Effects of 1,25-dihydroxyvitamin D3 on proliferation and differentiation of porcine preadipocyte in vitro

1,25-Dihydroxyvitamin D3, the physiologically active form of vitamin D3, exerts its functions through a receptor-mediated mechanism and plays an important role in the cell differentiation. This study investigated the effects of 1,25-dihydroxyvitamin D3 on the proliferation and differentiation of porcine preadipocyte. Stromal-vascular cells containing preadipocytes were prepared from dorsal subcutaneous adipose tissue of approximately 3-day-old Chinese male crossbred pigs. After confluence, the differentiation was induced by transferrin, dexamethasone and insulin for 2 days, and then subsequently cultured for 6 days. The cells were treated with 1,25-dihydroxyvitamin D3 during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. The terminal differentiation markers, such as glycerol-3-phosphate dehydrogenase activity and lipid accumulation were measured during the process of cultures. The treatment with 1,25-dihydroxyvitamin D3 severely affected the induction of all differentiation markers throughout the differentiation period. 1,25-Dihydroxyvitamin D3 suppressed the expression of peroxisome proliferator-activated receptor gamma mRNA and interfered with the induction of retinoid X receptor alpha mRNA. The mRNAs of the adipogenesis-related genes, lipoprotein lipase, stearoyl-CoA desaturase, phosphoenolpyruvate carboxykinase, glycerol-3-phosphate dehydrogenase and glucose transporter 4 were reduced when 1,25-dihydroxyvitamin D3 was added into differentiation medium. Also, 1,25-dihydroxyvitamin D3 inhibited preadipocyte differentiation in dose-dependent manner. These results suggested that 1,25-dihydroxyvitamin D3 inhibited porcine preadipocyte differentiation through suppressing PPAR gamma and RXR alpha mRNA expressions and then down regulating the expression of adipogenesis-related genes.     

The Hsp70 chaperone Ssa1 is essential for catabolite induced degradation of the gluconeogenic enzyme fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase (FBPase) is a key regulatory enzyme of gluconeogenesis. In the yeast Saccharomyces cerevisiae, it is only expressed when cells are grown in medium with nonfermentable carbon sources. Addition of glucose to cells leads to inactivation of FBPase and degradation via the ubiquitin-proteasome system. Polyubiquitination of FBPase is carried out by the Gid complex, a multi-subunit ubiquitin ligase. Using tandem affinity purification and subsequent mass spectrometry we identified the Hsp70 chaperone Ssa1 as a novel interaction partner of FBPase. Studies with the temperature-sensitive mutant ssa1-45^ts showed that Ssa1 is essential for polyubiquitination of FBPase by the Gid complex. Moreover, we show that degradation of an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is also affected in ssa1-45^ts cells demonstrating that Ssa1 plays a general role in elimination of gluconeogenic enzymes.     

Role of cysteine 306 in the catalytic mechanism of Ascaris suum phosphoenolpyruvate carboxykinase

Biochemical and metabolic data lead to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. The Ascaris suum PEPCK shares considerable homology with PEPCK from avian liver and is a good candidate for mutagenesis studies. The Cys306 substitution by Ser and Ala produced active enzymes and the two mutants are kinetically indistinguishable from each other. This substitution affects the catalytic affinity for the formation of the specific enzyme-nucleotide complex (k(cat)/K(m)) in the forward and reverse reactions. Studies with the substrate analogs 2(')dGDP and 2(')dGTP indicate that Cys306 in A. suum PEPCK is one of the residues important in nucleotide binding and may interact with the 2(')OH group in the ribose ring. Alternatively, mutation of this residue could cause protein changes that interfere with the proper conformation of the nucleotides for optimal catalysis to take place.     

A methyl-deficient diet fed to rats during the pre- and peri-conception periods of development modifies the hepatic proteome in the adult offspring

A methyl-deficient diet (MD) lacking folic acid and the associated methyl donors choline and methionine, fed to the laboratory rat during the periods of oocyte and embryo development, has been shown to programme glucose metabolism in the offspring. The hepatic proteome of the male offspring of female rats fed MD diets for 3 weeks prior to mating and for the first 5 days of gestation has been examined by 2-dimensional gel electrophoresis. Three groups of differentially abundant proteins associated with energy metabolism, amino acid metabolism and antioxidant defence were identified in the soluble proteins extracted from the liver from the MD offspring at both 6 and 12 months of age. Altered mitochondrial activity in other programming models leads to a similar pattern of differential protein abundance. Two of the differentially abundant proteins were identified as GAPDH and PGK-1 by mass spectrometry. Western blotting showed that there were multiple isoforms of both proteins with similar molecular weights but different isoelectric points. The differentially abundant spots reduced in the MD offspring corresponded to minor isoforms of GAPDH and PGK-1. The levels of PPAR-alpha, SREBP and glucocorticoid receptor mRNAs associated with other models of prenatal programming were unchanged in the MD offspring. The data suggest that a diet deficient in folic acid and associated methyl donors fed during the peri-conception and early preimplantation periods of mammalian development affects mitochondrial function in the offspring and that the posttranslational modification of proteins may be important.     

Effects of hyperhomocysteinemia and betaine–homocysteine S-methyltransferase inhibition on hepatocyte metabolites and the proteome

Both cardiovascular disease and liver injury are major public health issues. Hyperhomocysteinemia has been linked to cardiovascular diseases, and defects in methyl group metabolism, often resulting in hyperhomocysteinemia, are among the key molecular events postulated to play a role in liver injury. We employed proteomics and metabolomics analyses of human hepatocytes in primary cell culture to explore the spectrum of proteins and associated metabolites affected by the disruption of methyl group metabolism. We treated the hepatocytes with homocysteine (Hcy, 0.1 mM and 2 mM) to follow the impact of hyperhomocysteinemia, and in parallel, we used a specific inhibitor of betaine–homocysteine S-methyltransferase (BHMT) to extend our understanding of the physiological functions of the enzyme. The major effect of BHMT inhibition was a 50% decrease in S-adenosylmethionine levels. The treatments with Hcy resulted in multiple changes in the metabolite levels depending on the treatment modality. The BHMT inhibition and 0.1 mM Hcy treatment induced only moderate changes in the hepatocyte proteome and secretome, while the changes induced by the 2 mM Hcy treatment were extensive. Phosphatidylethanolamine carboxykinase and ornithine aminotransferase were up-regulated about two fold indicating an intervention into metabolism. Cellular proliferation was suspended, secretome composition was changed and signs of apoptosis were discernible. We have detected fibrinogen gamma dimers, which might have a role as a potentially new biomarker of early liver injury. Finally, we have demonstrated the failed maturation of apolipoprotein A1, which might be a new mechanism of disruption of cholesterol efflux from tissues.     

Daytime restricted feeding modifies the daily variations of liver gluconeogenesis: Adaptations in biochemical and endocrine regulators

Daytime restricted feeding (DRF) promotes circadian adaptations in the metabolic processing of nutrients. We explored the hepatic gluconeogenic response in DRF rats by the temporal profiles of the following: (1) the activity of glucose 6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), as well as the periportal and pericentral distribution of PEPCK; (2) conversion of alanine to glucose; (3) glycemia and liver glycogen content; (4) presence of glycogen synthase (GYS) and its phosphorylated form (at Ser641, pGYS); (5) circulating levels of corticosterone, glucagon and insulin; (6) glucose-tolerance test; and (7) sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-coactivator 1α (PGC-1α). The results showed that DRF promoted: (1) a phase shift in G6Pase activity and an increase in PEPCK activity as well as a change of PEPCK from periportal to pericentral hepatocytes, (2) a net conversion of alanine to circulating glucose, (3) a decrease in glycemic values and a phase shift in the liver glycogen content, (4) a phase shift in GYS and an increase of pGYS, (5) an increase in the daily levels of corticosterone and glucagon, but a reduction in the levels of insulin, (6) normal glucose homeostasis in all groups and (7) an enhanced presence of SIRT1 and PGC-1α. It is proposed that the increased gluconeogenic in DRF group promotes synthesis of hepatic glycogen and the production of glucose. These results could be a modulation of the gluconeogenic process due to rheostatic adaptations in the endocrine, metabolic and timing regulation of liver and could be associated with the physiology of the food entrained oscillator.     

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (− 386/− 374), non-perfect DR1 (− 118/− 106) and HNF4α-specific binding motif (H4-SBM) (− 26/− 14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.     

The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice

Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2 h and was 1.7-fold greater than that observed in the control group after 6 h. The up-regulation of GPD1 began 2 h after administering ethanol, and significantly increased 6 h later with the concomitant escalation in the glycolytic gene expression. The incorporation of ¹⁴C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.