Effect of starvation, diabetes and insulin on the casein kinase 2 from rat liver cytosol.

Starvation, diabetes and insulin did not alter the concentration of casein kinases in rat liver cytosol. However, the Km for casein of casein kinase 2 from diabetic rats was about 2-fold lower than that from control animals. Administration of insulin to control rats did not alter this parameter, but increased the Km for casein of casein kinase 2 in diabetic rats. Starvation did not affect the kinetic constants of casein kinases. The effect of diabetes on casein kinase 2 persisted after partial purification of the enzyme by glycerol-density-gradient centrifugation and affected also its activity on other protein substrates such as phosvitin, high-mobility-group protein 14 and glycogen synthase. The results indicate that rat liver cytosol casein kinase 2 is under physiological control.     

Insulin resistance in the liver in fasting and diabetes mellitus: the failure of insulin to stimulate the release of a chemical modulator of pyruvate dehydrogenase

To further define the mechanism(s) of insulin resistance in the liver associated with diabetes and fasting, we evaluated the ability of insulin to release an activator of pyruvate dehydrogenase activity from a liver particulate fraction. Insulin reproduceably and significantly enhanced the release of mediator from the liver particulate fraction of control animals. The particulate fractions from fasted and diabetic animals were resistant to this effect of insulin. Refeeding and insulin treatment, respectively, restored responsiveness to insulin. These data support the concept that alterations at or near the plasma membrane can be responsible for or accompany the insulin resistance observed in the liver in fasting and diabetes mellitus.     

Nutritional and hormonal control of lung and liver fatty acid synthesis.

During starvation and in streptozotocin-induced diabetes, the total activities of rat lung acetyl CoA carboxylase and fatty acid synthetase are reduced to one-third of the normal values. Refeeding of the starved animals or administration of insulin to diabetic animals restores the levels to the original values. The insulin effect is dose and time dependent. These data contrast with those in the liver, where a 30- to 50-fold depression of these enzymes is observed in the diabetic state and administration of insulin is actually followed by doubling of the activity over normal controls. Fat-free high-fructose diet (containing 60% fructose by weight) enhances the activities of liver enzymes 3- to 6-fold over the values of controls on laboratory diet but has no effect on the lung enzymes. Long-term feeding of fructose diet also increases the activities of liver enzymes from diabetic animals to twice the value of normal controls on laboratory diet. Insulin administration to fructose-fed diabetic animals restores the enzyme activities to those obtained with fructose-fed normal controls. However, the stimulation of lung enzymes of diabetic animals can be effected either by fructose or by insulin. Antigen-antibody titrations and measurements of the rate of protein synthesis show that the increased activity of the lung and liver fatty acid synthetase is due to enhanced content rather than increased specific activity. These data suggest that insulin or fructose effects on fatty acid-synthesizing enzymes are mediated through intermediate(s) whose concentration is affected in the experimental diabetes. Furthermore, all tissues may not have stringent insulin requirements since the lung enzymes can be stimulated by fructose alone.     

Insulin action on protein phosphatase-1 activation is enhanced by the antidiabetic agent pioglitazone in cultured diabetic hepatocytes

Effect of the antidiabetic agent pioglitazone on the insulin-mediated activation of protein phosphatase-1 was examined in diabetic hepatocytes. Streptozotocin-induced diabetes in Sprague Dawley rats caused a significant decrease in the activation of glycogen synthase in hepatocytes isolated from these animals. There was an inverse correlation between the in vivo hyperglycemic condition and the in vitro activation of glycogen synthase in liver cells (r = 0.93, p > 0.001). Long term incubation of diabetic hepatocytes with insulin and dexamethasone caused significant (p > 0.001) improvement in the activation of glycogen synthase activation. When incubated along with hormones, pioglitazone enhanced their action (p > 0.05-0.01). Diabetic hepatocytes were also characterized by 50% decrease in the activity of protein phosphatase-1, the enzyme which dephosphorylates and activates glycogen synthase. Pioglitazone potentiated the acute stimulatory effect of insulin on protein phosphatase-1 in normal hepatocytes but not in diabetic hepatocytes. Long term incubation of diabetic hepatocytes with insulin ameliorated the decrease in the protein phosphatase -1 activity in these cells. This stimulatory long-term effect of insulin was significantly (p > 0.05) enhanced by the antidiabetic agent pioglitazone.     

Metabolic and mitochondrial disturbances in streptozotocin-treated Sprague-Dawley and Sherman rats

This study provides explanation for conflicting evidence in the literature relating to changes in mitochondrial function and metabolic parameters during chemically induced diabetes. Diabetes of 3 days' duration (early ketosis) did not alter heart, kidney, or liver mitochondrial respiratory rates with glutamate or succinate even though serum glucose and triglycerides were elevated. Diabetes of 5 weeks' duration did not alter kidney or liver mitochondrial function in the fed adult rat although weight gain was depressed. The amount of kidney mitochondrial protein isolated per gram of tissue was increased by 30% in the diabetic. This increase was reversed by insulin treatment as were the other biochemical modalities measured. Superimposition of a 24-hr fast resulted in enhanced gluconeogenesis as measured by an animal weight loss of 17% within 24 hr (liver weight loss, 21%) and an elevation of serum urea nitrogen by 180% compared to fasted control. Respiratory rates of diabetic kidney mitochondria with glutamate were unaffected in the fasted animal whereas diabetic liver mitochondrial respiratory rates during succinate oxidation were reduced by 43%. Respiratory control was unchanged in the fasted diabetic rat. All the observed changes were reversed by insulin. Variation in the serum and liver metabolic indices (urea nitrogen, creatinine, glycerol, free fatty acids, free amino acids, triglycerides, and glucose) and liver mitochondrial responses to 7 weeks of chemically induced diabetes was affected by the rat strain, Sprague-Dawley versus Sherman, and rat weight, 72 g versus 222 g. Liver mitochondrial respirations in fed Sherman rats were not depressed by diabetes. Both rat strains had elevated liver free fatty acids and glutamate dehydrogenase activity in the diabetic state. Serum leucine, isoleucine, and valine were more elevated and serum lysine and arginine were more depressed in the diabetic Sprague-Dawley rat than in the Sherman rat. Conjectures on these results are presented in the text.     

Liver cytosolic fatty acids binding proteins in rats and Psammomys obesus: modulation in diabetes

The effect of insulin on [3H]oleate binding to delipidated liver cytosolic proteins was studied in four groups of animals: untreated rats, streptozotocin induced diabetic rats, Psammomys obesus fed salt bush diet, and Psammomys obesus fed ordinary laboratory chow. The distribution of the protein bound [3H]oleate between low and high molecular weight cytosolic proteins in Psammomys differed from the distribution found in rats. Diet induced high insulin diabetes in Psammomys and streptozotocin induced low insulin diabetes in rats, modulated [3H]oleate binding in the same manner.     

Hepatic glycoprotein synthesis in streptozotocin diabetic rats

In vitro incorporation of 3H-mannose into dolichol phosphate mannose, dolichol pyrophosphate oligosaccharides, and secretory and membrane glycoproteins was investigated in liver slices from streptozotocin diabetic rats. In addition, 14C-leucine incorporation into glycoproteins was studied. 3H-mannose incorporation was significantly less in secretory glycoproteins from diabetic rat liver slices than from control tissues, but 14C-leucine incorporation in these proteins was similar in both groups. Dolichol-phosphate mannose and dolichol-phosphate oligosaccharide synthesis were significantly down-regulated in diabetes. When incubated with insulin, mannosylation of secretory proteins, dolichol-phosphate mannose and dolichol-phosphate oligosaccharides reached control levels in three hours. Dolichol-phosphate mannosyltransferase activity was significantly less in diabetes, while in the presence of insulin, the enzyme activity reached control levels in three hours. These results indicate that key intermediates in glycoprotein biosynthesis are regulated by insulin.     

Regulation of ascorbic acid and of xylulose synthesis in rat-liver extracts. The effect of alloxan-diabetes on the glucuronic acid pathway.

1. The synthesis of l-ascorbic acid and of l-xylulose from d-glucuronolactone, d-glucuronate, l-gulonate and l-gulonolactone has been studied with liver extracts from normal and alloxan-diabetic rats. 2. In diabetic animals the synthesis of ascorbic acid is impaired, and more from glucuronolactone and glucuronate than from gulonate and gulonolactone, whereas the formation of xylulose from gulonate and gulonolactone is enhanced. These changes are reversed by insulin therapy. 3. The activity of the NAD-linked gulonate dehydrogenase is enhanced during diabetes.     

Role of ATP in specific binding of 125I-insulin to cytoplasmic receptors of the liver and muscle membranes of controls and diabetic rats

A study was made of the action of various concentrations of ATP on insulin ability to bind to the receptors of the liver and muscle membranes in control and streptozocin-induced diabetes animals. Specific binding of 125I-insulin to the receptors of the liver and muscle membranes was shown to rise in animals with streptozocin-induced diabetes as compared to control. This effect was most pronounced in the muscle membranes. Preincubation of the membranes with ATP did not affect insulin binding to the liver and muscle receptors of control animals. However, hormone binding to the liver receptors of diabetic rats was drastically suppressed by ATP (10(-3) M). Less ATP concentrations (10(12) M) produced an additional inhibitory action which was not marked. ATP led to decreased insulin binding to the muscle receptors of diabetic rats only at extremely low concentrations (10(-12) M). The data obtained may be of importance for regulation of membrane phosphorylation in the states characteristic of insulin resistance.     

Central adrenergic suppression augments the insulin and glucagon secretory, and the glycogenolytic responses in streptozotocin-diabetic rats.

It has been suggested that the increased activity of the sympathetic nervous system and the resultant increase in the tissue catecholamine levels contribute to the pathogenesis of diabetes. In this study we evaluated the effect of clonidine, a central adrenergic agonist that decreases sympathetic tone, on the serum levels of glucose, insulin, glucagon and norepinephrine and on the hepatic glycogen content in normal and streptozotocin-diabetic rats. The animals were treated with clonidine 25 micrograms/kg/day interperitoneally for 3 weeks to suppress the central adrenergic impulses. Clonidine treatment significantly increased the weight gain, but did not affect plasma glucose, insulin, glucagon and norepinephrine in the diabetic animals. Pancreatic insulin and liver glycogen contents were significantly higher in the clonidine-treated than in the untreated diabetic rats. However, clonidine did not affect pancreatic insulin and liver glycogen content of nondiabetic animals. The intravenous administration of glucagon increased plasma glucose in the clonidine-treated, but not in the saline-treated diabetic rats. Insulin-induced hypoglycemia significantly enhanced glucagon release in clonidine-treated but not in saline-treated diabetic rats. We conclude that the suppression of central adrenergic activity may ameliorate the effects of insulin insufficiency on pancreatic hormone secretion and hepatic glycogen content.     

The sensitivity of isoelectric focusing and electrophoresis in the detection of sequence differences in proteins

Fourteen myoglobins of known sequence were examined by isoelectric focusing with and without urea. The 14 sequences formed six distinct mobility classes on gels without urea and three classes on those with urea. For these proteins, isoelectric focusing provides no advantage over single, nonequilibrium, nondenaturing gels in the total number of distinguishable mobility classes. Only major charge differences, resulting from the changes in the total numbers of acidic and basic amino acids, can be detected on gels with urea, indicating that denaturation by urea alters proteins so that small differences in ionization are eliminated.We thank the Department of Biology, University of Utah, for financial support.     

金黄地鼠胰岛素抵抗和糖尿病动物模型的建立

目的建立胰岛素抵抗和糖尿病动物模型。方法给金黄地鼠喂以高脂果糖饲料,部分给小剂量链脲菌素(30mg/kg)腹腔注射,以正常金黄地鼠作为对照,测定动物体重、空腹血糖、血脂、胰岛素水平,计算胰岛素敏感指数,并对肝脏、胰腺及主动脉弓组织进行形态学比较。结果金黄地鼠经高脂果糖喂养6周制成胰岛素抵抗、肥胖和脂质代谢紊乱的动物模型。再经小剂量一次性腹腔注射链脲菌素后,约80%的胰岛素抵抗和肥胖的金黄地鼠出现显性糖尿病。结论高脂果糖饲料结合小剂量链脲菌素腹腔注射可以制成胰岛素抵抗的2型糖尿病金黄地鼠模型。     

Phosphorylase phosphatase activities of rat liver in streptozotocin-diabetes

Protein phosphatase-1 and 2A, accounting for all the hepatic activity regulating phosphorylase, were assayed in streptozotocin-induced (8 weeks) diabetic Wistar rats. Cytosolic protein phosphatase-1 and 2A were distinguished by chromatography on heparin-Sepharose and by inhibition with inhibitor-2. Approx. 25-35% increases in type-1 phosphorylase phosphatase activity measured in cytosols were registered in diabetic rats when compared with control and 24 h fasting animals. The enrichment of protein phosphatase-1 in the cytosol of streptozotocin-treated rat livers could not be attributed to the reduced glycogen content with the onset of diabetes, since this elevated level of type-1 phosphatase was not observed in fasting rats with low glycogen content. The translocation of type-1 phosphatase from the particulate fraction into the cytosol was also recorded in trypsin-treated samples of diabetic rat livers. The apparent molecular weight of type-1 phosphatase in the cytosol of control and fasted rats was 160,000 as judged by gel filtration. The type-1 phosphatase activity that was released from the particulate fraction by streptozotocin-induced diabetes identified a further enzyme species (Mr 110,000) in the cytosol. Our data imply that the higher levels of cytosolic protein phosphatase-1 in diabetic rat liver could be a consequence of the dissociation of the catalytic subunit of protein phosphatase-1 and the glycogen-binding subunit in rat livers.     

Effect of insulin and insulin-like growth factors I and II on phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate breakdown in liver from humans with and without type II diabetes

We have characterized a plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and a cytosolic phosphatidylinositol (PI)-specific PLC in human liver. Epinephrine, 1 x 10(-5) M, and vasopressin, 1 x 10(-8) M, stimulated PIP2-PLC which was enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). PI-PLC stimulation was not observed by these agents. Insulin and insulin-like growth factors (IGF-I and IGF-II) in the presence and absence of GTP gamma S did not stimulate PIP2-PLC or PI-PLC in plasma membranes and cytosol preparations nor phosphoinositide breakdown in isolated human hepatocytes. Furthermore, serendipitly we found that PIP2-PLC activity was increased in liver membranes from obese patients with type II diabetes when compared to obese and lean controls. We conclude that in human liver, insulin and IGFs are not members of the family of hormones generating inositol trisphosphate (IP3) as a second messenger. Furthermore, the increased PIP2-PLC in diabetic liver may result in: (a) increased intracellular concentrations of IP3 and thus increased Ca2+, which has been postulated to induce insulin resistance; and (b) increased diacylglycerol and thus increased protein kinase C which phosphorylates the insulin receptor at serine residues inactivating the insulin receptor kinase. While the mechanism of increased PIP2-PLC activity in diabetes is unknown, it may initiate a cascade of events that result in insulin resistance.     

Altered insulin signaling in retinal tissue in diabetic states

Both type 1 and type 2 diabetes can lead to altered retinal microvascular function and diabetic retinopathy. Insulin signaling may also play a role in this process, and mice lacking insulin receptors in endothelial cells are protected from retinal neovascularization. To define the role of diabetes in retinal function, we compared insulin signaling in the retinal vasculature of mouse models of type 1 (streptozotocin) and type 2 diabetes (ob/ob). In streptozotocin mice, in both retina and liver, insulin receptor (IR) and insulin receptor substrate (IRS)-2 protein and tyrosine phosphorylation were increased by insulin, while IRS-1 protein and its phosphorylation were maintained. By contrast, in ob/ob mice, there was marked down-regulation of IR, IRS-1, and IRS-2 protein and phosphorylation in liver; these were maintained or increased in retina. In both mice, Phosphatidylinositol 3,4,5-trisphosphate generation by acute insulin stimulation was enhanced in retinal endothelial cells. On the other hand, protein levels and phosphorylation of PDK1 and Akt were decreased in retina of both mice. Interestingly, phosphorylation of p38 mitogen-activated protein kinase and ERK1 were responsive to insulin in retina of both mice but were unresponsive in liver. HIF-1alpha and vascular endothelial growth factor were increased and endothelial nitric-oxide synthase was decreased in retina. These observations indicate that, in both insulin-resistant and insulin-deficient diabetic states, there are alterations in insulin signaling, such as impaired PDK/Akt responses and enhanced mitogen-activated protein kinases responses that could contribute to the retinopathy. Furthermore, insulin signaling in retinal endothelial cells is differentially altered in diabetes and is also differentially regulated from insulin signaling in classical target tissues such as liver.     

Insulin-like growth factor I levels decrease in the development of diabetes inMacaca nigra

Members of the monkey speciesMacaca nigra spontaneously develop impairments in insulin secretion and glucose clearance, and eventually become overtly diabetic. Changes in certain metabolic signals such as clearance of glucose and insulin increment secreted in an intravenous glucose tolerance test have allowed the identification of four stages in the progression from non-diabetes to diabetes in monkeys — non-diabetic, hormonally impaired, borderline diabetic, and diabetic. Recently, another metabolic stage, hyperinsulinemic, was also identified in these animals. In recent years, other factors besides those listed above have been implicated to be correlated with the metabolic progression from a nondiabetic to a diabetic state. One of these factors, is insulin like growth factor I (IGF-I). In diabetic humans who are in poor metabolic control, and in rats with streptozotocin induced ketotic diabetes, serum levels of IGF-I are lowered by as much as 40–50% of control non-diabetics. If indeed decreased IGF-I levels are correlated with the onset of diabetes then changes in IGF-I concentrations prior to the clinically diagnosed disease state would be expected. Using serum samples collected from different animals in a colony ofMacaca nigra in a variety of metabolic states, we have found that IGF-I and insulin levels decrease in each defined metabolic state as the animals progress from nondiabetic to diabetic. Since IGF-I and insulin levels decrease in a similar fashion in the progression of this disease then this maybe indicative of the coordinate expression of these two factors.     

The effect of insulin supplementation on diabetes-induced alterations in the extractable levels of functional mitochondrial anion transport proteins

The effect of insulin supplementation on diabetes-induced alterations in the levels of functional mitochondrial anion transport proteins has been determined. The experimental approach consisted of the extraction of the pyruvate, dicarboxylate, and citrate transport proteins from the mitochondrial inner membrane with Triton X-114 using rat liver mitoplasts (prepared from control, diabetic, or insulin-supplemented diabetic animals) as the starting material, followed by the reconstitution of the function of each transporter in a proteoliposomal system. This experimental strategy permitted the quantification of the functional levels of these three transporters in the absence of the complications that arise when such measurements are carried out with intact mitochondria (or mitoplasts). We found that treatment of diabetic rats (i.e., animals that were injected with streptozotocin 3 weeks earlier) on a daily basis with insulin for 3 weeks resulted in a reversal of the diabetes-induced (a) increase in the extractable and reconstitutable total (and specific) transport activities of the pyruvate and dicarboxylate transporters and (b) decrease in the activity of the citrate transporter. These findings indicate that diabetes-induced alterations in the functional levels of mitochondrial anion transport proteins are a direct consequence of the insulin insufficiency that characterizes this disease. Furthermore, this study provides the first demonstration that insulin participates in the regulation of the functional levels of liver mitochondrial anion transport proteins.