Pathogenic Potential of a PCK1 Gene Variant in Cytosolic PEPCK Deficiency: A Compelling Case Study.

BACKGROUND Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) deficiency is an extremely rare autosomal recessive inherited error of metabolism in which gluconeogenesis is impaired, resulting in life-threatening episodes of hypoglycemia and metabolic acidosis. The diagnosis of gluconeogenesis disorders is challenging. In the diagnostic pathway, the molecular test plays a paramount role. CASE REPORT The aim of the paper is to present the case report of a girl with recurrent episodes of severe hypoglycemia, in whom molecular diagnosis enabled the confirmation of PEPCK - C deficiency. The patient experienced 4 episodes of severe hypoglycemia. Most of them were accompanied by hyperlacticaemia, metabolic acidosis, and elevated liver enzymes. All of the metabolic decompensations were triggered by infectious agents. The episodes resolved after continuous infusion of high-dose glucose. Due to the recurrent character of the disease, a genetic condition was suspected. The differential diagnosis included metabolic and endocrinological causes of hypoglycemia. Two variants in the PCK1 gene were detected: c.265G>A p.(Glu89Lys) in exon 3 and c.925G>A p.(Gly309Arg) in exon 6. As c.925G>A p.(Gly309Arg) is a known pathogenic variant, the second variant was first described in June 2023 in the ClinVar database and described as "with unknown clinical significance". CONCLUSIONS According to the clinical symptoms observed in the presented case, the variant c.265G>A p.(Glu89Lys) in PCK1 gene should be considered likely pathogenic. We suggest considering molecular diagnostics in every patient presented with recurrent, severe hypoglycemia with accompanying liver damage as most accurate, feasible, and reliable method.

Antiobesity effects of intestinal gluconeogenesis are mediated by the brown adipose tissue sympathetic nervous system.

OBJECTIVE: Intestinal gluconeogenesis (IGN), via the initiation of a gut-brain nervous circuit, accounts for the metabolic benefits linked to dietary proteins or fermentable fiber in rodents and has been positively correlated with the rapid amelioration of body weight after gastric bypass surgery in humans with obesity. In particular, the activation of IGN moderates the development of hepatic steatosis accompanying obesity. In this study, we investigated the specific effects of IGN on adipose tissue metabolism, independent of its induction by nutritional manipulation. METHODS: We used two transgenic mouse models of suppression or overexpression of G6pc1, the catalytic subunit of glucose-6 phosphatase, which is the key enzyme of endogenous glucose production specifically in the intestine. RESULTS: Under a hypercaloric diet, mice overexpressing IGN showed lower adiposity and higher thermogenic capacities than wild-type mice, featuring marked browning of white adipose tissue (WAT) and prevention of the whitening of brown adipose tissue (BAT). Sympathetic denervation restricted to BAT caused the loss of the antiobesity effects associated with IGN. Conversely, IGN-deficient mice exhibited an increase in adiposity under a standard diet, which was associated with decreased expression of markers of thermogenesis in both BAT and WAT. CONCLUSIONS: IGN is sufficient to activate the sympathetic nervous system and prevent the expansion and the metabolic alterations of BAT and WAT metabolism under a high-calorie diet, thereby preventing the development of obesity. These data increase knowledge of the mechanisms of weight reduction in gastric bypass surgery and pave the way for new approaches to prevent or cure obesity.

Disruption of hepatic mitochondrial pyruvate and amino acid metabolism impairs gluconeogenesis and endurance exercise capacity in mice.

Exercise robustly increases the glucose demands of skeletal muscle. This demand is met by not only muscle glycogenolysis but also accelerated liver glucose production from hepatic glycogenolysis and gluconeogenesis to fuel mechanical work and prevent hypoglycemia during exercise. Hepatic gluconeogenesis during exercise is dependent on highly coordinated responses within and between muscle and liver. Specifically, exercise increases the rate at which gluconeogenic precursors such as pyruvate/lactate or amino acids are delivered from muscle to the liver, extracted by the liver, and channeled into glucose. Herein, we examined the effects of interrupting hepatic gluconeogenic efficiency and capacity on exercise performance by deleting mitochondrial pyruvate carrier 2 (MPC2) and/or alanine transaminase 2 (ALT2) in the liver of mice. We found that deletion of MPC2 or ALT2 alone did not significantly affect time to exhaustion or postexercise glucose concentrations in treadmill exercise tests, but mice lacking both MPC2 and ALT2 in hepatocytes (double knockout, DKO) reached exhaustion faster and exhibited lower circulating glucose during and after exercise. Use of 2H/1³C metabolic flux analyses demonstrated that DKO mice exhibited lower endogenous glucose production owing to decreased glycogenolysis and gluconeogenesis at rest and during exercise. Decreased gluconeogenesis was accompanied by lower anaplerotic, cataplerotic, and TCA cycle fluxes. Collectively, these findings demonstrate that the transition of the liver to the gluconeogenic mode is critical for preventing hypoglycemia and sustaining performance during exercise. The results also illustrate the need for interorgan cross talk during exercise as described by the Cahill and Cori cycles.NEW & NOTEWORTHY Martino and colleagues examined the effects of inhibiting hepatic gluconeogenesis on exercise performance and systemic metabolism during treadmill exercise in mice. Combined inhibition of gluconeogenesis from lactate/pyruvate and alanine impaired exercise endurance and led to hypoglycemia during and after exercise. In contrast, suppressing either pyruvate-mediated or alanine-mediated gluconeogenesis alone had no effect on these parameters. These findings provide new insight into the molecular nodes that coordinate the metabolic responses of muscle and liver during exercise.

The bloodstream form of Trypanosoma brucei displays non-canonical gluconeogenesis.

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.

Integration of metabolic flux with hepatic glucagon signaling and gene expression profiles in the conscious dog.

Glucagon rapidly and profoundly stimulates hepatic glucose production (HGP), but for reasons that are unclear, this effect normally wanes after a few hours, despite sustained plasma glucagon levels. This study characterized the time course of glucagon-mediated molecular events and their relevance to metabolic flux in the livers of conscious dogs. Glucagon was either infused into the hepato-portal vein at a sixfold basal rate in the presence of somatostatin and basal insulin, or it was maintained at a basal level in control studies. In one control group, glucose remained at basal, whereas in the other, glucose was infused to match the hyperglycemia that occurred in the hyperglucagonemic group. Elevated glucagon caused a rapid (30 min) and largely sustained increase in hepatic cAMP over 4 h, a continued elevation in glucose-6-phosphate (G6P), and activation and deactivation of glycogen phosphorylase and synthase activities, respectively. Net hepatic glycogenolysis increased rapidly, peaking at 15 min due to activation of the cAMP/PKA pathway, then slowly returned to baseline over the next 3 h in line with allosteric inhibition by glucose and G6P. Glucagon's stimulatory effect on HGP was sustained relative to the hyperglycemic control group due to continued PKA activation. Hepatic gluconeogenic flux did not increase due to the lack of glucagon's effect on substrate supply to the liver. Global gene expression profiling highlighted glucagon-regulated activation of genes involved in cellular respiration, metabolic processes, and signaling, as well as downregulation of genes involved in extracellular matrix assembly and development.NEW & NOTEWORTHY Glucagon rapidly stimulates hepatic glucose production, but these effects are transient. This study links the molecular and metabolic flux changes that occur in the liver over time in response to a rise in glucagon, demonstrating the strength of the dog as a translational model to couple findings in small animals and humans. In addition, this study clarifies why the rapid effects of glucagon on liver glycogen metabolism are not sustained.

Inhibition of Sodium-Glucose Cotransporter-2 during Serum Deprivation Increases Hepatic Gluconeogenesis via the AMPK/AKT/FOXO Signaling Pathway.

BACKGRUOUND: Sodium-dependent glucose cotransporter 2 (SGLT2) mediates glucose reabsorption in the renal proximal tubules, and SGLT2 inhibitors are used as therapeutic agents for treating type 2 diabetes mellitus. This study aimed to elucidate the effects and mechanisms of SGLT2 inhibition on hepatic glucose metabolism in both serum deprivation and serum supplementation states. METHODS: Huh7 cells were treated with the SGLT2 inhibitors empagliflozin and dapagliflozin to examine the effect of SGLT2 on hepatic glucose uptake. To examine the modulation of glucose metabolism by SGLT2 inhibition under serum deprivation and serum supplementation conditions, HepG2 cells were transfected with SGLT2 small interfering RNA (siRNA), cultured in serum-free Dulbecco's modified Eagle's medium for 16 hours, and then cultured in media supplemented with or without 10% fetal bovine serum for 8 hours. RESULTS: SGLT2 inhibitors dose-dependently decreased hepatic glucose uptake. Serum deprivation increased the expression levels of the gluconeogenesis genes peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), glucose 6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (PEPCK), and their expression levels during serum deprivation were further increased in cells transfected with SGLT2 siRNA. SGLT2 inhibition by siRNA during serum deprivation induces nuclear localization of the transcription factor forkhead box class O 1 (FOXO1), decreases nuclear phosphorylated-AKT (p-AKT), and p-FOXO1 protein expression, and increases phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK) protein expression. However, treatment with the AMPK inhibitor, compound C, reversed the reduction in the protein expression levels of nuclear p- AKT and p-FOXO1 and decreased the protein expression levels of p-AMPK and PEPCK in cells transfected with SGLT2 siRNA during serum deprivation. CONCLUSION: These data show that SGLT2 mediates glucose uptake in hepatocytes and that SGLT2 inhibition during serum deprivation increases gluconeogenesis via the AMPK/AKT/FOXO1 signaling pathway.

A conserved transcription factor controls gluconeogenesis via distinct targets in hypersaline-adapted archaea with diverse metabolic capabilities.

Timely regulation of carbon metabolic pathways is essential for cellular processes and to prevent futile cycling of intracellular metabolites. In Halobacterium salinarum, a hypersaline adapted archaeon, a sugar-sensing TrmB family protein controls gluconeogenesis and other biosynthetic pathways. Notably, Hbt. salinarum does not utilize carbohydrates for energy, uncommon among Haloarchaea. We characterized a TrmB-family transcriptional regulator in a saccharolytic generalist, Haloarcula hispanica, to investigate whether the targets and function of TrmB, or its regulon, is conserved in related species with distinct metabolic capabilities. In Har. hispanica, TrmB binds to 15 sites in the genome and induces the expression of genes primarily involved in gluconeogenesis and tryptophan biosynthesis. An important regulatory control point in Hbt. salinarum, activation of ppsA and repression of pykA, is absent in Har. hispanica. Contrary to its role in Hbt. salinarum and saccharolytic hyperthermophiles, TrmB does not act as a global regulator: it does not directly repress the expression of glycolytic enzymes, peripheral pathways such as cofactor biosynthesis, or catabolism of other carbon sources in Har. hispanica. Cumulatively, these findings suggest rewiring of the TrmB regulon alongside metabolic network evolution in Haloarchaea.

TOX3 deficiency mitigates hyperglycemia by suppressing hepatic gluconeogenesis through FoxO1.

BACKGROUND: Excessive hepatic glucose production is a hallmark that contributes to hyperglycemia in type 2 diabetes (T2D). The regulatory network governing this process remains incompletely understood. Here, we demonstrate that TOX3, a high-mobility group family member, acts as a major transcriptional driver for hepatic glucose production. METHODS: Tox3-overexpressed and knockout mice were constructed to explore its metabolic functions. Transcriptomic and chromatin-immunoprecipitation sequencing (ChIP-seq) were used to identify downstream targets of TOX3. Both FoxO1 silencing and inhibitor approaches were used to assess the contribution of FoxO1. TOX3 expression levels were examined in the livers of mice and human subjects. Finally, Tox3 was genetically manipulated in diet-induced obese mice to evaluate its therapeutic potential. RESULTS: Hepatic Tox3 overexpression activates the gluconeogenic program, resulting in hyperglycemia and insulin resistance in mice. Hepatocyte-specific Tox3 knockout suppresses gluconeogenesis and improves insulin sensitivity. Mechanistically, integrated hepatic transcriptomic and ChIP-seq analyses identify FoxO1 as a direct target of TOX3. TOX3 stimulates FoxO1 transcription by directly binding to and activating its promoter, whereas FoxO1 silencing abrogates TOX3-induced dysglycemia in mice. In human subjects, hepatic TOX3 expression shows a significant positive correlation with blood glucose levels under normoglycemic conditions, yet is repressed by high glucose during T2D. Importantly, hepatic Tox3 deficiency markedly protects against and ameliorates the hyperglycemia and glucose intolerance in diet-induced diabetic mice. CONCLUSIONS: Our findings establish TOX3 as a driver for excessive gluconeogenesis through activating hepatic FoxO1 transcription. TOX3 could serve as a promising target for preventing and treating hyperglycemia in T2D.

Endogenous renal adiponectin drives gluconeogenesis through enhancing pyruvate and fatty acid utilization.

Adiponectin is a secretory protein, primarily produced in adipocytes. However, low but detectable expression of adiponectin can be observed in cell types beyond adipocytes, particularly in kidney tubular cells, but its local renal role is unknown. We assessed the impact of renal adiponectin by utilizing male inducible kidney tubular cell-specific adiponectin overexpression or knockout mice. Kidney-specific adiponectin overexpression induces a doubling of phosphoenolpyruvate carboxylase expression and enhanced pyruvate-mediated glucose production, tricarboxylic acid cycle intermediates and an upregulation of fatty acid oxidation (FAO). Inhibition of FAO reduces the adiponectin-induced enhancement of glucose production, highlighting the role of FAO in the induction of renal gluconeogenesis. In contrast, mice lacking adiponectin in the kidney exhibit enhanced glucose tolerance, lower utilization and greater accumulation of lipid species. Hence, renal adiponectin is an inducer of gluconeogenesis by driving enhanced local FAO and further underlines the important systemic contribution of renal gluconeogenesis.

Cold-activated brown fat-derived extracellular vesicle-miR-378a-3p stimulates hepatic gluconeogenesis in male mice.

During cold exposure, activated brown adipose tissue (BAT) takes up a large amount of circulating glucose to fuel non-shivering thermogenesis and defend against hypothermia. However, little is known about the endocrine function of BAT controlling glucose homoeostasis under this thermoregulatory challenge. Here, we show that in male mice, activated BAT-derived extracellular vesicles (BDEVs) reprogram systemic glucose metabolism by promoting hepatic gluconeogenesis during cold stress. Cold exposure facilitates the selective packaging of miR-378a-3p-one of the BAT-enriched miRNAs-into EVs and delivery into the liver. BAT-derived miR-378a-3p enhances gluconeogenesis by targeting p110α. miR-378 KO mice display reduced hepatic gluconeogenesis during cold exposure, while restoration of miR-378a-3p in iBAT induces the expression of gluconeogenic genes in the liver. These findings provide a mechanistic understanding of BDEV-miRNA as stress-induced batokine to coordinate systemic glucose homoeostasis. This miR-378a-3p-mediated interorgan communication highlights a novel endocrine function of BAT in preventing hypoglycemia during cold stress.

Neprilysin deficiency reduces hepatic gluconeogenesis in high fat-fed mice.

Neprilysin is a peptidase that cleaves glucoregulatory peptides, including glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK). Some studies suggest that its inhibition in diabetes and/or obesity improves glycemia, and that this is associated with enhanced insulin secretion, glucose tolerance and insulin sensitivity. Whether reduced neprilysin activity also improves hepatic glucose metabolism has not been explored. We sought to determine whether genetic deletion of neprilysin suppresses hepatic glucose production (HGP) in high fat-fed mice. Nep+/+ and Nep-/- mice were fed high fat diet for 16 weeks, and then underwent a pyruvate tolerance test (PTT) to assess hepatic gluconeogenesis. Since glycogen breakdown in liver can also yield glucose, we assessed liver glycogen content in fasted and fed mice. In Nep-/- mice, glucose excursion during the PTT was reduced when compared to Nep+/+ mice. Further, liver glycogen levels were significantly greater in fasted but not fed Nep-/- versus Nep+/+ mice. Since gut-derived factors modulate HGP, we tested whether gut-selective inhibition of neprilysin could recapitulate the suppression of hepatic gluconeogenesis observed with whole-body inhibition, and this was indeed the case. Finally, the gut-derived neprilysin substrates, GLP-1 and CCK, are well-known to suppress HGP. Having previously demonstrated elevated plasma GLP-1 levels in Nep-/- mice, we now measured plasma CCK bioactivity and reveal an increase in Nep-/- versus Nep+/+ mice, suggesting GLP-1 and/or CCK may play a role in reducing HGP under conditions of neprilysin deficiency. In sum, neprilysin modulates hepatic gluconeogenesis and strategies to inhibit its activity may reduce HGP in type 2 diabetes and obesity.

Differential expression of gluconeogenesis-related transcripts in a freshwater zooplankton model organism suggests a role of the Cori cycle in hypoxia tolerance.

Gluconeogenesis (GNG) is the process of regenerating glucose and NAD+ that allows for continued ATP synthesis by glycolysis during fasting or in hypoxia. Recent data from C. elegans and crustaceans challenged with hypoxia show differential and tissue-specific expression of GNG-specific genes. Here we report differential expression of several GNG-specific genes in the head and body of a model organism, Daphnia magna, a planktonic crustacean, in normoxic and acute hypoxic conditions. We predict that GNG-specific transcripts will be enriched in the body, where most of the fat tissue is located, rather than in the head, where the tissues critical for survival in hypoxia, the central nervous system and locomotory muscles, are located. We measured the relative expression of GNG-specific transcripts in each body part by qRT-PCR and normalized them by either the expression of a reference gene or the rate-limiting glycolysis enzyme pyruvate kinase (PK). Our data show that of the three GNG-specific transcripts tested, pyruvate carboxylase (PC) showed no differential expression in either the head or body. Phosphoenolpyruvate carboxykinase (PEPCK-C), on the other hand, is upregulated in hypoxia in both body parts. Fructose-1,6-bisphosphatase (FBP) is upregulated in the body relative to the head and upregulated in hypoxia relative to normoxia, with a stronger body effect in hypoxia when normalized by PK expression. These results support our hypothesis that Daphnia can survive hypoxic conditions by implementing the Cori cycle, where body tissues supply glucose and NAD+ to the brain and muscles, enabling them to continuously generate ATP by glycolysis.

Chronic Glucocorticoid Exposure Induced an S1PR2-RORγ Axis to Enhance Hepatic Gluconeogenesis in Male Mice.

It is well established that chronic glucocorticoid exposure causes hyperglycemia. While glucocorticoid receptor (GR) stimulates hepatic gluconeogenic gene transcription, additional mechanisms are activated by chronic glucocorticoid exposure to enhance gluconeogenesis. We found that chronic glucocorticoid treatment activated sphingosine-1-phosphate (S1P)-mediated signaling. Hepatic knockdown of hepatic S1P receptor 1 (S1PR1) had no effect on chronic glucocorticoid-induced glucose intolerance but elevated fasting plasma insulin levels. In contrast, hepatic S1PR3 knockdown exacerbated chronic glucocorticoid-induced glucose intolerance without affecting fasting plasma insulin levels. Finally, hepatic S1PR2 knockdown attenuated chronic glucocorticoid-induced glucose intolerance and reduced fasting plasma insulin levels. Here, we focused on dissecting the role of S1PR2 signaling in chronic glucocorticoid response on glucose homeostasis. We found that chronic glucocorticoid-induced hepatic gluconeogenesis, gluconeogenic gene expression, and GR recruitment to the glucocorticoid response elements (GREs) of gluconeogenic genes were all reduced in hepatic S1PR2 knockdown male mice. Hepatic S1PR2 knockdown also enhanced glucocorticoid suppression of RAR-related orphan receptor γ (RORγ) expression. Hepatic RORγ overexpression in hepatic S1PR2 knockdown mice restored glucocorticoid-induced glucose intolerance, gluconeogenic gene expression, and GR recruitment to their GREs. Conversely, RORγ antagonist and the reduction of hepatic RORγ expression attenuated such glucocorticoid effects. Thus, chronic glucocorticoid exposure induces an S1PR2-RORγ axis to cooperate with GR to enhance hepatic gluconeogenesis. Overall, this work provides novel mechanisms of and pharmaceutical targets against steroid-induced hyperglycemia.

Advances in Small Molecules of Flavonoids for the Regulation of Gluconeogenesis.

Hyperglycemia resulting from over-gluconeogenesis is a prominent feature of type 2 diabetes mellitus (T2DM). Therefore, it is very important to reduce glucose output, especially liver glucose output, and maintain blood glucose homeostasis in the treatment of T2DM. It has been found that small molecules of natural flavonoids are able to act on various targets in the gluconeogenic pathways, interfering with rate-limiting enzyme activity or regulating the cascade of hormonal signaling and affecting all levels of transcription factors by limiting the transport of non-sugar substrates. As a result, gluconeogenesis is inhibited. Literature indicated that gluconeogenesis regulated by flavonoids could be divided into two pathways, namely the pre-translational pathway and the pro-translational pathway. The pre-translational pathway mainly interferes with the signaling pathway and transcription factors in gluconeogenesis and inhibits RNA transcription and the expression of gluconeogenic genes, while the post-translational pathway mainly regulates the transport of nonglucose substrates and directly inhibits four rate-limiting enzymes. This review describes the effects of small flavonoid molecules on different targets and signaling pathways during gluconeogenesis, as well as relevant validation methods, in the hope of providing references for similar studies and promoting the development of anti-diabetic drugs.

Role of serum- and glucocorticoid-inducible kinase 1 in the regulation of hepatic gluconeogenesis.

Excessive hepatic gluconeogenesis partially accounts for the occurrence of type 2 diabetes mellitus. Serum- and glucocorticoid inducible-kinase 1 (SGK1) is linked to the development of metabolic syndrome, such as obesity, hypertension, and hyperglycemia. However, the regulatory role of SGK1 in glucose metabolism of liver remains uncertain. Our microarray analysis showed that SGK1 expression was strongly induced by 8-Br-cAMP and suppressed by metformin in primary mouse hepatocytes. Hepatic SGK1 expression was markedly increased in obese and diabetic mice. Metformin treatment decreased hepatic SGK1 expression levels in db/db mice. Inhibition or knockdown of SGK1 suppressed gluconeogenesis in primary mouse hepatocytes, with decreased expressions of key gluconeogenic genes. Furthermore, SGK1 silencing in liver decreased hepatic glucose production in C57BL/6 mice. Knockdown of SGK1 had no impact on CREB phosphorylation level but increased AKT and FoxO1 phosphorylation levels with decreased expressions of transcription factors including FoxO1 and hepatocyte nuclear factors. Adenovirus-mediated expression of dominant-negative AMPK antagonized metformin-suppressed SGK1 expression induced by 8-Br-cAMP. These findings demonstrate that hepatic specific silence of SGK1 might be a potential therapeutic strategy for type 2 diabetes.

Reciprocal Regulation of Hepatic TGF-ß1 and Foxo1 Controls Gluconeogenesis and Energy Expenditure.

Obesity and insulin resistance are risk factors for the pathogenesis of type 2 diabetes (T2D). Here, we report that hepatic TGF-ß1 expression positively correlates with obesity and insulin resistance in mice and humans. Hepatic TGF-ß1 deficiency decreased blood glucose levels in lean mice and improved glucose and energy dysregulations in diet-induced obese (DIO) mice and diabetic mice. Conversely, overexpression of TGF-ß1 in the liver exacerbated metabolic dysfunctions in DIO mice. Mechanistically, hepatic TGF-ß1 and Foxo1 are reciprocally regulated: fasting or insulin resistance caused Foxo1 activation, increasing TGF-ß1 expression, which, in turn, activated protein kinase A, stimulating Foxo1-S273 phosphorylation to promote Foxo1-mediated gluconeogenesis. Disruption of TGF-ß1→Foxo1→TGF-ß1 looping by deleting TGF-ß1 receptor II in the liver or by blocking Foxo1-S273 phosphorylation ameliorated hyperglycemia and improved energy metabolism in adipose tissues. Taken together, our studies reveal that hepatic TGF-ß1→Foxo1→TGF-ß1 looping could be a potential therapeutic target for prevention and treatment of obesity and T2D. ARTICLE HIGHLIGHTS: Hepatic TGF-ß1 levels are increased in obese humans and mice. Hepatic TGF-ß1 maintains glucose homeostasis in lean mice and causes glucose and energy dysregulations in obese and diabetic mice. Hepatic TGF-ß1 exerts an autocrine effect to promote hepatic gluconeogenesis via cAMP-dependent protein kinase-mediated Foxo1 phosphorylation at serine 273, endocrine effects on brown adipose tissue action, and inguinal white adipose tissue browning (beige fat), causing energy imbalance in obese and insulin-resistant mice. TGF-ß1→Foxo1→TGF-ß1 looping in hepatocytes plays a critical role in controlling glucose and energy metabolism in health and disease.

REDD1 deletion and treadmill running increase liver hepcidin and gluconeogenic enzymes in male mice.

The iron-regulatory hormone hepcidin is transcriptionally up-regulated by gluconeogenic signals. Recent evidence suggeststhat increases in circulating hepcidin may decrease dietary iron absorption following prolonged exercise, however evidence is limited on whether gluconeogenic signals contribute to post-exercise increases in hepcidin. Mice with genetic knockout of regulated in development and DNA response-1 (REDD1) display greater glycogen depletion following exercise, possibly indicating greater gluconeogenesis. The objective of the present study was to determine liver hepcidin, markers of gluconeogenesis and iron metabolism in REDD1 knockout and wild-type mice following prolonged exercise. Twelve-week-old male REDD1 knockout and wild-type mice were randomised to rest or 60 min treadmill running with 1, 3 or 6 h recovery (n = 5-8/genotype/group). Liver gene expression of hepcidin (Hamp) and gluconeogenic enzymes (Ppargc1a, Creb3l3, Pck1, Pygl) were determined by qRT-PCR. Effects of genotype, exercise and their interaction were assessed by two-way ANOVAs with Tukey's post-hoc tests, and Pearson correlations were used to assess the relationships between Hamp and study outcomes. Liver Hamp increased 1- and 4-fold at 3 and 6 h post-exercise, compared to rest (P-adjusted < 0⋅009 for all), and was 50% greater in REDD1 knockout compared to wild-type mice (P = 0⋅0015). Liver Ppargc1a, Creb3l3 and Pck1 increased with treadmill running (P < 0⋅0001 for all), and liver Ppargc1a, Pck1 and Pygl were greater with REDD1 deletion (P < 0⋅02 for all). Liver Hamp was positively correlated with liver Creb3l3 (R = 0⋅62, P < 0⋅0001) and Pck1 (R = 0⋅44, P = 0⋅0014). In conclusion, REDD1 deletion and prolonged treadmill running increased liver Hamp and gluconeogenic regulators of Hamp, suggesting gluconeogenic signalling of hepcidin with prolonged exercise.

Hepatic ZBTB22 promotes hyperglycemia and insulin resistance via PEPCK1-driven gluconeogenesis.

Excessive gluconeogenesis can lead to hyperglycemia and diabetes through as yet incompletely understood mechanisms. Herein, we show that hepatic ZBTB22 expression is increased in both diabetic clinical samples and mice, being affected by nutritional status and hormones. Hepatic ZBTB22 overexpression increases the expression of gluconeogenic and lipogenic genes, heightening glucose output and lipids accumulation in mouse primary hepatocytes (MPHs), while ZBTB22 knockdown elicits opposite effects. Hepatic ZBTB22 overexpression induces glucose intolerance and insulin resistance, accompanied by moderate hepatosteatosis, while ZBTB22-deficient mice display improved energy expenditure, glucose tolerance, and insulin sensitivity, and reduced hepatic steatosis. Moreover, hepatic ZBTB22 knockout beneficially regulates gluconeogenic and lipogenic genes, thereby alleviating glucose intolerance, insulin resistance, and liver steatosis in db/db mice. ZBTB22 directly binds to the promoter region of PCK1 to enhance its expression and increase gluconeogenesis. PCK1 silencing markedly abolishes the effects of ZBTB22 overexpression on glucose and lipid metabolism in both MPHs and mice, along with the corresponding changes in gene expression. In conclusion, targeting hepatic ZBTB22/PEPCK1 provides a potential therapeutic approach for diabetes.

Hepatic p38α MAPK controls gluconeogenesis via FOXO1 phosphorylation at S273 during glucagon signalling in mice.

AIMS/HYPOTHESIS: Hyperglucagonaemia-stimulated hepatic glucose production (HGP) contributes to hyperglycaemia during type 2 diabetes. A better understanding of glucagon action is important to enable efficient therapies to be developed for the treatment of diabetes. Here, we aimed to investigate the role of p38 MAPK family members in glucagon-induced HGP and determine the underlying mechanisms by which p38 MAPK regulates glucagon action. METHODS: p38α, ß, γ and δ MAPK siRNAs were transfected into primary hepatocytes, followed by measurement of glucagon-induced HGP. Adeno-associated virus serotype 8 carrying p38α MAPK short hairpin RNA (shRNA) was injected into liver-specific Foxo1 knockout, liver-specific Irs1/Irs2 double knockout and Foxo1S273D knockin mice. Foxo1S273A knockin mice were fed a high-fat diet for 10 weeks. Pyruvate tolerance tests, glucose tolerance tests, glucagon tolerance tests and insulin tolerance tests were carried out in mice, liver gene expression profiles were analysed and serum triglyceride, insulin and cholesterol levels were measured. Phosphorylation of forkhead box protein O1 (FOXO1) by p38α MAPK in vitro was analysed by LC-MS. RESULTS: We found that p38α MAPK, but not the other p38 isoforms, stimulates FOXO1-S273 phosphorylation and increases FOXO1 protein stability, promoting HGP in response to glucagon stimulation. In hepatocytes and mouse models, inhibition of p38α MAPK blocked FOXO1-S273 phosphorylation, decreased FOXO1 levels and significantly impaired glucagon- and fasting-induced HGP. However, the effect of p38α MAPK inhibition on HGP was abolished by FOXO1 deficiency or a Foxo1 point mutation at position 273 from serine to aspartic acid (Foxo1S273D) in both hepatocytes and mice. Moreover, an alanine mutation at position 273 (Foxo1S273A) decreased glucose production, improved glucose tolerance and increased insulin sensitivity in diet-induced obese mice. Finally, we found that glucagon activates p38α through exchange protein activated by cAMP 2 (EPAC2) signalling in hepatocytes. CONCLUSIONS/INTERPRETATION: This study found that p38α MAPK stimulates FOXO1-S273 phosphorylation to mediate the action of glucagon on glucose homeostasis in both health and disease. The glucagon-induced EPAC2-p38α MAPK-pFOXO1-S273 signalling pathway is a potential therapeutic target for the treatment of type 2 diabetes.

Hepatic IDH2 regulates glycolysis and gluconeogenesis.

BACKGROUND AND AIMS: The liver plays a central role in controlling glucose and lipid metabolism. IDH2, a mitochondrial protein, controls TCA cycle flux. However, its role in regulating metabolism in obesity is still unclear. This study intends to investigate the impact of hepatic IDH2 expression on overnutrition-regulated glucose and lipid metabolism. METHODS: Hepatic IDH2 was knocked-out in mice by the approach of CRISPR-Cas9. Mice were subjected to starvation and refeeding for hepatic glucose and lipid studies in vivo. Primary hepatocytes and mouse normal liver cell line, AML12 cells were used for experiments in vitro. RESULTS: This study found that IDH2 protein levels were elevated in the livers of obese people and mice with high-fat diet consumption or hepatic steatosis. Liver IDH2-deletion mice (IDH2LKO) were resistant to high-fat diet-induced body weight gain, with lower serum glucose and TG levels, increased insulin sensitivity, and higher FGF21 secretion, despite the higher TG content in the liver. Consistently, overexpression of IDH2 in hepatocytes promoted gluconeogenesis and enhanced glycogenesis. By performing mass spectrometry and proteomics analyses, we further demonstrated that IDH2-deficiency in hepatocytes accelerated ATP production by increasing forward TCA cycle flux, thus promoting glycolysis pathway and decreasing glycogen synthesis at refeeding state, and inhibiting hepatic gluconeogenesis, increasing ß-oxidation during starvation. Moreover, experiments in vivo demonstrated that IDH2-knockout might not exacerbate hepatic inflammatory responses in the NASH model. CONCLUSIONS: Elevated hepatic IDH2 under over-nutrition state contributes to elevated gluconeogenesis and glycogen synthesis. Inhibition of IDH2 in the liver could be a potential therapeutic target for obesity and diabetes.