presence of normal concentrations of circulating glucose. On the other hand, the concentration of cyclic AMP in the liver increases when that of glucose in the medium is low. 8. These results suggest that, in the absence of hormonal factors, the regulation of phosphoenolpyruvate carboxykinase can be accomplished by glucose itself, inadequate concentrations of it resulting in the induction of the enzyme. The mediator in this regulation, as in hormonal regulation, seems to be cyclic AMP.     

Insulin replacement therapy in diabetic rats using an osmotic pump normalizes expression of enzymes key to hepatic carbohydrate metabolism.

Intensively treating type I diabetics with continuous subcutaneous insulin infusions or multiple daily insulin injections to normalize mean blood glucose concentrations significantly reduces the onset of secondary diabetic complications when compared to conventionally treated diabetics. Our studies focused on characterizing hepatic enzyme expression in intensively and conventionally treated diabetic rats. Alloxan-induced diabetic rats were conventionally treated with insulin injected twice daily or intensively treated with similar daily dosages of insulin administered via a surgically implanted osmotic pump. Our results demonstrate a significant difference in hepatic enzyme expression when these treatment regimes are compared. In conventionally treated diabetic rats, phosphoenolpyruvate carboxykinase (PEPCK) protein and mRNA levels remained slightly elevated when compared to normal animals, glycogen phosphorylase (GP) protein levels were still slightly decreased, and glycogen synthase (GS) protein and mRNA levels remained at the elevated levels observed in untreated diabetics. In contrast, the protein and mRNA levels of all three enzymes were normalized in the insulin pump-treated animals. These results suggest that intensive insulin therapy improves glycemia directly by normalizing hepatic gene expression while conventional insulin therapy normalizes plasma glucose concentrations indirectly.     

成纤维细胞生长因子21对1型糖尿病动物模型的肝糖代谢影响及机制研究

成纤维细胞生长因子(FGF)-21是FGF家族的成员之一.作为近年发现的一种新的糖代谢调节因子,大量研究表明,FGF-21是一种不依赖胰岛素,能够独立降糖的2型糖尿病治疗潜力型药物.但是,能否应用于1型糖尿病的治疗,国内外目前尚无报道.通过改良传统造模方法,诱导小鼠缓慢产生糖耐量异常,研究FGF-21对此类模型的糖代谢影响及肝糖代谢机制.通过检测FGF-21短期注射和长期注射后模型动物血糖的变化,研究FGF-21在模型动物上对血糖的调控效果.采用实时定量PCR检测FGF-21对模型动物肝脏中葡萄糖转运蛋白(GLUT)1、4 mRNA的表达影响.利用蒽酮法检测模型动物肝脏中糖原合成量.实验结果显示,FGF-21能够调节1型糖尿病动物的血糖水平,并呈剂量依赖性.同时,首次在1型糖尿病动物模型上证实了低剂量FGF-21(0.125 mg/kg)与胰岛素的协同作用效果优于相同剂量FGF-21和胰岛素单独注射的效果.治疗结果表明,FGF-21能够维持1型糖尿病动物模型血糖在正常范围,效果优于胰岛素.实时定量PCR结果发现,与胰岛素上调GLUT4 mRNA表达量不同的是,FGF-21作用动物模型8周后,GLUT1 mRNA表达量显著提高,长期的FGF-21与胰岛素协同注射使GLUT1、4 mRNA表达量同时显著提高.长期FGF-21与胰岛素协同注射组和高剂量FGF-21注射均可显著提高模型动物肝糖原的合成.结果表明,FGF-21促进动物模型糖代谢机制与增加GLUT1表达、增加糖原合成作用有关.为临床应用FGF-21治疗1型糖尿病,增加胰岛素敏感性提供了理论依据.     

Hepatic mechanisms of the Walnut antidiabetic effect in mice

The present study was designed to explore the mechanism of action of walnut (the seed of Juglans regia) leaf and ridge on hepatic glucose metabolism in diabetic mice. Experimental diabetes was induced by intravenous administration of streptozotocin (60 mg/kg)and confirmed with an increase of blood glucose, 90–100% of the control, 72 hours later. Isolated extracts from walnut leaf and ridges were administered in a single effective dose of 400 mg/kg orally. Firstly, blood glucose was determined every 1 hour until 5 hours post administration of extracts. In the second experiment, the liver was surgically removed, 2 hours post treatment of diabetic animals with extracts, homogenized and used for measurement of key enzymes of glycogenolysis (glycogen phosphorylase, GP) and gluconeogenesis (phosphoenolpyruvate carboxykinase, PEPCK). Treatment by both leaf and ridge extracts decreased blood glucose and liver PEPCK activity and increased blood insulin and liver GP activity. It is concluded that walnut is able to lower blood glucose through inhibition of hepatic gluconeogenesis and secretion of pancreatic insulin.     

Impact of interleukin-6 on the glucose metabolic capacity in rat liver

The actute phase reaction mediated by the proinflammatory cytokine IL6 initiates a number of metabolic changes in the liver, which may contribute to the pathogenesis of the septic shock during prolonged exposition. Here, the impact of IL6 on the hepatic glucose providing capacity was studied by monitoring glycogen degradation and the expression of the gluconeogenic phosphoenolpyruvate carboxykinase (PCK1) in rat livers during the daily feeding rhythm. Eight hours after i.p. injection of IL6, mRNA levels of α2-macroglobulin, a prominent acute phase reactant in rat liver, were elevated as shown by Northern blot analysis and in situ hybridization (ISH). PCK1 mRNA levels were decreased by IL6 to 50% of levels in untreated animals due to the reduction of PCK1 mRNA in the periportal zone of the liver as shown by ISH. PCK1 enzyme activity was not affected by IL6. Glycogen degradation was accelerated by IL6, which led to nearly complete depletion of glycogen pools in periportal areas 8 h after IL6 injection. This was very likely due to inhibition of glycogen pool replenishment. Thus, the depletion of glycogen stores in the liver might contribute to the impairment of hepatic glucose production during prolonged acute phase challenge.     

Effect of insulin-like growth factor-1 (IGF-1) on the gluconeogenesis in calf hepatocytes cultured in vitro

The major role of insulin-like growth factor-1 (IGF-1) in the liver is to mediate glucose uptake in hepatocytes to synthesize glycogen and maintain blood glucose homeostasis. In this study, to evaluate the role of IGF-1 on gluconeogenesis and nutrient metabolism in dairy cattle, pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK) expression and enzyme activity were evaluated in primary cultures of bovine hepatocytes treated with different concentrations of IGF-1 by quantitative polymerase chain reaction and spectrophotometry, respectively. The results showed that expression of PC and PEPCK were significantly lower in bovine hepatocytes by IGF-1 treatment in test group compare to the control group (P < 0.01). As IGF-1 concentration increased, PC and PEPCK enzyme activity in bovine hepatocytes decreased. Evaluating PC and PEPCK mRNA levels and enzyme activity may thus be useful to monitor subclinical ketosis in dairy cows.     

Different sensitivity of PPARalpha gene expression to nutritional changes in liver of suckling and adult rats

The amount of peroxisome proliferator-activated receptor-alpha (PPARalpha) protein was markedly augmented in the liver of suckling rats compared to adult rats. This different PPARalpha abundance was used to study the sensitivity to nutritional changes in the expression and activity of this receptor. Thus, 10-day-old and adult rats were orally given either glucose, Intralipid or a combination of both diets, and liver mRNA levels of PPARalpha and the PPAR related genes, acyl-CoA oxidase (ACO) and phosphoenolpyruvate carboxykinase (PEPCK), and plasma metabolites were measured. In neonates, the expression of PPARalpha and ACO was seen to increase when the level of FFA in plasma was also high, unless an elevated level of insulin was also present. However, this fatty acid-induced effect was not detected in adult rats. On the contrary, the hepatic expression of PEPCK was modulated by the nutritional changes similarly in both neonates and adult rats. Thus, it may be concluded that the expression of the PPARalpha gene in adult rats seems to be less sensitive to nutritional changes than in neonates.     

A novel glycogen-targeting subunit of protein phosphatase 1 that is regulated by insulin and shows differential tissue distribution in humans and rodents

Stimulation of glycogen-targeted protein phosphatase 1 (PP1) activity by insulin contributes to the dephosphorylation and activation of hepatic glycogen synthase (GS) leading to an increase in glycogen synthesis. The glycogen-targeting subunits of PP1, GL and R5/PTG, are downregulated in the livers of diabetic rodents and restored by insulin treatment. We show here that the mammalian gene PPP1R3E encodes a novel glycogen-targeting subunit of PP1 that is expressed in rodent liver. The phosphatase activity associated with R3E is slightly higher than that associated with R5/PTG and it is downregulated in streptozotocin-induced diabetes by 60-70% and restored by insulin treatment. Surprisingly, although mRNA for R3E is most highly expressed in rat liver and heart muscle, with only low levels in skeletal muscle, R3E mRNA is most abundant in human skeletal muscle and heart tissues with barely detectable levels in human liver. This species-specific difference in R3E mRNA expression has similarities to the high level of expression of GL mRNA in human but not rodent skeletal muscle. The observations imply that the mechanisms by which insulin regulates glycogen synthesis in liver and skeletal muscle are different in rodents and humans.     

Postmaturity in the rat: phosphorylase, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities in the fetal liver.

Liver glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities were increased in the postmature rat fetus (23.5 day old) when compared to term rat fetus (21.5 day old). Postmaturity was without effect on liver phosphorylase activity. The three liver enzyme activities were also determined in normal 2 day old neonates. These results are correlated with the mobilisation of fetal liver glycogen occuring during postmaturity in the rat and are discussed in relation to the secretory pattern of the pancreatic hormones.     

Growth and function of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium.

The properties of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose-free and in glucose-supplemented medium. Growth in glucose-free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose-free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose-free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D-glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium.     

Contrasting interactions between phorbol ester and insulin on the regulation of glycogen synthase activity and p33 mRNA accumulation in rat hepatoma cells

We have compared the effect of phorbol 12-myristate 13-acetate (PMA) with that of insulin on three targets of insulin action in H4IIEC3 (H4) rat hepatoma cells. These parameters are the phosphorylation state and tyrosine kinase activity of the insulin receptor, the activation state of glycogen synthase, and the accumulation of p33 mRNA. Under conditions where insulin treatment of H4 cells clearly activated receptor serine and tyrosine phosphorylation on the insulin receptor beta-subunit in situ, activated receptor tyrosine kinase activity in vitro, and activated glycogen synthase and p33 mRNA accumulation in situ, PMA alone did not influence the insulin receptor phosphorylation state or tyrosine kinase activity and did not affect glycogen synthase activity, but markedly increased p33 mRNA accumulation. When PMA was added in the presence of insulin, particularly if PMA was preincubated, the receptor phosphorylation state and the tyrosine kinase activity again were not affected, but insulin-activated glycogen synthase was significantly diminished or abolished. In contrast, increased p33 mRNA accumulation by PMA was additive with that of insulin. Thus, under conditions where no effect was observed on the insulin receptor phosphorylation state or the tyrosine kinase activity, PMA acted in an insulin-antagonistic manner on glycogen synthase and in an insulin-like manner on p33 mRNA accumulation, indicating that these actions of PMA are unrelated to early events in the pathway of the insulin action. Effects on glycogen synthase are most readily explained by an effect of protein kinase C-activated phosphorylation of glycogen synthase.