BACKGROUND: Under fasting conditions, the pathway converting gluconeogenesis precursors into muscle glycogen becomes crucial due to reduced glycogen reserves. However, there is limited research on skeletal muscle gluconeogenesis and the impact of fasting on gluconeogenic gene expression.
METHODS: Sheep fetal skeletal muscle cells cultured in vitro were used to study the effects of varying lactic acid concentrations (0 to 30 mM) and 2.5 mM glucose on the expression of gluconeogenesis-related genes after 6 h of fasting. The effects on mRNA and protein expression of key genes involved in skeletal muscle gluconeogenesis were measured by quantitative real time polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting at 48 h.
RESULTS: Fasting increased the expression of key gluconeogenic genes, fructose-1,6-bisphosphatase 2 (FBP2), glucose-6-phosphatase 3 (G6PC3), pyruvate kinase M (PKM), monocarboxylate transporter1 (MCTS1), glucose transporter type 4 (GLUT4), pyruvate carboxylase (PC), and lactate dehydrogenase A (LDHA). The mRNA levels of FBP2, G6PC3, and MCTS1 significantly decreased with glucose addition. Additionally, 10 mM lactic acid significantly promoted the expression of FBP2, PC, MCTS1, LDHA, GLUT4, and PKM while inhibiting phosphoenolpyruvate carboxykinase (PEPCK) expression. At the protein level, 10 mM lactic acid significantly increased FBP2 and PKM protein expression.
CONCLUSIONS: This study shows that fasting regulates key gluconeogenic gene expression in sheep skeletal muscle cells and highlights the role of lactic acid in inducing these gene expressions.

BACKGROUND: Extracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with sequence similarity 3 member A (FAM3A) in controlling hepatic glucolipid metabolism, islet β cell function, adipocyte differentiation, blood pressure, and other biological and pathophysiological processes. Although mitochondrial protein FAM3A plays crucial roles in the regulation of glucolipid metabolism via stimulating ATP release to activate P2 receptor pathways, its mechanism in promoting ATP release in hepatocytes remains unrevealed.
METHODS: db/db, high-fat diet (HFD)-fed, and global pannexin 1 (PANX1) knockout mice, as well as liver sections of individuals, were used in this study. Adenoviruses and adeno-associated viruses were utilized for in vivo gene overexpression or inhibition. To evaluate the metabolic status in mice, oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT), and magnetic resonance imaging (MRI) were conducted. Protein-protein interactions were determined by coimmunoprecipitation with mass spectrometry (MS) assays.
RESULTS: In livers of individuals and mice with steatosis, the expression of ATP-permeable channel PANX1 was increased (P < 0.01). Hepatic PANX1 overexpression ameliorated the dysregulated glucolipid metabolism in obese mice. Mice with hepatic PANX1 knockdown or global PANX1 knockout exhibited disturbed glucolipid metabolism. Restoration of hepatic PANX1 rescued the metabolic disorders of PANX1-deficient mice (P < 0.05). Mechanistically, ATP release is mediated by the PANX1-activated protein kinase B-forkhead box protein O1 (Akt-FOXO1) pathway to inhibit gluconeogenesis via P2Y receptors in hepatocytes. PANX1-mediated ATP release also activated calmodulin (CaM) (P < 0.01), which interacted with c-Jun N-terminal kinase (JNK) to inhibit its activity, thereby deactivating the transcription factor activator protein-1 (AP1) and repressing fatty acid synthase (FAS) expression and lipid synthesis (P < 0.05). FAM3A stimulated the expression of PANX1 via heat shock factor 1 (HSF1) in hepatocytes (P < 0.05). Notably, FAM3A overexpression failed to promote ATP release, inhibit the expression of gluconeogenic and lipogenic genes, and suppress gluconeogenesis and lipid deposition in PANX1-deficient hepatocytes and livers.
CONCLUSIONS: PANX1-mediated release of ATP plays a crucial role in maintaining hepatic glucolipid homeostasis, and it confers FAM3A\'s suppressive effects on hepatic gluconeogenesis and lipogenesis.

During the adaptive evolution of animals, the host and its gut microbiota co-adapt to different elevations. Currently, there are few reports on the rumen microbiota-hepato-intestinal axis of Tibetan sheep at different altitudes. Therefore, the purpose of this study was to explore the regulatory effect of rumen microorganism-volatile fatty acids (VFAs)-VFAs transporter gene interactions on the key enzymes and genes related to gluconeogenesis in Tibetan sheep. The rumen fermentation parameters, rumen microbial densities, liver gluconeogenesis activity and related genes were determined and analyzed using gas chromatography, RT-qPCR and other research methods. Correlation analysis revealed a reciprocal relationship among rumen microflora-VFAs-hepatic gluconeogenesis in Tibetan sheep at different altitudes. Among the microbiota, Ruminococcus flavefaciens (R. flavefaciens), Ruminococcus albus (R. albus), Fibrobactersuccinogenes and Ruminobacter amylophilus (R. amylophilus) were significantly correlated with propionic acid (p < 0.05), while propionic acid was significantly correlated with the transport genes monocarboxylate transporter 4 (MCT4) and anion exchanger 2 (AE2) (p < 0.05). Propionic acid was significantly correlated with key enzymes such as pyruvate carboxylase, phosphoenolpyruvic acid carboxylase and glucose (Glu) in the gluconeogenesis pathway (p < 0.05). Additionally, the expressions of these genes were significantly correlated with those of the related genes, namely, forkhead box protein O1 (FOXO1) and mitochondrial phosphoenolpyruvate carboxykinase 2 (PCK2) (p < 0.05). The results showed that rumen microbiota densities differed at different altitudes, and the metabolically produced VFA contents differed, which led to adaptive changes in the key enzyme activities of gluconeogenesis and the expressions of related genes.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases in the world, which begins with liver lipid accumulation and is associated with metabolic syndrome. Also, the name chosen to replace NAFLD was metabolic dysfunction-associated steatotic liver disease (MASLD). We performed focused drug screening and found that Cilostazol effectively ameliorated hepatic steatosis and might offer potential for NAFLD treatment. Our aim was to investigate the therapeutic effects of Cilostazol on the glycolipid metabolism and intestinal flora in NAFLD mice and explore the specific mechanism. In this study, 7-week-old male C57BL/6J mice were fed a high-fat diet (HFD) for 8 weeks to induce NAFLD, and then treated with intragastric administration for 12 weeks. The results showed that Cilostazol inhibited liver lipid de novo synthesis by regulating the AMPK-ACC1/SCD1 pathway and inhibited liver gluconeogenesis by the AMPK-PGC1α-G6P/PEPCK pathway. Cilostazol improved the intestinal flora diversity and intestinal microbial composition in the NAFLD mice, and specifically regulated Desulfovibrio and Akkermansia. In addition, Cilostazol increased the level of short-chain fatty acids in the NAFLD mice to a level similar to that in the blank Control group. Cilostazol reduces liver lipid accumulation in NAFLD mice by improving glucose and lipid metabolism disorders and intestinal dysfunction, thereby achieving the purpose of treating NAFLD.

Food proteins are considered an ideal source for the identification of bioactive peptides with the potential to intervene in nutrition-related chronic diseases such as cardiovascular disease, obesity, and diabetes. Egg white-derived peptides (EWPs) have been shown to improve glucose tolerance in insulin-resistant rats. However, underlying mechanisms are to be elucidated. Therefore, we hypothesized that EWP exerts a hypoglycemic effect by regulating hepatic glucose homeostasis. Our results showed that 7 weeks of EWP treatment reduced the fasting blood glucose in T2DM mice and the inhibition of the liver gluconeogenic pathway was involved in the mechanisms of actions. Using the untargeted metabolomics technique, we found that EWP treatment also altered the hepatic metabolic profile in T2DM mice, in which, the role of fatty acid esters of hydroxy fatty acids in mediating the hypoglycemic effect of EWPs might be pivotal.

Adenosine deaminase (ADA) catalyzes the irreversible deamination of adenosine (ADO) to inosine and regulates ADO concentration. ADA ubiquitously expresses in various tissues to mediate ADO-receptor signaling. A significant increase in plasma ADA activity has been shown to be associated with the pathogenesis of type 2 diabetes mellitus. Here, we show that elevated plasma ADA activity is a compensated response to high level of ADO in type 2 diabetes mellitus and plays an essential role in the regulation of glucose homeostasis. Supplementing with more ADA, instead of inhibiting ADA, can reduce ADO levels and decrease hepatic gluconeogenesis. ADA restores a euglycemic state and recovers functional islets in db/db and high-fat streptozotocin diabetic mice. Mechanistically, ADA catabolizes ADO and increases Akt and FoxO1 phosphorylation independent of insulin action. ADA lowers blood glucose at a slower rate and longer duration compared to insulin, delaying or blocking the incidence of insulinogenic hypoglycemia shock. Finally, ADA suppresses gluconeogenesis in fasted mice and insulin-deficient diabetic mice, indicating the ADA regulating gluconeogenesis is a universal biological mechanism. Overall, these results suggest that ADA is expected to be a new therapeutic target for diabetes.

Ginsenoside compound K (GCK) possesses a glucocorticoid (GC)-like structure and functions as an agonist of the glucocorticoid receptor (GR), thereby exerting anti-inflammatory effects through GR activation. However, it remains unclear whether GCK leads to hyperglycemia, which is a known adverse reaction associated with classical GCs. In this study, we have successfully demonstrated that GCK exerts its anti-inflammatory effects in a rat model of adjuvant arthritis without impacting gluconeogenesis and pentose phosphate pathways, thus avoiding any glucose metabolism disorders. By employing the GR mutant plasmid, we have identified the binding site between GCK and GR as GRM560T, which differs from the binding site shared by dexamethasone (DEX) and GR. Notably, compared to DEX, GCK induces distinct levels of phosphorylation at S211 on GR upon binding to activate steroid receptor coactivator 1 (SRC1)-a co-factor responsible for mediating anti-inflammatory effects-while not engaging peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)-an associated coactivator involved in gluconeogenesis.

Vitamin D3 (VD3) is a steroid hormone that plays pivotal roles in pathophysiology, and 1,25(OH)2D3 is the most active form of VD3. In the current study, the crucial role of VD3 in maintaining energy homeostasis under short-term fasting conditions was investigated. Our results confirmed that glucose-depriving pathways were inhibited while glucose-producing pathways were strengthened in zebrafish after fasting for 24 or 48 h. Moreover, VD3 anabolism in zebrafish was significantly suppressed in a time-dependent manner under short-fasting conditions. After fasting for 24 or 48 h, zebrafish fed with VD3 displayed a higher gluconeogenesis level and lower glycolysis level in the liver, and the serum glucose was maintained at higher levels, compared to those fed without VD3. Additionally, VD3 augmented the expression of fatty acids (FAs) transporter cd36 and lipogenesis in the liver, while enhancing lipolysis in the dorsal muscle. Similar results were obtained in cyp2r1-/- zebrafish, in which VD3 metabolism is obstructed. Importantly, it was observed that VD3 induced the production of gut GLP-1, which is considered to possess a potent gluconeogenic function in zebrafish. Meanwhile, the gene expression of proprotein convertase subtilisin/kexin type 1 (pcsk1), a GLP-1 processing enzyme, was also induced in the intestine of short-term fasted zebrafish. Notably, gut microbiota and its metabolite acetate were involved in VD3-regulated pcsk1 expression and GLP-1 production under short-term fasting conditions. In summary, our study demonstrated that VD3 regulated GLP-1 production in zebrafish by influencing gut microbiota and its metabolite, contributing to energy homeostasis and ameliorating hypoglycemia under short-term fasting conditions.

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.
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.

Liver cancer stem cells were found to rely on glycolysis as the preferred metabolic program. Phosphoenolpyruvate carboxylase 1 (PCK1), a gluconeogenic metabolic enzyme, is down-regulated in hepatocellular carcinoma and is closely related to poor prognosis. The oncogenesis and progression of tumors are closely related to cancer stem cells. It is not completely clear whether the PCK1 deficiency increases the stemness of hepatoma cells and promotes the oncogenesis of hepatocellular carcinoma. Herein, the results showed that PCK1 inhibited the self-renewal property of hepatoma cells, reduced the mRNA level of cancer stem cell markers, and inhibited tumorigenesis. Moreover, PCK1 increased the sensitivity of hepatocellular carcinoma cells to sorafenib. Furthermore, we found that PCK1 activated the Hippo pathway by enhancing the phosphorylation of YAP and inhibiting its nuclear translocation. Verteporfin reduced the stemness of hepatoma cells and promoted the pro-apoptotic effect of sorafenib. Thus, combined treatment with verteporfin and sorafenib may be a potential anti-tumor strategy in hepatocellular carcinoma.