PCK1 Research Articles

Gene–nutrient interactions on the phosphoenolpyruvate carboxykinase influence insulin sensitivity in metabolic syndrome subjects

Genetic background may interact with habitual dietary fat composition, and affect development of the metabolic syndrome (MetS). The phosphoenolpyruvate carboxykinase gene (PCK1) plays a significant role regulating glucose metabolism, and fatty acids are key metabolic regulators, which interact with transcription factors and influence glucose metabolism. We explored genetic variability at the PCK1 gene locus in relation to degree of insulin resistance and plasma fatty acid levels in MetS subjects. Moreover, we analyzed the PCK1 gene expression in the adipose tissue of a subgroup of MetS subjects according to the PCK1 genetic variants. Insulin sensitivity, insulin secretion, glucose effectiveness, plasma concentrations of C-peptide, fatty acid composition and three PCK1 tag-single nucleotide polymorphisms (SNPs) were determined in 443 MetS participants in the LIPGENE cohort. The rs2179706 SNP interacted with plasma concentration of n - 3 polyunsaturated fatty acids (n - 3 PUFA), which were significantly associated with plasma concentrations of fasting insulin, peptide C, and HOMA-IR. Among subjects with n - 3 PUFA levels above the population median, carriers of the C/C genotype exhibited lower plasma concentrations of fasting insulin (P = 0.036) and HOMA-IR (P = 0.019) as compared with C/C carriers with n - 3 PUFA below the median. Moreover, homozygous C/C subjects with n - 3 PUFA levels above the median showed lower plasma concentrations of peptide C as compared to individuals with the T-allele (P = 0.006). Subjects carrying the T-allele showed a lower gene PCK1 expression as compared with carriers of the C/C genotype (P = 0.015). The PCK1 rs2179706 polymorphism interacts with plasma concentration of n - 3 PUFA levels modulating insulin resistance in MetS subjects.

Genome-wide location analysis reveals an important overlap between the targets of the yeast transcriptional regulators Rds2 and Adr1

Upon glucose depletion, a massive reprogramming of gene expression occurs in the yeast Saccharomyces cerevisiae for the use of alternate carbon sources such as the nonfermentable compounds ethanol and glycerol. This process is mediated by the master kinase Snf1 that controls the activity of various targets including the transcriptional regulators Cat8, Sip4 and Adr1. We have recently identified Rds2 as an additional player in this pathway. Here, we have performed genome-wide location analysis of Rds2 in cells grown in the presence of glycerol. We show that Rds2 binds to promoters of genes involved in gluconeogenesis, the glyoxylate shunt, and the TCA cycle as well as some genes encoding mitochondrial components or some involved in the stress response. Interestingly, we also detected Rds2 at the promoters of SIP4, ADR1 and HAP4 which encodes the limiting subunit of the Hap2/3/4/5 complex, a regulator of respiration. Strikingly, we observed an important overlap between the targets of Rds2 and Adr1. Finally, we provide a model to account for the complex interplay among these transcriptional regulators.

Orphan Nuclear Receptor Estrogen-Related Receptor γ (ERRγ) Is Key Regulator of Hepatic Gluconeogenesis

Glucose homeostasis is tightly controlled by hormonal regulation of hepatic glucose production. Dysregulation of this system is often associated with insulin resistance and diabetes, resulting in hyperglycemia in mammals. Here, we show that the orphan nuclear receptor estrogen-related receptor γ (ERRγ) is a novel downstream mediator of glucagon action in hepatic gluconeogenesis and demonstrate a beneficial impact of the inverse agonist GSK5182. Hepatic ERRγ expression was increased by fasting-dependent activation of the cAMP-response element-binding protein-CRTC2 pathway. Overexpression of ERRγ induced Pck1 and G6PC gene expression and glucose production in primary hepatocytes, whereas abolition of ERRγ gene expression attenuated forskolin-mediated induction of gluconeogenic gene expression. Deletion and mutation analyses of the Pck1 promoter showed that ERRγ directly regulates the Pck1 gene transcription via ERR response elements of the Pck1 promoter as confirmed by ChIP assay and in vivo imaging analysis. We also demonstrate that GSK5182, an inverse agonist of ERRγ, specifically inhibits the transcriptional activity of ERRγ in a PGC-1α dependent manner. Finally, the ERRγ inverse agonist ameliorated hyperglycemia through inhibition of hepatic gluconeogenesis in db/db mice. Control of hepatic glucose production by an ERRγ-specific inverse agonist is a new potential therapeutic approach for the treatment of type 2 diabetes.

Modulation of Mouse Coagulation Gene Transcription following Acute In Vivo Delivery of Synthetic Small Interfering RNAs Targeting HNF4α and C/EBPα

Hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer-binding protein α (C/EBPα) are important for the transcriptional control of coagulation factors. To determine in vivo the direct role of HNF4α and C/EBPα in control of genes encoding coagulation factors, a synthetic small interfering (si)RNA approach was used that enabled strong reduction of mouse hepatic HNF4α and C/EBPα under conditions that minimized target-related secondary effects. For both HNF4α and C/EBPα, intravenous injection of specific synthetic siRNAs (siHNF4α and siC/EBPα) resulted in more than 75% reduction in their liver transcript and protein levels 2 days post-injection. For siHNF4α, this coincided with marked and significantly reduced transcript levels of the coagulation genes Hrg, Proz, Serpina5, F11, F12, F13b, Serpinf2, F5, and F9 (in order of magnitude of effect) as compared to levels in control siRNA injected animals. Significant decreases in HNF4α target gene mRNA levels were also observed at 5 days post-siRNA injection, despite a limited level of HNF4α knockdown at this time point. Compared to HNF4α, C/EBPα knockdown had a modest impact on genes encoding coagulation factors. A strong reduction in C/EBPα transcript and protein levels resulted in significantly affected transcript levels of the control genes Pck1 and Fasn and a modest downregulation for coagulation genes Fba, Fbg and F5. F5 and F11 were the sole coagulation genes that were significantly affected upon prolonged (5 day) C/EBPα knockdown. We conclude that in the mouse, HNF4α has a direct and essential regulatory role for multiple hepatic coagulation genes, while a role for C/EBPα is more restricted. In addition, this study demonstrates that synthetic siRNA provides a simple and fast means for determining liver transcription factor involvement in vivo.

Fatty acid elongase‐5 (Elovl5) regulates the mTORC2‐FoxO1 pathway in obese‐diabetic C57BL/6J mice

Elevated fatty acid elongase‐5 (Elovl5) activity in livers of high fat diet induced obese‐ diabetic C57BL/6J mice corrects fasting hyperglycemia by suppressing gluconeogenesis (GNG) through the regulation of Akt2 and FoxO1 phosphorylation status. This report examines Elovl5 regulated signaling mechanisms controlling Akt2 & FoxO1 phosphorylation. Elevated hepatic Elovl5 activity in obese‐diabetic mice and in HepG2 cells increased Akt2‐Serine473 (an mTORC2 site) phosphorylation. Increased Akt2‐Serine473 correlates with increased FoxO1 phosphorylation and suppressed Pck1 gene expression. In HepG2 cells, Akt inhibitor (Akt1/2i) blocked Elovl5 mediated FoxO1 phosphorylation while the PP2A inhibitor (mycrocystin‐LR) augmented FoxO1 phosphorylation. The mTORC1 and mTORC2 inhibitor (PP242), but not the mTORC1 inhibitor (rapamycin), blocked Elovl5 regulated Akt2‐Serine473 and FoxO1 phosphorylation. In vivo, high fat diets suppressed raptor (a mTORC1 component), but induced rictor (a mTORC2 component). Induction of hepatic Elovl5 activity augmented rictor protein abundance ~2‐fold and stimulated rictor‐mTor interaction. These studies establish rictor as a target for Elovl5 mediated suppression of GNG by regulating Akt2‐Serine473 and FoxO1 phosphorylation.Research support: NIH (DK43220) and NIFA/USDA (2009‐65200‐05846).

Expression of peroxisome proliferator activated receptor gamma 2 and its target genes in visceral fat tissue of growth restricted offspring in rats

Objective To investigate the expression of transcription factor peroxisome proliferator activated receptor gamma 2 （PPAR2） and its target genes--adipose differentiation-related protein （ADFP） and phosphoenolpyruvate carboxykinasel （PCK1） in visceral fat of growth restricted rat offspring, in order to explore their effects on the incidence of obesity and metabolism syndrome. Methods Fetal growth restriction （FGR） rat model was established by maternal low-protein diet and only male offsprings were studied. The mRNA expressions of PPAR2, ADFP and PCK1 in perirenalfat tissues were measured at 3 week （W3） and 8 week （W8）-old rats by real time quantitative reverse transcription-polymerase chain reaction （real-time-RT-PCR）. PPAR）2, ADFP and PCK1 protein levels in the perrenal fat tissues were determined by Western blot. Statistical analysis was performed with t-test and Spearman linear correlation analysis. Results The PPAR2 mRNA level in perrenal fat tissue of FGR rats at W3 and W8 were significantly elevated than that of the controls （W3：2.43 0.38 vs 1.034-O. 11,t=2.74,PO. O5;W8：2.170.34 vs 1.074-O. 14,t=2.49,PO. 05）, and PPAR/2 protein content was also increased （W3：l. 070.17 vs 0.41±0.06,t=3.01,P0.05;W8： 1.29±0.20 vs0.68--0.09,t=2.62,P0.05）, which equal to （261±31） and （19024） % of control, respectively. Increased mRNA and protein levels of the target genes ADFP and PCK1 in perrenal fat tissue were also found in the FGR rats at W3 and W8 compared with the same aged control rats （ADFP mRNA at W3..1. 874-0.29 vs 1.02=0. 12,t=2.20;ADFP mRNA at W8：1.64 0.31 vs 1.064-0.14,t=3.47; PCK1 mRNA at W3：2.030.26 vs 0.980.08,t=2.97;PCK1 mRNA at W8：l. 79±0.28 vs 1. 034-0.11,t=3.24; ADFP protein at W3 ： 0. 43 = 0. 06 vs 0. 14= 0. 03 , t= 3. 01; ADFP protein at W8：0.71=0.09 vs 0.38＋0.05,t=3.06;PCK1 protein at W3： 0.820. 10 vs 0. 394-0.05,t=2.65;PCK1 protein at WS： 0.85＋0.11 vs 0. 674-0.07,t=2.86; all P%0.05）. The protein and mRNA expressions of ADFP and PCK1 were positively correlated with expression the levels ofPPAR72 （mRNA：r 0.907 and 0.826,both P%0.01;protein：r=0.763 and 0. 805,both P0.05）. Conclusions Intrauterine programming＂ increases the expression of PPAR2 in perrenal adipose tissue of FGR individuals, which might contribute to the increased expressions of its target genes ADFP and PCK1, thereby causes the accumulation of adipose cells and subsequently promotes obesity and metabolism syndrome in adulthood. Key words: Fetal growth retardation; Adipose tissue; PPAR gamma; Membrane proteins; Phosphoenolpyruvate carboxykinase （GTP）; Intracellular signaling peptides andproteins

Genes implicated in insulin resistance are down-regulated in primary aldosteronism patients

Primary aldosteronism (PA) patients display an increased incidence of insulin resistance. Herein we demonstrate the decreased gene expression of lipid metabolism genes PCK1, PLIN, ADIPOQ and PPARG in the visceral adipose tissue (VAT) of PA patients compared to age-, sex- and BMI-matched controls. In VAT, the expression of PCK1, PLIN, ADIPOQ and PPARG was inversely correlated with aldosterone levels; furthermore, PLIN and ADIPOQ gene expression was correlated with potassium levels. Therefore, raised aldosterone and low potassium may contribute to the reduced expression of these genes in PA patients. Finally, incubation of primary cultures of human adipocytes with aldosterone resulted in a decrease in the expression of PCK1, PLIN and ADIPOQ and this effect was blocked by eplerenone. Therefore, the characteristic aldosterone excess of PA patients may mediate the down-regulation of PCK1, PLIN and ADIPOQ in VAT that in turn may contribute to the insulin resistance observed in PA patients.

Expression and manipulation of PHOSPHOENOLPYRUVATE CARBOXYKINASE 1 identifies a role for malate metabolism in stomatal closure

Malate, along with potassium and chloride ions, is an important solute for maintaining turgor pressure during stomatal opening. Although malate is exported from guard cells during stomatal closure, there is controversy as to whether malate is also metabolised. We provide evidence that phosphoenolpyruvate carboxykinase (PEPCK), an enzyme involved in malate metabolism and gluconeogenesis, is necessary for full stomatal closure in the dark. Analysis of the Arabidopsis PCK1 gene promoter indicated that this PEPCK isoform is specifically expressed in guard cells and trichomes of the leaf. Spatially distinct promoter elements were found to be required for post-germinative, vascular expression and guard cell/trichome expression of PCK1. We show that pck1 mutant plants have reduced drought tolerance, and show increased stomatal conductance and wider stomatal apertures compared with the wild type. During light-dark transients the PEPCK mutant plants show both increased overall stomatal conductance and less responsiveness of the stomata to darkness than the wild type, indicating that stomata get 'jammed' in the open position. These results show that malate metabolism is important during dark-induced stomatal closure and that PEPCK is involved in this process.

Control of Blood Glucose in the Absence of Hepatic Glucose Production During Prolonged Fasting in Mice

OBJECTIVESince the pioneering work of Claude Bernard, the scientific community has considered the liver to be the major source of endogenous glucose production in all postabsorptive situations. Nevertheless, the kidneys and intestine can also produce glucose in blood, particularly during fasting and under protein feeding. The aim of this study was to better define the importance of the three gluconeogenic organs in glucose homeostasis.RESEARCH DESIGN AND METHODSWe investigated blood glucose regulation during fasting in a mouse model of inducible liver-specific deletion of the glucose-6-phosphatase gene (L-G6pc−/− mice), encoding a mandatory enzyme for glucose production. Furthermore, we characterized molecular mechanisms underlying expression changes of gluconeogenic genes (G6pc, Pck1, and glutaminase) in both the kidneys and intestine.RESULTSWe show that the absence of hepatic glucose release had no major effect on the control of fasting plasma glucose concentration. Instead, compensatory induction of gluconeogenesis occurred in the kidneys and intestine, driven by glucagon, glucocorticoids, and acidosis. Moreover, the extrahepatic action of glucagon took place in wild-type mice.CONCLUSIONSOur study provides a definitive quantitative estimate of the capacity of extrahepatic gluconeogenesis to sustain fasting endogenous glucose production under the control of glucagon, regardless of the contribution of the liver. Thus, the current dogma relating to the respective role of the liver and of extrahepatic gluconeogenic organs in glucose homeostasis requires re-examination.

Chromatin occupancy of transcription factor 7-like 2 (TCF7L2) and its role in hepatic glucose metabolism

The mechanisms by which transcription factor 7-like 2 (TCF7L2) regulates the pathways that are important in the pathogenesis of type 2 diabetes are unknown. We therefore examined the role of TCF7L2 in hepatic glucose production (HGP) in vitro and characterised the whole-genome chromatin occupancy of TCF7L2 in hepatocytes. We investigated the effect of TCF7L2 silencing and overexpression on HGP from gluconeogenic precursors and used chromatin-immunoprecipitation (ChIP) combined with massively parallel DNA sequencing (ChIP-Seq) to investigate the DNA binding patterns of TCF7L2 across the whole genome. Silencing of TCF7L2 induced a marked increase in basal HGP, which was accompanied by significant increases in the expression of the gluconeogenic genes Fbp1, Pck1 and G6pc. Overexpression of Tcf7l2 reversed this phenotype and significantly reduced HGP. TCF7L2 silencing did not affect the half-maximal inhibitory concentration of insulin or metformin, but HGP remained elevated in TCF7L2-silenced cells due to the increased baseline HGP. Using ChIP-Seq, we detected 2,119 binding events across the genome. Pathway analysis demonstrated that diabetes genes were significantly over-represented in the dataset. Our results indicate that TCF7L2 binds directly to multiple genes that are important in regulation of glucose metabolism in the liver, including Pck1, Fbp1, Irs1, Irs2, Akt2, Adipor1, Pdk4 and Cpt1a. TCF7L2 is an important regulator of HGP in vitro and binds directly to genes that are important in pathways of glucose metabolism in the liver. These data highlight the possibility that TCF7L2 may affect fasting and postprandial hyperglycaemia in carriers of at-risk TCF7L2 genetic polymorphisms.

Disrupting the CH1 Domain Structure in the Acetyltransferases CBP and p300 Results in Lean Mice with Increased Metabolic Control

Opposing activities of acetyltransferases and deacetylases help regulate energy balance. Mice heterozygous for the acetyltransferase CREB binding protein (CBP) are lean and insulin sensitized, but how CBP regulates energy homeostasis is unclear. In one model, the main CBP interaction with the glucagon-responsive factor CREB is not limiting for liver gluconeogenesis, whereas a second model posits that Ser436 in the CH1 (TAZ1) domain of CBP is required for insulin and the antidiabetic drug metformin to inhibit CREB-mediated liver gluconeogenesis. Here we show that conditional knockout of CBP in liver does not decrease fasting blood glucose or gluconeogenic gene expression, consistent with the first model. However, mice in which the CBP CH1 domain structure is disrupted by deleting residues 342-393(ΔCH1) are lean and insulin sensitized, as are p300ΔCH1 mutants. CBP(ΔCH1/ΔCH1) mice remain metformin responsive. An intact CH1 domain is thus necessary for normal energy storage, but not for the blood glucose-lowering actions of insulin and metformin.

Insulin-Regulated Srebp-1c and Pck1 mRNA Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions

Insulin regulates the transcription of genes for hepatic glucose and lipid metabolism. We hypothesized that this action may be impaired in hepatocytes from insulin resistant animals. Primary hepatocytes from insulin sensitive Zucker lean (ZL) and insulin resistant Zucker fatty (ZF) rats in ad libitum or after an overnight fasting were isolated, cultured and treated with insulin and other compounds for analysis of gene expression using real-time PCR. The mRNA levels of one insulin-induced (Srebp-1c) and one insulin-suppressed (Pck1) genes in response to insulin, glucagon, and compactin treatments in hepatocytes from ad libitum ZL and ZF rats were analyzed. Additionally, the effects of insulin and T1317 on their levels in hepatocytes from ad libitum or fasted ZL or ZF rats were compared. The mRNA levels of Srebp-1c, Fas, and Scd1, but not that of Insr, Gck and Pck1, were higher in freshly isolated hepatocytes from ad libitum ZF than that from ZL rats. These patterns of Srebp-1c and Pck1 mRNA levels remained in primary hepatocyte cultured in vitro. Insulin's ability to regulate Srebp-1c and Pck1 expression was diminished in hepatocytes from ad libitum ZF, but not ZL rats. Glucagon or compactin suppressed Srebp-1c mRNA expression in lean, but not fatty hepatocytes. However, glucagon induced Pck1 mRNA expression similarly in hepatocytes from ad libitum ZL and ZF rats. Insulin caused the same dose-dependent increase of Akt phosphorylation in hepatocytes from ad libitum ZL and ZF rats. It synergized with T1317 to induce Srebp-1c, and suppressed Pck1 mRNA levels in hepatocytes from fasted, but not that from ad libitum ZF rats. We demonstrated that insulin was unable to regulate its downstream genes' mRNA expression in hepatocytes from ad libitum ZF rats. This impairment can be partially restored in hepatocytes from ZF rats after an overnight fasting, a phenomenon that deserves further investigation.

Perfiles genéticos de longevidad y envejecimiento saludable en nonagenarios del País Vasco

Introduction Currently there are notable differences in the aging of individuals in modern populations. While some of them enjoy a long healthy aging, others develop neurodegenerative diseases, such as Alzheimer's disease (AD). Environmental factors are critical, but genetics could explain the differences observed. It has recently been postulated that longevity genes might also be neuroprotective. Objectives To assess whether certain genetic variants associated with longevity might have a neuroprotective effect. Methods The subjects of this study are people older than 90 years. We will collect sociodemographic and clinical data and multiple assessments, cognitive, functional, anthropometric, nutritional, sensory and physical each participant. In addition, 64 SNPs loci distributed in 13 candidate genes FOXO3, SIRT1, TOMM40, APOE, PICALM, COMT, CETP, CLU, CR1, IL-6, PCK-1, ZNF224 and ACE will be analysed by Taqman array. Results It is hoped to gain more knowledge about under/over-represented alleles in nonagenarians. Furthermore, comparison of the genetic characteristics of nonagenarians with AD with those free of disease will enable links to be seen between certain alleles with protection or the risk of AD. Associated information on the participants will create subgroups showing the interactions between environment and genetic variation in relation to healthy aging and AD. Conclusion The study of the genetic variability of nonagenarians can give us information on the alleles associated with longevity and neuroprotection.

Retinoids induced Pck1 expression and attenuated insulin-mediated suppression of its expression via activation of retinoic acid receptor in primary rat hepatocytes

Insulin regulates the expression of genes involved in hepatic glucose and lipid metabolism, such as the cytosolic form of phosphoenolpyruvate carboxykinase gene (Pck1). We have reported that lipophilic molecules from rat livers induced Pck1 transcription and attenuated insulin-mediated suppression of its expression in primary rat hepatocytes. After identification of retinol and retinal as the active molecules, the present study was aimed to determine the effects of retinoids on insulin-mediated suppression of Pck1 expression in primary rat hepatocytes. Real-time PCR and reporter gene assays were designed to determine retinoid effects in the absence or presence of insulin on the expression levels of Pck1 mRNA and activation of its promoter constructs, respectively. The lipophilic extract from rat livers specifically induced the expression of Pck1, but not that of two other insulin-suppressed genes, glucose 6-phosphatase catalytic subunit and insulin-like growth factor-binding protein 1. Retinol, retinal, and retinoic acid (RA) induced Pck1 expression dose-dependently in primary hepatocytes. Specific activation of retinoic acid receptor (RAR), but not retinoid X receptor, attenuated insulin-mediated suppression of Pck1 expression. RARα antagonist (Ro41-5253) abolished the retinal-mediated induction of Pck1 expression and attenuation of insulin-mediated suppression of its expression. Disruption of the proximal, but not the distal, RA responsive element in the Pck1 promoter eliminated the RA response of Pck1 promoter reporter constructs in primary hepatocytes. The results of this study demonstrated for the first time that retinoid treatment attenuated insulin-mediated suppression of Pck1 expression in primary rat hepatocytes. It suggests that retinoid metabolism in hepatocytes may modulate hepatic insulin action.

The intestinal factor Tff3 and a miRNA network regulate murine caloric metabolism

Genetic impairment of the genes coding for three mammalian trefoil peptides resulted in severe gastrointestinal malfunctions. The trefoil peptides also appear involved in caloric metabolism. Monitoring global miRNA expression of Tff3 deficient mice points to an interplay of Tff3 with a miRNA regulatory network. We identified 21 miRNAs that were deregulated when compared to the wild type strain. In silico evaluation indicated that the majority of the 21 miRNA were connected with the metabolic pathway “glycolysis/gluconeogenesis’’ (p=0.032), a signaling pathway including nine target genes Aldh9a1, Aldh2, Pck1, Aldoc, Pgam2, Pck2, Adh4, Adh5, and Fbp1. Association of Tff3 with this metabolic pathway is further supported by the observation that the body mass of adult Tff3 KO mice (five months) showed a clearly reduced weight. Furthermore, the majority of the identified 21 miRNA genes are localized on murine chromosomes 2 and 5 in three clusters (2A1, 2B, 5B3) suggesting a coordinated expression control and function.

Suppressor of Cytokine Signaling-3 Is a Glucagon-inducible Inhibitor of PKA Activity and Gluconeogenic Gene Expression in Hepatocytes

SOCS3 is a cytokine-inducible negative regulator of cytokine receptor signaling. Recently, SOCS3 was shown to be induced by a cAMP-dependent pathway involving exchange protein directly activated by cAMP (Epac). We observed in livers of fasted mice that Socs3 mRNA was increased 4-fold compared with refed mice, suggesting a physiologic role for SOCS3 in the fasted state that may involve glucagon and Epac. Treating primary hepatocytes with glucagon resulted in a 4-fold increase in Socs3 mRNA levels. The Epac-selective cAMP analog 8-4-(chlorophenylthio)-2'-O-methyladenosine-3',5'-monophosphate, acetoxymethyl ester (cpTOME) increased Socs3 expression comparably. In gain-of-function studies, adenoviral expression of SOCS3 in primary hepatocytes caused a 50% decrease in 8-br-cAMP-dependent PKA phosphorylation of the transcription factor CREB. Induction of the gluconeogenic genes Ppargc1a, Pck1, and G6pc by glucagon or 8-br-cAMP was suppressed nearly 50%. In loss-of-function studies, hepatocytes from liver-specific SOCS3 knock-out mice responded to 8-br-cAMP with a 200% greater increase in Ppargc1a and Pck1 expression, and a 30% increase in G6pc expression, relative to wild-type cells. Suppression of SOCS3 by shRNA in hepatocytes resulted in a 60% increase in cAMP-dependent G6pc and Pck1 expression relative to control cells. SOCS3 expression also inhibited cAMP-dependent phosphorylation of the IP3 receptor but did not inhibit nuclear localization of the catalytic subunit of PKA. Using an in vitro kinase assay, cAMP-dependent PKA activity was reduced by 80% in hepatocytes expressing ectopic SOCS3. These data indicate that cAMP activates both the PKA and Epac pathways with induction of SOCS3 by the Epac pathway negatively regulating the PKA pathway.

CAMP-mediated regulation of HNF-4α depends on the level of coactivator PGC-1α

Hepatocyte nuclear factor-4 alpha (HNF-4α) is a member of the nuclear receptor superfamily with important roles in hepatic metabolism. Fasting induces the cAMP/protein kinase A (PKA)-signaling pathway. The mechanisms whereby cAMP regulates HNF-4α transcriptional activity are incompletely understood. We have therefore investigated the role of cAMP/PKA in regulation of HNF-4α in COS-1 cells and the hepatoma HepG2 cell line. cAMP/PKA inhibited the transcriptional activity of HNF-4α in COS-1 cells, whereas a stimulatory effect was observed in HepG2 cells. The cAMP-induced inhibition of HNF-4α in COS-1 cells was counteracted by overexpression of the nuclear receptor coactivator PGC-1α, and cAMP/PKA-dependent induction of the PGC1A gene in HepG2 cells seems to explain the cell specific differences. This was further supported by knock-down of PGC-1α in HepG2 cells, which abolished the stimulatory effect of PKA on HNF-4α transcriptional activity. Similar to the cAMP/PKA-mediated regulation of HNF-4α, overexpression of the cAMP-response element binding protein (CREB) inhibited the transcriptional activity of HNF-4α in COS-1 cells, regardless of cAMP/PKA activation and CREB phosphorylation. Moreover, activation of CREB by cAMP/PKA further stimulated HNF-4α transactivation in HepG2 cells. cAMP induced the expression of the HNF-4α target genes PCK1 and G6Pase in these cells. In conclusion, our results suggest that the level of PGC-1α determines whether the cAMP/PKA-pathway overall stimulates or inhibits HNF-4α transcriptional activation.

T-Cell Protein Tyrosine Phosphatase Attenuates STAT3 and Insulin Signaling in the Liver to Regulate Gluconeogenesis

OBJECTIVEInsulin-induced phosphatidylinositol 3-kinase (PI3K)/Akt signaling and interleukin-6 (IL-6)-instigated JAK/STAT3-signaling pathways in the liver inhibit the expression of gluconeogenic genes to decrease hepatic glucose output. The insulin receptor (IR) and JAK1 tyrosine kinases and STAT3 can serve as direct substrates for the T-cell protein tyrosine phosphatase (TCPTP). Homozygous TCPTP-deficiency results in perinatal lethality prohibiting any informative assessment of TCPTP's role in glucose homeostasis. Here we have used Ptpn2+/− mice to investigate TCPTP's function in glucose homeostasis.RESEARCH DESIGN AND METHODSWe analyzed insulin sensitivity and gluconeogenesis in chow versus high-fat–fed (HFF) Ptpn2+/− and Ptpn2+/+ mice and insulin and IL-6 signaling and gluconeogenic gene expression in Ptpn2+/− and Ptpn2+/+ hepatocytes.RESULTSHFF Ptpn2+/− mice exhibited lower fasted blood glucose and decreased hepatic glucose output as determined in hyperinsulinemic euglycemic clamps and by the decreased blood glucose levels in pyruvate tolerance tests. The reduced hepatic glucose output coincided with decreased expression of the gluconeogenic genes G6pc and Pck1 and enhanced hepatic STAT3 phosphorylation and PI3K/Akt signaling in the fasted state. Insulin-induced IR-β–subunit Y1162/Y1163 phosphorylation and PI3K/Akt signaling and IL-6–induced STAT3 phosphorylation were also enhanced in isolated Ptpn2+/− hepatocytes. The increased insulin and IL-6 signaling resulted in enhanced suppression of G6pc and Pck1 mRNA.CONCLUSIONSLiver TCPTP antagonises both insulin and STAT3 signaling pathways to regulate gluconeogenic gene expression and hepatic glucose output.

Reduced Milk Triglycerides in Mice Lacking Phosphoenolpyruvate Carboxykinase in Mammary Gland Adipocytes and White Adipose Tissue Contribute to the Development of Insulin Resistance in Pups

Obesity and type 2 diabetes are growing problems worldwide in adults and children. This study focused on understanding the patterning of insulin resistance as a result of altered perinatal nutrition. We analyzed mice in which the binding site for peroxisome proliferator‐activated receptor γ (PPARγ) was deleted from the promoter of the cytosolic phosphoenolpyruvate carboxykinase gene (Pck1) (PPARE−/−). We analyzed pups from dams with the same genotype as well as fostered and cross‐fostered pups. Pck1 expression and triglyceride content in the milk were measured. The PPARE mutation reduced Pck1 expression in WAT to 2.2% of wild type (WT) and reduced Pck1 expression in whole mammary gland tissue to 1% of WT. The female PPARE−/− mice presented reduced lipid storage in mammary gland adipocytes and in WAT, resulting in a 40% reduction of milk triglycerides during lactation. Pups from PPARE−/− dams exhibited insulin resistance as early as 14 days after birth, a condition that persisted into adulthood. Wild type pups fostered by PPARE−/− dams have lower body weights and plasma insulin concentrations compared to WT pups reared by WT dams. PPARE−/− pups fostered by WT dams have improved glucose clearance compared to pups raised by PPARE−/− dams. PPARE+/− and PPARE−/− dams patterned the newborn pup for reduced growth and insulin resistance in utero as well. Thus, in utero environment and altered nutrition in the perinatal period cause epigenetic changes that last into adulthood and contribute to the development of insulin resistance. J Nutr. 139:1–9, 2009.

Reduced Milk Triglycerides in Mice Lacking Phosphoenolpyruvate Carboxykinase in Mammary Gland Adipocytes and White Adipose Tissue Contribute to the Development of Insulin Resistance in Pups ,

Obesity and type 2 diabetes are growing problems worldwide in adults and children. In this study, we focused on understanding the patterning of insulin resistance as a result of altered perinatal nutrition. We analyzed mice in which the binding site for PPARγ was deleted from the promoter of the cytosolic phosphoenolpyruvate carboxykinase gene (Pck1) (PPARE−/−). We analyzed pups from dams with the same genotype as well as fostered and cross-fostered pups. Pck1 expression and triglyceride concentration in the milk were measured. The PPARE mutation reduced Pck1 expression in white adipose tissue (WAT) to 2.2% of wild type (WT) and reduced Pck1 expression in whole mammary gland tissue to 1% of WT. The female PPARE−/− mice had reduced lipid storage in mammary gland adipocytes and in WAT, resulting in a 40% reduction of milk triglycerides during lactation. Pups from PPARE−/− dams had insulin resistance as early as 14 d after birth, a condition that persisted into adulthood. WT pups fostered by PPARE−/− dams had lower body weights and plasma insulin concentrations compared with WT pups reared by WT dams. PPARE−/− pups fostered by WT dams had improved glucose clearance compared with pups raised by PPARE−/− dams. PPARE+/− and PPARE−/− dams also patterned newborn pups for reduced growth and insulin resistance in utero. Thus, the in utero environment and altered nutrition during the perinatal period cause epigenetic changes that persist into adulthood and contribute to the development of insulin resistance.