Gluconeogenic Enzymes Research Articles

Alanine alters the carbohydrate metabolism of rainbow trout: glucose flux and cell signaling.

In rainbow trout, dietary carbohydrates are poorly metabolized compared with other macronutrients. One prevalent hypothesis suggests that high dietary amino acid levels could contribute to the poor utilization of carbohydrates in trout. In mammals, alanine is considered an important gluconeogenic precursor, but has recently been found to stimulate AMP-activated protein kinase (AMPK) to reduce glucose levels. In trout, the effect of alanine on glucose flux is unknown. The goal of this study was to determine the effects of 4 h exogenous alanine infusion on glucose metabolism in rainbow trout. Glucose flux, and the rate of glucose appearance (Ra) and disposal (Rd) were measured in vivo. Key glycolytic and gluconeogenic enzyme expression and activity, and cell signaling molecules relevant to glucose metabolism were assessed in the liver and muscle. The results show that alanine inhibits glucose Ra (from 13.2±2.5 to 7.3±1.6 μmolkg-1 min-1) and Rd (from 13.2±2.5 to 7.4±1.5 μmolkg-1 min-1) and the slight mismatch between Ra and Rd caused a reduction in glycemia, similar to the effects of insulin in trout. The reduction in glucose Rd can be partially explained by a reduction in glut4b expression in red muscle. In contrast to mammals, trout alanine-dependent glucose-lowering effects did not involve hepatic AMPK activation, suggesting a different mechanistic basis. Interestingly, protein kinase B (AKT) activation increased only in muscle, similar to effects observed in insulin-infused trout. We speculate that alanine-dependent effects were probably mediated through stimulation of insulin secretion, which could indirectly promote alanine oxidation to provide the needed energy.

Contribution of fasting and postprandial glucose-lowering mechanisms to the acute hypoglycemic effect of traditionally used Eryngium cymosum F.Delaroche

Ethnopharmacological relevanceEryngium cymosum F. Delaroche was detected as a traditional remedy against type 2 diabetes consumed by patients of Tlanchinol in the state of Hidalgo, Mexico. Aim of the study. Assessing the hypoglycemic effect and safety of the traditional extract of E. cymosum and relating it to key glucose-lowering mechanisms both in fasting and postprandial state. Materials and methodsThe aqueous extract of E. cymosum was subjected to HPLC analysis to identify its main components. Hyperglycaemic STZ-NA Wistar rats were administered with the extract to evaluate its effect on blood glucose levels and a possible dose-dependence. Afterward, it was evaluated in both pyruvate and maltose tolerance tests in STZ-NA rats to characterize its effect on gluconeogenesis and carbohydrate breakdown, two of the main mechanisms responsible for fasting and postprandial hyperglycaemia in type 2 diabetes patients. In addition, the inhibitory capacity of the extract was evaluated on key enzymes involved in gluconeogenesis and a-glucosidases. Moreover, insulin concentrations were measured in normoglycemic rats in both conditions to establish a link between the hypoglycaemic effect of the extract with insulin release and functioning. ResultsCaffeic acid (1), chlorogenic acid (2), and rosmarinic acid (3) were identified as the main constituents of the aqueous extract of E. cymosum, which exerted a hypoglycaemic effect in hyperglycaemic STZ-NA rats. It has a significant antihyperglycemic effect in the pyruvate tolerance test, and it was able to reduce the postprandial hyperglycaemia in maltose tolerance tests significantly. Moreover, it effectively reduced the activity of both gluconeogenic enzymes reaching almost 100% of inhibition, while it presented a modest 32% inhibition of aglucosidases. On the other hand, the extract decreased insulin levels after its oral administration in healthy rats in both nutritional states, without affecting normoglycemia in normal curves and reducing the postprandial peak in glucose load curves. ConclusionsThe traditional consumed form of aerial parts of E. cymosum is safe and regulated glucose levels both in fasting and in postprandial state.

279-OR: Hydroxylation Regulation of Hepatic Gluconeogenesis via Prolyl-Hydroxylase 3 in Mice

Hepatic gluconeogenesis is critical for metabolic adaption in response to fasting, and its abnormal elevation contributes to hyperglycemia and type 2 diabetes. However, whether and how hydroxylation regulates hepatic gluconeogenesis remains largely unknown. Here we identified prolyl hydroxylase 3 (PHD3) of the hydroxylase family as a key regulator for hepatic gluconeogenesis. Liver-specific knockout of PHD3 (PHD3 LKO) mice show attenuated fasting gluconeogenic genes, glycaemia and hepatic capacity to produce glucose as evidenced by a pyruvate tolerance test. The efficacies of glucagon or adenylyl cyclase activator forskolin on stimulating the expression of gluconeogenic enzymes, such as PEPCK and G6Pase, and on glucose output are abolished in PHD3-/- mouse primary hepatocytes. To determine whether the prolyl hydroxylase activity is required for PHD3’s effects on promoting gluconeogenesis, liver-specific prolyl hydroxylase-deficient knockin mice with alanine mutation in PHD3 at Pro196 (PHD3 KI) were generated. Similar to PHD3 LKO mice, PHD3 KI mice exhibit attenuated fasting gluconeogenic genes, glycaemia and hepatic capacity to produce glucose, suggesting that PHD3 regulates hepatic gluconeogenesis in a proline hydroxylase-dependent manner. Moreover, several cellular proteins that associate with PHD3 were identified by comprehensive protein analysis using LC-MS/MS followed by bioinformatic analysis including GSK3β, IRS2 and OGT, which may act as PHD3 substrates and play roles in mediating PHD3’s effects on regulating glucose metabolism. Intriguingly, expression levels of PHD3 are increased in the liver diet-induced insulin resistant mice. Together, these results demonstrate that hydroxylation plays a critical role in the regulation of hepatic gluconeogenesis under physiological and insulin resistant conditions. Hyperactivation of PHD3 may underscore the potential role of PHD3 in the development of sustained gluconeogenesis, hyperglycemia and type 2 diabetes. Disclosure Y. Xue: None. J. Gao: None. X. Dai: None. A. Cui: None. Y. Li: None. G. Cai: None. F. Ma: None. Z. Liu: None. Y. Liu: None. W. Su: None. Z. Zheng: None. D. Ding: None. Y. Jiang: None. Funding National Key Research and Development Project of China (2019YFA0802502); National Natural Science Foundation of China (81925008); Shanghai Science and Technology Commission (19140903300)

1202-P: Hepatic NgBR Is a Potential Resilience Factor for Preventing Obesity-Caused Insulin Resistance

Adiponectin is an adipocyte-secreted protein hormone and prevents the development of insulin resistance. The adiponectin-mediated activation of AMP-activated protein kinase (AMPK) pathway is essential for decreasing the expression of hepatic gluconeogenic enzymes and enhancing the hepatic effects of insulin. However, the resilience factors required to maintain adiponectin-mediated AMPK activation signaling in the liver remain unclear. Recent studies from our laboratory suggest that NgBR plays a previously unrecognized role in preserving AMPK activation. The loss of NgBR in the obesity liver impairs adiponectin-dependent gluconeogenesis and insulin sensitivity. NgBR hepatocyte-specific knockout (hepKO) mice resemble many features of prediabetes, such as moderately increased fasting blood glucose levels and insulin resistance, as well as facilitating the high-fat diet (HFD)-induced onset of T2D. NgBR was identified as a cell surface receptor for soluble Nogo-B (sNogo-B). Also, NgBR binds the farnesylated form of liver kinase B1 (LKB1-farn), a key regulator of AMPK. NgBR depletion in hepatocytes abolishes the localization of LKB1 in the plasma membrane and impairs AMPK activation, the critical signal for adiponectin-mediated insulin sensitivity. NgBR expression is decreased in the liver of diabetic mice. Similar changes also occur in diabetes patients. The NgBR transcript level is reduced in T2D obese patients’ liver compared to non-T2D obese and lean subjects. Additional in vitro studies show that NgBR deficiency in hepatocyte prevents AMPK activation induced by AdipoRon (an agonist of adiponectin receptor) and increases the gluconeogenesis. Our data suggest that NgBR is a potential resilience factor required for preserving adiponectin-mediated insulin sensitivity and preventing the onset of obesity-caused T2D, and disruption of the NgBR-dependent regulation system leads to impairing gluconeogenesis regulation and insulin sensitivity in the liver. Disclosure W. Hu: None. X. Wang: None. Q. Miao: None. Funding National Institutes of Health (R01DK112971)

Novel regulation of renal gluconeogenesis by Atp6ap2 in response to high fat diet via PGC1-\u03b1/AKT-1 pathway

Recent studies suggested that renal gluconeogenesis is substantially stimulated in the kidney in presence of obesity. However, the mechanisms responsible for such stimulation are not well understood. Recently, our laboratory demonstrated that mice fed high fat diet (HFD) exhibited increase in renal Atp6ap2 [also known as (Pro)renin receptor] expression. We hypothesized that HFD upregulates renal gluconeogenesis via Atp6ap2-PGC-1α and AKT pathway. Using real-time polymerase chain reaction, western blot analysis and immunostaining, we evaluated renal expression of the Atp6ap2 and renal gluconeogenic enzymes, PEPCK and G6Pase, in wild type and inducible nephron specific Atp6ap2 knockout mice fed normal diet (ND, 12 kcal% fat) or a high-fat diet (HFD, 45 kcal% fat) for 8 weeks. Compared with ND, HFD mice had significantly higher body weight (23%) (P < 0.05), renal mRNA and protein expression of Atp6ap2 (39 and 35%), PEPCK (44 and 125%) and G6Pase (39 and 44%) respectively. In addition, compared to ND, HFD mice had increased renal protein expression of PGC-1α by 32% (P < 0.05) and downregulated AKT by 33% (P < 0.05) respectively in renal cortex. Atp6ap2-KO abrogated these changes in the mice fed HFD. In conclusion, we identified novel regulation of renal gluconeogenesis by Atp6ap2 in response to high fat diet via PGC1-α/AKT-1 pathway.

Cobalt Regulates Activation of Camk2α in Neurons by Influencing Fructose 1,6-bisphosphatase 2 Quaternary Structure and Subcellular Localization.

Fructose 1,6-bisphosphatase 2 (Fbp2) is a gluconeogenic enzyme and multifunctional protein modulating mitochondrial function and synaptic plasticity via protein-protein interactions. The ability of Fbp2 to bind to its cellular partners depends on a quaternary arrangement of the protein. NAD+ and AMP stabilize an inactive T-state of Fbp2 and thus, affect these interactions. However, more subtle structural changes evoked by the binding of catalytic cations may also change the affinity of Fbp2 to its cellular partners. In this report, we demonstrate that Fbp2 interacts with Co2+, a cation which in excessive concentrations, causes pathologies of the central nervous system and which has been shown to provoke the octal-like events in hippocampal slices. We describe for the first time the kinetics of Fbp2 in the presence of Co2+, and we provide a line of evidence that Co2+ blocks the AMP-induced transition of Fbp2 to the canonical T-state triggering instead of a new, non-canonical T-state. In such a state, Fbp2 is still partially active and may interact with its binding partners e.g., Ca2+/calmodulin-dependent protein kinase 2α (Camk2α). The Fbp2-Camk2α complex seems to be restricted to mitochondria membrane and it facilitates the Camk2α autoactivation and thus, synaptic plasticity.

GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme

SummaryHow are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.

Gluconeogenic enzyme PCK1 deficiency promotes CHK2 O-GlcNAcylation and hepatocellular carcinoma growth upon glucose deprivation.

Although cancer cells are frequently faced with a nutrient- and oxygen-poor microenvironment, elevated hexosamine-biosynthesis pathway (HBP) activity and protein O-GlcNAcylation (a nutrient sensor) contribute to rapid growth of tumor and are emerging hallmarks of cancer. Inhibiting O-GlcNAcylation could be a promising anticancer strategy. The gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 (PCK1) is downregulated in hepatocellular carcinoma (HCC). However, little is known about the potential role of PCK1 in enhanced HBP activity and HCC carcinogenesis under glucose-limited conditions. In this study, PCK1 knockout markedly enhanced the global O-GlcNAcylation levels under low-glucose conditions. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo uridine triphosphate synthesis contributing to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. Meanwhile, deletion of PCK1 also resulted in AMPK-GFAT1 axis inactivation, promoting UDP-GlcNAc synthesis for elevated O-GlcNAcylation. Notably, lower expression of PCK1 promoted CHK2 threonine 378 O-GlcNAcylation, counteracting its stability and dimer formation, increasing CHK2-dependent Rb phosphorylation and HCC cell proliferation. Moreover, aminooxyacetic acid hemihydrochloride and 6-diazo-5-oxo-L-norleucine blocked HBP-mediated O-GlcNAcylation and suppressed tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation.

Leaf extracts of Glyphaea brevis attenuate high blood glucose and lipids in diabetic rats induced with streptozotocin

Background: Diabetes is a non-communicable disease causing impairment of carbohydrate and lipid metabolisms. Previous ethnobotanical surveys showed that leaves of Glyphaea brevis (G. brevis) are used conventionally in the treatment of diabetes with limited scientific evidence. Objective: We aimed to determine the effects of aqueous (AE) and hydroethanolic extracts (HEE) of G. brevis leaves in attenuating diabetes-linked hyperglycemia and hyperlipidemia in an animal model. Materials and Methods: Thirty streptozotocin-induced diabetic male rats were divided into six groups receiving the following daily treatments for 4 weeks orally: control (distilled water), reference (tolbutamide 80 mg/kg) and 4 tests (AE 250 mg/kg, AE 500 mg/kg, HEE 250 mg/kg, and HEE 500 mg/kg). The effects of each treatment on postprandial hyperglycemia were assessed using oral glucose tolerance and oral starch tolerance tests. Blood was collected to assess the effects of treatments on fasting plasma glucose, glycated hemoglobin (HbA1c), and lipid profile. Liver glycogen and gluconeogenic enzyme activity were also measured. Results: AE- and HEE-treated rats had 36%–64% lower fasting blood glucose levels, 34%–73% lower postprandial glycemia, and 15%–75% lower HbA1c than rats from control group (P

Amelioration effect of black seed oil against high-fat diet-induced obesity in rats through Nrf2/HO-1 pathway.

Obesity is a chronic inflammatory disease that represents a risk factor for number of diseases including diabetes, steatohepatitis, and cancer. Using safe natural compounds to ameliorate obesity and its related metabolic syndrome is an interesting spot for research. We investigated the regulatory role and the underlying mechanism of black seed oil (BSO) on high-fat diet (HFD)-induced obesity in rats. The study included two models: the first one aimed to study the prophylactic effect of BSO (BSO administration for 10weeks along with HFD) while the second one aimed to study the treatment role of BSO (BSO administration starting from the 10th week for 4weeks along with HFD). BSO significantly decreased insulin resistance and body weight characteristics in both models. It also normalized lipid profile. Moreover, histopathological examination confirmed these results as BSO significantly decreased adipocyte size and hepatic lipid deposition. Besides, BSO alleviated HFD-induced oxidative stress as indicated by significant increase in the total antioxidant capacity and significant decrease in liver malondialdehyde. Moreover, BSO decreased significantly liver gluconeogenic enzymes mRNA expressions (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) and increased significantly heme oxygenase-1 (HO-1), nuclear factor erythroid-2-related factor-2 (Nrf2) and insulin receptor mRNA expressions. In conclusion, BSO represents a natural therapy that has the ability to prevent and treat HFD-induced obesity in rats that may be mediated through Nrf2/HO-1 pathway's activation and insulin receptor expression's increase. To our best knowledge, this study represents a novel study that investigates the regulatory role of BSO on Nrf2 pathway in preventing and treating HFD-induced obesity. PRACTICAL APPLICATIONS: Black seed oil is a natural available safe supplement, thus it can be used for prevention from obesity and even treatment of obesity and obesity related complications. Introducing of black seed oil in the treatment regimen of obese patients may be promising.

The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Glucose and Fatty Acid Metabolism of Dairy Cows in a Total Mixed Ration or Pasture-Based System During Lactation

In this study, we explored mechanisms related to glucose and fatty acid metabolism in Holstein–Friesian multiparous dairy cows during lactation under two feeding strategies. From 0 to 180 days postpartum, cows were fed total mixed ration (TMR) ad libitum (non-grazing group, G0) or grazed Festuca arundinacea or Medicago sativa and were supplemented with 5.4 kg DM/d of an energy-protein concentrate (grazing group, G1). From 180 to 250 days postpartum, all cows grazed F. arundinacea and were supplemented with TMR. Plasma samples and liver biopsies were collected at −14, 35, 60, 110, 180, and 250 days in milk (DIM) for metabolite, hormone, gene expression, and western blot analysis. Our results showed increased levels of negative energy balance markers: plasma non-esterified fatty acids (NEFA), liver triglyceride and plasma β-hydroxybutyrate (BHB) (P &amp;lt; 0.01), triglyceride and β-hydroxybutyrate concentration were especially elevated for G1 cows. Also, hepatic mRNA expression of gluconeogenic enzymes was upregulated during early lactation (P &amp;lt; 0.05). In particular, methymalonyl-CoA mutase expression was increased for G0 cows (P &amp;lt; 0.05) while pyruvate carboxylase (PC) expression was increased for G1 cows (P &amp;lt; 0.05), suggesting differential gluconeogenic precursors for different feeding strategies. Phosphorylation of AMP-activated protein kinase was increased in early lactation vs. late lactation (P &amp;lt; 0.01) and negatively correlated with PC mRNA levels. The positive association of gluconeogenic genes with proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) hepatic expression supported the importance of this transcription factor in glucose metabolism. The peroxisome proliferator-activated receptor alpha (PPARA) mRNA was increased during early lactation (P &amp;lt; 0.05), and was positively associated to PPARGC1A, carnitine palmitoyl-transferase 1, and hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) mRNA expression. Alongside, hepatic mRNA expression of FABP was decreased for G1 vs. G0 cows (P &amp;lt; 0.05), possibly linked to impaired fatty acid transport and related to accumulation of liver triglycerides, evidencing G1 cows fail to adapt to the demands of early lactation. In sum, our results showed that metabolic adaptations related to early lactation negative energy balance can be affected by feeding strategy and might be regulated by the metabolic sensors AMPK, SIRT1, and coordinated by transcription factors PPARGC1A and PPARA.

Roles of hepatic atypical protein kinase C hyperactivity and hyperinsulinemia in insulin-resistant forms of obesity and type 2 diabetes mellitus.

Diet‐induced obesity, the metabolic syndrome, type 2 diabetes (DIO/MetS/T2DM), and their adverse sequelae have reached pandemic levels. In mice, DIO/MetS/T2DM initiation involves diet‐dependent increases in lipids that activate hepatic atypical PKC (aPKC) and thereby increase lipogenic enzymes and proinflammatory cytokines. These or other hepatic aberrations, via adverse liver‐to‐muscle cross talk, rapidly impair postreceptor insulin signaling to glucose transport in muscle. The ensuing hyperinsulinemia further activates hepatic aPKC, which first blocks the ability of Akt to suppress gluconeogenic enzyme expression, and later impairs Akt activation, further increasing hepatic glucose production. Recent findings suggest that hepatic aPKC also increases a proteolytic enzyme that degrades insulin receptors. Fortunately, all hepatic aberrations and muscle impairments are prevented/reversed by inhibition or deficiency of hepatic aPKC. But, in the absence of treatment, hyperinsulinemia induces adverse events, some by using “spare receptors” to bypass receptor defects. Thus, in brain, hyperinsulinemia increases Aβ‐plaque precursors and Alzheimer risk; in kidney, hyperinsulinemia activates the renin–angiotensin–adrenal axis, thus increasing vasoconstriction, sodium retention, and cardiovascular risk; and in liver, hyperinsulinemia increases lipogenesis, obesity, hepatosteatosis, hyperlipidemia, and cardiovascular risk. In summary, increases in hepatic aPKC are critically required for development of DIO/MetS/T2DM and its adverse sequelae, and therapeutic approaches that limit hepatic aPKC may be particularly effective.

A bioinformatics and transcriptomics based investigation reveals an inhibitory role of Huanglian-Renshen-Decoction on hepatic glucose production of T2DM mice via PI3K/Akt/FoxO1 signaling pathway

BackgroundExcessive hepatic glucose production (HGP) largely promotes the development of type 2 diabetes mellitus (T2DM), and the inhibition of HGP significantly ameliorates T2DM. Huanglian-Renshen-Decoction (HRD), a classic traditional Chinese herb medicine, is widely used for the treatment of diabetes in clinic for centuries and proved effective. However, the relevant mechanisms of HRD are not fully understood. PurposeBased on that, this study was designed to identify the potential effects and underlying mechanisms of HRD on HGP by a comprehensive investigation that integrated in vivo functional experiments, network pharmacology, molecular docking, transcriptomics and molecular biology. MethodsAfter confirming the therapeutic effects of HRD on T2DM mice, the inhibitory role of HRD on HGP was evaluated by pyruvate and glucagon tolerance tests, liver positron emission tomography (PET) imaging and the detection of gluconeogenic key enzymes. Then, network pharmacology and transcriptomics approaches were used to clarify the underlying mechanisms. Molecular biology, computational docking analysis and in vitro experiments were applied for final mechanism verification. ResultsHere, our results showed that HRD can decrease weight gain and blood glucose, increase fasting insulin, glucose clearance and insulin sensitivity in T2DM mice. Dysregulated lipid profile was also corrected by HRD administration. Pyruvate, glucagon tolerance tests and liver PET imaging all indicated that HRD inhibited the abnormal HGP of T2DM, and the expressions of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) were significantly suppressed by HRD as expected. Network pharmacology and transcriptomics approaches illustrated that PI3K/Akt/FoxO1 signaling pathway may be responsible for the inhibitory effect of HRD on HGP. Afterward, further western blot and immunoprecipitation found that HRD did activate PI3K/Akt/FoxO1 signaling pathway in T2DM mice, which confirmed previous results. Additionally, the conclusion was further supported by molecular docking and in vitro experiments, in which identified HRD compound, oxyberberine, was proven to exert an obvious effect on Akt. ConclusionOur data demonstrated that HRD can treat T2DM by inhibiting hepatic glucose production, the underlying mechanisms were associated with the activation of PI3K/Akt/FoxO1 signaling pathway.

Long-Term Aspartame Administration Leads to Fibrosis, Inflammasome Activation, and Gluconeogenesis Impairment in the Liver of Mice.

Simple SummaryAspartame is an artificial sweetener used in foods and beverages worldwide to prevent increasing obesity and diabetes mellitus, acting as a tool to help the control of caloric intake. However, its chronic intake is controversial since it has been linked to some adverse effects including oxidative stress, inflammation, and liver damage through mechanisms that are not fully elucidated yet. Thus, this work aimed to investigate the effects of long-term administration of aspartame on the oxidative and inflammatory mechanisms associated with liver fibrosis progression in mice. Aspartame generated liver injury and fibrosis. It also decreased the activity of antioxidant enzymes and increased the levels of lipid peroxidation, thus, probably, triggering inflammation and cell death through the induction of protein 53 (p53). Finally, via p53 activation, aspartame inhibited a transcriptional coactivator, the peroxisome proliferator-activated receptor gamma coactivator 1 alpha, a master regulator of glucose and lipid metabolism, probably leading to changes in lipid profile in serum, total lipid accumulation, as well as an impairment in the gluconeogenesis in mouse liver, thus causing hypoglycemia. Therefore, this study provides new insights to understand the mechanisms related to aspartame-linked adverse effects, showing that its intake should be cautioned.Background: Aspartame is an artificial sweetener used in foods and beverages worldwide. However, it is linked to oxidative stress, inflammation, and liver damage through mechanisms that are not fully elucidated yet. This work aimed to investigate the effects of long-term administration of aspartame on the oxidative and inflammatory mechanisms associated with liver fibrosis progression in mice. Methods: Mice were divided into two groups with six animals each: control and aspartame. Aspartame (80 mg/kg, via oral) or vehicle was administrated for 12 weeks. Results: Aspartame caused liver damage and elevated serum transaminase levels. Aspartame also generated liver fibrosis, as evidenced by histology analysis, and pro-fibrotic markers’ upregulation, including transforming growth factor β 1, collagen type I alpha 1, and alpha-smooth muscle actin. Furthermore, aspartame reduced nuclear factor erythroid 2-related factor 2 (Nrf2) activation and enzymatic antioxidant activity and increased lipid peroxidation, which triggered NOD-like receptor containing protein 3 (NLRP3) inflammasome activation and p53 induction. Furthermore, aspartame reduced peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) levels, possibly through p53 activation. This PGC-1α deficiency could be responsible for the changes in lipid profile in serum, total lipid accumulation, and gluconeogenesis impairment in liver, evidenced by the gluconeogenic enzymes’ downregulation, thus causing hypoglycemia. Conclusions: This work provides new insights to understand the mechanisms related to the adverse effects of aspartame on liver tissue.

Lipocalin-2: a role in hepatic gluconeogenesis via AMP-activated protein kinase (AMPK).

Evidence is accumulating that lipocalin2 (LCN2) is implicated in insulin resistance and glucose homeostasis, but the underlying possible mechanisms remain unclear. This study is to investigate the possible linkage between LCN2 and AMP-activated protein kinase (AMPK) or forkhead transcription factor O1 (FoxO1), which influences insulin sensitivity and gluconeogenesis in liver. LCN2 knockout (LCN2KO) mice and wild-type littermates were used to evaluate the effect of LCN2 on insulin sensitivity and hepatic gluconeogenesis through pyruvate tolerance test (PTT), glucose tolerance test (ipGTT), insulin tolerance test (ITT), and hyperinsulinemic-euglycemic clamps, respectively. LCN2KO mice and WT mice in vivo, and in vitro HepG2 cells were co-transfected with adenoviral FoxO1-siRNA (Ad-FoxO1-siRNA) or adenovirus expressing constitutively active form of AMPK (Ad-CA-AMPK), or dominant negative adenovirus AMPK (Ad-DN-AMPK), the relative mRNA and protein levels of two key gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6P) were measured. Improved insulin sensitivity and inhibited gluconeogenesis in the LCN2KO mice were confirmed by pyruvate tolerance tests and hyperinsulinemic-euglycemic clamps. Nuclear FoxO1 and its downstream genes PEECK and G6P were decreased in the livers of the LCN2KO mice, and AMPK activity was stimulated and directly phosphorylated FoxO1. In vitro, AMPK activity was inhibited in HepG2 cells overexpressing LCN2 leading to a decrease in phosphorylated FoxO1 and an increase in nuclear FoxO1. The present study demonstrates that LCN2 regulates insulin sensitivity and glucose metabolism through inhibiting AMPK activity, and regulating FoxO1 and its downstream genes PEPCK/G6P, which regulate hepatic gluconeogenesis.

Expression of the ace operon in Escherichia coli is triggered in response to growth rate-dependent flux-signal of ATP.

The signal that triggers the expression of the ace operon and, in turn, the transition of central metabolism's architecture from acetogenic to gluconeogenic in Escherichia coli remains elusive despite extensive research both in vivo and in vitro. Here, with the aid of flux analysis together with measurements of the enzymic activity of isocitrate lyase (ICL) and its aceA-messenger ribonucleuc acid (mRNA) transcripts, we provide credible evidence suggesting that the expression of the ace operon in E. coli is triggered in response to growth rate-dependent threshold flux-signal of adenosine triphosphate (ATP). Flux analysis revealed that the shortfall in ATP supply observed as the growth rate ($\mu $) diminishes from µmax to≤0.43h-1 ($ \pm 0.02;n4)\ $is partially redressed by up-regulating flux through succinyl CoA synthetase. Unlike glycerol and glucose, pyruvate cannot feed directly into the two glycolytic ATP-generating reactions catalyzed by phosphoglycerokinase and pyruvate kinase. On the other hand, glycerol, which upon its conversion to D-glyceraldehyde, feeds into the phosphorylation and dephosphorylation parts of glycolysis including the substrate-level phosphorylation-ATP generating reactions, thus preventing ATP flux from dropping to the critical threshold signal required to trigger the acetate-diauxic switch until glycerol is fully consumed. The mRNA transcriptional patterns of key gluconeogenic enzymes, namely, ackA, acetate kinase; pta, phosphotransacetylase; acs, acetyl CoA synthetase and aceA, ICL, suggest that the pyruvate phenotype is better equipped than the glycerol phenotype for the switch from acetogenic to gluconeogenic metabolism.

Phosphoenolpyruvate Carboxykinase (PCK) in the Brain Gluconeogenic Pathway Contributes to Oxidative and Lactic Injury After Stroke.

To demonstrate the role of the rate-limiting and ATP-dependent gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) in oxidative and lactic stress and the effect of phenothiazine on PCK after stroke, a total of 168 adult male Sprague Dawley rats (3months old, 280-300g) underwent 2-h intraluminal middle cerebral artery occlusion (MCAO) and reperfusion for 6, 24, 48h, or 7days. Phenothiazine (chlorpromazine and promethazine (C+P)) (8mg/kg) and 3-mercaptopicolinic acid (3-MPA, a PCK inhibitor, 100μM) were administered at reperfusion onset. The effects of phosphoenolpyruvate, 3-MPA, or PCK knockdown were studied in neuronal cultures subjected to oxygen/glucose deprivation. Reactive oxygen species, lactate, phosphoenolpyruvate (PEP; a gluconeogenic product), mRNA, and protein of total PCK, PCK-1, and PCK-2 increased after MCAO and oxygen-glucose deprivation (OGD). Oxaloacetate (a gluconeogenic substrate) decreased, while PEP and glucose were increased, suggesting reactive gluconeogenesis. These changes were attenuated by phenothiazine, 3-MPA, or PCK shRNA. PCK-1 and -2 existed primarily in neurons, while the effects of ischemic stroke on the PCK expression were seen predominately in astrocytes. Thus, phenothiazine reduced infarction and oxidative/lactic stress by inhibiting PCKs, leading to functional recovery.

Role of small leucine zipper protein in hepatic gluconeogenesis and metabolic disorder.

Hepatic gluconeogenesis is the central pathway for glucose generation in the body. The imbalance between glucose synthesis and uptake leads to metabolic diseases such as obesity, diabetes, and cardiovascular diseases. Small leucine zipper protein (sLZIP) is an isoform of LZIP and it mainly functions as a transcription factor. Although sLZIP is known to regulate the transcription of genes involved in various cellular processes, the role of sLZIP in hepatic glucose metabolism is not known. In this study, we investigated the regulatory role of sLZIP in hepatic gluconeogenesis and its involvement in metabolic disorder. We found that sLZIP expression was elevated during glucose starvation, leading to the promotion of phosphoenolpyruvate carboxylase and glucose-6-phosphatase expression in hepatocytes. However, sLZIP knockdown suppressed the expression of the gluconeogenic enzymes under low glucose conditions. sLZIP also enhanced glucose production in the human liver cells and mouse primary hepatic cells. Fasting-induced cyclic adenosine monophosphate impeded sLZIP degradation. Results of glucose and pyruvate tolerance tests showed that sLZIP transgenic mice exhibited abnormal blood glucose metabolism. These findings suggest that sLZIP is a novel regulator of gluconeogenic enzyme expression and plays a role in blood glucose homeostasis during starvation.

Association of phosphoenolpyruvate carboxykinase 1 protein kinase activity-dependent sterol regulatory element-binding protein 1 activation with prognosis of oesophageal carcinoma

Metabolic enzymes have non-canonical functions and play vital roles in the regulation of various cellular activities. Phosphoenolpyruvate carboxykinase 1 (PCK1), a gluconeogenic enzyme, was recently identified as an AKT-dependent protein kinase and promoted sterol regulatory element-binding protein 1 (SREBP1)-dependent lipogenesis. However, association of this protein kinase activity of PCK1 with progression of oesophageal squamous cell carcinoma (ESCC) is unclear. We examined 200 ESCC patientsamples and prognosis using immunohistochemistry, multivariate Cox regressionand Kaplan-Meier Plot analyses. We show that the expression levels of AKT pS473, AKT-regulated PCK1 pS90, PCK1-mediated INSIG1 pS207/INSIG2 pS151and nuclear SREBP1 were higher in analysed 200 human ESCC specimens than in their adjacent non-tumour tissues; the expression levels of these proteins were significantly and positively correlated with each other in tumour specimens. In addition, the expression levels of PCK1 pS90, INSIG1 pS207/INSIG2 pS151and SREBP1 were associated with the tumour, nodeand metastasisstage and progression in ESCC. Importantly, levels of PCK1 pS90 or INSIG1 pS207/INSIG2 pS151 or nuclear SREBP1 were positively correlated with poor prognosis in patients with ESCC, and the combined expression values of PCK1 pS90, INSIG1 pS207/INSIG2 pS151and nuclear SREBP1 had a better prognostic value than that of each individual protein expression value and was an independent prognostic marker for ESCC. These findings reveal the role of PCK1 protein kinase activity-dependent SREBP1 activation in ESCC progression. The regulation of SREBP1 by AKT activation-dependent PCK1 protein kinase activity may provide the potential for the diagnosis and treatment of human ESCC.