Hormonal and acid-base regulation of phosphoenolpyruvate carboxykinase mRNA levels in rat kidney

Metabolic acidosis (6 days NH4Cl) causes a fourfold increase in the relative abundance of mRNA encoding phosphoenolpyruvate carboxykinase in rat kidney. Streptozotocin-diabetes (6 days) also results in an increased abundance of the mRNA but this increase can be prevented if the acidosis associated with bicarbonate is corrected by treatment with bicarbonate. The results confirm that renal phosphoenolpyruvate carboxykinase is regulated primarily by changes in acid-base status and that this control is at a pretranslational step. Isolated kidney tubules in short-term incubation have been used to identify which agents regulate levels of phosphoenolpyruvate carboxykinase mRNA. The relative abundance of the mRNA was increased by glucocorticoids and hormones which act via cAMP, or by cAMP analogues directly, but was not affected by hormones acting via Ca2+. Neither incubation at pH 7.1 nor the presence of serum from acidotic rats had any effect on the level of phosphoenolpyruvate carboxykinase mRNA. It is concluded that acidosis, glucocorticoids, and cAMP independently regulate expression of renal phosphoenolpyruvate carboxykinase.     

Differential regulation of the rat phosphoenolpyruvate carboxykinase gene expression in several tissues of transgenic mice.

The selective expression of a unique copy gene in several mammalian tissues has been approached by studying the regulatory sequences needed to control expression of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene in transgenic mice. A transgene containing the entire PEPCK gene, including 2.2 kb of the 5'-flanking region and 0.5 kb of the 3'-flanking region, exhibits tissue-specific expression in the liver, kidney, and adipose tissue, as well as the hormonal and developmental regulation inherent to endogenous gene expression. Deletions of the 5'-flanking region of the gene have shown the need for sequences downstream of position -540 of the PEPCK gene for expression in the liver and sequences downstream of position -362 for expression in the kidney. Additional sequences upstream of position -540 (up to -2200) are required for expression in adipose tissue. In addition, the region containing the glucocorticoid-responsive elements of the gene used by the kidney was identified. This same sequence was found to be needed specifically for developmental regulation of gene expression in the kidney and, together with upstream sequences, in the intestine. The apparently distinct sequence requirements in the various tissues indicate that the tissues use different mechanisms for expression of the same gene.     

Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression

A cDNA library from RNA of senescing cucumber cotyledons was screened for sequences also expressed in cotyledons during post-germinative growth. One clone encodes ATP-dependent phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.49), an enzyme of the gluconeogenic pathway. The sequence of a fulllength cDNA predicts a polypeptide of 74397 Da which is 43%, 49% and 57% identical to bacterial, trypanosome and yeast enzymes, respectively. The cDNA was expressed in Escherichia coli and antibodies raised against the resultant protein. The antibody recognises a single polypeptide of ca. 74 kDa, in extracts of cotyledons, leaves and roots. The cucumber genome contains a single pck gene. In the seven-day period after seed imbibition, PCK mRNA and protein steady-state levels increase in amount in cotyledons, peaking at days 2 and 3 respectively, and then decrease. Both accumulate again to a low level in senescing cotyledons. This pattern of gene expression is similar to that of isocitrate lyase (ICL) and malate synthase (MS). When green cotyledons are detached from seedlings and incubated in the dark, ICL and MS mRNAs increase rapidly in amount but PCK mRNA does not. Therefore it seems unlikely that the glyoxylate cycle serves primarily a gluconeogenic role in starved (detached) cotyledons, in contrast to post-germinative and senescing cotyledons where PCK, ICL and MS are coordinately synthesised. While exogenous sucrose greatly represses expression of icl and ms genes in dark-incubated cotyledons, it has a smaller effect on the level of PCK mRNA.     

Predominant periportal expression of the phosphoenolpyruvate carboxykinase and tyrosine aminotransferase genes in rat liver

Summary The zonal distribution of phosphoenolpyruvate carboxykinase (PCK) and tyrosine aminotransferase (TAT) mRNA in liver was studied by in situ hybridization with radiolabelled cRNA probes and the abundance of PCK and TAT mRNA was quantified by Northern blot analysis of total RNA with biotinylated cRNA probes. Livers were taken from rats during a normal 12 h day/night rhythm, when they had access to food only during the dark period from 7 pm to 7 am, or during refeeding, when they had access to food after having been starved for 60 h. 1. Daily feeding rhythm: High levels of PCK mRNA were distributed mainly in the periportal and intermediate zone during the fasting period at noon and 6 pm. Feeding caused a rapid decrease in PCK mRNA level and a restriction of PCK mRNA localization to the periportal area within the first 2 h. No further alterations were observed during the following hours of the feeding period. TAT mRNA was distributed also in the periportal and intermediate zone during the fasting period. Feeding first reduced the mRNA level without changing the distribution pattern. Then towards the end of the feeding period TAt mRNA increased again to half-maximal levels and became restricted mainly to the periportal area. 2. Starvation-refeeding cycle: High amounts of PCK mRNA as well as of TAT mRNA were localized predominantly in the periportal and intermediate zone after 60 h of starvation. PCK and TAT mRNA both decreased markedly during the first 2 h of refeeding and then remained almost constant. Whereas the alterations in the overall abundance of the two mRNAs were similar, the distribution patterns of both mRNAs differed. While PCK mRNA became more and more restricted to a small area of periportal cells towards the end of refeeding, TAT mRNA was first evenly distributed in the periportal and perivenous area with higher amounts in the intermediate zone and then again was predominantly located in the periportal area. The present data indicate that the predominant periportal localization of PCK and TAT activity and enzyme protein is regulated mainly at the pretranslational level.     

Hormonal control of renal phosphoenolpyruvate carboxykinase activity during development of the rat.

The effect of injections of hormones in utero on fetal rat kidney and liver extramitochondrial phosphoenolpyruvate carboxykinase activity has been studied. Glucagon and thyroxine induced the liber enzyme but none of the hormones tested affected the renal enzyme. In the postnatal rat, the hepatic phosphoenolpyruvate barboxykinase activity is increased after triamcinolone or thyroxine injection but only triamcinolone injection increases the activity of the kidney enzyme. It is suggested that the rise in renal phosphoenolpyruvate carboxykinase activity at about 10 days of age is due to the increase in blood corticosterone content occurring at the same age.     

Inflammation inhibits the expression of phosphoenolpyruvate carboxykinase in liver and adipose tissue

Inhibition of adipocyte triglyceride biosynthesis is required for fatty acid mobilization during inflammation. Triglyceride biosynthesis requires glycerol 3-phosphate and phosphoenolpyruvate carboxykinase (PEPCK) plays a key role. We demonstrate that LPS, zymosan, and TNF-α decrease PEPCK in liver and fat. Turpentine decreases PEPCK in liver, but not in fat. The LPS-induced decrease in PEPCK does not occur in TLR4 deficient animals, indicating that this receptor is required. The LPS-induced decrease in hepatic PEPCK does not occur in TNF receptor/IL-1 receptor knockout mice, but occurs in fat, indicating that TNF-α/IL-1 is essential for the decrease in liver but not fat. In 3T3-L1 adipocytes TNF-α, IL-1, IL-6, and IFNγ inhibit PEPCK indicating that there are multiple pathways by which PEPCK is decreased in adipocytes. The binding of PPARγ and RXRα to the PPARγ response element in the PEPCK promoter is markedly decreased in adipose tissue nuclear extracts from LPS treated animals. Lipopolysaccharide and zymosan reduce PPARγ and RXRα expression in fat, suggesting that a decrease in PPARγ and RXRα accounts for the decrease in PEPCK. Thus, there are multiple cytokine pathways by which inflammation inhibits PEPCK expression in adipose tissue which could contribute to the increased mobilization of fatty acids during inflammation.     

Regulation of the expression of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes by glucagon and insulin

The induction of phosphoenolpyruvate carboxykinase (PEPCK) by glucagon was studied in primary rat hepatocyte cultures by determining the time course of the sequential events, increases in the enzyme's mRNA abundance, synthesis rate, amount and activity, and by investigating the antagonistic action of insulin on the induction by glucagon. 1. The mRNA of PEPCK was induced maximally 2-3 h after addition of 10 nM glucagon, as detected by Northern-blot analysis after hybridization with a biotinylated antisense RNA of PEPCK. 2. The synthesis rate of PEPCK increased maximally 2-3 h after application of glucagon as revealed by pansorbin-linked immunoprecipitation of [35S]methionine-labelled PEPCK. 3. The enzyme amount and activity was maximally induced 4 h after glucagon application. 4. The mRNA of PEPCK was half-maximally induced by 0.1 nM and maximally by 1 nM and 10 nM glucagon. The half-maximal induction by 0.1 nM glucagon was antagonized almost totally, and the maximal induction by 1 nM glucagon partially, while the maximal induction by 10 nM glucagon remained unaffected by 10 nM insulin. The results show that in cultured rat hepatocytes physiological concentrations of glucagon stimulated the induction of PEPCK by an increase in mRNA, that the glucagon-dependent increase in mRNA and enzyme-synthesis rate occurred in parallel and preceded the increase of enzyme amount and activity by 1-1.5 h, and that physiological levels of insulin antagonized the induction by glucagon in the physiological concentration range, with glucagon being the dominant hormone.