Gluconeogenesis In Liver Cells Research Articles

Combinational administration of mesenchymal stem cell-derived exosomes and metformin reduces inflammatory responses in an in vitro model of insulin resistance in HepG2 cells

Diabetes is a highly common metabolic disorder in advanced societies. One of the causes of diabetes is insulin resistance, which is associated with a loss of sensitivity to insulin-sensitive cells. Insulin resistance develops in the body of a person prone to diabetes many years before diabetes development. Insulin resistance is associated with complications such as hyperglycemia, hyperlipidemia, and compensatory hyperinsulinemia and causes liver inflammation, which, if left untreated, can lead to cirrhosis, fibrosis, and even liver cancer. Metformin is the first line of treatment for patients with diabetes, which lowers blood sugar and increases insulin sensitivity by inhibiting gluconeogenesis in liver cells. The use of metformin has side effects, including a metallic taste in the mouth, vomiting, nausea, diarrhea, and upset stomach. For this reason, other treatments, along with metformin, are being developed. Considering the anti-inflammatory role of mesenchymal stem cells (MSCs) derived exosomes, their use seems to help improve liver tissue function and prevent damage caused by inflammation. This study investigated the anti-inflammatory effect of Wharton's jelly MSCs derived exosomes in combination with metformin in the HepG2 cells insulin resistance model induced by high glucose. This study showed that MSCs derived exosomes as an anti-inflammatory agent in combination with metformin could increase the therapeutic efficacy of metformin without needing to change metformin doses by decreasing inflammatory cytokines production, including IL-1, IL-6, and TNF-α and apoptosis in HepG2 cells.

Effective fraction from Simiao Wan prevents hepatic insulin resistant by inhibition of lipolysis via AMPK activation

Simiao Wan (SMW) is a traditional Chinese formula, including Atractylodis Rhizoma, Achyranthis Bidentatae Radix, Phellodendri Chinensis Cortex and Coicis Semen at the ratio of 1:1:2:2. It can be used to the treatment of diabetes. However, its bioactive compounds and underlying mechanism are unclear. This study aimed to screen the antilipolytic fraction from SMW and investigate its therapeutic mechanisms on hepatic insulin resistance. Different fractions of SMW were prepared by membrane separation combined with macroporous resin and their antilipolytic activities were screened in fasted mice. The effects of 60% ethanol elution (ESMW) on lipolysis were investigated in 3T3-L1 adipocytes stimulated by palmitic acid (PA) and high fat diet (HFD)-fed mice. In our study, ESMW is the bioactive fraction responsible for the antilipolytic activity of SMW and 13 compounds were characterized from ESMW by UHPLC-QTOF-MS/MS. ESMW suppressed protein kinase A (PKA)-hormone-sensitive lipase (HSL) related lipolysis and increased AMP-activated protein kinase (AMPK) phosphorylation in PA challenged 3T3-L1 adipocytes. AMPKα knockdown abolished the inhibitory effects of ESMW on IL-6 and HSL pSer-660, revealing that the antilipolytic and anti-inflammatory activities of ESMW are AMPK dependent. Furthermore, ESMW ameliorated insulin resistance and suppressed lipolysis in HFD-fed mice. It inhibited diacylglycerol accumulation in the liver and inhibited hepatic gluconeogenesis. Conditional medium collected from ESMW-treated 3T3-L1 cells ameliorated insulin action on hepatic gluconeogenesis in liver cells, demonstrating the antilipolytic activity contributed to ESMW beneficial effects on hepatic glucose production. In conclusion, ESMW, as the antilipolytic fraction of SMW, inhibited PKA-HSL related lipolysis by activating AMPK, thus inhibiting diacylglycerol (DAG) accumulation in the liver and thereby improving insulin resistance and hepatic gluconeogenesis.

H2S-induced S-sulfhydration of pyruvate carboxylase contributes to gluconeogenesis in liver cells

BackgroundCystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H2S) possesses diverse roles in the liver, affecting lipoprotein synthesis, insulin sensitivity, and mitochondrial biogenesis. H2S S-sulfhydration is now proposed as a major mechanism for H2S-mediated signaling. Pyruvate carboxylase (PC) is an important enzyme for gluconeogenesis. S-sulfhydration regulation of PC by H2S and its implication in gluconeogenesis in the liver have been unknown. MethodsGene expressions were analyzed by real-time PCR and western blotting, and protein S-sulfhydration was assessed by both modified biotin switch assay and tag switch assay. Glucose production and PC activity was measured with coupled enzyme assays, respectively. ResultsExogenously applied H2S stimulates PC activity and gluconeogenesis in both HepG2 cells and mouse primary liver cells. CSE overexpression enhanced but CSE knockout reduced PC activity and gluconeogenesis in liver cells, and blockage of PC activity abolished H2S-induced gluconeogenesis. H2S had no effect on the expressions of PC mRNA and protein, while H2S S-sulfhydrated PC in a dithiothreitol-sensitive way. PC S-sulfhydration was significantly strengthened by CSE overexpression but attenuated by CSE knockout, suggesting that H2S enhances glucose production through S-sulfhydrating PC. Mutation of cysteine 265 in human PC diminished H2S-induced PC S-sulfhydration and activity. In addition, high-fat diet feeding of mice decreased both CSE expression and PC S-sulfhydration in the liver, while glucose deprivation of HepG2 cells stimulated CSE expression. ConclusionsCSE/H2S pathway plays an important role in the regulation of glucose production through S-sulfhydrating PC in the liver. General significanceTissue-specific regulation of CSE/H2S pathway might be a promising therapeutic target of diabetes and other metabolic syndromes.

Positional isotopic analysis of 13C-labelled glucose by mass spectrometry: applications to the study of gluconeogenesis in liver cells.

The aim of the present investigation was to ascertain whether mass spectrometric analysis of glucose allows determination in small samples (0.01 nmol) of the sites and the extent of labelling of glucose produced by isolated liver cells from various gluconeogenic labelled precursors. The electron impact spectrum of the methyloxime pentatrimethylsilyl derivative of natural glucose affords fragment ions retaining specific carbon atoms, i.e. 1-2 (m/z 160), 1-2-3 (m/z 262), 3-4-5-6 (m/z 319), 4-5-6 (m/z 217), 5-6 (m/z 205), 6 (m/z 103). The mass fragmentography analysis of the same derivative of commercially available labelled glucose molecules (1-13C, 6-13C, 2-2H, 3-2H, 6,6-2H2) permitted evaluation of the degree of specificity of these fragment ions, and development of a calculation method for isotope incorporation. Using this methodology we found that incubation of hepatocytes with (2-13C)glycerol, (1,3-13C)glycerol or NaH13CO3 plus pyruvate or lactate produced (2,5-13C)glucose, (1,3,4,6-13C) glucose or (3,4-13C)glucose, respectively. The extent of labelling was measurable on individual carbon of the glucose molecule except for carbon 1. The lowest enrichment detectable on carbon 1-3 or 3 was found to be 0.5%. In conclusion, gas chromatography mass spectrometry is a reliable method for positional isotopic analysis of 13C-labelled glucose, and appears useful in the study of the gluconeogenic pathway.

Loss of glucagon control of gluconeogenesis in liver cells from rats with bile duct obstruction

Bile acids induce membrane alterations including reduced response to peptide hormones in vitro. Isolated liver cells from rats with bile duct obstruction were studied regarding gluconeogenesis and its hormonal control. While cells from shamoperated animals showed an 63% increase of glucose release in the presence of glucagon (1 μM), cells from cholestatic livers did not response regardless of the duration of obstruction. Cholestatic cells also showed other signs of membrane alterations, such as an increased enzyme leakage while redoxstatus and other metabolic responses were unchanged. These results suggest that a loss of hormonal control in the liver could contribute to disturbations of glucose homeostasis in cholestatic conditions.

In vivo and in vitro effects of beta-endorphin on glucose metabolism in the rat.

The effects of beta-endorphin (beta-Ep) on plasma glucose levels in rats and on glucose metabolism in isolated rat liver cells were examined. Intravenous injection of beta-Ep (5 micrograms/100 g BW) into ether-anaesthetized rats resulted in prompt and sustained hyperglycaemia with increases in the plasma glucagon and somatostatin levels and decrease in the plasma insulin level. When liver cells isolated from fed rats were incubated in the presence of beta-Ep at concentrations of 6 X 10(-8) M to 6 X 10(-7) M, glucose release into the medium increased within 15 min in a dose-related manner. Time course experiments showed that beta-Ep increased the level of cyclic AMP within 3 min. Significant increase in gluconeogenesis in liver cells isolated from fasted rats was also observed on addition of 10(-7) M beta-Ep in the presence of 10 mM L-lactate. These results suggest that the hyperglycaemia induced by beta-Ep may be caused, at least in part, by the effects of beta-Ep on releases of pancreatic hormones and glucose production in liver cells.

Rate-limiting steps of gluconeogenesis in liver cells as determined with the aid of fluoro-dicarboxylic acids.

Fluoromalate (an inhibitor of malate dehydrogenase and the malate carrier) or difluorooxaloacetate (an inhibitor of aspartate aminotransferase) inhibited gluconeogenesis from both pyruvate and lactate, but had no effect on glucose formation from fructose or endogenous substrates. When cells were incubated with both fluorocarboxylic acids simultaneously, an additive effect on the inhibition of glucose formation from lactate was observed, but the inhibition was not additive when pyruvate was the glucogenic precursor. Pyruvate removal was reduced 15% by difluorooxaloacetate and 44% by fluoromalate. Lactate production from pyruvate was inhibited 36% by fluoromalate but was almost unaffected by difluorooxaloacetate. The results suggest that both malate-oxaloacetate and glutamate-aspartate shuttles participate, but to different extents, in glucose formation from either pyruvate or lactate. The malateoxaloacetate shuttle predominates during gluconeogenesis from pyurvate whereas the glutamateaspartate shuttle is of greater importance when lactate is the gluconeogenic precursor. However, the inhibition by fluoromalate of glucose formation from lactate indicates that not all the reducing equivalents arising during lactate oxidation are directly available for the reductive step of gluconeogenesis. Neither of the two shuttles seems to compensate for inhibition of the alternative system. This lack of compensation implies that each shuttle is working close to or at its maximal capacity. Hence, transfer processes between mitochondria and cytoplasm must be considered as potential rate-limiting and regulatory sites for gluconeogenesis. The failure of fluoromalate to inhibit ethanol oxidation suggests that alternatives to the malate-oxaloacetate shuttle exist for the transfer to the mitochondria of reducing equivalents generated in the cytoplasmic compartment.