Overexpression of plasma membrane-associated sialidase attenuates insulin signaling in transgenic mice

Plasma membrane-associated sialidase is a key enzyme for ganglioside hydrolysis, thereby playing crucial roles in regulation of cell surface functions. Here we demonstrate that mice overexpressing the human ortholog (NEU3) develop diabetic phenotype by 18-22 weeks associated with hyperinsulinemia, islet hyperplasia, and increased beta-cell mass. As compared with the wild type, insulin-stimulated phosphorylation of the insulin receptor (IR) and insulin receptor substrate I was significantly reduced, and activities of phosphatidylinositol 3-kinase and glycogen synthase were low in transgenic muscle. IR phosphorylation was already attenuated in the younger mice before manifestation of hyperglycemia. Transient transfection of NEU3 into 3T3-L1 adipocytes and L6 myocytes caused a significant decrease in IR signaling. In response to insulin, NEU3 was found to undergo tyrosine phosphorylation and subsequent association with the Grb2 protein, thus being activated and causing negative regulation of insulin signaling. In fact, accumulation of GM1 and GM2, the possible sialidase products in transgenic tissues, caused inhibition of IR phosphorylation in vitro, and blocking of association with Grb2 resulted in reversion of impaired insulin signaling in L6 cells. The data indicate that NEU3 indeed participates in the control of insulin signaling, probably via modulation of gangliosides and interaction with Grb2, and that the mice can serve as a valuable model for human insulin-resistant diabetes.     

Catalytically inactive lipoprotein lipase overexpression increases insulin sensitivity in mice.

Abnormalities in lipoprotein lipase (LPL) function contribute to the development of hypertriglyceridemia, one of the characteristic disorders observed in the metabolic syndrome. In addition to the hydrolyzing activity of triglycerides, LPL modulates various cellular functions via its binding ability to the cell surface. Here we show the effects of catalytically inactive LPL overexpression on high-fat diet (HFD)-induced decreased systemic insulin sensitivity in mice. The binding capacity of catalytically inactive G188E-LPL to C2C12 skeletal muscle cells was not significantly different from that of wild type LPL. Insulin-stimulated IRS-1 phosphorylation and glucose uptake were increased by addition of wild type or mutant LPL in C2C12 cells. After 10 weeks' of HFD feeding, mice had significantly higher blood glucose levels than chow-fed mice in insulin tolerance tests. The blood glucose levels after insulin injection was significantly decreased in mutated LPL-overexpressing mice (G188E mice), as well as in wild type LPL-overexpressing mice (WT mice). Overexpression of catalytically inactive LPL, as well as wild type LPL, improved impaired insulin sensitivity in mice. These results show that decreased expression of LPL possibly causes the insulin resistance, in addition to hypertriglyceridemia, in metabolic syndrome.     

Hepatocyte CYP2E1 overexpression and steatohepatitis lead to impaired hepatic insulin signaling

Insulin resistance and increased cytochrome P450 2E1 (CYP2E1) expression are both associated with and mechanistically implicated in the development of nonalcoholic fatty liver disease. Although currently viewed as distinct factors, insulin resistance and CYP2E1 expression may be interrelated through the ability of CYP2E1-induced oxidant stress to impair hepatic insulin signaling. To test this possibility, the effects of in vitro and in vivo CYP2E1 overexpression on hepatocyte insulin signaling were examined. CYP2E1 overexpression in a hepatocyte cell line decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 in response to insulin. CYP2E1 overexpression was also associated with increased inhibitory serine 307 and 636/639 IRS-1 phosphorylation. In parallel, the effects of insulin on Akt activation, glycogen synthase kinase 3, and FoxO1a phosphorylation, and glucose secretion were all significantly decreased in CYP2E1 overexpressing cells. This inhibition of insulin signaling by CYP2E1 overexpression was partially c-Jun N-terminal kinase dependent. In the methionine- and choline-deficient diet mouse model of steatohepatitis with CYP2E1 overexpression, insulin-induced IRS-1, IRS-2, and Akt phosphorylation were similarly decreased. These findings indicate that increased hepatocyte CYP2E1 expression and the presence of steatohepatitis result in the down-regulation of insulin signaling, potentially contributing to the insulin resistance associated with nonalcoholic fatty liver disease.     

MiR-15b can target insulin receptor to regulate hepatic insulin signaling in mice

Now diabetes is growing to be a health problems globally. However, its specific pathogenesis still needs further exploration. Here we showed that miR-15b was upregulated in the palmitate-induced HepG2 cells and livers of hyperglycemic mice. At the same time, we confirmed that the insulin receptor was a direct target of miR-15b. Then we found that the manipulation of miR-15b expression level could affect the insulin signaling pathway of HepG2 cells and the inhibition of miR-15b in liver of ob/ob mice can improve insulin sensitivity of mice. Furthermore, our study demonstrated that palmitate could upregulate the expression of miR-15b by activating PPARα. Our findings established PPARα-responsive miR-15b as a critical regulator of hepatic insulin signaling, thus serving as a new potential therapeutic target for diabetes.     

Genetic overexpression of eNOS attenuates hepatic ischemia-reperfusion injury

Previous studies have shown that endothelial nitric oxide (NO) synthase (eNOS)-derived NO is an important signaling molecule in ischemia-reperfusion (I-R) injury. Deficiency of eNOS-derived NO has been shown to exacerbate injury in hepatic and myocardial models of I-R. We hypothesized that transgenic overexpression of eNOS (eNOS-TG) would reduce hepatic I-R injury. We subjected two strains of eNOS-TG mice to 45 min of hepatic ischemia and 5 h of reperfusion. Both strains were protected from hepatic I-R injury compared with wild-type littermates. Because the mechanism for this protection is still unclear, additional studies were performed by using inhibitors and activators of both soluble guanylyl cyclase (sGC) and heme oxygenase-1 (HO-1) enzymes. Blocking sGC with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and HO-1 with zinc (III) deuteroporphyrin IX-2,4-bisethyleneglycol (ZnDPBG) in wild-type mice increased hepatic I-R injury, whereas pharmacologically activating these enzymes significantly attenuated I-R injury in wild-type mice. Interestingly, ODQ abolished the protective effects of eNOS overexpression, whereas ZnDPBG had no effect. These results suggest that hepatic protection in eNOS-TG mice may be mediated in part by NO signaling via the sGC-cGMP pathway and is independent of HO-1 signal transduction pathways.     

Phosphoenolpyruvate carboxykinase is induced in growth-arrested hepatoma cells

Phosphoenolpyruvate carboxykinase (PEPCK) mRNA is elevated in H4IIEC3 rat hepatoma cells cultured at high density, suggesting that PEPCK expression and growth arrest may be coordinately regulated. Induction of growth arrest either by contact inhibition (high culture density) or by serum deprivation correlated with significant increases in PEPCK protein and its mRNA. The observation that PEPCK mRNA was induced by contact inhibition in the presence of serum indicates that the effect of high density is independent of insulin or any other serum component. The magnitudes of the changes in PEPCK expression during growth arrest were greatly enhanced in KRC-7 cells, an H4IIEC3 subclone that is much more sensitive to growth arrest than its parental cell line. Restimulation of proliferation in growth-arrested KRC-7 cells, either by addition of serum or insulin to serum-deprived cells or by replating contact-inhibited cells at low density, caused a rapid decrease in PEPCK expression. However, PEPCK mRNA is not always reduced in proliferating cells since treatment of serum-starved cells with epidermal growth factor stimulated entry into the cell cycle but did not affect PEPCK mRNA levels. Finally, dexamethasone induction of PEPCK mRNA was blunted in cells cultured at high density but was unaffected by the presence or absence of serum. Collectively, these data suggest the possibility of cross-talk between the control of PEPCK expression and growth arrest in KRC-7 cells.     

Phosphoenolpyruvate carboxykinase and gluconeogenesis in cotyledons of Cucurbita pepo

1. The aim of this work was to investigate the role of phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) in the conversion of fat to sugar by the cotyledons of seedlings of Cucurbita pepo. 2. The enzyme was partially purified from the cotyledons of 5-day-old seedlings. The Michaelis constants for oxaloacetate and ATP were 56 and 119 micron, respectively. The decarboxylation reaction was optimum at pH 7.4. A range of intermediary metabolites did not affect the activity of the enzyme, but 3-mercaptopicolinic acid at micron concentrations was an effective inhibitor. 3. Centrifugation of extracts of 5-day-old cotyledons sedimented appreciable proportions of the ribuloseibisphosphate carboxylase, isocitrate lyase and fumarate hydratase present but very little of the phosphoenolpyruvate carboxykinase. 4. Measurements of phosphoenolpyruvate carboxykinase of cotyledons during germination showed that the maximum catalytic activity exceeded, and changed coincidently with, the rate of gluconeogenesis. 5. 3-Mercaptopicolinic acid inhibited gluconeogenesis from [1-14C]- and [2-14C]acetate supplied to excised cotyledons. The detailed distribution of 14C indicated inhibition of the conversion of oxaloacetate to phosphoenolpyruvate. 6. It is concluded that in marrow cotyledons phosphoenolpyruvate carboxykinase is in the soluble phase of the cytoplasm and catalyses a component reaction of gluconeogenesis.     

Adipose-specific overexpression of GLUT4 reverses insulin resistance and diabetes in mice lacking GLUT4 selectively in muscle

Adipose tissue plays an important role in glucose homeostasis and affects insulin sensitivity in other tissues. In obesity and type 2 diabetes, glucose transporter 4 (GLUT4) is downregulated in adipose tissue, and glucose transport is also impaired in muscle. To determine whether overexpression of GLUT4 selectively in adipose tissue could prevent insulin resistance when glucose transport is impaired in muscle, we bred muscle GLUT4 knockout (MG4KO) mice to mice overexpressing GLUT4 in adipose tissue (AG4Tg). Overexpression of GLUT4 in fat not only normalized the fasting hyperglycemia and glucose intolerance in MG4KO mice, but it reduced these parameters to below normal levels. Glucose infusion rate during a euglycemic clamp study was reduced 46% in MG4KO compared with controls and was restored to control levels in AG4Tg-MG4KO. Similarly, insulin action to suppress hepatic glucose production was impaired in MG4KO mice and was restored to control levels in AG4Tg-MG4KO. 2-deoxyglucose uptake during the clamp was increased approximately twofold in white adipose tissue but remained reduced in skeletal muscle of AG4Tg-MG4KO mice. AG4Tg and AG4Tg-MG4KO mice have a slight increase in fat mass, a twofold elevation in serum free fatty acids, an approximately 50% increase in serum leptin, and a 50% decrease in serum adiponectin. In MG4KO mice, serum resistin is increased 34% and GLUT4 overexpression in fat reverses this. Overexpression of GLUT4 in fat also reverses the enhanced clearance of an oral lipid load in MG4KO mice. Thus overexpression of GLUT4 in fat reverses whole body insulin resistance in MG4KO mice without restoring glucose transport in muscle. This effect occurs even though AG4Tg-MG4KO mice have increased fat mass and low adiponectin and is associated with normalization of elevated resistin levels.     

Phosphoenolpyruvate carboxykinase in plants exhibiting crassulacean Acid metabolism

Phosphoenolpyruvate carboxykinase has been found in significant activities in a number of plants exhibiting Crassulacean acid metabolism. Thirty-five species were surveyed for phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase, ribulose diphosphate carboxylase, malic enzyme, and malate dehydrogenase (NAD). Plants which showed high activities of malic enzyme contained no detectable phosphoenolpyruvate carboxykinase, while plants with high activities of the latter enzyme contained little malic enzyme. It is proposed that phosphoenolpyruvate carboxykinase acts as a decarboxylase during the light period, furnishing CO₂ for the pentose cycle and phosphoenolpyruvate for gluconeogenesis.     

Phosphoenolpyruvate carboxykinase in mouse pancreatic islets. ATP-induced changes in sensitivity to Mn2+ activation

The presence of high phosphoenolpyruvate carboxykinase (EC 4.1.1.32) activity in mouse islet cytosol has been demonstrated. The enzyme was activated by Mn2+ with a Ka of 100 X 10(-6) mol/l. The mean total activity of the Mn2+-stimulated phosphoenolpyruvate carboxykinase in islet cytosol estimated at 22 degrees C with saturating concentrations of the substrates oxaloacetate and ITP was 146 pmol/min per micrograms DNA. Km was calculated to be 6 X 10(-6) mol/l for oxaloacetate and 140 X 10(-6) mol/l for ITP. The islet phosphoenolpyruvate carboxykinase activity was not increased after starvation of the animals for 48 h. Preincubation of the cytosol at 4 degrees C with Fe2+, quinolinate, ATP, Pi, glucose 6-phosphate, fructose 1,6-bisphosphate, NAD+, NADH, oxaloacetate, ITP, cyclic AMP and Ca2+ had no effect on the enzyme activity. However, preincubation of the cytosol at 37 degrees C with ATP-Mg inhibited the Mn2+-stimulated phosphoenolpyruvate carboxykinase activity progressively with time and in a concentration-dependent manner. A similar but weaker inhibitory effect was observed with p[NH]ppA, whereas p[CH2]ppA, ADP, AMP, adenosine and Pi had no effect. It is tentatively suggested that ATP and p[NH]ppA either by adenylation or otherwise affect the interaction between islet phosphoenolpyruvate carboxykinase and the recently discovered Mr = 29000 protein modulator of the enzyme in such a way - perhaps by causing a dissociation between them - that phosphoenolpyruvate carboxykinase loses its sensitivity to Mn2+ activation.     

Phosphoenolpyruvate carboxykinase is a nuclear enzyme in rats and chickens

The presence of phosphoenolpyruvate carboxykinase (PEPCK) in the nuclei of chicken liver cells was confirmed using two experimental designs. PEPCK was found to be enriched in the nuclear fraction of rat liver, a species whose hepatic PEPCK is reported to be predominantly cytosolic. We suggest that PEPCK plays a role in nuclear synthesis of N-acetyl-neuraminic acid.     

Enhanced gluconeogenesis and hepatic insulin resistance in insulin-like growth factor binding protein-1 transgenic mice

Fasting hyperglycemia is observed in transgenic mice which overexpress insulin-like growth factor binding protein-1. In an attempt to understand the mechanisms underlying this observation we have examined glycogenolysis and gluconeogenesis in isolated hepatocytes from wild-type and transgenic mice. Glucose production from pyruvate was significantly less responsive to inhibition by insulin in hepatocytes from transgenic mice compared to hepatocytes from wild-type mice. Serum from transgenic mice resulted in more glucose production by hepatocytes than serum from wild-type mice. Serum alanine was increased while serum lactate was significantly reduced in transgenic mice compared to wild-type mice. Serum free fatty acids and beta-hydroxybutyrate were similar in both groups of mice. These data suggest that fasting hyperglycemia is due to enhanced gluconeogenesis, hepatic insulin resistance and increased serum gluconeogenic substrate in transgenic mice.     

Altered renal morphology in transgenic mice with cholecystokinin overexpression

Although cholecystokinin is a regulatory peptide with a predominant role in the brain and the gastrointestinal tract, there is an increasing evidence for its role in the kidney. The aim of this study was to reveal morphological changes in the structure of kidney of mice with cholecystokinin overexpression by means of light, transmission and scanning electron microscope, and atomic force microscopy. Using immunohistochemistry the expression of important basement membrane proteins collagen IV, laminin and fibronectin, as well the distribution of cholecystokinin-8 in the renal structures was evaluated. The altered morphology of kidneys of mice with cholecystokinin overexpression was seen by all microscopic techniques used. The renal corpuscles were relatively small with narrow capsular lumen. The basement membranes of renal tubules were thickened and the epithelial cells were damaged, which was more pronounced for distal tubules. Characteristic feature was the increased number of vesicles seen throughout the epithelial cells of proximal and especially in distal tubules reflecting to the enhanced cellular degeneration. The relative expression of laminin but not collagen IV in the glomerular basement membrane was higher than in the tubular basement membranes. The content of fibronectin, in opposite, was higher in tubular membranes. Cholecystokinin-8 was clearly expressed in the glomeruli, in Bowman’s capsule, in proximal and distal tubules, and in collecting ducts. Ultrastructural studies showed irregularly thickened glomerular basement membranes to which elongated cytopodia of differently shaped podocytes were attached. As foot processes were often fused the number of filtration pores was decreased. In conclusion, cholecystokinin plays important role in renal structural formation and in functioning as different aspects of urine production in mice with cholecystokinin overexpression are affected-the uneven glomerular basement membrane thickening, structural changes in podocytes and in filtration slits affect glomerular filtration, while damaged tubular epithelial cells and changed composition of thickened tubular basement membranes affect reabsorption.     

Defective beta-adrenergic receptor signaling precedes the development of dilated cardiomyopathy in transgenic mice with calsequestrin overexpression.

Calsequestrin is a high capacity Ca(2+)-binding protein in the junctional sarcoplasmic reticulum that forms a quaternary complex with junctin, triadin, and the ryanodine receptor. Transgenic mice with cardiac-targeted calsequestrin overexpression show marked suppression of Ca(2+)-induced Ca(2+) release, myocyte hypertrophy, and premature death by 16 weeks of age (Jones, L. R., Suzuki, Y. J., Wang, W., Kobayashi, Y. M., Ramesh, V., Franzini-Armstrong, C., Cleemann, L., and Morad, M. (1998) J. Clin. Invest. 101, 1385-1393). To investigate whether alterations in intracellular Ca(2+) trigger changes in the beta-adrenergic receptor pathway, we studied calsequestrin overexpressing transgenic mice at 7 and 14 weeks of age. As assessed by echocardiography, calsequestrin mice at 7 weeks showed mild left ventricular enlargement, mild decreased fractional shortening with increased wall thickness. By 14 weeks, the phenotype progressed to marked left ventricular enlargement and severely depressed systolic function. Cardiac catheterization in calsequestrin mice revealed markedly impaired beta-adrenergic receptor responsiveness in both 7- and 14- week mice. Biochemical analysis in 7- and 14-week mice showed a significant decrease in total beta-adrenergic receptor density, adenylyl cyclase activity, and the percent high affinity agonist binding, which was associated with increased beta-adrenergic receptor kinase 1 levels. Taken together, these data indicate that alterations in beta-adrenergic receptor signaling precede the development of overt heart failure in this mouse model of progressive cardiomyopathy.     

Neonatal FcR overexpression boosts humoral immune response in transgenic mice

The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation. We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum. The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger spleens containing a higher number of Ag-specific B cells and plasma cells, as well as many more granulocytes and dendritic cells, analyzed by ELISPOT and flow cytometric studies. The neutrophils from these Tg mice expressed the Tg FcRn and phagocytosed IgG immune complexes more efficiently than did those from wt mice. These results show that FcRn overexpression not only extends the IgG half-life but also enhances the expansion of Ag-specific B cells and plasma cells. Although both effects increase the level of Ag-specific IgG, the increase in immune response and IgG production seems to be more prominent compared with the reduced IgG clearance.     

Tributyrin attenuates obesity-associated inflammation and insulin resistance in high-fat-fed mice

The aim of this study was to investigate whether treatment with tributyrin (Tb; a butyrate prodrug) results in protection against diet-induced obesity and associated insulin resistance. C57BL/6 male mice fed a standard chow or high-fat diet were treated with Tb (2 g/kg body wt, 10 wk) and evaluated for glucose homeostasis, plasma lipid profile, and inflammatory status. Tb protected mice against obesity and obesity-associated insulin resistance and dyslipidemia without food consumption being affected. Tb attenuated the production of TNFα and IL-1β by peritoneal macrophages and their expression in adipose tissue. Furthermore, in the adipose tissue, Tb reduced the expression of MCP-1 and infiltration by leukocytes and restored the production of adiponectin. These effects were associated with a partial reversion of hepatic steatosis, reduction in liver and skeletal muscle content of phosphorylated JNK, and an improvement in muscle insulin-stimulated glucose uptake and Akt signaling. Although part of the beneficial effects of Tb are likely to be secondary to the reduction in body weight, we also found direct protective actions of butyrate reducing TNFα production after LPS injection and in vitro by LPS- or palmitic acid-stimulated macrophages and attenuating lipolysis in vitro and in vivo. The results, reported herein, suggest that Tb may be useful for the treatment and prevention of obesity-related metabolic disorders.