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In most actinomycetes, GlnR governs both nitrogen and non-nitrogen metabolisms (e.g., carbon, phosphate, and secondary metabolisms). Although GlnR has been recognized as a global regulator, its regulatory role in central carbon metabolism [e.g., glycolysis, gluconeogenesis, and the tricarboxylic acid (TCA) cycle] is largely unknown. In this study, we characterized GlnR as a direct transcriptional repressor of the pckA gene that encodes phosphoenolpyruvate carboxykinase, catalyzing the conversion of the TCA cycle intermediate oxaloacetate to phosphoenolpyruvate, a key step in gluconeogenesis. Through the transcriptomic and quantitative real-time PCR analyses, we first showed that the pckA transcription was upregulated in the glnR null mutant of Amycolatopsis mediterranei . Next, we proved that the pckA gene was essential for A. mediterranei gluconeogenesis when the TCA cycle intermediate was used as a sole carbon source. Furthermore, with the employment of the electrophoretic mobility shift assay and DNase I footprinting assay, we revealed that GlnR was able to specifically bind to the pckA promoter region from both A. mediterranei and two other representative actinomycetes ( Streptomyces coelicolor and Mycobacterium smegmatis ). Therefore, our data suggest that GlnR may repress pckA transcription in actinomycetes, which highlights the global regulatory role of GlnR in both nitrogen and central carbon metabolisms in response to environmental nutrient stresses., Importance: The GlnR regulator of actinomycetes controls nitrogen metabolism genes and many other genes involved in carbon, phosphate, and secondary metabolisms. Currently, the known GlnR-regulated genes in carbon metabolism are involved in the transport of carbon sources, the assimilation of short-chain fatty acid, and the 2-methylcitrate cycle, although little is known about the relationship between GlnR and the TCA cycle and gluconeogenesis. Here, based on the biochemical and genetic results, we identified GlnR as a direct transcriptional repressor of pckA , the gene that encodes phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, thus highlighting that GlnR plays a central and complex role for dynamic orchestration of cellular carbon, nitrogen, and phosphate fluxes and bioactive secondary metabolites in actinomycetes to adapt to changing surroundings., Competing Interests: The authors declare no conflict of interest.

Gluconeogenesis is one of the key processes through which the kidney contributes to glucose homeostasis. Urinary exosomes (uE) have been used to study renal gene regulation noninvasively in humans and rodents. Recently, we demonstrated fast-fed regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme for gluconeogenesis, in human uE. The regulation was impaired in subjects with early insulin resistance. Here, we studied primary human proximal tubule cells (hPT) and human uE to elucidate a potential link between insulin resistance and fast-fed regulation of renal PEPCK. We demonstrate that fasted hPTs had higher PEPCK and insulin receptor substrate-2 (IRS2) mRNA and protein levels, relative to fed cells. The fast-fed regulation was, however, attenuated in insulin receptor knockdown (IRKO) hPTs. The IRKO was confirmed by the blunted insulin-induced response on PEPCK, PGC1α, p-IR, and p-AKT expression in IRKO cells. Exosomes secreted by the wild-type or IRKO hPT showed similar regulation to the respective hPT. Similarly, in human uE, the relative abundance of IRS-2 mRNA (to IRS1) was higher in the fasted state relative to the fed condition. However, the fast-fed difference was absent in subjects with early insulin resistance. These subjects had higher circulating glucagon levels relative to subjects with optimal insulin sensitivity. Furthermore, in hPT cells, glucagon significantly induced PEPCK and IRS2 gene, and gluconeogenesis. IR knockdown in hPT cells further increased the gene expression levels. Together the data suggest that reduced insulin sensitivity and high glucagon in early insulin resistance may impair renal gluconeogenesis via IRS2 regulation., (© 2022 Wiley Periodicals LLC.)

Postoperative fatigue (POF) is the most common and long-lasting complication after surgery, which brings heavy burden to individuals and society. Recently, hastening postoperative recovery receives increasing attention, but unfortunately, the mechanisms underlying POF remain unclear. Propofol is a wildly used general anesthetic in clinic, and inspired by the rapid antidepressant effects induced by ketamine at non-anesthetic dose, the present study was undertaken to investigate the anti-fatigue effects and underlying mechanisms of propofol at a non-anesthetic dose in 70% hepatectomy induced POF model in rats. We first showed here that single administration of propofol at 0.1 mg/kg ameliorated acute POF in hepatectomy induced POF rats. Based on metabonomics analysis, we hypothesized that propofol exerted anti-fatigue activity in POF rats by facilitating free fatty acid (FFA) oxidation and gluconeogenesis. We further confirmed that propofol restored the deficit in FFA oxidation and gluconeogenesis in POF rats, as evidenced by the elevated FFA utilization, acetyl coenzyme A content, pyruvic acid content, phosphoenolpyruvic acid content, hepatic glucose output and glycogen storage. Moreover, propofol stimulated glucagon secretion and up-regulated expression of cAMP-response element binding protein (CREB), phosphorylated CREB, peroxlsome prolifeator-activated receptor-γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinade1 and carnitine palmitoltransferase 1A. In summary, our study suggests for the first time that propofol ameliorates acute POF by promoting glucagon-regulated gluconeogenesis via CREB/PGC-1α signaling and accelerating FFA beta-oxidation., Competing Interests: Declaration of competing interest The author have declared that no competing interest exists., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The mosquito Aedes aegypti undertakes a shift in carbohydrate metabolism during embryogenesis, including an increase in the activity of phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, at critical steps of embryo development. All eukaryotes studied to date present two PEPCK isoforms, namely PEPCK-M (mitochondrial) and PEPCK-C (cytosolic). In A. aegypti, however, these proteins are so far uncharacterized. In the present work we describe two A. aegypti PEPCK isoforms by sequence alignment, protein modeling, and transcription analysis in different tissues, as well as PEPCK enzymatic activity assays in mitochondrial and cytoplasmic compartments during oogenesis and embryogenesis. First, we characterized the protein sequences compared to other organisms, and identified conserved sites and key amino acids. We also performed structure modeling for AePEPCK(M) and AePEPCK(C), identifying highly conserved structural sites, as well as a signal peptide in AePEPCK(M) localized in a very hydrophobic region. Moreover, after blood meal and during mosquito oogenesis and embryogenesis, both PEPCKs isoforms showed different transcriptional profiles, suggesting that mRNA for the cytosolic form is transmitted maternally, whereas the mitochondrial form is synthesized by the zygote. Collectively, these results improve our understanding of mosquito physiology and may yield putative targets for developing new methods for A. aegypti control., (Copyright © 2021. Published by Elsevier Inc.)

High glucose diets are unhealthy, although the mechanisms by which elevated glucose is harmful to whole animal physiology are not well understood. In Caenorhabditis elegans, high glucose shortens lifespan, while chemically inflicted glucose restriction promotes longevity. We investigated the impact of glucose metabolism on aging quality (maintained locomotory capacity and median lifespan) and found that, in addition to shortening lifespan, excess glucose negatively impacts locomotory healthspan. Conversely, disrupting glucose utilization by knockdown of glycolysis-specific genes results in large mid-age physical improvements via a mechanism that requires the FOXO transcription factor DAF-16. Adult locomotory capacity is extended by glycolysis disruption, but maximum lifespan is not, indicating that limiting glycolysis can increase the proportion of life spent in mobility health. We also considered the largely ignored role of glucose biosynthesis (gluconeogenesis) in adult health. Directed perturbations of gluconeogenic genes that specify single direction enzymatic reactions for glucose synthesis decrease locomotory healthspan, suggesting that gluconeogenesis is needed for healthy aging. Consistent with this idea, overexpression of the central gluconeogenic gene pck-2 (encoding PEPCK) increases health measures via a mechanism that requires DAF-16 to promote pck-2 expression in specific intestinal cells. Dietary restriction also features DAF-16-dependent pck-2 expression in the intestine, and the healthspan benefits conferred by dietary restriction require pck-2. Together, our results describe a new paradigm in which nutritional signals engage gluconeogenesis to influence aging quality via DAF-16. These data underscore the idea that promotion of gluconeogenesis might be an unappreciated goal for healthy aging and could constitute a novel target for pharmacological interventions that counter high glucose consequences, including diabetes., Competing Interests: The authors have declared that no competing interests exist.

The phosphoenolpyruvate-pyruvate-oxaloacetate node is a major branch within the central carbon metabolism and acts as a connection point between glycolysis, gluconeogenesis, and the TCA cycle. Phosphoenolpyruvate carboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malic enzymes, and pyruvate kinase, among others, are enzymes included in this node. We determined the mRNA levels and specific activity profiles of some of these genes and enzymes in Streptomyces coelicolor M-145. The results obtained in the presence of glucose demonstrated that all genes studied of the phosphoenolpyruvate-pyruvate-oxaloacetate node were expressed, although at different levels, with 10- to 100-fold differences. SCO3127 (phosphoenolpyruvate carboxylase gene) and SCO5261 (NADP + -dependent malic enzyme gene) showed the highest expression in the rapid growth phase, and the mRNA levels corresponding to SCO5896 (phosphoenolpyruvate-utilizing enzyme gene), and SCO0546 (pyruvate carboxylase gene) increased 5- to 10-fold towards the stationary phase. In casamino acids, in general mRNA levels of S. coelicolor were lower than in glucose, however, results showed greater mRNA expression of SCO4979 (PEP carboxykinase), SCO0208 (pyruvate phosphate dikinase gene), and SCO5261 (NADP + -dependent malic enzyme). These results suggest that PEP carboxylase (SCO3127) is an important enzyme during glucose catabolism and oxaloacetate replenishment. On the other hand, phosphoenolpyruvate carboxykinase, pyruvate phosphate dikinase, and NADP + -malic enzyme could have an important role in gluconeogenesis in S. coelicolor.

Approximately 15 to 50% of short-chain fatty acids (SCFA) reach the ruminant small intestine. Previous research suggests that activation of small intestinal gluconeogenesis induced by propionate has beneficial effects on energy homeostasis. However, the regulatory effect of propionate on key gluconeogenic genes in enterocytes of the bovine small intestine remains less known. Therefore, the purpose of this study was to establish the long-term cultures of bovine intestinal epithelial cells (BIEC) from bovine jejunum tissue using SV40T (1:200; Santa Cruz, Shanghai, China) and investigate the regulatory effect of propionate on the key gluconeogenic genes in BIEC. Our study showed that long-term BIEC cultures were established by SV40T-induced immortalization. Immortal BIEC were distinguished by the expression of cytokeratin 18, villin, fatty acid binding protein 2, and small intestine peptidase. The mRNA expression of genes involved in the SCFA transporters, monocarboxylate transporter 4, and Na + /H + exchanger isoforms 1 were significantly elevated with 20 mM SCFA compared with untreated controls. In addition, BIEC exhibited significant uptake of propionate and butyrate from the culture medium. Remarkably, 3 mM propionate induced profound changes in mRNA level of key genes involved in gluconeogenesis, including phosphoenolpyruvate carboxykinase 2, pyruvate carboxylase, fructose-1,6-bisphosphatase 1, and peroxisome proliferator-activated receptor-γ coactivator 1α. Additionally, 3 mM propionate enhanced the expression of PGC1A mRNA at 3, 6, 12, and 24 h of incubation. These findings suggest that propionate controls the mRNA expression of genes involved in key enzymes for gluconeogenesis in the enterocytes of bovines., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Impaired insulin-induced suppression of renal gluconeogenesis could be a risk for hyperglycemia. Diabetes is associated with elevated renal gluconeogenesis; however, its regulation in early insulin resistance is unclear in humans. A noninvasive marker of renal gluconeogenesis would be helpful. Here, we show that human urine exosomes (uE) contain three gluconeogenic enzymes: phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase, and glucose 6-phosphatase. Their protein levels were positively associated with whole body insulin sensitivity. PEPCK protein in uE exhibited a meal-induced suppression. However, subjects with lower insulin sensitivity had blunted meal-induced suppression. Also, uE from subjects with prediabetes and diabetic rats had higher PEPCK relative to nondiabetic controls. Moreover, uE-PEPCK was higher in drug-naïve subjects with diabetes relative to drug-treated subjects with diabetes. To determine whether increased renal gluconeogenesis is associated with hyperglycemia or PEPCK expression in uE, acidosis was induced in rats by 0.28 M NH 4 Cl with 0.5% sucrose in drinking water. Control rats were maintained on 0.5% sucrose. At the seventh day posttreatment, gluconeogenic enzyme activity in the kidneys, but not in the liver, was higher in acidotic rats. These rats had elevated PEPCK in their uE and a significant rise in blood glucose relative to controls. The induction of gluconeogenesis in human proximal tubule cells increased PEPCK expression in both human proximal tubules and human proximal tubule-secreted exosomes in the media. Overall, gluconeogenic enzymes are detectable in human uE. Elevated PEPCK and its blunted meal-induced suppression in human urine exosomes are associated with diabetes and early insulin resistance.

During the peripartum period, dairy cows experience both an increase in circulating fatty acid (FA) profile and a change in circulating FA profile, which have been shown to alter regulation of gluconeogenic genes. The objective was to quantify gene expression of key enzymes involved in gluconeogenesis and FA transport into the mitochondria in primary hepatocytes in response to exposure to an FA mixture mimicking what is circulating in a transition dairy cow with or without enrichment of C16:0, C18:0, and C18:1. Primary hepatocytes were isolated from 4 Holstein bull calves 3 d of age (± standard deviation 2 d) and cultured. Twenty-four hours after plating, treatments were applied to the cells for 24-h incubation. Treatments consisted of (1) control (1% BSA), (2) 0.75 mM FA cocktail (3% C14:0, 27% C16:0, 23% C18:0, 31% C18:1, 8% C18:2, and 8% C18:3 to mimic the FA profile of dairy cattle at calving), (3) 0.90 mM FA cocktail, (4) 0.75 mM FA cocktail + 0.15 mM C16:0, (5) 0.75 mM FA cocktail + 0.15 mM C18:0, and (6) 0.75 mM FA cocktail + 0.15 mM C18:1. After harvest in Trizol (Life Technologies, Carlsbad, CA), samples were stored at -80°C until RNA extraction, purification, and reverse transcription. Abundance of mRNA was measured using quantitative real-time PCR. Expression of genes of interest [carnitine palmitoyltransferase 1A, pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase] was calculated relative to the average abundance of 2 reference genes (ribosomal protein L32 and glyceraldehyde 3-phosphate dehydrogenase), which were the most stable out of 3 tested. Data were analyzed using PROC MIXED (SAS version 9.4; SAS Institute, Cary, NC) with the fixed effect of treatment and calf in the random statement. The addition of FA compared with the 1% BSA treatment increased the expression of carnitine palmitoyltransferase 1A and cytosolic PCK1. Enrichment with individual FA did not further regulate pyruvate carboxylase or PCK1 beyond that achieved by the basal profile. These results suggest that shifts in circulating FA profile within a biological range, without a difference in the total FA concentration, have minimal effects on transcriptional regulation of hepatic gluconeogenic genes in primary bovine hepatocytes., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Cancer cells constantly face a fluctuating nutrient supply and interference with adaptive responses might be an effective therapeutic approach. It has been discovered that in the absence of glucose, cancer cells can synthesize crucial metabolites by expressing phosphoenolpyruvate carboxykinase (PEPCK, PCK1 or PCK2) using abbreviated forms of gluconeogenesis. Gluconeogenesis, which in essence is the reverse pathway of glycolysis, uses lactate or amino acids to feed biosynthetic pathways branching from glycolysis. PCK1 and PCK2 have been shown to be critical for the growth of certain cancers. In contrast, fructose-1,6-bisphosphatase 1 (FBP1), a downstream gluconeogenesis enzyme, inhibits glycolysis and tumor growth, partly by non-enzymatic mechanisms. This review sheds light on the current knowledge of cancer cell gluconeogenesis and its role in metabolic reprogramming, cancer cell plasticity, and tumor growth., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The kidneys are as involved as the liver in gluconeogenesis which can significantly contribute to hyperglycemia in the diabetic condition. Substantial evidence has demonstrated the overexpression of rate-limiting gluconeogenic enzymes, especially phosphoenolpyruvate carboxykinase and glucose 6 phosphatase, and the accelerated glucose release both in the isolated proximal tubular cells and in the kidneys of diabetic animal models and diabetic patients. The aim of this review is to provide an insight into the mechanisms that accelerate renal gluconeogenesis in the diabetic conditions and the therapeutic approaches that could affect this process in the kidney. Increase in gluconeogenic substrates, reduced insulin concentration or insulin resistance, downregulation of insulin receptors and insulin signaling, oxidative stress, and inappropriate activation of the renin-angiotensin system are likely to participate in enhancing renal gluconeogenesis in the diabetic milieu. Several studies have suggested that controlling glucose metabolism at the renal level favors effective overall glycemic control in both type 1 and type 2 diabetes. Therefore, renal gluconeogenesis may be a promising target for effective glycemic control as a therapeutic strategy in diabetes., (© 2018 Wiley Periodicals, Inc.)

Hepatic gluconeogenesis is the major contributor to hyperglycemia in diabetes. Long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been shown to promote hepatic insulin resistance; however, the underlying mechanism involving hepatic gluconeogenesis remains unclear. This study aims to investigate the potential role of MEG3 in hepatic gluconeogenesis. Mouse primary hepatocytes were used in this study. Cell transfection was performed for the overexpression or knockdown of specific genes. Expressions of MEG3, miR-302a-3p, CREB-regulated transcriptional coactivator 2 (CRTC2), protein kinase A (PKA), cAMP-response element binding protein (CREB), PPARγ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pc) were determined by quantitative real-time polymerase chain reaction (qRT-qPCR) and Western blot analysis, respectively. The association among MEG3, miR-302a-3p, and CRTC2 was disclosed by dual-luciferase reporter assay. MEG3 was highly expressed in high glucagon-treated mouse primary hepatocytes. CREB-induced MEG3 upregulation increased gluconeogenic gene expression in high glucagon-treated primary hepatocytes, while MEG3 interference led to an opposite effect. MEG3 served as a competing endogenous RNA (ceRNA) to upregulate CRTC2 by targeting miR-302a-3p in primary hepatocytes, thereby increasing PGC-1α-PEPCK/G6Pc. CREB-upregulated MEG3-enhanced hepatic gluconeogenesis via mediating miR-302a-3p-CRTC2 axis, revealing that MEG3 might be a potential target and therapeutic strategy for diabetes., (© 2018 Wiley Periodicals, Inc.)

Tribbles related homolog 1 is the mammalian ortholog of Tribbles, which controls cell division and migration during development in Drosophila. TRB1 is a pseudokinase and functions as a scaffold protein. Recent findings suggest that TRB1 plays important roles in hepatic lipid metabolism and participates in insulin resistance. However, the underlying mechanisms have not yet been elucidated. Here, we demonstrate that TRB1 suppresses FOXO1 transcriptional activity to downregulate the expression of G6Pase and PEPCK, which encode gluconeogenic rate-limiting enzymes. TRB1 knockdown enhances FOXO1 binding to the gluconeogenic gene promoters. It also increases FOXO1 acetylation and recruits CBP to the binding sequence of FOXO1. These results suggest that TRB1 suppresses the expression of G6Pase and PEPCK by attenuating FOXO1 transcriptional activity and negatively regulates gluconeogenesis., (© 2018 Federation of European Biochemical Societies.)

Cancer cells display a high rate of glycolysis in the presence of oxygen to promote proliferation. Gluconeogenesis, the reverse pathway of glycolysis, can antagonize aerobic glycolysis in cancer via three key enzymes - phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), and glucose-6-phosphatase (G6Pase). Recent studies have revealed that, in addition to metabolic regulation, these enzymes also play a role in signaling, proliferation, and the cancer stem cell (CSC) tumor phenotype. Multifaceted regulation of PEPCK, FBPase, and G6Pase through transcription, epigenetics, post-translational modification, and enzymatic activity is observed in different cancers. We review here the function and regulation of key gluconeogenic enzymes and new therapeutic opportunities., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Red pepper seed, a by-product of red pepper, has been reported to have antioxidant and antiobesity activities. However, its role in diabetes has not yet been highly investigated. Glucose homeostasis is mainly maintained by insulin, which suppresses glucose production in the liver and enhances glucose uptake in peripheral tissues. In this study, we investigated the underlying mechanisms through which red pepper seed extract (RPSE) affects glucose production in AML12 hepatocytes and glucose uptake in C2C12 myotubes. RPSE reduced glucose production in a dose-dependent manner in AML12 cells. The levels of glucose 6 phosphatase, phosphoenolpyruvate carboxykinase, and critical enzymes for hepatic gluconeogenesis were decreased by RPSE. Gluconeogenesis regulating proteins, Akt and forkhead box protein O1, were also activated by RPSE. In addition, RPSE increased glucose uptake in C2C12 via inducing translocation of glucose transporter type 4 from cytosol to plasma membrane. Analysis of the insulin-dependent pathway showed that the activities of insulin receptor substrate 1, phosphatidylinositol 3-kinase, and Akt were significantly stimulated by RPSE. In conclusion, RPSE might improve glucose homeostasis by reducing hepatic gluconeogenesis and increasing peripheral glucose uptake. Results obtained also suggest that RPSE can be a compelling antidiabetic nutraceutical.

As a member of miR-17-92 miRNA clusters, miR‑19a has been considered to regulate hepatic glycogenesis by mediating the PI3K/AKT signaling pathway. However, whether miR‑19a serves an important role in gluconeogenesis in hepatocytes remains unknown. In the present study, the impact of miR‑19a on gluconeogenesis in HEP1‑6 cells and its mechanisms of action were investigated. It was observed that overexpression of miR‑19a led to decreased glucose production, accompanied by increased activation of the AKT/FOXO1 signaling pathway and downregulated expression of gluconeogenesis‑associated genes, including peroxisome proliferator‑activated receptor γ coactivator 1α, phosphoenolpyruvate carboxykinase and glucose 6‑phosphatase in the HEP1‑6 cells transfected with the miR‑19a mimic. In contrast, suppression of miR‑19a impaired the activation of the AKT/FOXO1 signaling pathway and increased the expression of gluconeogenesis‑associated genes, accompanied by an elevated glucose production. Additionally, phosphatase and tensin homolog (PTEN) was identified as a target of miR‑19a and participated in the miR‑19a‑mediated gluconeogenesis in hepatocytes. These findings provide mechanistic insight into the effects of miR‑19a on the regulation of the AKT/FOXO1 signaling pathway and the expression of gluconeogenesis‑associated genes. MiR‑19a may mediate gluconeogenesis in hepatocytes by downregulating PTEN expression.

Hypothesis: Palmitate causes insulin resistance (IR) in insulin target tissue. Pioglitazone (an anti-hyperglycemic agent) and epigallocatechin gallate (EGCG, a dietary supplement) can be used for the treatment of type 2 diabetes. However, their molecular effects on gluconeogenesis remain unclear., Objective: Hence, we aimed to investigate the simultaneous effect of these anti-hyperglycemic agents on gluconeogenesis through in vitro experiments., Methods: HepG2 cells were treated with 0.5 mM palmitate, 10 μM pioglitazone, and 40 μM epigallocatechin gallate (EGCG). Gene expression assay was used to investigate the underlying mechanism. Glucose production assay was applied in culture medium to evaluate the activity of gluconeogenesis pathway., Results: Palmitate induced IR could significantly increase G6Pase and PEPCK gene expressions by 58 and 30%, respectively, compared to the control. EGCG reduced the expression of PEPCK and G6Pase by 53 and 67%, respectively. Pioglitazone reduced the mRNA level of PEPCK and G6Pase by 58 and 62% respectively. Combined treatment of insulin-resistant cells with EGCG and pioglitazone significantly decreased the mRNA level of PEPCK and G6Pase by 73 and 80%, respectively. Treatment with palmitate increased glucose production by 50% in HepG2 cells. When the insulin resistant HepG2 cells were treated alone with EGCG and pioglitazone, the glucose production reduced by 50 and 55%, respectively. The combined treatment with EGCG and pioglitazone resulted in 69% reduction in glucose production compared to the palmitate treated HepG2 cells., Conclusions: These data suggest the additive inhibitory effect of co-treatment with pioglitazone and EGCG on the gluconeogenesis pathway in palmitate-induced insulin resistance HepG2 cells.

Maintenance of whole-body glucose homeostasis is critical to glycemic function. Genetic variants mapping to chromosome 8p23.1 in genome-wide association studies have been linked to glycemic traits in humans. The gene of known function closest to the mapped region, PPP1R3B (protein phosphatase 1 regulatory subunit 3B), encodes a protein (G L ) that regulates glycogen metabolism in the liver. We therefore sought to test the hypothesis that hepatic PPP1R3B is associated with glycemic traits. We generated mice with either liver-specific deletion ( Ppp1r3b Δ hep ) or liver-specific overexpression of Ppp1r3b The Ppp1r3b deletion significantly reduced glycogen synthase protein abundance, and the remaining protein was predominantly phosphorylated and inactive. As a consequence, glucose incorporation into hepatic glycogen was significantly impaired, total hepatic glycogen content was substantially decreased, and mice lacking hepatic Ppp1r3b had lower fasting plasma glucose than controls. The concomitant loss of liver glycogen impaired whole-body glucose homeostasis and increased hepatic expression of glycolytic enzymes in Ppp1r3b Δ hep mice relative to controls in the postprandial state. Eight hours of fasting significantly increased the expression of two critical gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, above the levels in control livers. Conversely, the liver-specific overexpression of Ppp1r3b enhanced hepatic glycogen storage above that of controls and, as a result, delayed the onset of fasting-induced hypoglycemia. Moreover, mice overexpressing hepatic Ppp1r3b upon long-term fasting (12-36 h) were protected from blood ketone-body accumulation, unlike control and Ppp1r3b Δ hep mice. These findings indicate a major role for Ppp1r3b in regulating hepatic glycogen stores and whole-body glucose/energy homeostasis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cytoplasmic and mitochondrial isoforms of phosphoenolpyruvate carboxykinase (PEPCK-C and PEPCK-M) regulate hepatic gluconeogenesis to control systemic glucose homeostasis. Transcriptional and post-transcriptional mechanisms may govern synthesis, maintenance and cooperative function of compartmentalized PEPCK enzymes. In a comparative analysis, we show that tumor cells consistently transcribe and translate higher levels of enzymatically active PEPCK-C than PEPCK-M and both the isoforms were present at lower levels in normal fibroblasts. Unlike in PEPCK-M, absence of glucose reduced the PEPCK-C mRNA and protein levels only in HepG2 cells. Interestingly, isoflavone genistein significantly increased PEPCK-C mRNA and protein levels in normal fibroblasts indicating cell type specific control mechanisms. Genistein also significantly affected RNA stability of PEPCK-C but not PEPCK-M in HepG2 cells. This was due to the conserved and functional mRNA destabilizing AU rich sequences at the 3'-UTR region of PEPCK-C gene and was confirmed by luciferase reporter assays suggesting that glucose deprivation and genistein targets these sequences for mRNA degradation in HepG2 cells but not in fibroblasts. Analysis of promoter methylation by luciferase reporter assays and bisulfite DNA sequencing suggested that PEPCK-C but not PEPCK-M promoter was activated by 5-aza-2-deoxycytidine by inducing cytosine demethylation at the specific CpG dinucleotides of 5'-UTR region. Taken together, our data suggests stable PEPCK-M activity and identifies intricate relationship between (1) mRNA stability and (2) promoter DNA methylation as two mechanisms of gene expression that distinguishes PEPCK-C and PEPCK-M enzyme activities in a context and cell type dependent manner during gluconeogenesis. J. Cell. Biochem. 117: 2506-2520, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

MicroRNAs (miRNAs) are a new class of regulatory molecules implicated in type 2 diabetes, which is characterized by insulin resistance and hepatic glucose overproduction. We show that miRNA-451 (miR-451) is elevated in the liver tissues of dietary and genetic mouse models of diabetes. Through an adenovirus-mediated gain- and loss-of-function study, we found that miR-451 negatively regulates hepatic gluconeogenesis and blood glucose levels in normal mice and identified glycerol kinase (Gyk) as a direct target of miR-451. We demonstrate that miR-451 and Gyk regulate hepatic glucose production, the glycerol gluconeogenesis axis, and the AKT-FOXO1-PEPCK/G6Pase pathway in an opposite manner; Gyk could reverse the effect of miR-451 on hepatic gluconeogenesis and AKT-FOXO1-PEPCK/G6Pase pathway. Moreover, overexpression of miR-451 or knockdown of Gyk in diabetic mice significantly inhibited hepatic gluconeogenesis, alleviated hyperglycemia, and improved glucose tolerance. Further studies showed that miR-451 is upregulated by glucose and insulin in hepatocytes; the elevation of hepatic miR-451 in diabetic mice may contribute to inhibiting Gyk expression. This study provides the first evidence that miR-451 and Gyk regulate the AKT-FOXO1-PEPCK/G6Pase pathway and play critical roles in hepatic gluconeogenesis and glucose homeostasis and identifies miR-451 and Gyk as potential therapeutic targets against hyperglycemia in diabetes., (© 2016 by the American Diabetes Association.)

Nasturtium (Tropaeolum majus L.) contains high concentrations of benzylglcosinolate. We found that a hydrolysis product of benzyl glucosinolate-the benzyl isothiocyanate (BITC)-modulates the intracellular localization of the transcription factor Forkhead box O 1 (FOXO1). FoxO transcription factors can antagonize insulin effects and trigger a variety of cellular processes involved in tumor suppression, longevity, development and metabolism. The current study evaluated the ability of BITC-extracted as intact glucosinolate from nasturtium and hydrolyzed with myrosinase-to modulate i) the insulin-signaling pathway, ii) the intracellular localization of FOXO1 and, iii) the expression of proteins involved in gluconeogenesis, antioxidant response and detoxification. Stably transfected human osteosarcoma cells (U-2 OS) with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein) were used to evaluate the effect of BITC on FOXO1. Human hepatoma HepG2 cell cultures were selected to evaluate the effect on gluconeogenic, antioxidant and detoxification genes and protein expression. BITC reduced the phosphorylation of protein kinase B (AKT/PKB) and FOXO1; promoted FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect; was able to down-regulate the gene and protein expression of gluconeogenic enzymes; and induced the gene expression of antioxidant and detoxification enzymes. Knockdown analyses with specific siRNAs showed that the expression of gluconeogenic genes was dependent on nuclear factor (erythroid derived)-like2 (NRF2) and independent of FOXO1, AKT and NAD-dependent deacetylase sirtuin-1 (SIRT1). The current study provides evidence that BITC might have a role in type 2 diabetes T2D by reducing hepatic glucose production and increasing antioxidant resistance., Competing Interests: The authors have declared that no competing interests exist.

MicroRNAs (miRNAs or miRs) are known to play a vital role in type 2 diabetes, and peroxisome proliferator-activated receptor-γ (PPARγ) coactivator-1α (PGC-1α) is involved in the pathogenesis of hepatic insulin resistance. However, the correlation, if any, between PGC-1α and miRNAs in the disease has not yet been determined. Thus, in the present study, we aimed to examine the correlation between PGC-1α and miRNAs in diabetes. For this purpose, we used primary hepatocytes isolated from C57BL/6 mice and ob/ob mice. First, we found an inverse correlation between miR‑696 and PGC-1α protein levels in vivo. Second, in vitro evidence demonstrated that PGC-1α expression was significantly decreased by infection with pre-miR‑696-LV, whereas infection with anti-miR‑696-LV increased the PGC-1α protein levels. Third, a luciferase reporter assay confirmed that miR‑696 directly recognizes a specific location within the 3'-untranslated region of PGC-1α transcripts. Furthermore, the biological consequences of miR‑696 targeting PGC-1α were determined by measuring the expression levels of the characteristic hepatic gluconeogenic enzyme, PEPCK, which is regulated by PGC-1α in the liver via the coactivation of transcription factors. Taken together, our findings demonstrate that miR‑696 plays an important role in the development of hepatic gluconeogenesis and insulin resistance through the inhibition of PGC-1α translation in the liver.

The constitutive androstane receptor (CAR) is a key sensor in xenobiotic detoxification and endobiotic metabolism. Increasing evidence suggests that CAR also plays a role in energy metabolism by suppressing the hepatic gluconeogenesis and lipogenesis. In this study, we investigated the effects of two evodia alkaloids, rutaecarpine (Rut) and evodiamine (Evo), on gluconeogenesis and lipogenesis through their activation of the human CAR (hCAR). We found that both Rut and Evo exhibited anti-lipogenic and anti-gluconeogenic effects in the hyperlipidemic HepG2 cells. Both compounds can potently activate hCAR, and treatment of cells with hCAR antagonists reversed the anti-lipogenic and anti-gluconeogenic effects of Rut and Evo. The anti-gluconeogenic effect of Rut and Evo was due to the CAR-mediated inhibition of the recruitment of forkhead box O1 (FoxO1) and hepatocyte nuclear factor 4α (HNF4α) onto the phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) gene promoters. In vivo, we showed that treatment of mice with Rut improved glucose tolerance in a CAR-dependent manner. Our results suggest that the evodia alkaloids Rut and Evo may have a therapeutic potential for the treatment of hyperglycemia and type 2 diabetes. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Both epidemiologic and experimental animal studies demonstrate that chronic psychological stress exerts adverse effects on the initiation and/or progression of many diseases. However, intergenerational effects of this environmental information remains poorly understood. Here, using a C57BL/6 mouse model of restraint stress, we show that offspring of stressed fathers exhibit hyperglycemia due to enhanced hepatic gluconeogenesis and elevated expression of PEPCK. Mechanistically, we identify an epigenetic alteration at the promoter region of the Sfmbt2 gene, a maternally imprinted polycomb gene, leading to a downregulation of intronic microRNA-466b-3p, which post-transcriptionally inhibits PEPCK expression. Importantly, hyperglycemia in F1 mice is reversed by RU486 treatment in fathers, and dexamethasone administration in F0 mice phenocopies the roles of restraint stress. Thus, we provide evidence showing the effects of paternal psychological stress on the regulation of glucose metabolism in offspring, which may have profound implications for our understanding of health and disease risk inherited from fathers., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Class IIa histone deacetylases (HDACs), such as HDAC4, HDAC5, and HDAC7, provide critical mechanisms for regulating glucose homeostasis. Here we report that HDAC9, another class IIa HDAC, regulates hepatic gluconeogenesis via deacetylation of a Forkhead box O (FoxO) family transcription factor, FoxO1, together with HDAC3. Specifically, HDAC9 expression can be strongly induced upon hepatitis C virus (HCV) infection. HCV-induced HDAC9 upregulation enhances gluconeogenesis by promoting the expression of gluconeogenic genes, including phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, indicating a major role for HDAC9 in the development of HCV-associated exaggerated gluconeogenic responses. Moreover, HDAC9 expression levels and gluconeogenic activities were elevated in livers from HCV-infected patients and persistent HCV-infected mice, emphasizing the clinical relevance of these results. Our results suggest HDAC9 is involved in glucose metabolism, HCV-induced abnormal glucose homeostasis, and type 2 diabetes., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Elevated needs for glucose in lactating dairy cows are met through a combination of increased capacity for gluconeogenesis and increased supply of gluconeogenic precursors, primarily propionate. This study evaluated the effects of propionate on mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC), key gluconeogenic enzymes, and capacity for glucose synthesis in liver of dairy cattle. In experiment 1, six multiparous mid-lactation Holstein cows were used in a replicated 3×3 Latin square design consisting of a 6-d acclimation or washout phase followed by 8h of postruminal infusion of either propionate (1.68mol), glucose (0.84mol), or an equal volume (10mL/min) of water. In experiment 2, twelve male Holstein calves [39±4 kg initial body weight (BW)] were blocked by birth date and assigned to receive, at 7d of age, either propionate [2mmol·h(-1)·(BW(0.75))(-1)], acetate [3.5mmol·h(-1)·(BW(.75))(-1)], or an equal volume (4mL/min) of saline. In both experiments, blood samples were collected at 0, 2, 4, 6, and 8h relative to the start of infusion and liver biopsy samples were collected at the end of the infusion for mRNA analysis. Liver explants from experiment 1 were used to measure tricarboxylic acid cycle flux and gluconeogenesis using (13)C mass isotopomer distribution analysis from (13)C3 propionate. Dry matter intake and milk yield were not altered by infusions in cows. Serum insulin concentration in cows receiving propionate was elevated than cows receiving water, but was not different from cows receiving glucose. Hepatic expression of PCK1 and G6PC mRNA and glucose production in cows receiving propionate were not different from cows receiving water, but tended to be higher compared with cows receiving glucose. Hepatic expression of PCK2 and PC mRNA was not altered by propionate infusion in cows. Blood glucose, insulin, and glucagon in calves receiving propionate were not different than controls. Calves receiving propionate had increased PCK1 mRNA, tended to have increased G6PC mRNA, and had similar PC mRNA compared with saline controls. These data indicate a tendency for in vivo effects of propionate to alter hepatic gene expression in mid-lactation cows and neonatal calves, which are consistent with a feed-forward effect of propionate to regulate its own metabolism toward gluconeogenesis through changes in hepatic PCK1 mRNA., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Increased glucose production and reduced hepatic glycogen storage contribute to metabolic abnormalities in diabetes. Irisin, a newly identified myokine, induces the browning of white adipose tissue, but its effects on gluconeogenesis and glycogenesis are unknown. In the present study, we investigated the effects and underlying mechanisms of irisin on gluconeogenesis and glycogenesis in hepatocytes with insulin resistance, and its therapeutic role in type 2 diabetic mice. Insulin resistance was induced by glucosamine (GlcN) or palmitate in human hepatocellular carcinoma (HepG2) cells and mouse primary hepatocytes. Type 2 diabetes was induced by streptozotocin/high-fat diet (STZ/HFD) in mice. In HepG2 cells, irisin ameliorated the GlcN-induced increases in glucose production, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) expression, and glycogen synthase (GS) phosphorylation; it prevented GlcN-induced decreases in glycogen content and the phosphoinositide 3-kinase (PI3K) p110α subunit level, and the phosphorylation of Akt/protein kinase B, forkhead box transcription factor O1 (FOXO1) and glycogen synthase kinase-3 (GSK3). These effects of irisin were abolished by the inhibition of PI3K or Akt. The effects of irisin were confirmed in mouse primary hepatocytes with GlcN-induced insulin resistance and in human HepG2 cells with palmitate-induced insulin resistance. In diabetic mice, persistent subcutaneous perfusion of irisin improved the insulin sensitivity, reduced fasting blood glucose, increased GSK3 and Akt phosphorylation, glycogen content and irisin level, and suppressed GS phosphorylation and PEPCK and G6Pase expression in the liver. Irisin improves glucose homoeostasis by reducing gluconeogenesis via PI3K/Akt/FOXO1-mediated PEPCK and G6Pase down-regulation and increasing glycogenesis via PI3K/Akt/GSK3-mediated GS activation. Irisin may be regarded as a novel therapeutic strategy for insulin resistance and type 2 diabetes., (© 2015 Authors; published by Portland Press Limited.)

Cancer cells adapt metabolically to proliferate under nutrient limitation. Here we used combined transcriptional-metabolomic network analysis to identify metabolic pathways that support glucose-independent tumor cell proliferation. We found that glucose deprivation stimulated re-wiring of the tricarboxylic acid (TCA) cycle and early steps of gluconeogenesis to promote glucose-independent cell proliferation. Glucose limitation promoted the production of phosphoenolpyruvate (PEP) from glutamine via the activity of mitochondrial PEP-carboxykinase (PCK2). Under these conditions, glutamine-derived PEP was used to fuel biosynthetic pathways normally sustained by glucose, including serine and purine biosynthesis. PCK2 expression was required to maintain tumor cell proliferation under limited-glucose conditions in vitro and tumor growth in vivo. Elevated PCK2 expression is observed in several human tumor types and enriched in tumor tissue from non-small-cell lung cancer (NSCLC) patients. Our results define a role for PCK2 in cancer cell metabolic reprogramming that promotes glucose-independent cell growth and metabolic stress resistance in human tumors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Statin therapy is known to increase blood glucose levels in humans. Statins utilize pregnane X receptor (PXR) and serum/glucocorticoid regulated kinase 2 (SGK2) to activate phosphoenolpyruvate carboxykinase 1 (PEPCK1) and glucose-6-phosphatase (G6Pase) genes, thereby increasing glucose production in human liver cells. Here, the novel statin/PXR/SGK2-mediated signaling pathway has now been characterized for hepatic gluconeogenesis. Statin-activated PXR scaffolds the protein phosphatase 2C (PP2C) and SGK2 to stimulate PP2C to dephosphorylate SGK2 at threonine 193. Non-phosphorylated SGK2 co-activates PXR-mediated trans-activation of promoters of gluconeogenic genes in human liver cells, thereby enhancing gluconeogenesis. This gluconeogenic statin-PXR-SGK2 signal is not present in mice, in which statin treatment suppresses hepatic gluconeogenesis. These findings provide the basis for statin-associated side effects such as an increased risk for Type 2 diabetes.

Objective: Inhibition of intestinal NPC1L1 by ezetimibe has been demonstrated to improve glucose metabolism in rodent models; however, the role of hepatic NPC1L1 in glucose metabolism has not been elucidated. In this study, we analyzed the effects of hepatic NPC1L1 on glucose metabolism., Material and Methods: We overexpressed NPC1L1 in the livers of lean wild type mice, diet-induced obesity mice and db/db mice with adenoviral gene transfer., Results: We found that in all three mouse models, hepatic NPC1L1 overexpression lowered fasting blood glucose levels as well as blood glucose levels on ad libitum; in db/db mice, hepatic NPC1L1 overexpression improved blood glucose levels to almost the same as those found in lean wild type mice. A pyruvate tolerance test revealed that gluconeogenesis was suppressed by hepatic NPC1L1 overexpression. Further analyses revealed that hepatic NPC1L1 overexpression decreased the expression of FoxO1, resulting in the reduced expression of G6Pase and PEPCK, key enzymes in gluconeogenesis., Conclusions: These results indicate that hepatic NPC1L1 might have distinct properties of suppressing gluconeogenesis via inhibition of FoxO1 pathways., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In mammalian cells, tumor suppressor p53 plays critical roles in the regulation of glucose metabolism, including glycolysis and oxidative phosphorylation, but whether and how p53 also regulates gluconeogenesis is less clear. Here, we report that p53 efficiently down-regulates the expression of phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC), which encode rate-limiting enzymes in gluconeogenesis. Cell-based assays demonstrate the p53-dependent nuclear exclusion of forkhead box protein O1 (FoxO1), a key transcription factor that mediates activation of PCK1 and G6PC, with consequent alleviation of FoxO1-dependent gluconeogenesis. Further mechanistic studies show that p53 directly activates expression of the NAD(+)-dependent histone deacetylase sirtuin 6 (SIRT6), whose interaction with FoxO1 leads to FoxO1 deacetylation and export to the cytoplasm. In support of these observations, p53-mediated FoxO1 nuclear exclusion, down-regulation of PCK1 and G6PC expression, and regulation of glucose levels were confirmed in C57BL/J6 mice and in liver-specific Sirt6 conditional knockout mice. Our results provide insights into mechanisms of metabolism-related p53 functions that may be relevant to tumor suppression.

cAMP receptor protein (CRP) is the best characterized global regulator of Escherichia coli. After genomic SELEX screening, a total of minimum 378 promoters have been identified as its regulation targets on the E. coli genome. Among a number of promoters carrying two CRP-binding sites, several promoters carry two CRP-binding sites, one upstream but another downstream of transcription initiation sites. The regulatory role of downstream CRP site remains unsolved. Using the pck gene encoding phosphoenolpyruvate carboxykinase as a model promoter, we analyzed the role of CRP-associated downstream of the transcription initiation site. Gel shift assay and AFM observation indicate that CRP binds to both the promoter-distal site (CRP box-1) at -90.5 and the site (CRP box-2) at +13.5 downstream of transcription initiation site. The binding affinity is higher for CRP box-1. Roles of two CRP sites were examined using in vitro transcription assay and in vivo reporter assay. In both cases, transcription repression was observed in the presence of high concentrations of CRP. Taken together, we propose that cAMP-CRP associated at downstream CRP box-2 plays as a repressor for pck transcription only in the presence of high levels of cAMP-CRP., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Liver gluconeogenesis is essential to provide energy to glycolytic tissues during fasting periods. However, aberrant up-regulation of this metabolic pathway contributes to the progression of glucose intolerance in individuals with diabetes. Phosphoenolpyruvate carboxykinase (PEPCK) expression plays a critical role in the modulation of gluconeogenesis. Several pathways contribute to the regulation of PEPCK, including the nuclear receptor Rev-erbα and the histone deacetylase SIRT1. Deleted in breast cancer 1 (DBC1) is a nuclear protein that binds to and regulates both Rev-erbα and SIRT1 and, therefore, is a candidate to participate in the regulation of PEPCK. In this work, we provide evidence that DBC1 regulates glucose metabolism and the expression of PEPCK. We show that DBC1 levels decrease early in the fasting state. Also, DBC1 KO mice display higher gluconeogenesis in a normal and a high-fat diet. DBC1 absence leads to an increase in PEPCK mRNA and protein expression. Conversely, overexpression of DBC1 results in a decrease in PEPCK mRNA and protein levels. DBC1 regulates the levels of Rev-erbα, and manipulation of Rev-erbα activity or levels prevents the effect of DBC1 on PEPCK. In addition, Rev-erbα levels decrease in the first hours of fasting. Finally, knockdown of the deacetylase SIRT1 eliminates the effect of DBC1 knockdown on Rev-erbα levels and PEPCK expression, suggesting that the mechanism of PEPCK regulation is, at least in part, dependent on the activity of this enzyme. Our results point to DBC1 as a novel regulator of gluconeogenesis.

Background: Exposure to maternal obesity or hyperglycemia increases the risk of obesity and poor glucose tolerance in the offspring. We hypothesized that maternal overnutrition in late pregnancy would result in (i) lower methylation in the promoter region of the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-C; PCK1) and (ii) higher expression of hepatic gluconeogenic factors in the fetal and postnatal lamb., Methods: Ewes were fed 100% (n = 18) or ~155% (n = 17) of energy requirements from 115 d gestation, and livers were collected at ~140 d gestation or 30 d postnatal age., Results: Maternal overnutrition resulted in a decrease in hepatic expression of the mitochondrial form of PEPCK (PEPCK-M; PCK2) but not of PEPCK-C or glucose-6-phosphatase (G6PHOS) before and after birth. Hepatic expression of peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1), peroxisome proliferator-activated receptor α (PPARα), PEPCK-C, G6PHOS, and 11β hydroxysteroid dehydrogenase type 1 (11βHSD1), but not PEPCK-M, was higher in the postnatal lamb compared with that in the fetal lamb. The level of PCK1 methylation was paradoxically approximately twofold higher in the postnatal liver compared with that in the fetal liver., Conclusion: Maternal overnutrition programs a decrease in hepatic PEPCK-M in the offspring and as ~50% of total hepatic PEPCK is PEPCK-M, the longer-term consequences of this decrease may be significant.

Sodium meta-arsenite (SA) is implicated in the regulation of hepatic gluconeogenesis-related genes in vitro; however, the effects in vivo have not been studied. We investigated whether SA has antidiabetic effects in a type 2 diabetic mouse model. Diabetic db/db mice were orally intubated with SA (10 mg kg(-1) body weight/day) for 8 weeks. We examined hemoglobin A1c (HbA1c), blood glucose levels, food intake, and body weight. We performed glucose, insulin, and pyruvate tolerance tests and analyzed glucose production and the expression of gluconeogenesis-related genes in hepatocytes. We analyzed energy metabolism using a comprehensive animal metabolic monitoring system. SA-treated diabetic db/db mice had reduced concentrations of HbA1c and blood glucose levels. Exogenous glucose was quickly cleared in glucose tolerance tests. The mRNA expressions of genes for gluconeogenesis-related enzymes, glucose 6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK) were significantly reduced in the liver of SA-treated diabetic db/db mice. In primary hepatocytes, SA treatment decreased glucose production and the expression of G6Pase, PEPCK, and hepatocyte nuclear factor 4 alpha (HNF-4α) mRNA. Small heterodimer partner (SHP) mRNA expression was increased in hepatocytes dependent upon the SA concentration. The expression of Sirt1 mRNA and protein was reduced, and acetylated forkhead box protein O1 (FoxO1) was induced by SA treatment in hepatocytes. In addition, SA-treated diabetic db/db mice showed reduced energy expenditure. Oral intubation of SA ameliorates hyperglycemia in db/db mice by reducing hepatic gluconeogenesis through the decrease of Sirt1 expression and increase in acetylated FoxO1.

Maf1 is a negative regulator of RNA polymerase III (Pol III) in yeast. Maf1-depleted cells manifest elevated tRNA transcription and inability to grow on non-fermentable carbon source, such as glycerol. Using genomic microarray approach, we examined the effect of Maf1 deletion on expression of Pol II-transcribed genes in yeast grown in medium containing glycerol. We found that transcription of FBP1 and PCK1, two major genes controlling gluconeogenesis, was decreased in maf1Δ cells. FBP1 is located on chromosome XII in close proximity to a tRNA-Lys gene. Accordingly we hypothesized that decreased FBP1 mRNA level could be due to the effect of Maf1 on tgm silencing (tRNA gene mediated silencing). Two approaches were used to verify this hypothesis. First, we inactivated tRNA-Lys gene on chromosome XII by inserting a deletion cassette in a control wild type strain and in maf1Δ mutant. Second, we introduced a point mutation in the promoter of the tRNA-Lys gene cloned with the adjacent FBP1 in a plasmid and expressed in fbp1Δ or fbp1Δ maf1Δ cells. The levels of FBP1 mRNA were determined by RT-qPCR in each strain. Although the inactivation of the chromosomal tRNA-Lys gene increased expression of the neighboring FBP1, the mutation preventing transcription of the plasmid-born tRNA-Lys gene had no significant effect on FBP1 transcription. Taken together, those results do not support the concept of tgm silencing of FBP1. Other possible mechanisms are discussed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background & Aims: Hepatic gluconeogenesis helps maintain systemic energy homeostasis by compensating for discontinuities in nutrient supply. Liver-specific deletion of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) abolishes gluconeogenesis from mitochondrial substrates, deregulates lipid metabolism and affects TCA cycle. While the mouse liver almost exclusively expresses PEPCK-C, humans equally present a mitochondrial isozyme (PEPCK-M). Despite clear relevance to human physiology, the role of PEPCK-M and its gluconeogenic potential remain unknown. Here, we test the significance of PEPCK-M in gluconeogenesis and TCA cycle function in liver-specific PEPCK-C knockout and WT mice., Methods: The effects of the overexpression of PEPCK-M were examined by a combination of tracer studies and molecular biology techniques. Partial PEPCK-C re-expression was used as a positive control. Metabolic fluxes were evaluated in isolated livers by NMR using (2)H and (13)C tracers. Gluconeogenic potential, together with metabolic profiling, was investigated in vivo and in primary hepatocytes., Results: PEPCK-M expression partially rescued defects in lipid metabolism, gluconeogenesis and TCA cycle function impaired by PEPCK-C deletion, while ∼10% re-expression of PEPCK-C normalized most parameters. When PEPCK-M was expressed in the presence of PEPCK-C, the mitochondrial isozyme amplified total gluconeogenic capacity, suggesting autonomous regulation of oxaloacetate to phosphoenolpyruvate fluxes by the individual isoforms., Conclusions: We conclude that PEPCK-M has gluconeogenic potential per se, and cooperates with PEPCK-C to adjust gluconeogenic/TCA flux to changes in substrate or energy availability, hinting at a role in the regulation of glucose and lipid metabolism in the human liver., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Lectins are proteins that bind specifically to the carbohydrate moiety of glyco-conjugates. Japanese mistletoe lectin given intragastrically affected cytokine gene expression in the mouse intestine. This study examines the actions of Lens culinaris agglutinin (LCA) on the gene expression of gluconeogenic enzymes in the intestine., Results: The results of quantitative real-time reverse transcription-polymerase chain reaction indicated that LCA caused an up-regulation of the gene expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). This change was correlated with an increase in the expression of two transcription factors, HNF1α and HNF4α. Experiments using human colonic cancer Caco-2 cells demonstrated that LCA up-regulated the gene expression of G6Pase and PEPCK whereas insulin had the opposite effect. In addition, the observed up-regulation of HNF4α gene expression in the duodenum raises the possibility that the lectin promotes the colorectal cancer., Conclusion: Lentil beans should be cooked well to avoid unfavourable effects of LCA., (Copyright © 2011 Society of Chemical Industry.)

Tea has many beneficial effects. We have previously reported that green tea and a catechin-rich green tea beverage modulated the gene expression of the gluconeogenic enzymes glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the normal murine liver. In the present study, we examined the effects of oral administration of oolong tea on the hepatic expression of gluconeogenesis-related genes in the mouse. The intake of oolong tea for 4 weeks reduced the hepatic expression of G6Pase and PEPCK together with that of the transcription factor hepatocyte nuclear factor (HNF) 4α. When rat hepatoma H4IIE cells were incubated in the presence of oolong tea, the expression of these genes was repressed in accordance with the findings in vivo. The reduced protein expression of PEPCK and HNF4α was also demonstrated. We then fractionated oolong tea by sequential extraction with three organic solvents to give three fractions and the residual fraction (Fraction IV). In addition to organic fractions, Fraction IV, which was devoid of low-molecular-weight catechins such as (-)-epigallocatechin gallate (EGCG), had effects similar to those of oolong tea on H4IIE cells. Fraction IV repressed the gene expression of insulin-like growth factor binding protein 1, as insulin did. This activity was different from that of EGCG. The present findings suggest that drinking oolong tea may help to prevent diabetes and that oolong tea contains a component or components with insulin-like activity distinguishable from EGCG. Identification of such component(s) may open the way to developing a new drug for diabetes.

Many biological activities of green tea have been attributed to a major constituent, (-)-epigallocatechin gallate (EGCG). We previously reported that EGCG and a catechin-rich green tea beverage modulated the gene expression of gluconeogenic enzymes, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), in the mouse liver. However, it remains to be examined whether or not a constituent other than EGCG contributes to the change in gene expression of these enzymes. In this study, we separated the hot water infusion of green tea leaves (GT) into an ethanol-soluble fraction (GT-E) and an EGCG-free water-soluble fraction (GT-W), and examined their effects using rat hepatoma H4IIE cells. The inclusion of GT, GT-E, and GT-W in the culture medium reduced the gene expression of G6Pase and PEPCK. GT-W caused a decrease in expression of the transcription factor HNF4α. Reduced levels of PEPCK and HNF4α proteins were demonstrated in the cells treated with GT-W. GT-W showed an activity similar to insulin, but different from EGCG. Administration of GT-W to mice for 4 weeks reduced the hepatic expression of G6Pase, PEPCK, and HNF4α. These results suggest that green tea contains some component(s) with insulin-like activity distinguishable from EGCG and that drinking green tea may help to prevent diabetes.

In this study, we investigated the effects of a petroleum ether fraction of Artemisia sacrorum Ledeb. (Compositae) (PEASL) on glucose production through AMP-activated protein kinase (AMPK) activation in human HepG2 cells. PEASL significantly inhibited glucose production in a concentration-dependent manner, and this effect was reversed in the presence of compound C, a selective AMPK inhibitor. PEASL markedly induced the phosphorylation of AMPK and downstream acetyl-CoA carboxylase (ACC) in a time- and concentration-dependent manner. In addition, it markedly increased the phosphorylations of glycogen synthase kinase 3β (GSK3β) in a concentration-dependent manner. In contrast, cAMP response element binding protein (CREB), a key transcription factor for gluconeogenic enzyme phosphorylation, decreased in a concentration-dependent manner. PEASL downregulated the gluconeogenesis gene expression of peroxisome proliferation activated receptor-γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase) in a concentration-dependent manner. In addition, the gene expression of orphan nuclear receptor small heterodimer partner (SHP) increased, also in a concentration-dependent manner. These effects were also abolished by pretreatment with compound C, an AMPK inhibitor. This indicates that PEASL inhibited glucose production via the AMPK-GSK-CREB pathway in HepG2 cells, and these effects appeared to be capable of revealing anti-diabetic mechanism of PEASL in HepG2 cells.

Orphan nuclear receptor small heterodimer partner (SHP) plays a key role in transcriptional repression of gluconeogenic enzyme gene expression. Here, we show that SHP inhibited protein kinase A-mediated transcriptional activity of cAMP-response element-binding protein (CREB), a major regulator of glucose metabolism, to modulate hepatic gluconeogenic gene expression. Deletion analysis of phosphoenolpyruvate carboxykinase (PEPCK) promoter demonstrated that SHP inhibited forskolin-mediated induction of PEPCK gene transcription via inhibition of CREB transcriptional activity. In vivo imaging demonstrated that SHP inhibited CREB-regulated transcription coactivator 2 (CRTC2)-mediated cAMP-response element-driven promoter activity. Furthermore, overexpression of SHP using adenovirus SHP decreased CRTC2-dependent elevations in blood glucose levels and PEPCK or glucose-6-phosphatase (G6Pase) expression in mice. SHP and CREB physically interacted and were co-localized in vivo. Importantly, SHP inhibited both wild type CRTC2 and S171A (constitutively active form of CRTC2) coactivator activity and disrupted CRTC2 recruitment on the PEPCK gene promoter. In addition, metformin or overexpression of a constitutively active form of AMPK (Ad-CA-AMPK) inhibited S171A-mediated PEPCK and G6Pase gene expression, and hepatic glucose production and knockdown of SHP partially relieved the metformin- and Ad-CA-AMPK-mediated repression of hepatic gluconeogenic enzyme gene expression in primary rat hepatocytes. In conclusion, our results suggest that a delayed effect of metformin-mediated induction of SHP gene expression inhibits CREB-dependent hepatic gluconeogenesis.

Metformin is widely used to treat hyperglycemia in individuals with type 2 diabetes. Recently the LKB1/AMP-activated protein kinase (LKB1/AMPK) pathway was proposed to mediate the action of metformin on hepatic gluconeogenesis. However, the molecular mechanism by which this pathway operates had remained elusive. Surprisingly, here we have found that in mice lacking AMPK in the liver, blood glucose levels were comparable to those in wild-type mice, and the hypoglycemic effect of metformin was maintained. Hepatocytes lacking AMPK displayed normal glucose production and gluconeogenic gene expression compared with wild-type hepatocytes. In contrast, gluconeogenesis was upregulated in LKB1-deficient hepatocytes. Metformin decreased expression of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), while cytosolic phosphoenolpyruvate carboxykinase (Pepck) gene expression was unaffected in wild-type, AMPK-deficient, and LKB1-deficient hepatocytes. Surprisingly, metformin-induced inhibition of glucose production was amplified in both AMPK- and LKB1-deficient compared with wild-type hepatocytes. This inhibition correlated in a dose-dependent manner with a reduction in intracellular ATP content, which is crucial for glucose production. Moreover, metformin-induced inhibition of glucose production was preserved under forced expression of gluconeogenic genes through PPARgamma coactivator 1alpha (PGC-1alpha) overexpression, indicating that metformin suppresses gluconeogenesis via a transcription-independent process. In conclusion, we demonstrate that metformin inhibits hepatic gluconeogenesis in an LKB1- and AMPK-independent manner via a decrease in hepatic energy state.

Rat hepatoma H4IIE cells were stimulated with dexamethasone and dibutyryl cAMP to increase gene expressions of gluconeogenic enzymes, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Inclusion of catechin-rich green tea beverage (GTB) in the culture medium reduced the up-regulation of these genes as well as that of hepatocyte nuclear factor 4 alpha (HNF4alpha) gene. GTB was fractionated into chloroform-soluble (Fraction I), ethyl acetatesoluble (Fraction II), methanol-soluble (Fraction III) and residual (Fraction IV) fractions. Fractions II and III containing catechins caused an attenuation of the up-regulated expression of these genes as well as the down-regulation of HNF4alpha gene expression. Fraction IV had a synergistic effect on the up-regulation by dexamethasone/dibutyryl cAMP of the PEPCK gene expression and upregulated HNF4alpha gene expression. These results suggest that GTB down-regulated the expression of the HNF4alpha gene to cause the down-regulated gene expression of gluconeogenic enzymes. One reason why GTB did not down-regulate hepatic PEPCK gene expression in previous animal experiments may be that the component(s) acting to up-regulate PEPCK gene expression was more effective in vivo than in cultured cells.

The regulation of expression of gluconeogenic genes including glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the liver plays an important role in glucose homeostasis, because aberrant expression of these genes contributes to the development of type 2 diabetes. Previous reports demonstrate that signal transducer and activator of transcription 3 (STAT3) plays a key role in regulating gluconeogenic gene expression, but the mechanism remains unclear. Herein we demonstrate that phosphorylated STAT3 is required for repression of G6Pase expression by IL-6 in both HepG2 cells and mouse liver. Interestingly, PEPCK expression is regulated by STAT3 independent of IL-6 activation. Using in vivo chromatin immunoprecipitation, we demonstrate that STAT3 binds to the promoters of the G6Pase, PEPCK, and suppressor of cytokine signaling (SOCS)3 genes, and its recruitment increases at the G6Pase and SOCS3 promoters with IL-6 treatment. Whereas persistent recruitment of RNA polymerase II is seen on the SOCS3 promoter, consistent with its induction by IL-6, a decrease in polymerase II recruitment and histone H4 acetylation is seen at the G6Pase promoter with IL-6 treatment. Thus STAT3 mediates negative regulation of hepatic gluconeogenic gene expression in vivo by interacting with regulatory regions of these genes.

Hepatic gluconeogenesis is elevated in diabetes and a major contributor to hyperglycemia. Stromal cell-derived factor-1 (SDF-1) is a chemokine and an activator of Akt. In this study, we tested the hypothesis that SDF-1 suppresses hepatic gluconeogenesis through Akt. Our results from isolated primary hepatocytes show that SDF-1alpha and SDF-1beta inhibited glucose production via gluconeogenesis and reduced transcript levels of key gluconeogenic genes glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Additionally, SDF-1alpha and SDF-1beta both inhibited activation of the PEPCK promoter. In examining the mechanism by which SDF-1 inhibits gluconeogenesis, we found that SDF-1 promoted phosphorylation of Akt, FoxO1, and c-Src, but did not activate insulin receptor substrate-1-like insulin. Blockade of Akt activation by LY294002, FoxO1 translocation by constitutively nuclear FoxO1 mutant, or c-Src activation by the chemical inhibitor PP2, respectively, blunted SDF-1 suppression of gluconeogenesis. Finally, our results show that knocking down the level of SDF-1 receptor CXCR4 mRNA blocked SDF-1 suppression of gluconeogenesis. Together, our results demonstrate that SDF-1 is capable of inhibiting gluconeogenesis in primary hepatocytes through a signaling pathway distinct from the insulin signaling.

Ethnopharmacological Relevance: Fructus Corni, the fruits of Cornus officinalis Sieb. et Zucc., is one important ingredient in Quei Fu Di Huang Wan, a Chinese herbal mixture., Aim of the Study: In the present study, additional anti-diabetic actions of Fructus Corni on transcriptional regulation of hepatic gluconeogenesis or beta-cell functions were investigated., Materials and Methods: Insulin mimetic action of Fructus Corni on dexamethasone and 8-bromo-cAMP induced phosphoenolpyruvate carboxykinase (PEPCK) expression in H4IIE cells was investigated. Besides, BRIN-BD11 cells were used to evaluate both insulinotropic and beta-cell protective effect of Fructus Corni., Results: Firstly, both methanol extract (CO-W-M) and fraction (CO-W-M2) had potent insulin mimic activity on PEPCK expression. Secondly, possibility of both loganin and ursolic acid as the responsible compounds was excluded. Moreover, indication of the existence of phenolic compounds in CO-W-M2 was noticed. In the presence of CO-W-M2, not only was the viability of BRIN-BD11 cells treated with alloxan, streptozotcin, or cytokine mix all significantly increased but also glucose-stimulated insulin secretion was potentiated., Conclusions: The ability of CO-W-M2 to reduce gene expression for hepatic gluconeogenesis, to protect beta-cell against toxic challenge, and to enhance insulin secretion strengthen the role of Fructus Corni in diabetes therapy.

Objective: To observe the effect of calorie restriction on the high fat diet rats mRNA expressions of liver forkhead box O1(FoxO1), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G-6-P) and to explore the possible mechanisms., Methods: 24 normal 6-week-old male Wistar rats were randomly divided into three groups: normal chow group (NC, n = 7), high fat diet group (HF, n = 9) and calorie restriction group (CR, n = 8). They were fed for 12 weeks. At the end of the experiment, the rats were sacrificed and their fasting blood glucose (FBG), insulin (INS), triglycerides (TG), total cholesterol (TC) were measured. Their visceral fat (VF) and body weight (BW) were also measured and VF/BW was calculated. Gene expression was investigated by using semi-quantitative RT-PCR methods. Liver histology was studied with HE stained slides., Results: Compared with the NC group, HF group rats developed visceral obesity which was accompanied by higher FBG, plasma INS, TG, and TC. The levels of FoxO1, PEPCK, and G-6-P increased by 18.9%, 33.8%, and 24.6%, respectively (P less than 0.01). Liver steatosis was observed with microscopy. The BW, VF FBG, INS, TG and TC of the CR group rats were lower in comparison to those of the HF group. The levels of FoxO1, PEPCK and G-6-P were lower by 26.6%, 35.0%, 34.3% (P less than 0.01). Meanwhile, liver steatosis was also milder., Conclusion: Calorie restriction can inhibit the expressions of FoxO1, PEPCK and G-6-P, strengthen insulin signal conduction, suppress gluconeogenesis and thus regulate glycometabolism.

Isoleucine, a branched chain amino acid, plays an important role in the improvement of glucose metabolism as evidenced by the increase of insulin-independent glucose uptake in vitro. This study evaluated the effect of isoleucine on glucose uptake and oxidation in fasted rats and on gluconeogenesis in vivo and in vitro. Oral administration of isoleucine decreased the plasma glucose level by 20% and significantly increased muscle glucose uptake by 71% without significant elevation of the plasma insulin level compared with controls at 60 min after administration. Furthermore, expiratory excretion of 14CO2 from [U-14C]glucose in isoleucine-administered rats was increased by 19% compared with controls. Meanwhile, isoleucine decreased AMP levels in the liver but did not affect hepatic glycogen synthesis. Under insulin-free conditions, isoleucine significantly inhibited glucose production when alanine was used as a glucogenic substrate in isolated hepatocytes. This inhibition by isoleucine was also associated with a decline in mRNA levels for phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase) and a decreased activity of G6Pase in isolated hepatocytes. These findings suggest that a reduction of gluconeogenesis in liver, along with an increase of glucose uptake in the muscle, is also involved in the hypoglycemic effect of isoleucine. In conclusion, isoleucine administration stimulates both glucose uptake in the muscle and whole body glucose oxidation, in addition to depressing gluconeogenesis in the liver, thereby leading to the hypoglycemic effect in rats.

SHP (short heterodimer partner) is an orphan nuclear receptor that plays an important role in regulating glucose and lipid metabolism. A variety of transcription factors are known to regulate transcription of the PEPCK (phosphoenolpyruvate carboxykinase) gene, which encodes a rate-determining enzyme in hepatic gluconeogenesis. Previous reports identified glucocorticoid receptor and Foxo1 as novel downstream targets regulating SHP inhibition [Borgius, Steffensen, Gustafsson and Treuter (2002) J. Biol. Chem. 277, 49761-49796; Yamagata, Daitoku, Shimamoto, Matsuzaki, Hirota, Ishida and Fukamizu (2004) J. Biol. Chem. 279, 23158-23165]. In the present paper, we show a new molecular mechanism of SHP-mediated inhibition of PEPCK transcription. We also show that the CRE1 (cAMP regulatory element 1; -99 to -76 bp relative to the transcription start site) of the PEPCK promoter is also required for the inhibitory regulation by SHP. SHP repressed C/EBPalpha (CCAAT/enhancer-binding protein alpha)-driven transcription of PEPCK through direct interaction with C/EBPalpha protein both in vitro and in vivo. The formation of an active transcriptional complex of C/EBPalpha and its binding to DNA was inhibited by SHP, resulting in the inhibition of PEPCK gene transcription. Taken together, these results suggest that SHP might regulate a level of hepatic gluconeogenesis driven by C/EBPalpha activation.