PCK1 and PCK2 as candidate diabetes and obesity genes

The PCK1 gene (Pck1 in rodents) encodes the cytosolic isozyme of phosphoenolpyruvate carboxykinase (PEPCK-C), which is well-known for its function as a gluconeogenic enzyme in the liver and kidney. Mouse studies involving whole body and tissue-specific Pck1 knockouts as well as tissue-specific over-expression of PEPCK-C have resulted in type 2 diabetes as well as several surprising phenotypes including obesity, lipodystrophy, fatty liver, and death. These phenotypes arise from perturbations not only in gluconeogenesis but in two additional metabolic functions of PEPCK-C: (1) cataplerosis which maintains metabolic flux through the Krebs cycle by removing excess oxaloacetate, and (2) glyceroneogenesis which produces glycerol-3-phosphate as a precursor for fatty acid esterification into triglycerides. PEPCK-C catalyzes the conversion of oxaloacetate + GTP to phosphoenolpyruvate + GDP + CO₂. It is in part the tissue-specificity of this simple reaction that results in the variety of phenotypes listed above. Briefly: (1) A 7-fold over-expression of PEPCK-C in the livers of mice causes excessive glucose production. (2) Mice with a whole-body knockout of Pck1 die within 2–3 days of birth, not from hypoglycemia, but probably because the Krebs cycle slows to approximately 10% of normal in the absence of cataplerosis. (3) Mice with a liver-specific knockout have an inability to remove oxaloacetate from the Krebs cycle, which leads to a fatty liver following a fast. (4) An adipose-specific knockout of Pck1 results in a fraction of the mice developing lipodystrophy due to lost glyceroneogenesis and a consequent decrease in fatty acid re-esterification. (5) Finally, disregulated over-expression of PEPCK-C in adipose tissue increases fatty acid re-esterification leading to obesity. These varied experimental phenotypes in mice have led us to postulate that abnormal production of PEPCK isozymes encoded by two PEPCK genes, PCK1 and PCK2, in humans could have similar consequences (Beale, E. G. et al. (2004). Trends in Endocrinology and Metabolism, 15, 129–135). The purpose of this review is to further explore these possibilities.     

Glucose Tolerance and Skeletal Muscle Gene Expression in Response to Alternate Day Fasting

Objective: Alternate day fasting may extend lifespan in rodents and is feasible for short periods in nonobese humans. The aim of this study was to examine the effects of 3 weeks of alternate day fasting on glucose tolerance and skeletal muscle expression of genes involved in fatty acid transport/oxidation, mitochondrial biogenesis, and stress response. Research Methods and Procedures: Glucose and insulin responses to a standard meal were tested in nonobese subjects (eight men and eight women; BMI, 20 to 30 kg/m²) at baseline and after 22 days of alternate day fasting (36 hour fast). Muscle biopsies were obtained from a subset of subjects (n = 11) at baseline and on day 21 (12‐hour fast). Results: Glucose response to a meal was slightly impaired in women after 3 weeks of treatment (p < 0.01), but insulin response was unchanged. However, men had no change in glucose response and a significant reduction in insulin response (p < 0.03). There were no significant changes in the expression of genes involved in mitochondrial biogenesis or fatty acid transport/oxidation, although a trend toward increased CPT1 expression was observed (p < 0.08). SIRT1 mRNA expression was increased after alternate day fasting (p = 0.01). Discussion: Alternate day fasting may adversely affect glucose tolerance in nonobese women but not in nonobese men. The gene expression results indicate that fatty acid oxidation and mitochondrial biogenesis are unaffected by alternate day fasting. However, the increased expression in SIRT1 suggests that alternate day fasting may improve stress resistance, a commonly observed feature of calorie‐restricted rodents.     

Non‐synonymous SNPs in MC1R gene are associated with the extended black variant in domestic ducks (Anas platyrhynchos)

In this study, we performed a sequence characterization of the duck melanocortin 1 receptor (alpha‐melanocyte stimulating hormone receptor) (MC1R) gene to analyze the relationship between MC1R polymorphism and the extended black variant in domestic ducks based on the extended black (E) and non‐extended black (e⁺) allele hypothesis of the duck MC1R gene. Both c.52G>A and c.376G>A substitutions are highly associated with the duck extended black variant (P < 0.01), but the novel c.52G>A substitution is more of a fit for the allele hypothesis of the duck MC1R gene.     

Is the hepatic metabolism of glucose and linoleic acid influenced by species in overfed ducks?

There are genetic differences in the hepatic glucose and linoleic acid metabolisms between Muscovy and Pekin ducks ad libitum-fed. To understand the effect of overfeeding on the hepatic metabolisms in these two species of ducks, we compared the different pathways of glucose and linoleic acid reaching the liver of Muscovy (Cairina moschata) (n = 6) and Pekin (Anas platyrhynchos) (n = 6) ducks overfed for 1 week and sacrificed 2–4 h after their last meal by using the ex vivo method of liver slices incubated for 16 h with [U-¹⁴C]-glucose, [1-¹⁴C]-linoleic acid and [³⁵S]-methionine added to the survival medium. The glucose was the main precursor of triacylglycerol synthesis in the liver of these two species and its hepatic metabolism was similar between species. The hepatic uptake of linoleic acid was 1.7-fold higher (P = 0.020) in the Muscovy duck than in the Pekin duck leading to a 1.9-fold higher (P = 0.017) esterification of this fatty acid in the liver of the Muscovy duck than in that of the Pekin duck. Finally, both species after 1 week of overfeeding exhibited the same capacity to secrete VLDL remaining insufficient to avoid hepatic steatosis.     

Identification and characterization of regulatory elements in the phosphoenolpyruvate carboxykinase gene PCK1 of Saccharomyces cerevisiae

Phosphoenolpyruvate carboxykinase is a key enzyme in gluconeogenesis. The expression of the PCK1 gene in Saccharomyces cerevisiae is strictly regulated and dependent on the carbon source provided. Two upstream activation sites (UAS1_PCK1 and UAS2_PCK1) and one upstream repression site (URS_PCK1) were localized by detailed deletion analysis. The efficacy of these three promoter elements when separated from each other was confirmed by investigations using heterologous promoter test plasmids. Activation mediated by UAS1_PCK1 or UAS2_PCK1 did not occur in the presence of glucose, indicating that these elements are essential for glucose derepression. The repressing effect caused by URS_PCK1 was much stronger in glucose-grown cells than in ethanol-grown cells.     

The gluconeogenic enzyme fructose-1,6-bisphosphatase is dispensable for growth of the yeast Yarrowia lipolytica in gluconeogenic substrates

The genes encoding gluconeogenic enzymes in the nonconventional yeast Yarrowia lipolytica were found to be differentially regulated. The expression of Y. lipolytica FBP1 (YlFBP1) encoding the key enzyme fructose-1,6-bisphosphatase was not repressed by glucose in contrast with the situation in other yeasts; however, this sugar markedly repressed the expression of YlPCK1, encoding phosphoenolpyruvate carboxykinase, and YlICL1, encoding isocitrate lyase. We constructed Y. lipolytica strains with two different disrupted versions of YlFBP1 and found that they grew much slower than the wild type in gluconeogenic carbon sources but that growth was not abolished as happens in most microorganisms. We attribute this growth to the existence of an alternative phosphatase with a high K_m (2.3 mM) for fructose-1,6-bisphosphate. The gene YlFBP1 restored fructose-1,6-bisphosphatase activity and growth in gluconeogenic carbon sources to a Saccharomyces cerevisiae fbp1 mutant, but the introduction of the FBP1 gene from S. cerevisiae in the Ylfbp1 mutant did not produce fructose-1,6-bisphosphatase activity or growth complementation. Subcellular fractionation revealed the presence of fructose-1,6-bisphosphatase both in the cytoplasm and in the nucleus.     

Regulatory regions in the yeast FBP1 and PCK1 genes.

By deletion analysis of the fusion genes FBP1-lacZ and PCK1-lacZ we have identified a number of strong regulatory regions in the genes FBP1 and PCK1 which encode fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase. Lack of expression of beta-galactosidase in fusions lacking sequences from the coding regions suggests the existence of downstream activating elements. Both promoters have several UAS and URS regions as well as sites implicated in catabolite repression. We have found in both genes consensus sequences for the binding of the same regulatory proteins, such as yAP1, MIG1 or the complex HAP2/HAP3/HAP4. Neither deletion nor overexpression of the MIG1 gene affected the regulated expression of the FBP1 or PCK1 genes.     

The dominant MHC class I gene is adjacent to the polymorphic<Emphasis Type="Italic"> TAP2</Emphasis> gene in the duck,<Emphasis Type="Italic"> Anas platyrhynchos</Emphasis>

We are investigating the expression and linkage of major histocompatibility complex (MHC) class I genes in the duck (Anas platyrhynchos) with a view toward understanding the susceptibility of ducks to two medically important viruses: influenza A and hepatitis B. In mammals, there are multiple MHC class I loci, and alleles at a locus are polymorphic and co-dominantly expressed. In contrast, in lower vertebrates the expression of one locus predominates. Southern-blot analysis and amplification of genomic sequences suggested that ducks have at least four loci encoding MHC class I. To identify expressed MHC genes, we constructed an unamplified cDNA library from the spleen of a single duck and screened for MHC class I. We sequenced 44 positive clones and identified four MHC class I sequences, each sharing approximately 85% nucleotide identity. Allele-specific oligonucleotide hybridization to a Northern blot indicated that only two of these sequences were abundantly expressed. In chickens, the dominantly expressed MHC class I gene lies adjacent to the transporter of antigen processing (TAP2) gene. To investigate whether this organization is also found in ducks, we cloned the gene encoding TAP2 from the cDNA library. PCR amplification from genomic DNA allowed us to determine that the dominantly expressed MHC class I gene was adjacent to TAP2. Furthermore, we amplified two alleles of the TAP2 gene from this duck that have significant and clustered amino acid differences that may influence the peptides transported. This organization has implications for the ability of ducks to eliminate viral pathogens.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AY294416–22     

High-protein diets prevent steatosis and induce hepatic accumulation of monomethyl branched-chain fatty acids

The hallmark of nonalcoholic fatty liver disease is steatosis of unknown etiology. To test how dietary protein decreases steatosis, we fed female C57BL/6 J mice low-fat (8 en%) or high-fat (42 en%) combined with low-protein (11 en%), high-protein (HP; 35 en%) or extra-high-protein (HPX; 58 en%) diets for 3 weeks. The 35 en% protein diets reduced hepatic triglyceride, free fatty acid, cholesterol and phospholipid contents to ~50% of that in 11 en% protein diets. Every additional 10 en% protein reduced hepatic fat content ~1.5 g%. HP diets had no effect on lipogenic or fatty acid-oxidizing genes except Ppargc1α (+30%), increased hepatic PCK1 content 3- to 5-fold, left plasma glucose and hepatic glycogen concentration unchanged, and decreased inflammation and cell stress (decreased Fgf21 and increased Gsta expression). The HP-mediated decrease in steatosis correlated inversely with plasma branched-chain amino-acid (BCAA) concentrations and hepatic content of BCAA-derived monomethyl branched-chain fatty acids (mmBCFAs) 14-methylpentadecanoic (14-MPDA; valine-derived) and, to a lesser extent, 14-methylhexadecanoic acid (isoleucine-derived). Liver lipid content was 1.6- to 1.8-fold higher in females than in males, but the anti-steatotic effect of HP diets was equally strong. The strong up-regulation of PCK1 and literature data showing an increase in phosphoenolpyruvate and a decline in tricarboxylic acid cycle intermediates in liver reveal that an increased efflux of these intermediates from mitochondria represents an important effect of an HP diet. The HP diet-induced increase in 14-MPDA and the dietary response in gene expression were more pronounced in females than males. Our findings are compatible with a facilitating role of valine-derived mmBCFAs in the antisteatotic effect of HP diets.     

A metabolic engineering strategy for producing free fatty acids by the Yarrowia lipolytica yeast based on impairment of glycerol metabolism

In recent years, bio‐based production of free fatty acids from renewable resources has attracted attention for their potential as precursors for the production of biofuels and biochemicals. In this study, the oleaginous yeast Yarrowia lipolytica was engineered to produce free fatty acids by eliminating glycerol metabolism. Free fatty acid production was monitored under lipogenic conditions with glycerol as a limiting factor. Firstly, the strain W29 (Δgpd1), which is deficient in glycerol synthesis, was obtained. However, W29 (Δgpd1) showed decreased biomass accumulation and glucose consumption in lipogenic medium containing a limiting supply of glycerol. Analysis of substrate utilization from a mixture of glucose and glycerol by the parental strain W29 revealed that glycerol was metabolized first and glucose utilization was suppressed. Thus, the Δgpd1Δgut2 double mutant, which is deficient also in glycerol catabolism, was constructed. In this genetic background, growth was repressed by glycerol. Oleate toxicity was observed in the Δgpd1Δgut2Δpex10 triple mutant strain which is deficient additionally in peroxisome biogenesis. Consequently, two consecutive rounds of selection of spontaneous mutants were performed. A mutant released from growth repression by glycerol was able to produce 136.8 mg L^?1 of free fatty acids in a test tube, whereas the wild type accumulated only 30.2 mg L^?1. Next, an isolated oleate‐resistant strain produced 382.8 mg L^?1 of free fatty acids. Finely, acyl‐CoA carboxylase gene (ACC1) over‐expression resulted to production of 1436.7 mg L^?1 of free fatty acids. The addition of dodecane promoted free fatty acid secretion and enhanced the level of free fatty acids up to 2033.8 mg L^?1 during test tube cultivation.