Hepatic gluconeogenesis is essential for glucose homeostasis and also a therapeutic target for type 2 diabetes, but its mechanism is incompletely understood. Here, we report that Sam68, an RNA-binding adaptor protein and Src kinase substrate, is a novel regulator of hepatic gluconeogenesis. Both global and hepatic deletions of Sam68 significantly reduce blood glucose levels and the glucagon-induced expression of gluconeogenic genes. Protein, but not mRNA, levels of CRTC2, a crucial transcriptional regulator of gluconeogenesis, are >50% lower in Sam68-deficient hepatocytes than in wild-type hepatocytes. Sam68 interacts with CRTC2 and reduces CRTC2 ubiquitination. However, truncated mutants of Sam68 that lack the C- (Sam68ΔC) or N-terminal (Sam68ΔN) domains fails to bind CRTC2 or to stabilize CRTC2 protein, respectively, and transgenic Sam68ΔN mice recapitulate the blood-glucose and gluconeogenesis profile of Sam68-deficient mice. Hepatic Sam68 expression is also upregulated in patients with diabetes and in two diabetic mouse models, while hepatocyte-specific Sam68 deficiencies alleviate diabetic hyperglycemia and improves insulin sensitivity in mice. Thus, our results identify a role for Sam68 in hepatic gluconeogenesis, and Sam68 may represent a therapeutic target for diabetes.

Fasting hyperglycemia in diabetes mellitus is caused by unregulated glucagon secretion that activates gluconeogenesis (GNG) and increases the use of pyruvate, lactate, amino acids, and glycerol. Studies of GNG in hepatocytes, however, tend to test a limited number of substrates at nonphysiologic concentrations. Therefore, we treated cultured primary hepatocytes with three identical substrate mixtures of pyruvate/lactate, glutamine, and glycerol at serum fasting concentrations, where a different U-13C- or 2-13C-labeled substrate was substituted in each mix. In the absence of glucagon stimulation, 80% of the glucose produced in primary hepatocytes incorporated either one or two 13C-labeled glycerol molecules in a 1:1 ratio, reflecting the high overall activity of this pathway. In contrast, glucose produced from 13C-labeled pyruvate/lactate or glutamine rarely incorporated two labeled molecules. While glucagon increased the glycerol and pyruvate/lactate contributions to glucose carbon by 1.6- and 1.8-fold, respectively, the glutamine contribution to glucose carbon was increased 6.4-fold in primary hepatocytes. To account for substrate 13C carbon loss during metabolism, we also performed a metabolic flux analysis, which confirmed that the majority of glucose carbon produced by primary hepatocytes was from glycerol. In vivo studies using a PKA-activation mouse model that represents elevated glucagon activity confirmed that most circulating lactate carbons originated from glycerol, but very little glycerol was derived from lactate carbons, reflecting glycerol's importance as a carbon donor to GNG. Given the diverse entry points for GNG substrates, hepatic glucagon action is unlikely to be due to a single mechanism.

The liver performs a central role in regulating energy homeostasis by increasing glucose output during fasting. Recent studies on Argonaute2 (Ago2), a key RNA-binding protein mediating the microRNA pathway, have illustrated its role in adaptive mechanisms according to changes in metabolic demand. Here we sought to characterize the functional role of Ago2 in the liver in the maintenance of systemic glucose homeostasis.

Metformin has been widely used to alleviate hyperglycemia in patients with type 2 diabetes mainly via suppressing hepatic gluconeogenesis. However, the underlying mechanism remains incompletely clear. Here, we aimed to explore the role of PPP1R3C in metformin-mediated inhibition of hepatic gluconeogenesis.

The physiological and metabolic functions of PIMT/TGS1, a third-generation transcriptional apparatus protein, in glucose homeostasis sustenance are unclear. Here, we observed that the expression of PIMT was upregulated in the livers of short-term fasted and obese mice. Lentiviruses expressing Tgs1-specific shRNA or cDNA were injected into wild-type mice. Gene expression, hepatic glucose output, glucose tolerance, and insulin sensitivity were evaluated in mice and primary hepatocytes. Genetic modulation of PIMT exerted a direct positive impact on the gluconeogenic gene expression program and hepatic glucose output. Molecular studies utilizing cultured cells, in vivo models, genetic manipulation, and PKA pharmacological inhibition establish that PKA regulates PIMT at post-transcriptional/translational and post-translational levels. PKA enhanced 3'UTR-mediated translation of TGS1 mRNA and phosphorylated PIMT at Ser656, increasing Ep300-mediated gluconeogenic transcriptional activity. The PKA-PIMT-Ep300 signaling module and associated PIMT regulation may serve as a key driver of gluconeogenesis, positioning PIMT as a critical hepatic glucose sensor.

Hepatic gluconeogenesis is required for maintaining blood glucose homeostasis; yet, in diabetes mellitus, this process is unrestrained and is a major contributor to fasting hyperglycemia. To date, the impacts of chromatin modifying enzymes and chromatin landscape on gluconeogenesis are poorly understood. Through catalyzing the removal of methyl groups from specific lysine residues in the histone tail, histone demethylases modulate chromatin structure and, hence, gene expression. Here we perform an RNA interference screen against the known histone demethylases and identify a histone H3 lysine 36 (H3K36) demethylase, Jhdm1a, as a key negative regulator of gluconeogenic gene expression. In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level. Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity. Mechanistically, we find that Jhdm1a regulates the expression of a major gluconeogenic regulator, C/EBPα. This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus. Our work provides compelling evidence that links histone demethylation to transcriptional regulation of gluconeogenesis and has important implications for the treatment of diabetes.

For rapid tumor growth, cancer cells often reprogram the cellular metabolic processes to obtain enhanced anabolic precursors and energy. The molecular changes of such metabolic rewiring are far from established. Here we explored the role of mTOR (mechanistic target of rapamycin), which serves as a key regulator of cell growth, proliferation and survival, in the metabolic reprograming of cancer cells. When we inhibited mTOR in human hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) cells, using pharmacologic inhibitors or by RNA interference, we noticed shuttle of the glycolytic flux to gluconeogenesis pathway along with reduction in cellular proliferation and survival. Augmentation of gluconeogenesis was mechanistically linked to upregulation of the key gluconeogenic enzymes PCK1 and G6PC expressions, enhanced lactate dehydrogenase activity and glucose-derived lipogenesis without causing any attenuation in mitochondrial function. Interestingly, concomitant knocking down of PCK1 and not G6PC along with mTOR pathway could overcome the inhibition of cancer cell proliferation and survival. These observations were validated by identifying distinctive diminution of PCK1 and G6PC expressions in human HCC and RCC transcriptome data. Significant correlation between mTOR-dependent upregulation of PCK1 and cell death in different cancer cell lines further emphasizes the physiological relevance of this pathway. We reveal for the first time that inhibition of mTORC2 and consequent redistribution of glycolytic flux can have a prosurvival role in HCC and RCC cancer cells only in the presence of downregulation of gluconeogenesis pathway genes, thus identifying novel pivots of cancer cell metabolic rewiring and targets for therapy.

Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin's blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes.

In the postabsorptive state, certain tissues, including the brain, require glucose as the sole source of energy. After an overnight fast, hepatic glycogen stores are depleted, and gluconeogenesis becomes essential for preventing life-threatening hypoglycemia. Mice with a targeted deletion of KLF15, a member of the Krüppel-like family of transcription factors, display severe hypoglycemia after an overnight (18 hr) fast. We provide evidence that defective amino acid catabolism promotes the development of fasting hypoglycemia in KLF15-/- mice by limiting gluconeogenic substrate availability. KLF15-/- liver and skeletal muscle show markedly reduced mRNA expression of amino acid-degrading enzymes. Furthermore, the enzymatic activity of alanine aminotransferase (ALT), which converts the critical gluconeogenic amino acid alanine into pyruvate, is decreased (approximately 50%) in KLF15-/- hepatocytes. Consistent with this observation, intraperitoneal injection of pyruvate, but not alanine, rescues fasting hypoglycemia in KLF15-/- mice. We conclude that KLF15 plays an important role in the regulation of gluconeogenesis.

Epidemiological evidence indicates that thyroid stimulating hormone (TSH) is positively correlated with abnormal glucose levels. We previously reported that TSH has direct effects on gluconeogenesis. However, the underlying molecular mechanism remains unclear. In this study, we observed increased fasting blood glucose and glucose production in a mouse model of subclinical hypothyroidism (only elevated TSH levels). TSH acts via the classical cAMP/PKA pathway and CRTC2 regulates glucose homeostasis. Thus, we explore whether CRTC2 is involved in the process of TSH-induced gluconeogenesis. We show that TSH increases CRTC2 expression via the TSHR/cAMP/PKA pathway, which in turn upregulates hepatic gluconeogenic genes. Furthermore, TSH stimulates CRTC2 dephosphorylation and upregulates p-CREB (Ser133) in HepG2 cells. Silencing CRTC2 and CREB decreases the effect of TSH on PEPCK-luciferase, the rate-limiting enzyme of gluconeogenesis. Finally, the deletion of TSHR reduces the levels of the CRTC2:CREB complex in mouse livers. This study demonstrates that TSH activates CRTC2 via the TSHR/cAMP/PKA pathway, leading to the formation of a CRTC2:CREB complex and increases hepatic gluconeogenesis.

FoxO1 and FoxO3a (collectively FoxO1/3a) proteins regulate a wide array of cellular processes, including hepatic gluconeogenesis. Phosphorylation of FoxO1/3a is a key event that determines its subcellular location and transcriptional activity. During glucose synthesis, the activity of FoxO1/3a is negatively regulated by Akt-mediated phosphorylation, which leads to the cytoplasmic retention of FoxO1/3a. However, the nuclear phosphatase that directly regulates FoxO1/3a remains to be identified. In this study, we discovered a nuclear phosphatase, SCP4/CTDSPL2 (SCP4), that dephosphorylated FoxO1/3a and promoted FoxO1/3a transcription activity. We found that SCP4 enhanced the transcription of FoxO1/3a target genes encoding PEPCK1 and G6PC, key enzymes in hepatic gluconeogenesis. Ectopic expression of SCP4 increased, while knockdown of SCP4 inhibited, glucose production. Moreover, we demonstrated that gene ablation of SCP4 led to hypoglycemia in neonatal mice. Consistent with the positive role of SCP4 in gluconeogenesis, expression of SCP4 was regulated under pathophysiological conditions. SCP4 expression was induced by glucose deprivation in vitro and in vivo and was elevated in obese mice caused by genetic (Avy) and dietary (high-fat) changes. Thus, our findings provided experimental evidence that SCP4 regulates hepatic gluconeogenesis and could serve as a potential target for the prevention and treatment of diet-induced glucose intolerance and type 2 diabetes.

Gluconeogenesis is a complex metabolic process that involves multiple enzymatic steps regulated by myriad factors, including substrate concentrations, the redox state, activation and inhibition of specific enzyme steps, and hormonal modulation. At present, the most widely accepted technique to determine gluconeogenesis is by measuring the incorporation of deuterium from the body water pool into newly formed glucose. However, several techniques using radioactive and stable-labeled isotopes have been used to quantitate the contribution and regulation of gluconeogenesis in humans. Each method has its advantages, methodological assumptions, and set of propagated errors. In this review, we examine the strengths and weaknesses of the most commonly used stable isotopes methods to measure gluconeogenesis in vivo. We discuss the advantages and limitations of each method and summarize the applicability of these measurements in understanding normal and pathophysiological conditions.

The regulation of energy metabolism, such as calorie restriction (CR), is a major determinant of cellular longevity. Although augmented gluconeogenesis is known to occur in aged yeast cells, the role of enhanced gluconeogenesis in aged cells remains undefined. Here, we show that age-enhanced gluconeogenesis is suppressed by the deletion of the tdh2 gene, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein that is involved in both glycolysis and gluconeogenesis in yeast cells. The deletion of TDH2 restores the chronological lifespan of cells with deletions of both the HST3 and HST4 genes, which encode yeast sirtuins, and represses the activation of gluconeogenesis. Furthermore, the tdh2 gene deletion can extend the replicative lifespan in a CR pathway-dependent manner. These findings demonstrate that the repression of enhanced gluconeogenesis effectively extends the cellular lifespan.

1. Experiments performed on isolated hepatocytes and perfused liver of starved chickens showed that gluconeogenesis from lactate, glycerol and fructose was inhibited by 22-100% on addition of urate precursors. 2. The inhibition was associated with an increased rate of urate formation. 3. 2,4-Dinitrophenol (40 microM), 2-bromooctanoate (2 mM) and 3-mercaptopicolinate (3MPA) (0.5 mM) were inhibitory with respect to gluconeogenesis but did not significantly affect the rate of urate formation. 4. The possible interrelationships between gluconeogenesis and uricogenesis are considered in terms of a competition for ATP and for other metabolites between the two pathways. 5. An interplay of both pathways at the level of anion transfer across the inner mitochondrial membrane is also discussed.

Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation-all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment-reversing hepatic steatosis and glucose intolerance-were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes.

No abstract available

The aim of this study was to determine whether resveratrol (Rev) affects the expression, phosphorylation, and nuclear and cytoplasmic distribution of histone deacetylase 4 (HDAC4), which in turn affects gluconeogenesis in hepatocytes under an insulin-resistant state.

Butyrate is a major short-chain fatty acid (SCFA) produced by microbial fermentation of dietary fiber in the gastrointestinal tract. Butyrate is also a well-known broad-spectrum histone deacetylase (HDAC) inhibitor. Butyrate has been reported to improve energy metabolism in rodents, which is associated with its beneficial effects on skeletal muscle, brown fat tissue and pancreatic β-cells. The present study investigated the direct effect of butyrate on hepatic gluconeogenesis in mouse primary hepatocytes and the underlying mechanism. Isolated mouse primary hepatocytes were incubated with sodium butyrate, other HDAC inhibitors and other SCFAs. Hepatic glucose production was measured and gluconeogenic gene expression was detected by polymerase chain reaction analysis. The phosphorylation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) was assessed by western blot analysis. The results revealed that sodium butyrate dose-dependently increased hepatic glucose production and gluconeogenic gene expression in isolated mouse primary hepatocytes. Trichostatin A, a potent broad-spectrum HDAC inhibitor, had the opposite effect. Similar to sodium butyrate, propionate, which is another SCFA, promoted hepatic glucose production and gluconeogenic gene expression in the presence or absence of gluconeogenic substrates, which were further enhanced by cAMP. Furthermore, sodium butyrate also increased the accumulation of intracellular ATP and induced the phosphorylation of CREB in mouse hepatocytes. In conclusion, the present study suggested that butyrate stimulates hepatic gluconeogenesis and induces gluconeogenic gene expression as a substrate and cAMP/CREB signaling activator.

Excessive glucose production by the liver is a key factor in the hyperglycemia observed in type 2 diabetes mellitus (T2DM). Here, we highlight a novel role of liver kinase B1 (Lkb1) in this regulation. We show that mice with a hepatocyte-specific deletion of Lkb1 have higher levels of hepatic amino acid catabolism, driving gluconeogenesis. This effect is observed during both fasting and the postprandial period, identifying Lkb1 as a critical suppressor of postprandial hepatic gluconeogenesis. Hepatic Lkb1 deletion is associated with major changes in whole-body metabolism, leading to a lower lean body mass and, in the longer term, sarcopenia and cachexia, as a consequence of the diversion of amino acids to liver metabolism at the expense of muscle. Using genetic, proteomic and pharmacological approaches, we identify the aminotransferases and specifically Agxt as effectors of the suppressor function of Lkb1 in amino acid-driven gluconeogenesis.

Mitochondria provide essential metabolites and adenosine triphosphate (ATP) for the regulation of energy homeostasis. For instance, liver mitochondria are a vital source of gluconeogenic precursors under a fasted state. However, the regulatory mechanisms at the level of mitochondrial membrane transport are not fully understood. Here, we report that a liver-specific mitochondrial inner-membrane carrier SLC25A47 is required for hepatic gluconeogenesis and energy homeostasis. Genome-wide association studies found significant associations between SLC25A47 and fasting glucose, HbA1c, and cholesterol levels in humans. In mice, we demonstrated that liver-specific depletion of SLC25A47 impaired hepatic gluconeogenesis selectively from lactate, while significantly enhancing whole-body energy expenditure and the hepatic expression of FGF21. These metabolic changes were not a consequence of general liver dysfunction because acute SLC25A47 depletion in adult mice was sufficient to enhance hepatic FGF21 production, pyruvate tolerance, and insulin tolerance independent of liver damage and mitochondrial dysfunction. Mechanistically, SLC25A47 depletion leads to impaired hepatic pyruvate flux and malate accumulation in the mitochondria, thereby restricting hepatic gluconeogenesis. Together, the present study identified a crucial node in the liver mitochondria that regulates fasting-induced gluconeogenesis and energy homeostasis.