Phosphoenolpyruvate Carboxykinase Enzyme Activity Research Articles

Antioxidant Capacity, Enzyme Activities Related to Energy Metabolism, and Transcriptome Analysis of Crassostrea hongkongensis Exposed to Hypoxia.

Crassostrea hongkongensis (C. hongkongensis) is one of the three most commonly cultivated oyster species in China. Seasonal hypoxia is one of the most serious threats to its metabolism, reproductive behavior, and survival. To investigate the effects of hypoxia stress on the antioxidant capacity and energy metabolism of C. hongkongensis, the total antioxidant capacity (T-AOC), glycogen content, and enzyme activities (phosphofructokinase, PFK; pyruvate kinase, PK; phosphoenolpyruvate carboxykinase, PEPCK) of oysters were determined under normoxic (DO 6 ± 0.2 mg/L) and hypoxic (DO 1.5 mg/L) conditions at 0 h, 6 h, 48 h, and 72 h. We also determined the T-AOC, glycogen content, and enzyme activities of oysters under reoxygenation (recovered to normoxia for 24 h). To further examine the potential molecular regulatory mechanism of hypoxic adaptation, a transcriptome analysis was conducted on the gill of C. hongkongensis under normoxia (N, 72 h), hypoxia (H, 72 h), and reoxygenation (R). After being exposed to hypoxia for 6 h, the T-AOC, glycogen content, and enzyme activities of PK, PFK, and PEPCK in C. hongkongensis were significantly decreased. However, after prolonging the duration of hypoxia exposure for 72 h, the T-AOC, glycogen content, and enzyme activities increased compared to that of 48 h. After 24 h reoxygenation, the T-AOC, glycogen content, and enzyme activity of PK and PFK returned to close to initial levels. In addition, a transcriptome analysis discovered 6097 novel genes by mapping the C. hongkongensis genome with the clean reads. In total, 352 differentially expressed genes (DEGs) were identified in the H vs. N comparison group (235 upregulated and 117 downregulated genes). After recovery to normoxia, 292 DEGs (134 upregulated and 158 downregulated genes) were identified in the R vs. N comparison group, and 632 DEGs were identified (253 upregulated and 379 downregulated genes) in the R vs. H comparison group. The DEGs included some hypoxia-tolerant genes, such as phosphoenolpyruvate carboxykinase (PEPCK), mitochondrial (AOX), tyramine beta-hydroxylase (TBH), superoxide dismutase (SOD), glutathione S-transferase (GST), and egl nine homolog 1 isoform X2 (EGLN1). Additionally, DEGs were significantly enriched in the KEGG pathways that are involved in hypoxia tolerance, including the metabolism of xenobiotics by cytochrome P450 pathways and the HIF-1 signaling pathway. Then, we selected the five hypoxic-tolerant candidate DEGs for real-time quantitative polymerase chain reaction (RT-qPCR) validation, and the results were consistent with the transcriptome sequencing data. These discoveries have increased our understanding of hypoxia tolerance, recovery ability after reoxygenation, and molecular mechanisms governing the responses to hypoxia in C. hongkongensis.

Carpachromene Ameliorates Insulin Resistance in HepG2 Cells via Modulating IR/IRS1/PI3k/Akt/GSK3/FoxO1 Pathway.

Insulin resistance contributes to several disorders including type 2 diabetes and cardiovascular diseases. Carpachromene is a natural active compound that inhibits α-glucosidase enzyme. The aim of the present study is to investigate the potential activity of carpachromene on glucose consumption, metabolism and insulin signalling in a HepG2 cells insulin resistant model. A HepG2 insulin resistant cell model (HepG2/IRM) was established. Cell viability assay of HepG2/IRM cells was performed after carpachromene/metformin treatment. Glucose concentration and glycogen content were determined. Western blot analysis of insulin receptor, IRS1, IRS2, PI3k, Akt, GSK3, FoxO1 proteins after carpachromene treatment was performed. Phosphoenolpyruvate carboxykinase (PEPCK) and hexokinase (HK) enzymes activity was also estimated. Viability of HepG2/IRM cells was over 90% after carpachromene treatment at concentrations 6.3, 10, and 20 µg/mL. Treatment of HepG2/IRM cells with carpachromene decreased glucose concentration in a concentration- and time-dependant manner. In addition, carpachromene increased glycogen content of HepG2/IRM cells. Moreover, carpachromene treatment of HepG2/IRM cells significantly increased the expression of phosphorylated/total ratios of IR, IRS1, PI3K, Akt, GSK3, and FoxO1 proteins. Furthermore, PEPCK enzyme activity was significantly decreased, and HK enzyme activity was significantly increased after carpachromene treatment. The present study examined, for the first time, the potential antidiabetic activity of carpachromene on a biochemical and molecular basis. It increased the expression ratio of insulin receptor and IRS1 which further phosphorylated/activated PI3K/Akt pathway and phosphorylated/inhibited GSK3 and FoxO1 proteins. Our findings revealed that carpachromene showed central molecular regulation of glucose metabolism and insulin signalling via IR/IRS1/ PI3K/Akt/GSK3/FoxO1 pathway.

Sex and sex-related differences in gamete development progression impinge on biomarker responsiveness in sentinel mussels

In marine pollution monitoring, the biomarkers recorded in sentinel organisms are influenced by natural confounding factors that may jeopardise their interpretation. Among these confounding factors, little is known about the influence of sex along the annual reproductive cycle. The present investigation aims at contributing to understand how sex and sex-related differences in gamete development progression impinge on biomarker baseline values and on biomarker responsiveness to pollution in sentinel mussels. Mussels (Mytilus galloprovincialis) were collected from a relatively clean locality and from a chronically polluted site in the Basque Coast (Bay of Biscay) in January, April, August and November. Sex and gametogenesis stages were determined for each mussel. Tissue concentration of metals and PAHs was analysed. A battery of biomarkers was investigated: cytochrome c oxidase, pyruvate kinase and phosphoenolpyruvate carboxykinase enzyme activities; levels of protein carbonyls, malondialdehyde and 4-hydroxy-2-nonenal; lysosomal enlargement and membrane stability; intracellular neutral lipid accumulation; cell type composition and thinning of the digestive gland epithelium; and survival-in-air. Sex- and reproductive stage-related differences were found in bioaccumulation and in the values and responsiveness of most of the biomarkers. However, the patterns of sex-related differences were not consistent across all biomarkers. The differences in the biomarker responses between females and males also depended on the season, reflecting the progression of the gametogenesis cycle. Thus, selecting mussels of one specific sex does not seem to be a crucial requisite to carry out biomarker-based monitoring; yet, it is highly recommended to identify sex condition and gamete developmental stage of each mussel to test for the potentially confounding effects of sex, reproductive status and sex-related variability along the reproductive cycle.

Mulberry anthocyanin extract ameliorates insulin resistance by regulating PI3K/AKT pathway in HepG2 cells and db/db mice

This study evaluated the capacity of mulberry anthocyanin extract (MAE) on insulin resistance amelioration in HepG2 cells induced by high glucose and palmitic acid and diabetes-related metabolic changes in type 2 diabetic mice. In vitro, MAE alleviated insulin resistance in HepG2 cells and increased glucose consumption, glucose uptake and glycogen content. Enzyme activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were decreased due to PPARγ coactivator 1α (PGC-1α) and forkhead box protein O1 (FOXO1) inhibition. Furthermore, phosphorylation of protein kinase B (AKT) and glycogen synthase kinase-3β (GSK3β) in model cells was recovered after treated with MAE, leading to an up-regulation of glycogen synthase 2 (GYS2), and this effect was blocked by the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. In vivo, MAE supplementation (50 and 125 mg/kg body weight per day) markedly decreased fasting blood glucose, serum insulin, leptin, triglyceride and cholesterol levels and increased adiponectin levels in db/db mice. The improvement of related metabolic parameters was in part associated with the impact of MAE on activating AKT and downstream targets in liver, skeletal muscle and adipose tissues. In summary, these findings suggest that MAEs have potential benefits on improving dysfunction in diabetic mice and mitigating insulin resistance in HepG2 cells via activation of PI3K/AKT pathways.

Knockdown of phosphoenolpyruvate carboxykinase increases carbon flux to lipid synthesis in Phaeodactylum tricornutum

The tricarboxylic acid (TCA) cycle is a key metabolic pathway that unifies carbohydrate, lipid and protein metabolism. In Phaeodactylum tricornutum, accumulation of triacylglycerols (TAGs) under nitrogen stress is a consequence of re-allocation of carbon mainly from the intermediates of TCA cycle. Oxaloacetate, an intermediate of the TCA cycle, can flux out of the cycle by the decarboxylation enzyme phosphoenolpyruvate carboxykinase (PEPCK), thus decreasing the carbon flow directed to fatty acid biosynthesis. We assayed the expression of genes encoding mitochondrially-localized decarboxylation enzymes, essential for putative C4 metabolism, in P. tricornutum. Targeted knockdown of PEPCK was performed using RNA-interference and the photosynthetic characteristics and TAG contents of knockdown lines were characterized. PEPCK was significantly upregulated at both the mRNA and protein levels during TAG accumulation in P. tricornutum, and both PEPCK transcript abundance and enzyme activity were reduced significantly in two RNAi lines. The mutants exhibited lower photosynthetic activity, while TAG content was markedly increased. Meanwhile, cell density was comparable with that of wild-type. We propose that carbon from decarboxylation of C4 metabolites in the mitochondria plays a minor role in carbon concentration in this diatom. Downregulated decarboxylation succeeded in increasing lipid accumulation by decreasing the loss of carbon skeletons without compromising cell biomass.

Mulberry anthocyanin extract regulates glucose metabolism by promotion of glycogen synthesis and reduction of gluconeogenesis in human HepG2 cells.

Mulberry has been demonstrated to possess important biological activities such as antioxidation and antiinflammation. However, research on the ability of mulberry for diabetes improvement mainly focuses on the leaves and less on the fruit. This study showed that a mulberry anthocyanin extract (MAE) had a significant effect on increasing the glucose consumption in HepG2 cells. The MAE enhanced the glycogen content and suppressed levels of glucose production. The enzyme activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were decreased in HepG2 cells after MAE treatment due to PPARγ coactivator 1α (PGC-1α) and forkhead box protein O1 (FOXO1) inhibition. Moreover, the phosphorylation of protein kinase B (AKT) and glycogen synthase kinase-3β (GSK-3β) was increased by the MAE, leading to an expression enhancement of glycogen synthase 2 (GYS2). And this effect was blocked by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. In summary, our results suggested that the MAE regulates glucose metabolism by activating the PI3K/AKT pathway that relates to glycogen synthesis as well as through the inhibition of key molecules that promote gluconeogenesis.

Geniposide Suppresses Hepatic Glucose Production via AMPK in HepG2 Cells.

Geniposide is one of the main compounds in Gardenia jasminoides ELLIS and has many pharmacological activities, but its anti-hyperglycemic activity has not yet been fully explored. This study was designed to determine, for the first time, how geniposide from G. jasminoides regulates hepatic glucose production, and the underlying mechanisms. During in vitro study, we found the inhibitory effect of geniposide on the hepatic glucose production is partly through AMP-activated protein kinase (AMPK) activation in HepG2 cells. Geniposide significantly inhibited hepatic glucose production in a dose-dependent manner. AMPK, acetyl coenzyme A synthetase (ACC) and forkhead box class O1 (FoxO1) phosphorylation were stimulated by different concentrations of geniposide. In addition, the enzyme activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were all significantly suppressed. What is important is that these effects were partly reversed by (1) inhibition of AMPK activity by compound C, a selective AMPK inhibitor, and by (2) suppression of AMPKα expression by small interfering RNA (siRNA). In summary, geniposide potentially ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis by modulation of the AMPK-FoxO1 signaling pathway. Geniposide or geniposide-containing medicinal plants could represent a promising therapeutic agent to prevent type 2 diabetes on gluconeogenesis.

Effects of heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase on organic acid production in Aspergillus carbonarius.

Aspergillus carbonarius has a potential as a cell factory for production of various organic acids. In this study, the organic acid profile of A. carbonarius was investigated under different cultivation conditions. Moreover, two heterologous genes, pepck and ppc, which encode phosphoenolpyruvate carboxykinase in Actinobacillus succinogenes and phosphoenolpyruvate carboxylase in Escherichia coli, were inserted individually and in combination in A. carbonarius to enhance the carbon flux toward the reductive TCA branch. Results of transcription analysis and measurement of enzyme activities of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase in the corresponding single and double transformants demonstrated that the two heterologous genes were successfully expressed in A. carbonarius. The production of citric acid increased in all the transformants in both glucose- and xylose-based media at pH higher than 3 but did not increase in the pH non-buffered cultivation compared with the wild type.Electronic supplementary materialThe online version of this article (doi:10.1007/s10295-015-1688-4) contains supplementary material, which is available to authorized users.

Effect of leptin on the gluconeogenesis in calf hepatocytes cultured in vitro

We have investigated the effect of leptin on gluconeogenesis in the liver. Hepatocytes were cultured and treated with 0, 2.5, 5, 10, 50, 100 ng/mL of leptin in groups I, II, III, IV, V, and VI, respectively. mRNA expression and enzyme activity of pyruvate carboxylase and phosphoenolpyruvate carboxykinase were determined by real-time fluorescence quantitative RT-PCR and biochemical kits, respectively. Compared with group I, mRNA expression of pyruvate carboxylase and phosphoenolpyruvate carboxykinase in groups III, IV, V, and VI were significantly lower (P < 0.01). Pyruvate carboxylase and phosphoenolpyruvate carboxykinase enzyme activity decreased significantly (P < 0.05) when leptin concentration exceeded 5 and 10 ng/mL, respectively. These results indicate that leptin markedly downregulated mRNA expression and enzyme activity of pyruvate carboxylase and phosphoenolpyruvate carboxykinase in hepatocytes, which suggests that high concentrations of LP inhibit hepatocyte gluconeogenesis, thus making negative energy balance more serious.

The effect of in vivo PBDE treatment on hepatic phosphoenolpyruvate carboxykinase (PEPCK) enzyme kinetics in male wistar rats

Xenobiotics such as phenobarbital, TCDD and PCB significantly suppress on the activity of a key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), suggesting that xenobiotics could disrupt glucose metabolism. The effect of polybrominated diphenyl ethers (PBDEs), a family of synthetic flame‐retardant chemicals, on PEPCK is unknown. This study investigated the effect of PBDE treatment on PEPCK. Forty‐eight, one month‐old, male Wistar rats were gavaged daily with corn oil or corn oil containing 14 mg/kg DE‐71 for 3, 14 or 28 days (N = 8 per group). At each time point, fasting plasma glucose, insulin and C‐peptide were measured and hepatic PEPCK and CYP enzyme activities were assayed. PBDE treatment significantly decreased PEPCK Vmax (μmol/min/g liver weight) by 22% (3 days) to 44% (28 days). CYP1A, −2B and −3A Vmax were increased by 6‐, 6‐ and 30‐fold, respectively, in treated rats compared to control. There was a significant inverse and temporal correlation (r = −0.74) between CYP3A and PEPCK Vmax for the treatment group. Fasting plasma glucose, insulin and C‐pep levels were unaffected by treatment, but the glucose:insulin ratio tended (P=0.07) to be higher in treated compared to control rats. These data suggest that 3 days of in vivo PBDE treatment is sufficient to compromise liver glucose metabolism and 28 days of treatment may influence insulin sensitivity. Supported by NH Agr. Exp. Stn. grant #NH00541.

Effect of insulin-like growth factor-1 (IGF-1) on the gluconeogenesis in calf hepatocytes cultured in vitro

The major role of insulin-like growth factor-1 (IGF-1) in the liver is to mediate glucose uptake in hepatocytes to synthesize glycogen and maintain blood glucose homeostasis. In this study, to evaluate the role of IGF-1 on gluconeogenesis and nutrient metabolism in dairy cattle, pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK) expression and enzyme activity were evaluated in primary cultures of bovine hepatocytes treated with different concentrations of IGF-1 by quantitative polymerase chain reaction and spectrophotometry, respectively. The results showed that expression of PC and PEPCK were significantly lower in bovine hepatocytes by IGF-1 treatment in test group compare to the control group (P < 0.01). As IGF-1 concentration increased, PC and PEPCK enzyme activity in bovine hepatocytes decreased. Evaluating PC and PEPCK mRNA levels and enzyme activity may thus be useful to monitor subclinical ketosis in dairy cows.

An Afferent Vagal Nerve Pathway Links Hepatic PPARα Activation to Glucocorticoid-Induced Insulin Resistance and Hypertension

Glucocorticoid excess causes insulin resistance and hypertension. Hepatic expression of PPARalpha (Ppara) is required for glucocorticoid-induced insulin resistance. Here we demonstrate that afferent fibers of the vagus nerve interface with hepatic Ppara expression to disrupt blood pressure and glucose homeostasis in response to glucocorticoids. Selective hepatic vagotomy decreased hyperglycemia, hyperinsulinemia, hepatic insulin resistance, Ppara expression, and phosphoenolpyruvate carboxykinase (PEPCK) enzyme activity in dexamethasone-treated Ppara(+/+) mice. Selective vagotomy also decreased blood pressure, adrenergic tone, renin activity, and urinary sodium retention in these mice. Hepatic reconstitution of Ppara in nondiabetic, normotensive dexamethasone-treated PPARalpha null mice increased glucose, insulin, hepatic PEPCK enzyme activity, blood pressure, and renin activity in sham-operated animals but not hepatic-vagotomized animals. Disruption of vagal afferent fibers by chemical or surgical means prevented glucocorticoid-induced metabolic derangements. We conclude that a dynamic interaction between hepatic Ppara expression and a vagal afferent pathway is essential for glucocorticoid induction of diabetes and hypertension.

Effects of Short-Term Glucagon Administration on Gluconeogenic Enzymes in the Liver of Midlactation Dairy Cows

During lactation, the dairy cow experiences an increased demand for glucose to support milk production. Increased glucose demand can be met through increased capacity for gluconeogenesis, increased supply of glucose precursors, or a combination of both processes. Glucagon, a key hormone in glucose homeostasis, acts to promote gluconeogenesis and increase glucose output from liver. The objective of this study was to determine the effect of short-term administration of glucagon on expression of gluconeogenic enzymes in lactating dairy cattle. Sixteen multiparous Holstein cows were selected from the Purdue University Animal Sciences Dairy Research Center herd. Cows were stratified on the basis of milk production and days in milk and randomly assigned to either a saline or glucagon injection group (n=8 per group). Cows were injected subcutaneously at −21, −14, −7, and 0h relative to final glucagon and saline injections with either 3.75mg of lyophilized bovine glucagon (15 mg/d) dissolved in 60mL of 0.15 M NaCl (pH 10.25) or 60mL of 0.15 M NaCl. Liver biopsy samples were obtained 1 wk before injection to establish baseline values and at 3h after cows received final glucagon and saline injections. Biopsy samples were analyzed for mRNA abundance, enzyme activity, protein abundance, and in vitro measures of gluconeogenesis. Glucagon did not alter pyruvate carboxylase or cytosolic phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance, enzyme activity, or protein abundance, although there was a tendency for greater mRNA expression with the glucagon treatment (4.69 vs. 6.78, arbitrary units). Glucagon injections did not change mitochondrial PEPCK mRNA expression. Gluconeogenesis from 2.5mM [2-14C]propionate and 2.0mM [U-14C]lactate was similar in liver biopsy samples from glucagon-treated and control cows. There was no effect of glucagon on dry matter intake and milk production. Glucose, nonesterified fatty acids, β-hydroxybutyrate acid, and insulin were not altered by glucagon. Blood glucagon was elevated, 76.09 vs. 96.14 pg/mL, for cows receiving glucagon injections. The data indicate that 24-h administration of glucagon does not alter cytosolic PEPCK mRNA expression or result in immediate alterations in total PEPCK enzyme activity and gluconeogenic capacity.

Adult rats prenatally exposed to ethanol have increased gluconeogenesis and impaired insulin response of hepatic gluconeogenic genes

Rat offspring exposed to ethanol (EtOH rats) during pregnancy are insulin resistant, but it is unknown whether they have increased gluconeogenesis. To address this issue, we determined blood glucose and liver gluconeogenic genes, proteins, and enzyme activities before and after insulin administration in juvenile and adult EtOH rats and submitted adult EtOH rats to a pyruvate challenge. In juvenile rats, basal glucose; peroxisome proliferator-activated receptor-coactivator-1alpha protein and mRNA; and phosphoenolpyruvate carboxykinase enzyme activity, protein, and mRNA were similar between groups. After insulin injection, these parameters failed to decrease in EtOH rats, but glucose decreased by 30% and gluconeogenic enzymes, proteins, and mRNAs decreased by 50-70% in control rats. In adult offspring, basal peroxisome proliferator-activated receptor-coactivator-1alpha protein and mRNA levels were 40-80% higher in EtOH rats than in controls. Similarly, basal phosphoenolpyruvate carboxykinase activity, protein, and mRNA were approximately 1.8-fold greater in EtOH rats than in controls. These parameters decreased by approximately 50% after insulin injection in control rats, but they remained unchanged in EtOH rats. After insulin injection in the adult rats, glucose decreased by 60% in controls but did not decrease significantly in EtOH rats. A subset of adult EtOH rats had fasting hyperglycemia and an exaggerated glycemic response to pyruvate compared with controls. The data indicate that, after prenatal EtOH exposure, the expression of gluconeogenic genes is exaggerated in adult rat offspring and is insulin resistant in both juvenile and adult rats, explaining increased gluconeogenesis. These alterations persist through adulthood and may contribute to the pathogenesis of Type 2 diabetes after exposure to EtOH in utero.

Receptor/gene-mediated pharmacodynamic effects of methylprednisolone on phosphoenolpyruvate carboxykinase regulation in rat liver.

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting enzyme for gluconeogenesis. To investigate underlying mechanisms of corticosteroid (CS) action in regulating glucose, temporal patterns of hepatic PEPCK gene expression, enzyme activity, and cAMP content were examined in adrenalectomized rats receiving acute and chronic methylprednisolone (MPL) treatments. After single MPL intravenous doses, PEPCK mRNA showed a fast increase, reaching a maximum at around 0.75 h, which was followed by an immediate decline to below baseline after 4 h, an apparent acute tolerance/rebound phenomenon. However, PEPCK enzyme showed continuous hyperactivity for over 72 h. This may be the result of generation of cAMP, an important inducer of PEPCK activity, which peaked at around 6 h. During 7-day subcutaneous infusion of MPL, PEPCK mRNA showed profiles consistent with single-dose results, whereas PEPCK activity increased to a comparable maximum followed by a slow decline. However, the extent of cAMP induction was markedly higher during infusion, which could be attributed to amplification of cAMP synthesis and/or a stabilizing effect of MPL on cAMP degradation. A pharmacokinetic/pharmacodynamic model was developed based on receptor/gene mechanisms of CS action. It successfully described the dual effects of MPL on regulating PEPCK message and the post-transcriptional control by cAMP. Our results exemplify the importance of the extent and duration of steroid exposure in mediating pharmacological effects. The model provides quantitation of multiple controlling factors regulating PEPCK and presents insights into its function in glucose metabolism.

Inhibition by recombinant human interleukin-6 of the glucagon-dependent induction of phosphoenolpyruvate carboxykinase and of the insulin-dependent induction of glucokinase gene expression in cultured rat hepatocytes: regulation of gene transcription and messenger RNA degradation.

The influence of recombinant human interleukin-6, the major mediator of the inflammatory response in liver, on the glucagon- and insulin-dependent induction of the phosphoenolpyruvate carboxykinase and glucokinase gene, respectively, was monitored on the level of gene transcription, mRNA abundance and enzyme activity in cultured rat hepatocytes. As control markers of the interleukin-6-induced acute-phase response the mRNA levels of the acute phase proteins alpha 2-macroglobulin and beta-fibrinogen were determined. In cultured rat hepatocytes, recombinant human interleukin-6, added simultaneously with glucagon and insulin, lowered the maximal increase in glucagon-induced phosphoenolpyruvate carboxykinase mRNA levels after 2 hr and the maximal increase in glucokinase mRNA levels after 3 hr to about 30%, respectively. It inhibited the glucagon-induced increase in phosphoenolpyruvate carboxykinase gene transcription and phosphoenolpyruvate carboxykinase enzyme activity, as well as the insulin-induced increases in glucokinase gene transcription and glucokinase enzyme activity. Recombinant human interleukin-6 increased the mRNA levels of the acute-phase proteins alpha 2-macroglobulin and beta-fibrinogen gradually over 4 to 6 hr. Recombinant human interleukin-6, added 2 hr after glucagon or 3 hr after insulin at the maximum of the hormone-induced enzyme mRNA levels, almost doubled the decay rate of phosphoenolpyruvate carboxykinase mRNA and glucokinase mRNA. The results show that interleukin-6 induced the expression of inflammatory proteins and simultaneously inhibited the hormone-induced expression of enzymes of intermediary metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of prolonged hyperglycemia on metabolic alterations in the subtotally pancreatectomized rat.

A delayed onset of diabetes is characteristic of subtotally pancreatectomized patients in whom persistent hyperglycemia per se is documented to lead to the development of insulin resistance. This study was conducted to elucidate the metabolic alterations seen during transition of the acute to chronic phase after subtotal pancreatectomy (SP). Eight male Sprague-Dawley rats were studied 2 weeks after surgery in the acute phase, and the other eight at 4 weeks in the chronic phase. Phosphoenolpyruvate carboxykinase (PEPck) for gluconeogenesis and the malic enzyme for de novo fatty acid synthesis in the liver showed a reciprocal change, the former activity being increased, while the latter was suppressed. Both alterations were more pronounced in the chronic phase. In the acute phase, lipoprotein lipase (LPL) for triglyceride clearance decreased in the adipose tissue, while that in the cardiac and skeletal muscle became significantly elevated. The latter elevations were decreased in the chronic phase. Sustained hyperglycemia in the SP rats not only increased the changes in PEPck and malic enzyme activities but reversed the tissue-specific muscle LPL elevations. These changes might help to explain the wasting condition seen in surgically induced diabetic patients.

Impaired suppression of gluconeogenesis induced by overexpression of a noninsulin-responsive phosphoenolpyruvate carboxykinase gene

Despite detailed knowledge of the regulation of individual steps in the gluconeogenic pathway, the relative importance of each step to the overall control of gluconeogenesis by insulin is not known. The aim of this study was to determine the role of phosphoenolpyruvate carboxykinase (PEPCK) in the regulation of gluconeogenesis by insulin. Clones of the rat hepatoma cell line H4IIE-C3 were produced, overexpressing a PEPCK gene, driven by a promoter not responsive to insulin. In these cells basal gluconeogenesis from 2-[14C]pyruvate was increased 2.1-fold compared to controls (4.63 +/- 0.49 nmol/10(5) cells vs. 2.21 +/- 0.24 nmol/10(5) cells after 3 h, P < 0.05, n = 5). Increased gluconeogenesis was associated with an increase in basal PEPCK mRNA levels (1.9-fold) and enzyme activity (2.8-fold). Insulin (10(-7) M) suppressed basal gluconeogenesis, PEPCK mRNA levels, and enzyme activity in control cells, but no detectable decrease was observed in PEPCK-transfected cells. These experiments provide direct evidence in intact cells that PEPCK is the rate-limiting enzyme in gluconeogenesis from pyruvate and show that insulin's action to inhibit gluconeogenesis is predominantly on the inhibition of PEPCK transcription.

Impaired suppression of gluconeogenesis induced by overexpression of a noninsulin-responsive phosphoenolpyruvate carboxykinase gene.

Despite detailed knowledge of the regulation of individual steps in the gluconeogenic pathway, the relative importance of each step to the overall control of gluconeogenesis by insulin is not known. The aim of this study was to determine the role of phosphoenolpyruvate carboxykinase (PEPCK) in the regulation of gluconeogenesis by insulin. Clones of the rat hepatoma cell line H4IIE-C3 were produced, overexpressing a PEPCK gene, driven by a promoter not responsive to insulin. In these cells basal gluconeogenesis from 2-[14C]pyruvate was increased 2.1-fold compared to controls (4.63 +/- 0.49 nmol/10(5) cells vs. 2.21 +/- 0.24 nmol/10(5) cells after 3 h, P < 0.05, n = 5). Increased gluconeogenesis was associated with an increase in basal PEPCK mRNA levels (1.9-fold) and enzyme activity (2.8-fold). Insulin (10(-7) M) suppressed basal gluconeogenesis, PEPCK mRNA levels, and enzyme activity in control cells, but no detectable decrease was observed in PEPCK-transfected cells. These experiments provide direct evidence in intact cells that PEPCK is the rate-limiting enzyme in gluconeogenesis from pyruvate and show that insulin's action to inhibit gluconeogenesis is predominantly on the inhibition of PEPCK transcription.

Identification of tumor necrosis factor as a transcriptional regulator of the phosphoenolpyruvate carboxykinase gene following endotoxin treatment of mice.

The decreased synthesis of hepatic phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme of gluconeogenesis, that occurs during endotoxemia was shown previously in rats to occur at the transcriptional level. In the current study, the exogenous administration of human recombinant tumor necrosis factor (TNF), a proximal mediator of endotoxic shock, reduced the PEPCK transcription rate, mRNAPEPCK levels, and PEPCK enzyme activity in a time- and dose-dependent manner in CD-1 mice. Comparable amounts of circulating TNF were measured in mice 2 h after injection of human recombinant TNF (10(5) U) or a 50% lethal dose of Escherichia coli endotoxin (20 mg/kg). Direct action of TNF to decrease the PEPCK transcription rate was confirmed in vitro with H-4-II-E Reuber hepatoma cells, in which a dose-dependent inhibition of PEPCK transcription was observed with 1 to 100 U of TNF per ml. A role for TNF-elicited changes in PEPCK gene expression during endotoxemia was confirmed by the protective effect of rabbit polyclonal antibodies to recombinant murine TNF. C57BL/6 mice passively immunized with anti-TNF 4 h prior to endotoxin challenge exhibited normal PEPCK enzyme activity. Neutralization of circulating TNF with anti-TNF failed, however, to prevent the hypoglycemia commonly observed during endotoxemia, suggesting the participation of other mediators. Anti-TNF treatment reduced circulating interleukins 1 and 6 at 3 and 6 h after endotoxin treatment, respectively. These results suggest that during endotoxemia, the development of hypoglycemia is multifaceted and that several cytokines are most likely involved. The findings from the Reuber hepatoma cell model afford an opportunity in future work to map putative cytokine response elements in the PEPCK promoter responsible for perturbed hormonal regulation of the gene during endotoxemia.