Liver-specific expression of carboxylesterase 1g/esterase-x reduces hepatic steatosis,counteracts dyslipidemia and improves insulin signaling

Ces1g/Es-x deficiency in mice results in weight gain, insulin resistance, fatty liver and hyperlipidemia through upregulation of de novo lipogenesis and oversecretion of triacylglycerol (TG)-rich lipoproteins. Here, we show that restoration of Ces1g/Es-x expression only in the liver significantly reduced hepatic TG concentration accompanied by decreased size of lipid droplets, reduced secretion of very low-density lipoproteins and improved insulin-mediated signal transduction in the liver. Collectively, these results demonstrate that hepatic Ces1g/Es-x plays a critical role in limiting hepatic steatosis, very low-density lipoprotein assembly and in augmenting insulin sensitivity.     

Socs1 deficiency enhances hepatic insulin signaling

Suppressor of cytokine signaling 1 (SOCS1) is an intracellular inhibitor of cytokine, growth factor, and hormone signaling. Socs1-/- mice die before weaning from a multiorgan inflammatory disease. Neonatal Socs1-/- mice display severe hypoglycemia and hypoinsulinemia. Concurrent interferon gamma gene deletion (Ifng-/-) prevented inflammation and corrected the hypoglycemia. In hyperinsulinemic clamp studies, however, Socs1-/- Ifng-/- mice had enhanced hepatic insulin sensitivity demonstrated by greater suppression of endogenous glucose production compared with controls with no difference in glucose disposal. Socs1-/- Ifng-/- mice had elevated liver insulin receptor substrate 2 expression (IRS-2) and IRS-2 tyrosine phosphorylation. This was associated with lower phosphoenolpyruvate carboxykinase mRNA expression. These effects were not associated with elevated hepatic AMP-activated protein kinase activity. Hepatic insulin sensitivity and IRS-2 levels play central roles in the pathogenesis of type 2 diabetes. Socs1 deficiency increases IRS-2 expression and enhances hepatic insulin sensitivity in vivo indicating that inhibition of SOCS1 may be a logical strategy in type 2 diabetes.     

Chronic treatment with epoxyeicosatrienoic acids modulates insulin signaling and prevents insulin resistance in hepatocytes

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites produced by cytochrome P450 epoxygenases which are highly expressed in hepatocytes. The functions of EETs in hepatocytes are not well understood. In this study, we investigated the effects of 14,15-EETs treatment on the insulin signal transduction pathway in hepatocytes. We report that chronic treatment, not acute treatment, with 30 μM 14,15-EETs prevents palmitate induced insulin resistance and potentiates insulin action in cultured HepG2 hepatocytes. 14,15-EETs increase Akt phosphorylation at S473, activating Akt, in an insulin dependent manner in HepG2 cells. Under insulin resistant conditions induced by palmitate, 14,15-EETs restore the insulin response by increasing S473-phosphorylated Akt. 8,9-EETs and 11,12-EETs demonstrated similar effects to 14,15-EETs. Furthermore, 14,15-EETs potentiate insulin-suppression of gluconeogenesis in cultured H4IIE hepatocytes. To elucidate the mechanism of EETs function, we analyzed the insulin signaling factors upstream of Akt. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 attenuated the 14,15-EETs-induced activating phosphorylation of Akt. 14,15-EETs reduced palmitate-stimulated phosphorylation of IRS-1 on S312 and phosphorylation of c-Jun N-terminal kinase (JNK) at threonine 183 and tyrosine 185 residues. The regulation of insulin sensitivity in cultured hepatocytes by chronic 14,15-EETs treatment appears to involve the JNK-IRS-PI3K pathway. The requirement of chronic treatment with EETs suggests that the effects of EETs on insulin response may be indirect.     

Effects of bile acids on hepatocellular signaling and secretion.

Bile acids modulate hepatocellular signaling pathways in vitro at physiological concentrations. The present paper provides a brief overview of the effects of bile acids on three key messengers in liver cells: cytosolic free calcium, protein kinase A and protein kinase C.     

Effects of feeding bile acids and a bile acid sequestrant on hepatic bile acid composition in mice

An improved ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method was established for the simultaneous analysis of various bile acids (BA) and applied to investigate liver BA content in C57BL/6 mice fed 1% cholic acid (CA), 0.3% deoxycholic acid (DCA), 0.3% chenodeoxycholic acid (CDCA), 0.3% lithocholic acid (LCA), 3% ursodeoxycholic acid (UDCA), or 2% cholestyramine (resin). Results indicate that mice have a remarkable ability to maintain liver BA concentrations. The BA profiles in mouse livers were similar between CA and DCA feedings, as well as between CDCA and LCA feedings. The mRNA expression of Cytochrome P450 7a1 (Cyp7a1) was suppressed by all BA feedings, whereas Cyp7b1 was suppressed only by CA and UDCA feedings. Gender differences in liver BA composition were observed after feeding CA, DCA, CDCA, and LCA, but they were not prominent after feeding UDCA. Sulfation of CA and CDCA was found at the 7-OH position, and it was increased by feeding CA or CDCA more in male than female mice. In contrast, sulfation of LCA and taurolithocholic acid (TLCA) was female-predominant, and it was increased by feeding UDCA and LCA. In summary, the present systematic study on BA metabolism in mice will aid in interpreting BA-mediated gene regulation and hepatotoxicity.     

Impaired insulin signaling in an animal model of Niemann-Pick Type C disease

Brain fatty acid-binding protein (FABP7) and PAX6 are both expressed in radial glial cells and have been implicated in neurogenesis and glial cell differentiation. FABP7 and PAX6 have also been postulated to play a role in malignant glioma cell growth and invasion. Here, we address the role of PAX6 in regulating FABP7 gene expression in malignant glioma cells. We report that PAX6 and FABP7 RNA are generally co-expressed in malignant glioma cell lines, tumors and tumor neurospheres. Using the CAT reporter gene assay, we show that FABP7 promoter activity is upregulated by PAX6. Sequential deletion analysis of the FABP7 promoter, combined with gel shift and supershift assays demonstrate the presence of a PAX6 responsive region located upstream of the FABP7 gene, at -862 to -1033 bp. Inclusion of sequences between -1.2 and -1.8 kb reduced CAT activity, suggesting the presence of a repressor element within this region. While PAX6 overexpression did not induce endogenous FABP7 expression in FABP7-negative cells, knock-down of PAX6 in PAX6-positive malignant glioma cells resulted in reduced FABP7 levels. These data provide the first evidence of direct transactivation of the FABP7 proximal promoter by PAX6 and suggest a synergistic mechanism for PAX6 and other co-factor(s) in regulating FABP7 expression in malignant glioma.     

Impaired miR-146a expression links subclinical inflammation and insulin resistance in Type 2 diabetes

Molecular and Cellular Biochemistry - Type 2 diabetes patients exhibit subclinical inflammation but the regulatory mechanisms are poorly understood. We sought to evaluate the role of miR-146a...     

Selective reduction of oxo bile acids: synthesis of 3 beta-, 7 beta-, and 12 beta-hydroxy bile acids

Preparation of some biologically important keto bile acids is described. Advantage is taken of the preferential ketalization of 3-oxo group in bile acids over 7- and 12-oxo groups for the selective reduction of these keto groups. The method was found to be specially useful for preparation of 7 beta-, 12 alpha, and 12 beta-[3H]-3-oxo bile acids. Improved methods are also described for the preparation of epimers of naturally occurring bile acids at C-3, C-7, and C-12. 3 beta-Hydroxy bile acids (iso-bile acids) were prepared with the use of diethylazodicarboxylate/triphenylphosphine/formic acid. Iso-bile acids were obtained in excellent yields (80-95%) except during synthesis of isoursodeoxycholic acid (yield, 50%). Isoursodeoxycholic acid was, however, prepared in very good yield via epimerization of 3 alpha-hydroxyl group in 7-oxolithocholic acid followed by stereoselective reduction of 7-oxo group. A highly efficient method for the reduction of 7-oxo and 12-oxo groups was developed. Thus, 7-oxolithocholic acid and 7-oxoisolithocholic acid on reduction with potassium/tertiary amyl alcohol yielded ursodeoxycholic acid and isoursodeoxycholic acid in yields of 96% and 94%, respectively, while reduction of 7-oxodeoxycholic acid resulted in ursocholic acid in 93% yield. In a similar manner, reduction of 12-oxolithocholic acid and 12-oxochenodeoxycholic acid yielded 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acid (lagodeoxycholic acid; 92% yield) and 3 alpha, 7 alpha, 12 beta-trihydroxy-5 beta-cholanoic acid (lagocholic acid, 86% yield).     

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.     

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.     

Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.     

Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction

The liver plays a central role in the control of glucose homeostasis and is subject to complex regulation by substrates, insulin, and other hormones. To investigate the effect of the loss of direct insulin action in liver, we have used the Cre-loxP system to inactivate the insulin receptor gene in hepatocytes. Liver-specific insulin receptor knockout (LIRKO) mice exhibit dramatic insulin resistance, severe glucose intolerance, and a failure of insulin to suppress hepatic glucose production and to regulate hepatic gene expression. These alterations are paralleled by marked hyperinsulinemia due to a combination of increased insulin secretion and decreased insulin clearance. With aging, the LIRKO liver exhibits morphological and functional changes, and the metabolic phenotype becomes less severe. Thus, insulin signaling in liver is critical in regulating glucose homeostasis and maintaining normal hepatic function.     

Membrane bilayer properties of sphingomyelins with amide-linked 2- or 3-hydroxylated fatty acids

The bilayer properties and interactions with cholesterol of N-acyl hydroxylated sphingomyelins (SM) were examined, and results were compared to nonhydroxylated chain-matched SM. The natural OH(D)-enantiomer of hydroxylated SM (with 16:0 or 22:0 acyl chain lengths) analogs was synthesized. Measuring steady-state diphenylhexatriene anisotropy, we observed that pure 2OH-SM bilayers always showed higher (5-10 °C) gel-liquid transition temperatures (T(m)) compared to their nonhydroxylated chain-matched analogs. Bilayers made from 3OH(D)-palmitoyl SM, however, had lower T(m) (5 °C) than palmitoyl SM. These data show that hydroxylation in a position-dependent manner directly affected SM interactions and gel state stability. From the c-laurdan emission spectra, we could observe that 2OH-palmitoyl SM bilayers showed a redshift in the emission compared to nonhydroxylated palmitoyl SM bilayers, whereas the opposite was true for c-laurdan emission in 3OH-palmitoyl SM bilayers. All hydroxylated SM analogs were able to form sterol-enriched ordered domains in a fluid phospholipid bilayer. 2-Hydroxylation appeared to increase domain thermostability compared to nonhydroxylated SM, whereas 3-hydroxylation appeared to decrease domain stability. When sterol affinity to bilayers containing SM analogs was determined (cholestatrienol partitioning), the affinity for hydroxylated SM analog bilayers was clearly reduced compared to the nonhydroxylated SM bilayers. Our results with hydroxylated SM analogs clearly show that hydroxylation affects interlipid interactions in a position-dependent manner.     

Role of insulin signaling impairment,adiponectin and dyslipidemia in peripheral and central neuropathy in mice

One of the tissues or organs affected by diabetes is the nervous system, predominantly the peripheral system (peripheral polyneuropathy and/or painful peripheral neuropathy) but also the central system with impaired learning, memory and mental flexibility. The aim of this study was to test the hypothesis that the pre-diabetic or diabetic condition caused by a high-fat diet (HFD) can damage both the peripheral and central nervous systems. Groups of C57BL6 and Swiss Webster mice were fed a diet containing 60% fat for 8 months and compared to control and streptozotocin (STZ)-induced diabetic groups that were fed a standard diet containing 10% fat. Aspects of peripheral nerve function (conduction velocity, thermal sensitivity) and central nervous system function (learning ability, memory) were measured at assorted times during the study. Both strains of mice on HFD developed impaired glucose tolerance, indicative of insulin resistance, but only the C57BL6 mice showed statistically significant hyperglycemia. STZ-diabetic C57BL6 mice developed learning deficits in the Barnes maze after 8 weeks of diabetes, whereas neither C57BL6 nor Swiss Webster mice fed a HFD showed signs of defects at that time point. By 6 months on HFD, Swiss Webster mice developed learning and memory deficits in the Barnes maze test, whereas their peripheral nervous system remained normal. In contrast, C57BL6 mice fed the HFD developed peripheral nerve dysfunction, as indicated by nerve conduction slowing and thermal hyperalgesia, but showed normal learning and memory functions. Our data indicate that STZ-induced diabetes or a HFD can damage both peripheral and central nervous systems, but learning deficits develop more rapidly in insulin-deficient than in insulin-resistant conditions and only in Swiss Webster mice. In addition to insulin impairment, dyslipidemia or adiponectinemia might determine the neuropathy phenotype.KEY WORDS: Glucose, High-fat diet, Insulin, Neuropathy     

A bioluminescent assay for 12-alpha-hydroxy bile acids using immobilized enzymes

A bioluminescent assay for 12-alpha-hydroxy bile acids was developed using enzymes coimmobilized onto Sepharose 4B. The immobilized enzymes used were a bacterial 12-alpha-hydroxysteroid dehydrogenase, bacterial luciferase, and NADPH:FMN oxidoreductase or bacterial diaphorase. The assay was specific for 12-alpha-hydroxy bile acids and the lower limit of detection was 4 pmol/0.5 ml assay volume with a linear range of 4 to 2000 pmol. Intraassay precision was from 7.8 to 8.2%. Values obtained with this assay showed good agreement with those obtained by gas-liquid chromatography. The system using diaphorase was not stable at 4 degrees C in the absence of added thiol compounds, but could be stabilized by the addition of glutathione (0.5 mM). The assay is a convenient, a rapid, and an extremely sensitive method for the measurement of 12-alpha-hydroxy bile acid concentrations in the serum of patients or experimental animals.     

Proteomic profiling of hepatic endoplasmic reticulum-associated proteins in an animal model of insulin resistance and metabolic dyslipidemia

Hepatic insulin resistance and lipoprotein overproduction are common features of the metabolic syndrome and insulin-resistant states. A fructose-fed, insulin-resistant hamster model was recently developed to investigate mechanisms linking the development of hepatic insulin resistance and overproduction of atherogenic lipoproteins. Here we report a systematic analysis of protein expression profiles in the endoplasmic reticulum (ER) fractions isolated from livers of fructose-fed hamsters with the intention of identifying new candidate proteins involved in hepatic complications of insulin resistance and lipoprotein dysregulation. We have profiled hepatic ER-associated proteins from chow-fed (control) and fructose-fed (insulin-resistant) hamsters using two-dimensional gel electrophoresis and mass spectrometry. A total of 26 large scale two-dimensional gels of hepatic ER were used to identify 34 differentially expressed hepatic ER protein spots observed to be at least 2-fold differentially expressed with fructose feeding and the onset of insulin resistance. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-quadrupole time of flight (MALDI-Q-TOF), MALDI-TOF-postsource decay, and database mining using ProteinProspector MS-fit and MS-tag or the PROWL ProFound search engine using a focused rodent or mammalian search. Hepatic ER proteins ER60, ERp46, ERp29, glutamate dehydrogenase, and TAP1 were shown to be more than 2-fold down-regulated, whereas alpha-glucosidase, P-glycoprotein, fibrinogen, protein disulfide isomerase, GRP94, and apolipoprotein E were all found to be up-regulated in the hepatic ER of the fructose-fed hamster. Seven isoforms of ER60 in the hepatic ER were all shown to be down-regulated at least 2-fold in hepatocytes from fructosefed/insulin-resistant hamsters. Implications of the differential expression of positively identified protein factors in the development of hepatic insulin resistance and lipoprotein abnormalities are discussed.     

Effective glucuronidation of 6 alpha-hydroxylated bile acids by human hepatic and renal microsomes

The glucuronidation of bile acids is an established metabolic pathway in different human organs. The hepatic and renal UDP-glucuronyltransferase activities vary according to the bile acids concerned. Thus, hyodeoxycholic acid is clearly differentiated from other bile acids by its high rate of glucuronidation and elevated urinary excretion in man. To determine whether such in vivo observations are related to variations in bile acid structure, human hepatic and renal microsomes were prepared and time courses of bile acid glucuronidation measured with the bile acids possessing hydroxyl groups in different positions. Eleven [24-14C]bile acids were chosen or synthesized in respect of their specific combination of hydroxyl and oxo groups at the 3, 6, 7 and 12 positions and of their alpha or beta hydroxyl configurations. The results clearly demonstrate that bile acids with an hydroxyl group in the 6 alpha position underwent a high degree of glucuronidation. Apparent kinetic Km and Vmax values for UDP-glucuronyltransferase activities ranged over 78-66 microM and 1.8-3.3 nmol.min-1.mg-1 protein in the liver and over 190-19 microM and 0.5-9.2 nmol.min-1.mg-1 protein in the kidney. All the other bile acids tested, each of which possessed a 3 alpha-hydroxyl group and whose second or third hydroxyl was bound at the 6 beta, 7 or 12 positions, were glucuronidated to a degree far below that of the 6 alpha-hydroxylated bile acids. We conclude that an active and highly specific UDP-glucuronyltransferase activity for 6 alpha-hydroxylated bile acids exists in human liver and kidneys. Moreover, this activity results in the linkage of glucuronic acid to the 6 alpha-hydroxyl group and not to the usual 3 alpha-hydroxyl group of bile acids.     

Munc18c phosphorylation by the insulin receptor links cell signaling directly to SNARE exocytosis

How the Sec1/Munc18-syntaxin complex might transition to form the SNARE core complex remains unclear. Toward this, Munc18c tyrosine phosphorylation has been correlated with its dissociation from syntaxin 4. Using 3T3-L1 adipocytes subjected to small interfering ribonucleic acid reduction of Munc18c as a model of impaired insulin-stimulated GLUT4 vesicle exocytosis, we found that coordinate expression of Munc18c-wild type or select phosphomimetic Munc18c mutants, but not phosphodefective mutants, restored GLUT4 vesicle exocytosis, suggesting a requirement for Munc18c tyrosine phosphorylation at Tyr219 and Tyr521. Surprisingly, the insulin receptor (IR) tyrosine kinase was found to target Munc18c at Tyr521 in vitro, rapidly binding and phosphorylating endogenous Munc18c within adipocytes and skeletal muscle. IR, but not phosphatidylinositol 3-kinase, activation was required. Altogether, we identify IR as the first known tyrosine kinase for Munc18c as part of a new insulin-signaling step in GLUT4 vesicle exocytosis, exemplifying a new model for the coordination of SNARE assembly and vesicle mobilization events in response to a single extracellular stimulus.