Glucose In HepG2 Cells Research Articles

The protective role of resveratrol against high glucose-induced oxidative stress and apoptosis in HepG2 cells.

High glucose concentrations result in oxidative stress, leading to damage of cellular constituents like DNA, proteins, and lipids, ultimately resulting in apoptosis. Resveratrol, a polyphenol phytoalexin, has been studied for its potential therapeutic effects on diabetes. This study investigated the influence of high glucose (HG) on HepG2 cells and assessed resveratrol's effect on high-glucose-induced oxidative stress and apoptosis. HepG2 cells were cultured for 48 and 72 h with high glucose (40 mM), low resveratrol (25 μM), high resveratrol (50 μM), high glucose + low resveratrol, and high glucose + high resveratrol. After exposure, oxidative and apoptosis-related gene expression was evaluated using quantitative polymerase chain reaction (qPCR), and lactate dehydrogenase (LDH) release was measured using the supernatant. In HepG2 cells cultured with high glucose, all antioxidant enzymes (SOD, superoxide dismutase; GPx1, glutathione peroxidase 1; CAT, catalase; Nrf2, nuclear factor erythroid 2-related factor 2; and NQO1, NAD(P)H quinone oxidoreductase 1) were significantly reduced; however, when HepG2 cells were cultured with resveratrol (25 and 50 μM) and high glucose, the expression levels of all antioxidant enzymes were increased. The anti-apoptotic gene (B-cell lymphoma 2; Bcl2) and the DNA repair gene (Oxoguanine glycosylase-1, OGG1) were significantly decreased following high glucose exposure to HepG2 cells. Surprisingly, the expression levels of Bcl2 and OGG1 were notably elevated after resveratrol treatment. Furthermore, high glucose levels increased the LHD release in HepG2 cells, whereas resveratrol treatment reduced the LDH release. Our results demonstrate that resveratrol provides protection against oxidative stress and apoptosis induced by high glucose in HepG2 cells. Hence, resveratrol shows potential as an effective approach to address the impaired antioxidant response resulting from elevated glucose levels commonly observed in diabetes and metabolic disorders.

Diosgenin promoted glucose uptake by attenuation of high glucose induced blockade of PI3K-Akt-GLUT4 pathway and ER stress

Introduction and Aim: Hyperglycemia is the signature style of type II diabetes (T2D) which impairs pathways of insulin signaling and endoplasmic reticulum (ER) stress. Diosgenin is a popular saponin known for its antidiabetic and anti-obesity properties predominantly found in Dioscorea species. However, the mechanism of hypoglycemic effects related to pathways of ER stress, insulin signaling, and uptake of glucose in the liver gained less attention. Accordingly, this study was aimed to assess the effect of Diosgenin on the uptake of glucose, insulin signaling as well as on ER stress in human HepG2 cells cultured in hyperglycemic or high glucose (HG) condition. Materials and Methods: Viability studies on HepG2 cells, assay for studying the uptake and consumption of glucose, gene expression, and docking studies were carried out. Results: The treatment with Diosgenin was found to elevate the uptake as well as consumption of glucose in HepG2 cells under hyperglycemic conditions. Diosgenin prevented inactivation of the PI3K Akt pathway as well as GLUT4 levels induced by high glucose. Furthermore, endoplasmic reticulum stress elements viz., inositol requiring enzyme 1 (IRE1), protein kinase like endoplasmic reticulum kinase (PERK), C/EBP homologous protein (CHOP) and sXBP1 were reduced by diosgenin treatment provoked by high glucose returning to normal ER homeostasis. Moreover, the effects are supported by the binding efficiency of diosgenin with insulin signaling proteins using bioinformatic tools. Conclusion: Our findings suggest that diosgenin elevated the uptake of glucose by hindering the inactivation of PI3K Akt GLUT4 pathway and ER stress caused by high glucose, thus, preventing further hepatic dysfunction.

Evaluation of in vitro glucose metabolism potential of sterols and essential oil extracts from Erythroxylum monogynum Roxb. using cell lines and enzyme inhibition models

The present research purports screening of extracts, essential oils and isolated sterols from Erythroxylum monogynum for in vitro anti-diabetic activity through in vitro cell line model and also by in vitro enzyme inhibition models. The MTT assay for the cytotoxic studies in vitro of isolated sterols on HepG2 cells at varied concentrations from 0-200 µg/ml revealed that all the tested extracts and compounds displayed low level of toxicity at all concentrations in a dose-dependent manner. The 4-methyl ergosta-7, 23-dien-3β-ol and 4-methyl ergosta-7, 24 (28)-dien-3β-ol exhibited considerable increase in the uptake of glucose in HepG2 cells at both the tested concentration (dose dependent behaviour) compared to the negative control (untreated) but not with the positive control, metformin. Further, the two sterols offered a safety profile to the cells as revealed from the assay wherein, at 25 and 100 µg/ml were also not toxic and exhibited <10% cell death. Moreover, the sterols also showed marked increase in triglyceride accumulation of 127% and 125% at 50 µg/ml when compared to the negative control (100%) but less than the positive control, Rosiglitazone (145%). No substantial increase in the glucose metabolism of INS-1 cells and a significant increase in the total cells (108-114%) were observed. Surprisingly, the sterols inhibited all the investigated enzymes such as sucrase, lipase, dipeptidyl peptidase IV, collagenase and protein tyrosine phosphatase 1B with IC50 values comparable to that of standard compounds. The results obtained were remarkable suggesting that these sterols and extracts might be the future lead molecules for the management of diabetes.

HM-chromanone reverses the blockade of insulin signaling induced by high glucose levels in human HepG2 cells

This study investigated whether (E)-5-hydroxy-7-methoxy-3-(2′-hydroxybenzyl)-4-chromanone (HM-chromanone) could counteract the high glucose level-induced blockade of insulin signaling in human HepG2 cells. Cells were pre-incubated with glucose (5.5 or 33 mM) and then incubated with a medium containing various concentrations of HM-chromanone. Assays for glucose uptake, glycogen synthesis, and glucose production were performed. Western blotting helped elucidate the underlying molecular mechanisms. High glucose concentration (33 mM) significantly increased p-IRS-1ser307 levels and decreased p-Akt levels. However, HM-chromanone significantly decreased p-IRS-1ser307 levels while increasing p-IRS-1tyr612 and Akt levels, which restored insulin signaling disturbed by high glucose concentration. HM-chromanone significantly increased p-AMPK levels, which were reduced by high glucose in HepG2 cells. Knockdown of AMPK using siRNA increased p-IRS-1ser307 and decreased p-Akt levels, even after treatment with HM-chromanone in high glucose concentration-treated cells. HM-chromanone stimulated glycogen synthesis by increasing p-GSK3βser9 and decreasing p-GSser641 levels in HepG2 cells under high glucose concentration; this effect was blocked by AMPK siRNA. HM-chromanone significantly decreased PEPCK, G6Pase, and hepatic glucose production, which were also blocked by AMPK siRNA. These results suggest that HM-chromanone could reverse insulin signaling blockade (induced by high glucose levels) through the activation of AMPK and stimulation of glucose uptake and glycogen synthesis in HepG2 cells.

Pyrroloquinoline quinone alleviates oxidative damage induced by high glucose in HepG2 cells.

Hyperglycemia as a common metabolic disorder in diabetes led to oxidative stress, inflammation and other complications. Natural and manufactured antioxidants alleviates the side effects of diabetes. The purpose of current study is to investigate the effect of pyrroloquinoline quinine (PQQ) as an antioxidant on the content of glucose-induced oxidative stress generation in the cells of the human hepatocellular liver carcinoma (HepG2) by inhibiting advanced glycation end products (AGEs) formation. The HepG2 cells were exposed to high dose (50 mM) of glucose (HG) only and with PQQ (HG + PQQ). Treatment with high dose increased AGEs formation, expression of receptor for advanced glycation endproducts (RAGE), reactive oxygen species ROS production, and oxidative stress markers in treated HepG2 cells. Interestingly, PQQ significantly reduced AGEs formation and (RAGE) expression, ROS formation, and inflammation induced by glucose. In conclusion, PQQ has a potentiail role as an antioxidant to reduce the oxidative damage during hyperglycemia by AGEs inhibition.

Glucosamine regulates hepatic lipid accumulation by sensing glucose levels or feeding states of normal and excess

Dose-dependent lipid accumulation was induced by glucose in HepG2 cells. GlcN also exerted a promotory effect on lipid accumulation in HepG2 cells under normal glucose conditions (NG, 5 mM) and liver of normal fed zebrafish larvae. High glucose (HG, 25 mM)-induced lipid accumulation was suppressed by l-glutamine-d-fructose 6-phosphate amidotransferase inhibitors. ER stress inhibitors did not suppress HG or GlcN-mediated lipid accumulation. HG and GlcN stimulated protein expression, DNA binding and O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP). Furthermore, both HG and GlcN increased nuclear sterol regulatory element-binding protein-1 (SREBP-1) levels in HepG2 cells. In contrast to its stimulatory effect under NG, GlcN suppressed lipid accumulation in HepG2 cells under HG conditions. Similarly, GlcN suppressed lipid accumulation in livers of overfed zebrafish. In addition, GlcN activity on DNA binding and O-GlcNAcylation of ChREBP was stimulatory under NG and inhibitory under HG conditions. Moreover, GlcN enhanced ChREBP, SREBP-1c, ACC, FAS, L-PK and SCD-1 mRNA expression under NG but inhibited HG-induced upregulation in HepG2 cells. The O-GlcNAc transferase inhibitor, alloxan, reduced lipid accumulation by HG or GlcN while the O-GlcNAcase inhibitor, PUGNAc, enhanced lipid accumulation in HepG2 cells and liver of zebrafish larvae. GlcN-induced lipid accumulation was inhibited by the AMPK activator, AICAR. Phosphorylation of AMPK (p-AMPK) was suppressed by GlcN under NG while increased by GlcN under HG. PUGNAc downregulated p-AMPK while alloxan restored GlcN- or HG-induced p-AMPK inhibition. Our results collectively suggest that GlcN regulates lipogenesis by sensing the glucose or energy states of normal and excess fuel through AMPK modulation.

Edgeworthia gardneri (Wall.) Meisn. Water Extract Ameliorates Palmitate Induced Insulin Resistance by Regulating IRS1/GSK3β/FoxO1 Signaling Pathway in Human HepG2 Hepatocytes.

The flower of Edgeworthia gardneri (Wall.) Meisn is commonly used in beverage products in Tibet and has potential health benefits for diabetes. However, the mechanisms underlying anti-insulin resistance (IR) action of the flower of E. gardneri are not fully understood. This study aims to investigate the effects of the water extract of the flower of E. gardneri (WEE) on IR in palmitate (PA)-exposed HepG2 hepatocytes. WEE was characterized by UPLC analysis. PA-treated HepG2 cells were selected as the IR cell model. The cell viability was determined using MTT assay. Moreover, the glucose consumption and production were measured by glucose oxidase method. The glucose uptake and glycogen content were determined by the 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose) glucose uptake assay and anthrone-sulfuric acid assay, respectively. The intracellular triglyceride content was detected by oxidative enzymic method. Protein levels were examined by Western blotting. Nuclear localization of FoxO1 was detected using immunofluorescence analyses and Western blotting. The expression of FoxO1 target genes was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The viability of PA-treated HepG2 cells was concentration-dependently increased by incubation with WEE for 24 h. WEE treatment remarkably increased the consumption and uptake of glucose in PA-exposed HepG2 cells. Moreover, treatment with WEE significantly decreased the PA-induced over-production of glucose in HepG2 cells. After exposure of HepG2 cells with PA and WEE, the glycogen content was significantly elevated. The phosphorylation and total levels of IRβ, IRS1, and Akt were upregulated by WEE treatment in PA-exposed HepG2 cells. The phosphorylation of GSK3β was elevated after WEE treatment in PA-treated cells. WEE treatment also concentration-dependently downregulated the phosphorylated CREB, ERK, c-Jun, p38 and JNK in PA-exposed HepG2 cells. Furthermore, the nuclear protein level and nuclear translocation of FoxO1 were also suppressed by WEE. Additionally, PA-induced changes of FoxO1 targeted genes were also attenuated by WEE treatment. The GLUT2 and GLUT4 translocation were also promoted by WEE treatment in PA-treated HepG2 cells. Taken together, WEE has potential anti-IR effect in PA-exposed HepG2 cells; the underlying mechanism of this action may be associated with the regulation of IRS1/GSK3β/FoxO1 signaling pathway. This study provides a pharmacological basis for the application of WEE in the treatment of metabolic diseases such as type 2 diabetes mellitus.

Can Galactose Be Converted to Glucose in HepG2 Cells? Improving the in Vitro Mitochondrial Toxicity Assay for the Assessment of Drug Induced Liver Injury.

Human hepatocellular carcinoma cells, HepG2, are often used for drug mediated mitochondrial toxicity assessments. Glucose in HepG2 culture media is replaced by galactose to reveal drug-induced mitochondrial toxicity as a marked shift of drug IC50 values for the reduction of cellular ATP. It has been postulated that galactose sensitizes HepG2 mitochondria by the additional ATP consumption demand in the Leloir pathway. However, our NMR metabolomics analysis of HepG2 cells and culture media showed very limited galactose metabolism. To clarify the role of galactose in HepG2 cellular metabolism, U-13C6-galactose or U-13C6-glucose was added to HepG2 culture media to help specifically track the metabolism of those two sugars. Conversion to U-13C3-lactate was hardly detected when HepG2 cells were incubated with U-13C6-galactose, while an abundance of U-13C3-lactate was produced when HepG2 cells were incubated with U-13C6-glucose. In the absence of glucose, HepG2 cells increased glutamine consumption as a bioenergetics source. The requirement of additional glutamine almost matched the amount of glucose needed to maintain a similar level of cellular ATP in HepG2 cells. This improved understanding of galactose and glutamine metabolism in HepG2 cells helped optimize the ATP-based mitochondrial toxicity assay. The modified assay showed 96% sensitivity and 97% specificity in correctly discriminating compounds known to cause mitochondrial toxicity from those with prior evidence of not being mitochondrial toxicants. The greatest significance of the modified assay was its improved sensitivity in detecting the inhibition of mitochondrial fatty acid β-oxidation (FAO) when glutamine was withheld. Use of this improved assay for an empirical prediction of the likely contribution of mitochondrial toxicity to human DILI (drug induced liver injury) was attempted. According to testing of 65 DILI positive compounds representing numerous mechanisms of DILI together with 55 DILI negative compounds, the overall prediction of mitochondrial mechanism-related DILI showed 25% sensitivity and 95% specificity.

Flavone glycosides from Sicyos angulatus and their inhibitory effects on hepatic lipid accumulation

A library of extracted natural materials (Korea Bioactive Natural Material Bank) have been screened to discover candidates for the treatment of non-alcoholic liver disease (NAFLD), and the 70% ethanol extract of Sicyos angulatus was found to inhibit hepatic lipid accumulation. Bioassay–guided fractionation of this bioactive extract yielded five previously undescribed flavonoid glycosides and one previously undescribed flavonolignan glycoside along with seven known flavonoid glycosides. The chemical structures of these compounds were elucidated by a combination of extensive spectroscopic analysis, including MS, NMR and UV techniques. Eight compounds of all isolated compounds showed inhibitory effects on the lipid accumulation induced by high concentrations of palmitic acid and glucose in HepG2 cells. Four selected compounds were tested for lipid content in a dose-dependent manner (10, 20 and 40 μM), and among those compounds, kaempferol 3-O-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside showed the strongest inhibition of hepatic lipid production in HepG2 cells. In an oil-red O staining assay, five compounds were shown to reduce hepatic lipid accumulation better than what was observed in the vehicle control group. The present study suggests a new class of chemical entities for developing bioactive agents for the treatment of diseases caused by fat accumulation in the liver.

Quantitative 13C Traces of Glucose Fate in Hepatitis B Virus-Infected Hepatocytes.

Quantitative characterization of 13C-labeled metabolites is an important part of the stable isotope tracing method widely used in metabolic flux analysis. Given the long relaxation time and low sensitivity of 13C nuclei, direct measurement of 13C-labeled metabolites using one-dimensional 13C NMR often fails to meet the demand of metabolomics studies, especially with large numbers of samples and metabolites having low abundance. Although HSQC-based 2D NMR methods have improved sensitivity with inversion detection, they are time-consuming and thus unsuitable for high-throughput absolute quantification of 13C-labeled metabolites. In this study, we developed a method for absolute quantification of 13C-labeled metabolites using naturally abundant TSP as a reference with the first increment of the HMQC pulse sequence, taking polarization transfer efficiencies into consideration. We validated this method using a mixture of 13C-labeled alanine, methionine, glucose, and formic acid together with a mixture of alanine, lactate, glycine, uridine, cytosine, and hypoxanthine, which have natural 13C abundance with known concentrations. We subsequently applied this method to analyze the flux of glucose in HepG2 cells infected with hepatitis B virus (HBV). The results showed that HBV infection increased the cellular uptake of glucose, stimulated glycolysis, and enhanced the pentose phosphate and hexosamine pathways for biosynthesis of RNA and DNA and nucleotide sugars to facilitate HBV replication. This method saves experimental time and provides a possibility for absolute quantitative tracking of the 13C-labeled metabolites for high-throughput studies.

Protective effects of marein on high glucose-induced glucose metabolic disorder in HepG2 cells

BackgroundOur previous study has shown that Coreopsis tinctoria increases insulin sensitivity and regulates hepatic metabolism in high-fat diet (HFD)-induced insulin resistance rats. However, it is unclear whether or not marein, a major compound of C. tinctoria, could improve insulin resistance. Here we investigate the effect and mechanism of action of marein on improving insulin resistance in HepG2 cells. MethodsWe investigated the protective effects of marein in high glucose-induced human liver carcinoma cell HepG2.In kinase inhibitor studies, genistein, LY294002, STO-609 and compound C were added to HepG2 cells 1h before the addition of marein. Transfection with siRNA was used to knock down LKB1, and 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG), an effective tracer, was used to detect glucose uptake. ResultsThe results showed for the first time that marein significantly stimulates the phosphorylation of AMP-activated protein kinase (AMPK) and the Akt substrate of 160kDa (AS160) and enhanced the translocation of glucose transporter 1 (GLUT1) to the plasma membrane. Further study indicated that genistein (an insulin receptor tyrosine kinase inhibitor) altered the effect of marein on glucose uptake, and both LY294002 (a phosphatidylinositol 3-kinase inhibitor) and compound C (an AMP-activated protein kinase inhibitor) significantly decreased marein-stimulated 2-NBDG uptake. Additionally, marein-stimulated glucose uptake was blocked in the presence of STO-609, a CaMKK inhibitor; however, marein-stimulated AMPK phosphorylation was not blocked by LKB1 siRNA in HepG2 cells. Marein also inhibited the phosphorylation of insulin receptor substrate (IRS-1) at Ser 612, but inhibited GSK-3β phosphorylation and increased glycogen synthesis. Moreover, marein significantly decreased the expression levels of FoxO1, G6Pase and PEPCK. ConclusionsConsequently, marein improved insulin resistance induced by high glucose in HepG2 cells through CaMKK/AMPK/GLUT1 to promote glucose uptake, through IRS/Akt/GSK-3β to increase glycogen synthesis, and through Akt/FoxO1 to decrease gluconeogenesis. Marein could be a promising leading compound for the development of hypoglycemic agent or developed as an adjuvant drug for diabetes mellitus.

Hepatic DGAT2 gene expression is regulated by the synergistic action of ChREBP and SP1 in HepG2 cells

ABSTRACTDiacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triglyceride synthesis and plays a critical role in the development of fatty liver disease and insulin resistance. The expression of DGAT2 is mostly induced by dietary carbohydrate in the mammalian liver; however, the transcription factors that regulate DGAT2 expression have yet to be identified. In this study, we investigated the molecular mechanism underlying the glucose-induced transcriptional regulation of human DGAT2 in HepG2 cells. Human DGAT2 expression was increased by glucose in HepG2 cells. Transfection studies of the DGAT2 promoter-luciferase reporter construct in vitro and in silico analysis identified two glucose-responsive regions in the DGAT2 promoter. Each region contains one ChREBP/MLX (carbohydrate response element binding protein/Max-like protein) binding site (ChoRE, −539 to −551 and −6067 to −6083) and one specificity protein 1 (SP1) binding site (GC-rich motif, −556 to −572 and −6016 to −6032). Mutational analysis showed that both ChREBP/MLX and SP1 sites are required for the glucose-induced transcription of DGAT2. Gel shift assays and chromatin immunoprecipitation assays demonstrated that ChREBP and SP1 bind directly to ChoRE and the GC-rich motif, respectively. High glucose promoted the recruitment of ChREBP to ChoRE, whereas SP1 was recruited to the GC-rich motif even under low-glucose conditions. These data demonstrate that both ChREBP and SP1 are key factors to regulate the expression of human DGAT2 by glucose.

Development of in vitro model of insulin receptor cleavage induced by high glucose in HepG2 cells

Soluble insulin receptor (sIR), the ectodomain of IR, has been detected in human plasma, and its concentration parallels that of blood glucose in patients with diabetes. IR has a pivotal role in glucose homeostasis and diabetes development; therefore, cleavage of IR promoted by hyperglycemia is involved in insulin resistance and glucose toxicity. To elucidate the physiology of sIR, we developed an in vitro model mimicking the changes in sIR levels in plasma from patients with diabetes. Among four human cell lines that expressed IR, spontaneous cleavage of IR occurred only in HepG2 cells. The molecular characteristics of sIR derived from HepG2 cells were similar to those of sIR detected in human plasma. The concentration of sIR in the medium did not differ between basal and high-glucose conditions in the initial 24-h period, but increasing the duration of pre-stimulation (>48h) led to a significant increase in sIR levels in cells exposed to high glucose. Additionally, glucose-dependent increment of sIR was reversible in this model. These results are consistent with the observation of plasma sIR in patients with diabetes. Using this model, O-linked N-acetylglucosamine modification was determined to be involved in high-glucose-induced IR cleavage. A calcium-dependent protease was shown to cleave IR extracellularly. These findings show that this in vitro model could be useful for determining the molecular mechanism underlying IR cleavage.

Apoptosis in HepG2 cells exposed to high glucose

Hyperglycemia which characterizes diabetes, leads to several abnormalities in the cellular pathways. We examined the toxicity of glucose in human hepatoma HepG2 cells. HepG2 cells when incubated with 50 mM glucose for 72 h showed altered morphology i.e. presence of detached and shrunken rounded cells. Glucose treated HepG2 cells also exhibited a significant decrease in viability. Caspase-3 activity and Annexin V staining were significantly increased in glucose treated HepG2 cells, suggesting an apoptotic mode of cell death. Glucose induced apoptosis in HepG2 cells was a consequence of increased oxidative stress as evidenced by the increased reactive oxygen species (ROS) level, lipid peroxidation, protein carbonyl and 3-nitrotyrosine adduct formation. The intracellular antioxidant glutathione was found to be increased in HepG2 cells treated with glucose, possibly to aid the cells to overcome the persistent oxidative stress elicited by glucose in HepG2 cells. N-Acetyl cysteine, a precursor of glutathione and an antioxidant was effective in reversing the morphological changes, increasing the viability, decreasing the ROS level and 4-hydroxynonenal and 3-nitrotyrosine adduct formation, thus validating the role of oxidative stress as a major mechanism for glucose induced apoptosis in HepG2 cells. These results suggest that glucose induces apoptosis in liver cells through increased oxidative stress.

A Combination of Grape Seed‐Derived Procyanidins and Gypenosides Alleviates Insulin Resistance in Mice and HepG2 Cells

This study investigated the effects of grape seed-derived procyanidins (GSP), gypenosides (GPE), and combination procyanidins/gypenosides on insulin resistance in mice and HepG2 cells. ICR mice were randomly divided into 2 control and 4 treatment groups. The control mice were to receive either normal diet (ND) or high-fat diet (HFD), and the treatment groups were fed high-fat diet with either 80 mg/kg of GSP (GSP80), GPE (GPE80), GSP + GPE (1: 1, GSP40 + GPE40), or 500 mg/kg of metformin for a 6-wk period. All the groups of mice except the normal control were on high-fat diet along with fructose (15%) administered in drinking water throughout the period of treatment. An insulin-resistant HepG2 cell model was developed after 24 h of 5 x 10(-7) mol/L insulin incubation. The treatment of GPE80 could significantly reduce the index of insulin resistance (HOMA-IR) and increase hepatic glycogen concentration, compared with HFD group (P < 0.05). When GSP and GPE were administered simultaneously, synergic effects were observed in decreasing the HOMA-IR index and serum total cholesterol (TC) level and enhancing glucose tolerance. All treatment groups showed considerable raise of hepatic glucokinase activity (P < 0.05 compared with HFD group). GSP application increased the consumption of extracellular glucose in HepG2 cells. Our data suggest that the combination of GSP and GPE may have functional efficacy in consumers with insulin resistance.

Regulation of mammalian pyruvate dehydrogenase alpha subunit gene expression by glucose in HepG2 cells.

We report the effect of glucose on the expression of the gene encoding the pyruvate dehydrogenase (E1) alpha subunit (E1alpha) in human hepatoma (HepG2) cells. Total pyruvate dehydrogenase complex activity as well as the levels of protein and mRNA of the E1alpha subunit were significantly increased in HepG2 cells cultured in medium containing 16.7 mM glucose compared with 1.0 mM glucose for a period of 4 weeks. The level of E1alpha mRNA was elevated approx. 2-fold in HepG2 cells cultured for 24 h in medium containing 16.7 mM glucose compared with 1 mM glucose. This effect was specific to glucose and independent of insulin. Nuclear run-on assays and promoter analysis indicate that the glucose-induced increases in the levels of E1alpha mRNA in HepG2 cells are due to increased transcription of the human E1alpha (PDHA1) gene. Mutational analysis of the E1alpha promoter region has identified two regions, from -78 to -73 bp (CCCCTG) and from -8 to -3 bp (GCGGTG), that are responsible for the effect of glucose on promoter activity; the former exhibits a larger effect. These two sequences represent new variations of the carbohydrate-response element that has been identified in other genes. The stimulation of E1alpha promoter activity by glucose was abolished by okadaic acid at 100 nM but not at 5 nM, suggesting that glucose-mediated regulation of pyruvate dehydrogenase complex E1alpha gene transcription involves a phosphorylation/dephosphorylation mechanism, possibly involving protein phosphatase-1.