Levels Of Phosphoenolpyruvate Carboxykinase Research Articles

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

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. 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. 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. 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.

Development of a Zebrafish Larvae Model for Diabetic Heart Failure With Reduced Ejection Fraction

Diabetes mellitus (DM)-associated heart failure (HF) causes high morbidity and mortality. In this study, we established a zebrafish larvae model for in vivo research on diabetic HF. DM-like phenotypes were induced by treating zebrafish larvae with a combination of D-glucose (GLU) and streptozotocin (STZ). HF was induced by treatment with terfenadine (TER), a potassium channel blocker. Additionally, myocardial contractility, motility, and viability were evaluated. The zebrafish larvae treated with a combination of GLU and STZ showed significantly higher whole-body glucose concentrations, lower insulin levels, and higher phosphoenolpyruvate carboxykinase levels, which are markers of abnormal glucose homeostasis, than the group treated with only GLU, with no effect on viability. When treated with TER, DM zebrafish showed significantly less myocardial fractional shortening and more irregular contractions than the non-DM zebrafish. Furthermore, in DM-HF with reduced ejection fraction (rEF) zebrafish, a significant increase in the levels of natriuretic peptide B, a HF biomarker, markedly reduced motility, and reduced survival rates were observed. We established a DM-HFrEF zebrafish model by sequentially treating zebrafish larvae with GLU, STZ, and TER. Our findings indicate the potential utility of the developed zebrafish larvae model not only in screening studies of new drug candidates for DM-HFrEF but also in mechanistic studies to understand the pathophysiology of DM-HFrEF.

DNA methylation of pck1 might contribute to the programming effects of early high-carbohydrate diets feeding to the glucose metabolism across two generations in zebrafish (Danio rerio).

The purpose of this study is to assess the effects of early high-carbohydrate stimulus on glucose metabolism in zebrafish (Danio rerio) over two generations and explore the mechanisms that explain those nutritional programming effects via epigenetic modifications. The larvae were delivered a high-carbohydrate diet (53.66%) that was used as an early nutritional stimulus from the first feeding to the end of the yolk sac (FF) and 5days after yolk-sac exhaustion (YE). The larvae (F0) and their offspring (F1) were then both fed the control diet (22.69%) until adulthood (15weeks), and they were challenged with a high-carbohydrate diet (35.36%) at the 16th week. The results indicated that early stimulus immediately raised the mRNA levels of genes involved in glycolysis and gluconeogenesis. At the end of F0 challenge, both treatment groups decreased the plasma glucose levels, increased the expression levels of glucokinase (gck), and inhibited the mRNA during gluconeogenesis. When challenged in F1, the glucose levels were lower in FF (F1), and the mRNA levels of phosphoenolpyruvate carboxykinase 1 (pck1) were decreased in FF (F1) and YE (F1). Besides, in both experimental groups (F0 and F1), the CpG island of pck1 maintained lower levels of hypermethylated expression from F0 adult, 24h post-fertilization embryo, to F1 adult. In conclusion, these results indicated that an early high-carbohydrate stimulus could significantly reprogram glucose metabolism in adult zebrafish, that those modifications could be partially transmitted to the next generation, and that the DNA methylation of pck1 might work as a stable epigenetic marker to contribute to those processes.

Effect of quercetin and Abelmoschus esculentus (L.) Moench on lipids metabolism and blood glucose through AMPK-α in diabetic rats (HFD/STZ).

Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme in the glyconeogenesis pathway. The AMP-activated protein kinase alpha (AMPK-α) pathway regulates PEPCK, which itself is activated by the AMP/ATP ratio and liver kinase B1 (KB1). The Abelmoschus esculentus (L.) Moench (okra) plant contains a large amount of quercetin that can function as an agonist or an antagonist. The aim of this study was to examine the effects of quercetin flavonoid and A. esculentus extract on the level of AMPK-α expression and associated metabolic pathways. The findings demonstrate that metformin, quercetin, and okra extract may significantly raise AMPK-α levels while significantly lowering PEPCK and hormone-sensitive lipase (HSL) levels, in addition to improving glucose and lipid profiles. By stimulating KB1, these substances increased AMPK-α activation. Additionally, AMPK-α activation improved insulin resistance and Glucose transporter type 4 (GLUT4) gene expression levels. Since AMPK-α maintains energy balance and its activity has not been reported to be inhibited so far, it could be a potent therapeutic target. PRACTICAL APPLICATIONS: The development of effective AMPK-α agonists and antagonists holds promise for the treatment of metabolic disorders like diabetes. Dietary polyphenols are a valuable source for developing new drugs. However, due to the lack of understanding of the underlying mechanisms of their effect on cells, their use in the treatment of diabetes is controversial. In addition to chemicals that have medicinal benefits, chemists are searching for less harmful substances. Using plants containing bioactive chemicals for this purpose can be a good alternative to chemical drugs.

Organic chromium derived from the chelation of Ganoderma lucidum polysaccharide and chromium (III) alleviates metabolic syndromes and intestinal microbiota dysbiosis induced by high-fat and high-fructose diet

Organic chromium is of great interest and has become an important chromium supplement resource in recent years because of its low toxicity and easy absorption. In our previous study, we synthesized a novel organic chromium [GLP-Cr] through the chelation of Ganoderma lucidum polysaccharide and chromium (III). The purpose of this study was to investigate the beneficial effects of GLP-Cr on the improvement of metabolic syndromes (MetS) in mice fed with a high-fat and high-fructose diet (HFHFD) and its mechanism of action. The results indicated that oral administration of GLP-Cr inhibited the excessive exaltation of body weight, glucose tolerance, fasting blood glucose and lipid levels, hepatic total cholesterol (TC), triglyceride (TG) levels caused by HFHFD. Besides, 16S rRNA amplicon sequencing showed that GLP-Cr intervention evidently ameliorated intestinal microbiota dysbiosis by changing the proportions of some intestinal microbial phylotypes. In addition, correlation network-based analysis indicated that the key intestinal microbial phylotypes were closely related to biochemical parameters associated with MetS under GLP-Cr intervention. Liver metabolomics analysis suggested that GLP-Cr intervention significantly regulated the levels of some biomarkers involved in alpha-linolenic acid metabolism, fatty acid biosynthesis, steroid hormone biosynthesis, glycerophospholipid metabolism, glycerolipid metabolism, steroid hormone biosynthesis, primary bile acid biosynthesis, and so on. Moreover, GLP-Cr intervention regulated liver mRNA levels of key genes associated with glucose and lipid metabolism. The mRNA level of glucose transporter type 4 (Glut4) was markedly increased by GLP-Cr intervention, and the mRNA levels of phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6Pase) in the liver were significantly decreased. Meanwhile, GLP-Cr intervention significantly decreased hepatic mRNA levels of cluster of differentiation 36 (Cd36), acetyl-CoA carboxylase 1 (Acc1) and sterol regulatory element binding protein-1c (Srebp-1c), indicating that GLP-Cr intervention inhibited the excessive accumulation of free fatty acids in the liver. These findings suggest that the prevention of hyperglycemia and dyslipidemia by GLP-Cr may be closely related to the regulation of gut microbial composition and hepatic metabolic pathways, thus GLP-Cr can be serving as a functional component in the prevention of MetS.

IPMK modulates hepatic glucose production and insulin signaling.

Hepatic glucose production (HGP) is crucial for the maintenance of normal glucose homeostasis. Although hepatic insulin resistance contributes to excessive glucose production, its mechanism is not well understood. Here, we show that inositol polyphosphate multikinase (IPMK), a key enzyme in inositol polyphosphate biosynthesis, plays a role in regulating hepatic insulin signaling and gluconeogenesis both in vitro and in vivo. IPMK-deficient hepatocytes exhibit decreased insulin-induced activation of Akt-FoxO1 signaling. The expression of messenger RNA levels of phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose 6-phosphatase (G6pc), key enzymes mediating gluconeogenesis, are increased in IPMK-deficient hepatocytes compared to wild type hepatocytes. Importantly, re-expressing IPMK restores insulin sensitivity and alleviates glucose production in IPMK-deficient hepatocytes. Moreover, hepatocyte-specific IPMK deletion exacerbates hyperglycemia and insulin sensitivity in mice fed a high-fat diet, accompanied by an increase in HGP during pyruvate tolerance test and reduction in Akt phosphorylation in IPMK deficient liver. Our results demonstrate that IPMK mediates insulin signaling and gluconeogenesis and may be potentially targeted for treatment of diabetes.

Long-noncoding RNA HOXA transcript at the distal tip ameliorates the insulin resistance and hepatic gluconeogenesis in mice with gestational diabetes mellitus via the microRNA-423-5p/wingless-type MMTV integration site family member 7A axis

ABSTRACT Long-noncoding RNA HOXA transcript at the distal tip (HOTTIP) has been probed to exert essential effects on diabetes progression, while its function in gestational diabetes mellitus (GDM) remains unclear. This study was committed to unravel the effects of HOTTIP on GDM progression via the microRNA (miR)-423-5p/wingless-type MMTV integration site family member 7A (WNT7A) axis. The GDM mouse model was established. HOTTIP, miR-423-5p and WNT7A levels in GDM mice were examined. The saline with dissolved various constructs altering HOTTIP, miR-423-5p and WNT7A expression was injected into GDM mice to detect the levels of GDM‐related biochemical indices, HOMA indices, liver gluconease: expression levels of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G-6-Pase), glucose transporter 2 (GLUT2) and pathological changes of pancreatic tissues, and the apoptosis rate of pancreatic cells in GDM mice. The relations among HOTTIP, miR-423-5p and WNT7A were validated. HOTTIP and WNT7A levels were decreased while miR-423-5p was elevated in GDM mice. The enriched HOTTIP or silenced miR-423-5p alleviated the levels of GDM‐relatedbiochemical indices, enhanced the insulin homeostasis, elevated GLUT2 expression and decreased G-6-pase and PEPCK expression, mitigated the pathological changes of pancreatic tissues, and hindered the apoptosis of pancreatic cells. MiR-143-5p upregulation abrogated the effects of elevated HOTTIP on repressing GDM; whereas WNT7A deletion reversed the therapeutic effects of reduced miR-423-5p. HOTTIP sponged miR-423-5p that targeted WNT7A. HOTTIP ameliorates insulin resistance and hepatic gluconeogenesis in GDM mice via the modulation of the miR-423-5p/WNT7A axis. This study affords novel therapeutic modalities for GDM treatment.

Ethanolic extract of Salacca zalacca peel reduce IL-1β and apoptosis in high glucose induced zebrafish embryo

Gestational diabetes is a glucose intolerance level first identified during pregnancy. This condition is caused by hormone secretion that hinders insulin action from the placenta which caused insulin resistance that will cause hyperglycemia. This condition will increase oxidative stress, and in consequence increasing inflammation and cell death which can hinder fetal growth and development. Salacca zalacca peel is a food waste that contains antioxidants and anti-inflammatory properties. This research aimed to study the effect of SZ peel ethanolic extract in the expression of IL-1β and cell death marker (BAX and Apaf-1) in glucose 3% induced zebrafish embryo. Glucose metabolism is measured by the level of Phosphoenolpyruvate carboxykinase (PEPCK) at the age of 3 dpf. The expression of PEPCK, IL-1β, Bax, and Apaf-1 is measured using reverse transcriptase PCR (RT-PCR) method. Salacca zalacca peel extract is given in the concentration of 0,1; 0,2; and 0,4 mg/mL. The result of this research proves that PEPCK decreases in the group which was given glucose 3%. Salacca zalacca peel extract significantly decreases the expression of IL-1β and Bax in the concentration of 0,4 mg/mL, and also able to decrease Apaf-1 expression with the result that most closely resemble the negative control in the concentration of 0,4 mg/mL. In conclusion, Salacca zalacca peel extract protects from gestational diabetes condition through the decrease of IL-1β, Bax, and Apaf-1.

Mentha cordifolia Leaf Extract Improves Hepatic Glucose and Lipid Metabolism in Obese Mice Fed with High-Fat Diet.

Mentha cordifolia (MC) is a popular herb used to flavor food in Thailand that exhibits several biological effects. The present study aimed to determine the role of MC in regulating glucose and lipid metabolism in mice fed a high-fat diet (HFD). ICR obese mice were fed an HFD (45 kcal% lard fat) for 12 weeks, with MC (100 and 200 mg/kg/d) treatment from Week 7. After treatment with MC for 6 weeks, mice showed significantly lower rates of hyperglycemia, hyperinsulinemia, hyperleptinemia, and hyperlipidemia, and increased amounts of serum adiponectin. Furthermore, in mice treated with MC, serum interleukin-6 and tumor necrosis factor alpha were significantly inhibited and liver histology results showed decreased lipid accumulation and liver triglyceride content vs. untreated mice. In addition, MC treatment was associated with smaller fat cells and lower gene expression of liver sterol regulatory element binding protein 1c, acetyl-CoA carboxylase, and fatty acid synthase. However, MC treatment was associated with higher carnitine palmitoyltransferase 1a gene expression and significantly higher rates of adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in liver, but lower levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. These results indicate MC regulates glucose and lipid metabolism in a HFD-induced obese mouse model, possibly via activation of AMPK signaling pathway.

A potential probiotic bacterium for antipsychotic-induced metabolic syndrome: mechanisms underpinning how Akkermansia muciniphila subtype improves olanzapine-induced glucose homeostasis in mice.

Olanzapine (OLZ) is one of the most effective atypical antipsychotics but is associated with severe metabolic side effects, in which the gut microbiota plays an important role. Akkermansia muciniphila (A. muciniphila; Akk), a Gram-negative anaerobic bacterium in the intestine, can potentially improve metabolic syndrome. This study investigated the effect and underlying mechanisms of an A. muciniphila subtype (A. muciniphilasub; Akksub) on OLZ-induced metabolic dysfunction in lean and obese mice. C57BL/6 female mice were fed a high-fat diet to induce obesity or normal chow for 8weeks before OLZ treatment for 16weeks. During the treatment period, mice in each group were orally administrated A. muciniphilasub. Weight gain, glucose and lipid metabolism, and inflammation were evaluated. A. muciniphilasub decreased OLZ-related weight gain only at week 16 in lean mice and significantly alleviated OLZ-induced hyperglycemia irrespective of diet. This was accompanied by reduced levels of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK)-key enzymes in hepatic gluconeogenesis-and OLZ-associated insulin resistance. Moreover, OLZ-induced increases in serum interleukin (IL)-6 and tumor necrosis factor (TNF)-α levels were improved by A. muciniphilasub in both obese and lean mice. OLZ did not increase serum lipid levels or hepatic fat accumulation. A. muciniphilasub improves OLZ-related hyperglycemia via regulation of G6Pase and PEPCK levels and insulin resistance. Moreover, A. muciniphilasub alleviates systemic inflammation caused by OLZ. A. muciniphilasub is a promising probiotic treatment for OLZ-induced metabolic dysfunction.

Low-level lead exposure promotes hepatic gluconeogenesis and contributes to the elevation of fasting glucose level

BackgroundLead (Pb) is considered an endocrine-disrupting chemical. However, few studies have investigated the effects of low-level Pb exposure on plasma glucose levels. Herein, we aimed to investigate whether low-level Pb exposure causes elevated plasma glucose levels and the possible mechanisms involved. MethodsWe conducted a cross-sectional study of 5747 participants from 16 sites in China. The participants underwent measurements of anthropometric factors, blood lead level (BLL) and fasting plasma glucose (FPG). Wistar rats were exposed to 0.05% Pb through drinking water or fed with a high-fat diet (HFD) for 28 weeks. The relevant parameters of glucose homeostasis, hepatic glucose production (HGP) and gene expression levels of hepatic gluconeogenesis enzymes, including phosphoenolpyruvate carboxy kinase (PEPCK), glucose-6-phosphatase (G6PC) and fructose-1,6-bisphosphatase (FBP1), were measured. In addition, gene expression levels of gluconeogenesis enzymes were also measured in HepG2 cells administered with different concentrations of lead acetate for 24 h. ResultsIn humans, after adjusting for confounders, the odds of having High_FPG (≥5.6 mmol/L) were significantly increased by 25% in the participants in the fourth BLL quartile (OR 1.25, 95% CI 1.05, 1.49). In the animals exposed to 0.05% Pb, FPG, HGP and hepatic gene expression levels of PEPCK, G6PC and FBP1 were increased. In addition, the mRNA expression levels of PEPCK, G6PC and FBP1 in HepG2 cells were also increased in response to Pb exposure. ConclusionsThese findings support the possibility that low-level Pb exposure may increase HGP by affecting key enzymes of hepatic gluconeogenesis, eventually resulting in impaired FPG and hyperglycemia.

SL010110, a lead compound, inhibits gluconeogenesis via SIRT2-p300-mediated PEPCK1 degradation and improves glucose homeostasis in diabetic mice.

Suppression of excessive hepatic gluconeogenesis is an effective strategy for controlling hyperglycemia in type 2 diabetes (T2D). In the present study, we screened our compounds library to discover the active molecules inhibiting gluconeogenesis in primary mouse hepatocytes. We found that SL010110 (5-((4-allyl-2-methoxyphenoxy) methyl) furan-2-carboxylic acid) potently inhibited gluconeogenesis with 3 μM and 10 μM leading to a reduction of 45.5% and 67.5%, respectively. Moreover, SL010110 caused suppression of gluconeogenesis resulted from downregulating the protein level of phosphoenolpyruvate carboxykinase 1 (PEPCK1), but not from affecting the gene expressions of PEPCK, glucose-6-phosphatase, and fructose-1,6-bisphosphatase. Furthermore, SL010110 increased PEPCK1 acetylation, and promoted PEPCK1 ubiquitination and degradation. SL010110 activated p300 acetyltransferase activity in primary mouse hepatocytes. The enhanced PEPCK1 acetylation and suppressed gluconeogenesis caused by SL010110 were blocked by C646, a histone acetyltransferase p300 inhibitor, suggested that SL010110 inhibited gluconeogenesis by activating p300. SL010110 decreased NAD+/NADH ratio, inhibited SIRT2 activity, and further promoted p300 acetyltransferase activation and PEPCK1 acetylation. These effects were blocked by NMN, an NAD+ precursor, suggested that SL010110 inhibited gluconeogenesis by inhibiting SIRT2, activating p300, and subsequently promoting PEPCK1 acetylation. In type 2 diabetic ob/ob mice, single oral dose of SL010110 (100 mg/kg) suppressed gluconeogenesis accompanied by the suppressed hepatic SIRT2 activity, increased p300 activity, enhanced PEPCK1 acetylation and degradation. Chronic oral administration of SL010110 (15 or 50 mg/kg) significantly reduced the blood glucose levels in ob/ob and db/db mice. This study reveals that SL010110 is a lead compound with a distinct mechanism of suppressing gluconeogenesis via SIRT2-p300-mediated PEPCK1 degradation and potent anti-hyperglycemic activity for the treatment of T2D.

Is serum biotinidase enzyme activity a potential marker of perturbed glucose and lipid metabolism?

Glycogen storage diseases (GSDs) belong to the group of inborn errors of carbohydrate metabolism. Hepatic GSDs predominantly involve the liver and most present with hepatomegaly. Biochemically they show known disturbances in glucose and fatty acids metabolism, namely fasting hypoglycaemia and increased triglycerides. Additionally, increased biotinidase (BTD) enzyme activity has been shown to be associated with many GSD types, whereas the mechanism by which BTD enzyme activity is altered remains unknown so far. We aimed to delineate changes in gluconeogenesis and fatty acid synthesis, potentially explaining raised BTD enzyme activity, by using liver (specimens from 2 patients) and serum samples of GSD Ia and GSD IV patients. By expression analysis of genes involved in gluconeogenesis, we ascertained increased levels of phosphoenolpyruvate carboxykinase and fructose‐1,6‐biphosphatase, indicating an increased flux through the gluconeogenic pathway. Additionally, we found increased gene expression of the biotin‐dependent pyruvate and acetyl‐CoA carboxylases, providing substrate for gluconeogenesis and increased fatty acid synthesis. We also observed a significant linear correlation between BTD enzyme activity and triglyceride levels in a cohort of GSD Ia patients. The results of this pilot study suggest that enhancement of BTD activity might serve the purpose of providing extra cofactor to the carboxylase enzymes as an adjustment to disturbed glucose and fatty acid metabolism. Future studies involving a higher number of samples should aim at confirming the here proposed mechanism, which extends the application of BTD enzyme activity measurement beyond its diagnostic purpose in suspected GSD, and opens up possibilities for its use as a sensor for increased gluconeogenesis and fatty acid synthesis.

Effects of microRNA-106b on hepatic gluconeogenesis and its underlying mechanism

Objective: To investigate the potential effects of microRNA-106b (miR-106b) on gluconeogenesis in normal human liver cell line L02 and its underlying mechanisms. Methods: Normal human liver L02 cells were cultured in DMEM containing 10% FBS and transfected with 20 nmol/L of miR-106b mimic or antagomiR-106b, respectively. Twenty-four hours later after transfection, Western blot was performed to detect the levels of proteins or phosphorylated proteins. Quantitative RT-PCR was carried out to measure the mRNA expressions of gluconeogenesis-related genes. Glucose Assay Kit was used to detect the glucose contents in the medium. Results: MiR-106b mimic significantly increased the protein abundances of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase)(P＜0.01,P＜0.01, respectively), enhanced the mRNA expression of phosphoenolpyruvate carboxykinase 1 (PCK1) (P＜0.01), and decreased the mRNA level of glucokinase (GCK) (P＜0.01). AntagomiR-106b dramatically reduced the protein levels of PEPCK and G6Pase (P＜0.01,P＜0.01, respectively), suppressed the mRNA level of PCK1 (P＜0.01), and increased the mRNA level of GCK(P＜0.01). In addition,miR-106b mimic or antagomiR-106b significantly reduced or enhanced the protein levels of signal transducer and activator of transcription 3 (STAT3)(P＜0.01,P＜0.01, respectively).The inhibition of STAT3 by its specific inhibitor abolished the inhibitory effects of antagomiR-106b on hepatic gluconeogenesis. Conclusion: miR-106b increases hepatic gluconeogenesis by inhibiting the STAT3 signaling pathway.

Modulation of glucose transporter proteins by polyphenolic extract of Ichnocarpus frutescens (L.) W. T. Aiton in experimental type 2 diabetic rats

Traditionally, in India, the decoction of Black creeper, Ichnocarpus frutescens (L.) W. T. Aiton leaves is used to treatment diabetes mellitus. However, its molecular mechanisms of antihyperglycemic effects have not been completely studied. Due to the potential antidiabetic effect of I. frutescens , we hypothesized that the polyphonic extract might add to glucose uptake through improvement in the expression of genes of the glucose transporter (GLUT) family messenger RNA (mRNA) in the liver and adipose tissues. Experimentally, diabetes mellitus was induced in Wistar rats through i.p. injection of freshly prepared solution of streptozotocin (45 mg/kg). This was done 15 minutes after the administration of nicotinamide (120 mg/kg, ip). Serum level of insulin and C-peptide were analyzed using standard methods. Glucose metabolism by the hepatocytes and adipocytes were also analyzed by quantitative RT-PCR mRNA expression levels of phosphoenolpyruvate carboxykinase 1 (PCK1), GLUT2 in the hepatocytes, and GLUT4 in the adipocytes. The hemidiaphragm were also isolated and processed to study in-vitro peripheral glucose utilization. Results of the present investigation suggest that STZ-NA induced diabetes is associated with hyperglycemia, altered levels of PCK1 and glucose transporters gene expression as well as decreased levels of insulin and C-peptide. On the other hand, the outcome of the daily oral administration of PPE to STZ-NA induced diabetic rats at different doses (150 and 300 mg/kg bodywt.) for 30 days supports our hypothesis by showing significant improvement of insulin levels, C-peptide level, downregulation of PCK1 and upregulation of GLUT (2, 4) mRNA expression levels when compared to those of diabetic rats. The administration of PPE had also increased the uptake of glucose by rat hemidiaphragm significantly. Findings from this study demonstrate that PPE enhances peripheral glucose uptake through glycogenesis pathway, mediated by upregulation of GLUT2 and GLUT4, and downregulation of PCK1. Our study suggests that the leaf of I. frutescens is a rich source of polyphenolic compounds, including those with an insulin-sensitizing function that may have the potential for treating or managing diabetes or insulin resistance.

Circular RNA HIPK3 contributes to hyperglycemia and insulin homeostasis by sponging miR-192-5p and upregulating transcription factor forkhead box O1.

It has been shown that circular RNAs, a class of non-coding RNA molecules, play an important role in the regulation of glucose and lipid homeostasis. In the present study, we sought to investigate the function of circular RNA HIPK3 (circHIPK3) in diabetes-associated metabolic disorders, including hyperglycemia and insulin resistance. Results show that oleate stimulated circHIPK3 increase, and that circHIPK3 enhanced the stimulatory effect of oleate on adipose deposition, triglyceride (TG) content, and cellular glucose content in HepG2 cells. MiR-192-5p was the potential target of circHIPK3, since circHIPK3 significantly decreased miR-192-5p mRNA level, whereas anti-circHIPK3 significantly increased miR-192-5p mRNA level. Further study shows that transcription factor forkhead box O1 (FOXO1) was a downstream regulator of miR-192-5p, since miR-192-5p significantly decreased FOXO1 expression, whereas circHIPK3 significantly increased FOXO1 expression. Notably, the inhibitory effect of miR-192-5p was significantly reversed by circHIPK3. In vivo study shows that anti-miR-192-5p significantly increased blood glucose content, which was significantly inhibited by FOXO1 shRNA. MiR-192-5p significantly decreased adipose deposition and TG content in HepG2 cells, which was significantly reversed by the co-treatment with circHIPK3. Forskolin/dexamethasone (FSK/DEX) significantly increased cellular glucose, mRNA level of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), and this stimulatory effect of FSK/DEX was significantly inhibited by miR-192-5p. In the presence of circHIPK3, however, the inhibitory effect of miR-192-5p was totally lost. In summary, the present study demonstrated that circHIPK3 contributes to hyperglycemia and insulin resistance by sponging miR-192-5p and up-regulating FOXO1.

FXR activation alleviates tacrolimus-induced post-transplant diabetes mellitus by regulating renal gluconeogenesis and glucose uptake

BackgroundTacrolimus (FK506)-induced diabetes mellitus is one of the most important factors of post-transplant diabetes mellitus (PTDM). However, the detailed mechanisms underlying PTDM are still unclear. Farnesoid X receptor (FXR) regulates glycolipid metabolism. The objective of this study was to explore whether FXR is involved in the development of tacrolimus-induced diabetes mellitus.MethodsAfter C57BL/6J mice were treated with tacrolimus (FK506) for 3 months, the fasting blood glucose levels, body weights, renal morphological alterations, and mRNA expression levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose transporter 2 (GLUT2) among the control group, the FK506 group and the FK506 + GW4064 (a FXR agonist) group (n = 7) were measured. The intracellular location of peroxisome proliferator activated receptor γ coactivator-1α (PGC1α) and forkhead box O1 (FOXO1) was detected by immunofluorescence. Human renal cortex proximal tubule epithelial cells (HK-2) were treated with 15 μM FK506 or 4 μM FXR agonist (GW4064) for 24, 48 and 72 h, and the expression levels of FXR, gluconeogenesis and glucose uptake, representing the enzymes PEPCK and GLUT2, were detected with real-time PCR and western blot analyses. Finally, the mRNA levels of PEPCK and GLUT2 in HK-2 cells were measured after FXR was upregulated.ResultsFK506 significantly inhibited the mRNA and protein levels of FXR at 48 h and 72 h in HK-2 cells (P < 0.05). Meanwhile, FK506 promoted gluconeogenesis and inhibited glucose uptake in HK-2 cells (P < 0.05). However, overexpression of FXR in transfected HK-2 cell lines significantly inhibited gluconeogenesis and promoted glucose uptake (P < 0.05). The FXR agonist GW4064 significantly decreased the fasting blood glucose in mice challenged with FK506 for 3 months (P < 0.05), inhibited gluconeogenesis (P < 0.05) and significantly promoted glucose uptake (P < 0.05). Immunofluorescence staining and western blot analyses further revealed that FXR activation may affect the translocation of PGC1α and FOXO1 from the nucleus to the cytoplasm.ConclusionsFXR activation may mitigate tacrolimus-induced diabetes mellitus by regulating gluconeogenesis as well as glucose uptake of renal cortex proximal tubule epithelial cells in a PGC1α/FOXO1-dependent manner, which may be a potential therapeutic strategy for the prevention and treatment of PTDM.

Dietary methionine affects growth and the expression of key genes involved in hepatic lipogenesis and glucose metabolism in cobia (Rachycentron canadum)

Juvenile cobia (Rachycentron canadum), a carnivorous fish, was chosen to examine the effects of methionine (Met) supplementation of low fishmeal diets on growth and the expression of key genes related to hepatic lipogenesis and glucose metabolism. Seven isonitrogenous and isolipidic diets were formulated to contain 7.2, 9.0, 10.0, 12.4, 14.1, 16.3 and 18.6 g/kg dietary Met levels. Healthy cobia (initial average weight ± SE = 9.79 ± 0.04 g) were randomly assigned into seven groups in triplicate with 40 individuals per sea cage for 8 weeks. The results showed that the weight gain rate (WGR) of fish increased as the dietary Met was increased up to 12.4 g/kg and then decreased. The expression of insulin‐like growth factor‐I (IGF‐I) and target of rapamycin (TOR) genes was inhibited by the Met‐deficient diets. The mRNA levels of fatty acid synthase (FAS) and sterol regulatory element‐binding protein‐1 (SREBP1) genes increased with increasing dietary Met until 12.4 g/kg after which the former remained constant, whereas the latter decreased from 12.4 to 18.6 g/kg Met. The mRNA levels of pyruvate kinase (PK) increased as the dietary Met levels increased, reaching a peak at 12.4 g/kg, and then decreased with further increases in Met. However, the mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) decreased as the dietary Met was increased up to 12.4 g/kg and then remained constant from 12.4 to 18.6 g/kg Met. In conclusion, based on the straight broken‐line analysis of WGR against dietary Met levels, the optimal dietary Met requirement for juvenile cobia was estimated to be 12.4 g/kg (26.9 g/kg dietary protein). A diet with the optimal level of Met induces fish growth and is associated with increased IGF‐I and TOR.

Dexmedetomidine alleviates insulin resistance in hepatocytes by reducing endoplasmic reticulum stress

PurposeDexmedetomidine (DEX) stabilizes intraoperative blood glucose levels and reduces insulin resistance (IR), a common perioperative complication. However, the molecular mechanisms underlying these effects remain unclear. Since endoplasmic reticulum stress (ERS) is a mechanism of IR, this study sought to examine whether DEX can effectively alleviate IR by reducing ERS.MethodsHepG2 and LO2 cells were treated with different concentrations of insulin. The glucose content assay and Cell Counting Kit-8 (CCK-8) were then employed to determine the optimal insulin concentration capable of inducing IR without affecting cell viability. Insulin-resistant hepatocytes were cultured with different concentrations of DEX for 24 h, and the glucose concentration in the supernatant was measured. ERS was assessed by qPCR and western blotting. The latter was also used to quantify the expression of phosphorylated protein kinase B (p-AKT), phosphoenolpyruvate carboxykinase (PEPCK), and glucose 6 phosphatase (G6Pase), which are key proteins involved in the action of insulin.ResultsAfter 48-h of culturing with 10 μg/mL insulin, glucose consumption in hepatocytes was found to be reduced. IR hepatocytes cultured with 10, 100, or 1000 ng/ml DEX for 24 h showed a concentration-dependent increase in glucose consumption. Elevated mRNA and protein levels of ERS markers binding immunoglobulin protein (BIP) and ER protein 29 (ERp29), were reversed by DEX treatment. Moreover, reduced p-AKT and increased PEPCK and G6Pase protein levels in IR hepatocytes were also restored following DEX treatment.ConclusionDEX may alleviate IR in hepatocytes by reducing ERS serving to restore insulin action via the IRS-1/PI3K/AKT pathway.

SUN-140 EFFECTS OF PARATHYROID HORMONE ON GLUCOSE METABOLISM IN RENAL PROXIMAL TUBULES

Gluconeogenesis in renal proximal tubules (PTs) is markedly enhanced in prolonged fasting and even in diabetic patients, which is comparable to the liver. However, its pathophysiological significance and hormonal regulation are not well known. We have recently found that cAMP increases mRNA expression levels of gluconeogenic enzyme, and insulin suppresses PT gluconeogenesis via insulin receptor substrate (IRS)1/Akt2 pathway (ASN Kidney Week 2017). On the other hand, parathyroid hormone (PTH) has also been reported to enhance PT gluconeogenesis along with the fact that the high level of PTH is associated with abnormal glucose metabolism. Therefore, in this study we investigated the details of PTH-enhanced gluconeogenesis in PTs. PTs were freshly isolated from rat kidneys or human kidneys, which were surgically obtained from consented patients of kidney cancer. To analyze the direct effect and mechanism of PTH on PT gluconeogenesis, PTs treated with 10 µM PKA (protein kinase A) inhibitor H89 or 100 nM PKC (protein kinase C) inhibitor Gö6983 were incubated with 1 nM PTH in DMEM overnight. To examine the relationship to insulin signaling, PTs were stimulated by 10 nM insulin for 4-hr after overnight incubation with PTH or 0.2 mM cAMP. We also performed siRNA against FoxO1 (forkhead box protein O1). During the whole process, PT gluconeogenesis was evaluated by measuring the mRNA expression levels of gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6P) by quantitative PCR. The mRNA expression levels of FoxO1 were also measured by quantitative PCR. Further, we performed western blotting in both of rat and human kidney cortex to analyze Akt phosphorylation in insulin signaling pathway. In PTs, PTH significantly increased the mRNA expression levels of PEPCK and G6P by 6 and 4 times, respectively. Not PKA inhibitor but PKC inhibitor completely abolished this PTH effect, which was consistent with PKC-mediated PTH signaling pathway. Interestingly, insulin failed to inhibit this PTH-enhanced PT gluconeogenesis, while insulin completely inhibited cAMP-enhanced PT gluconeogenesis. Moreover, siRNA against FoxO1 completely dissipated the PTH effect. In the kidney cortex, insulin induced Akt phosphorylation, which was preserved after incubation with PTH. Our results, for the first time to our knowledge, revealed that PTH increases mRNA expression levels of gluconeogenic enzymes in PTs via PKC pathway, and induces insulin resistance in PTs. Considering these results, the high level PTH may cause poor glucose control in these states through the direct enhancement of PT gluconeogenesis or exacerbating insulin resistance.