Hepatic Gluconeogenesis Pathway Research Articles

Effects of low-protein-high-starch diet on growth performance, glucose and lipid metabolism of Amur sturgeon (Acipenser schrenckii) during feeding and starvation phases

An 8-week growth (8 w) and 1-week starvation (S1 w) trial were conducted to investigate the effects of low-protein-high-starch diet on the growth performance, glucose and lipid metabolism in Amur sturgeon (initial body weight, 69.99 ± 0.06 g). Two diets with equal protein-to-energy ratios were fed, one diet contained 43.0% protein and 11.5% starch (HPLS) and the other contained 38.9% protein and 23.6% starch (LPHS). The results showed that LPHS diet did not compromise growth performance of Amur sturgeon, while the feeding rate increased significantly suggesting the keep of energy supplement. The LPHS diet also promoted the deposition of whole-body protein in Amur sturgeon after S1 w. An effective glucose metabolism response was observed that the transcript levels of glycolysis-related genes (gk) was upregulated significantly, suggesting that glucose catabolism was accelerated to maintain normal glycemic homeostasis and avoid hyperglycemia and glycogen accumulation during long-term feeding phase. Moreover, hepatic gluconeogenesis pathway (PEPCK, G6P) was upregulated to maintain normal plasma glucose levels of sturgeon in the LPHS group during S1 w phase. In addition, activated chrebp by high-starch intake induced excess glucose conversion to TG in the liver, which led to inhibit lipogenesis and accelerate lipolysis to prevent excessive fat accumulation after fed LPHS diet during feeding phase. Hepatic lipolysis (HSL) and β-oxidation (PPARα) were upregulated to satisfy energetic requirements, thereby promoting whole-body protein deposition. Our findings suggested that low-protein-high-starch diet could conserve dietary protein, and mitigate the excessive consumption of whole-body protein during starvation.

Design, synthesis and structural-activity relationship studies of phanginin A derivatives for regulating SIK1-cAMP/CREB signaling to suppress hepatic gluconeogenesis

Persistent activation of hepatic gluconeogenesis is a main cause of fasting hyperglycemia in patients with type 2 diabetes (T2D), and the salt-induced kinase 1 (SIK1) acts as a key modulator in regulating hepatic gluconeogenesis. Recently, we first reported phanginin A (PA, 1), a natural cassane diterpenoid isolated from the seeds of Caesalpinia sappan, exhibited potent anti-diabetic effect through activation of SIK1 and increasing PDE4 activity to inhibit hepatic gluconeogenesis pathway by suppressing the cAMP/PKA/CREB pathway in the liver. In present study, we designed and prepared 25 PA derivatives and their structure−activity relationship (SAR) for gluconeogenesis inhibitory activity were established. Among them, compound 7 exhibited remarkable inhibitory activity on hepatic gluconeogenesis by enhancing the SIK1 phosphorylation and ameliorated the hyperglyceamia of type 2 diabetic mice. Our results supported that compound 7 could be served as a potential candidate for the treatment of T2D.

Effects of stanniocalcin hormones on rat hepatic glucose homeostasis under fed and fasted conditions

To test the hypothesis of conservation of stanniocalcin 1 and 2 (STC-1; STC-2) metabolic functions in vertebrates, we performed an in vitro study to determine if these hormones are implicated in regulation of the gluconeogenesis pathway, glycogen synthesis, and 14C-glucose conversion to 14CO2 in livers from fed and fasting rats (Rattus norvegicus). Stc1 and Stc2 gene expressions increased in the liver after fasting. STC-1 participated in the regulation of the hepatic gluconeogenesis pathway in rats when the precursor was 14C-lactate. STC-2 demonstrated variational signaling on rat hepatic gluconeogenesis activity and Pck1 gene expression, decreasing levels in the fed state when the substrate was 14C-alanine and increasing levels during fasting when the substrate was 14C-lactate. At the concentrations used in this study, STC-1 and STC-2 did not affect glycogen concentration and synthesis from 14C-glucose or 14C-glucose conversion to 14CO2 in the livers from fed or fasting rats. These findings highlight the role of stanniocalcins in the hepatic gluconeogenesis pathway in mammals and confirm the conservation of STC-1 and STC-2 metabolic functions in the vertebrates.

Development of a robust functional cell-based assay for replacing the rabbit blood sugar bioidentity test of insulin glargine drug substance

A rabbit blood sugar bioidentity assay is required by the FDA to evaluate biological activity for all insulin and its analogs per USP<121> guideline. Not only are a large number of live animals used, but the rabbit blood sugar method is also highly variable and expensive. Our goal is to develop a functional cell-based assay to replace rabbit blood sugar method. An H4IIE G6P-Luc reporter assay was developed by utilizing insulin’s role in regulating hepatic gluconeogenesis pathway. It is known that Glucose 6-phosphatase is a rate-limiting enzyme in the gluconeogenesis pathway, and the mRNA expression of its catalytic subunit, G6PC, is highly regulated by insulin. A G6P-Luc stable cell line in H4IIE hepatocytes was first generated by stably expressing luciferase reporter gene driven by human G6PC promoter via lentivirus technology. The cell-based assay was developed and optimized to demonstrate good dose-dependent responsiveness to insulin. We further qualified the assay with two analysts through multiple runs, and demonstrated excellent performance characteristics of linearity, accuracy, and precision. A robustness study was then conducted to define critical factors for assay performance. We compared this newly developed assay with a previously established cell-based pIR MSD assay, which measures insulin receptor phosphorylation (pIR) in HepG2 cell line using Meso-Scale Discovery (MSD) technology. The comparability study was conducted to compare the two assays using samples generated from forced degradation. This study showed high correlation between assays, and both are stability indicating. Compared with the pIR MSD assay, the G6P-Luc assay not only has a significantly lower variability in qualification studies, but also offers many other advantages, including ease of use in a quality control laboratory with fewer steps, lower cost, and does not depend on a single vendor. In conclusion, we have developed a physiologically relevant and robust functional cell-based assay that is suitable to replace rabbit blood sugar method.

Nutritional regulation of gene expression and enzyme activity of phosphoenolpyruvate carboxykinase in the hepatic gluconeogenesis pathway in golden pompano (Trachinotus ovatus)

Phosphoenolpyruvate carboxykinase (PEPCK) is known to exist as mitochondrial (PEPCK-M) and cytosolic isoforms (PEPCK-C). They are involved in glucose production and play vital roles in regulating glucose homeostasis. This study aimed to (1) clone and characterize PEPCK-C cDNA from golden pompano fish (Trachinotus ovatus), which are known to have poor utilization of dietary carbohydrates, and (2) analyze the regulation of its expression by varying dietary carbohydrate-to-lipid ratios and nutritional status. A full length golden pompano PEPCK-C cDNA fragment of 2,652 bp was cloned, which contains an open reading frame of 1,875 bp encoding 624 amino acids and shows a high homology to cobia (Rachycentron canadum) PEPCK-C sequence (92% similarity). The analysis of tissue distributions of PEPCK-C mRNA showed that a high abundance of PEPCK-C was expressed in golden pompano liver, followed by kidney; while lower expression levels were found in the heart and intestine. In liver, PEPCK is under the regulation of dietary composition and nutritional status at the enzymatic and molecular levels. Our results indicate that PEPCK-C plays a modulating role in the adaptation of hepatic gluconeogenesis to different nutrient conditions. Thus, the absence of molecular inhibition of gluconeogenic PEPCK gene expression in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), which can at least partially account for their poor utilization of dietary carbohydrate, is specific to these species and is not observed in golden pompano. Further studies that examine, at the enzymatic and molecular levels, other key enzymes involved in hepatic glucose metabolism are necessary to understand the poor utilization of carbohydrates in golden pompano.

Metabolic reprogramming by PCK1 promotes TCA cataplerosis, oxidative stress and apoptosis in liver cancer cells and suppresses hepatocellular carcinoma.

Phosphoenolpyruvate carboxykinase (PEPCK or PCK) catalyzes the first rate-limiting step in hepatic gluconeogenesis pathway to maintain blood glucose levels. Mammalian cells express two PCK genes, encoding for a cytoplasmic (PCPEK-C or PCK1) and a mitochondrial (PEPCK-M or PCK2) isoforms, respectively. Increased expressions of both PCK genes are found in cancer of several organs, including colon, lung and skin, and linked to increased anabolic metabolism and cell proliferation. Here, we report that the expressions of both PCK1 and PCK2 genes are downregulated in primary hepatocellular carcinoma (HCC) and low PCK expression was associated with poor prognosis in patients with HCC. Forced expression of either PCK1 or PCK2 in liver cancer cell lines results in severe apoptosis under the condition of glucose deprivation and suppressed liver tumorigenesis in mice. Mechanistically, we show that the proapoptotic effect of PCK1 requires its catalytic activity. We demonstrate that forced PCK1 expression in glucose-starved liver cancer cells induced TCA cataplerosis, leading to energy crisis and oxidative stress. Replenishing TCA intermediate α-ketoglutarate or inhibition of reactive oxygen species production blocked the cell death caused by PCK expression. Taken together, our data reveal that PCK1 is detrimental to malignant hepatocytes and suggest activating PCK1 expression as a potential treatment strategy for patients with HCC.

Effect of two different diets on liver gene expression associated with glucose metabolism in dairy cows

Ruminant animals depend mainly on hepatic gluconeogenesis pathway to maintain glucose homeostasis. Effects of diets with different ingredients on gene expression profile related to bovine hepatic glucose metabolism are unknown. Objective of this study was to examine gene expression coding for rate-limiting enzymes in hepatic glucose metabolism in response to two different diets. In this study, two kinds of isoenergetic and isonitrogenous diets were designed. Group I diets consisted mainly of corn straw (37.1%), corn (33.5%) and soybean meal (23.6%). Group II diets consisted mainly of alfalfa hay (28.4%), corn silage (26.5%) and corn (22.8%). After 30-day experimental period, biopsy section of liver and blood samples were obtained before feeding. Expression of 28 genes coding for rate-limiting enzymes in hepatic gluconeogenesis pathway, pentose phosphate pathway, glycogen synthesis pathway, glycogen decomposition pathway, glycolysis pathway, glucose aerobic oxidation pathway, and glucose transporters was detected with reverse transcription quantitative PCR (RT-qPCR). Results showed that Group II led to marked up-regulation of phosphoenolpyruvate carboxykinase (PEPCK) and marked up-regulation tendency of glucose-6-phosphatase (G6P), both involved in hepatic gluconeogenesis pathway (P<0.05). No significant differences were observed in mRNA expression of genes involved in other glucose metabolism pathways. Higher relative percentage mRNA abundance of G6P (22.6%), representing the important gluconeogenic enzyme and also catalyzing the last step in glycogenolysis, underscored its importance for ruminant hepatic glucose metabolism. It was concluded that different dietary ingredients could change gene expression of hepatic gluconeogenesis pathway, especially PEPCK and G6P. Dietary ingredients had little effect on other bovine hepatic glucose metabolism pathways and glucose transporter protein. Results indicate that in order to respond to different diet ingredients, genes involved in gluconeogenesis pathway play an important role.

Investigation of hepatic gluconeogenesis pathway in non-diabetic Asian Indians with non-alcoholic fatty liver disease using in vivo (31P) phosphorus magnetic resonance spectroscopy

ObjectiveTo study hepatic gluconeogenesis pathway in non-diabetic Asian Indian males having non-alcoholic fatty liver disease (NAFLD) using in vivo (31P) phosphorous magnetic resonance spectroscopy (MRS) and correlate these data with anthropometry and insulin resistance. Research design and methodsForty non-diabetic patients with NAFLD and 20 healthy controls were divided into (i) obese with NAFLD (group I, n=20), (ii) non-obese with NAFLD (group II, n=20) and (iii) non-obese without NAFLD (group III, n=20). Anthropometric and biochemical profiles, short insulin tolerance test (SITT), liver ultrasound, and 31P MRS (to determine hepatic gluconeogenesis metabolite; phosphomonoesters (PMEs), inorganic phosphate (Pi) and their ratios with respect to ATP) were done. ResultsInsulin resistance (Kitt value) was highest in group I (p<0.05; compared to other two groups), but was also higher in group II as compared to group III (p=ns). The values of PME/Pi, PME/γATP, PME/βATP, PME/tATP ratios were higher (p<0.05) in group I compared to other two groups. Interestingly, non-obese subjects with NAFLD also showed more derangements of hepatic gluconeogenesis metabolites than non-obese subjects without NAFLD. Positive correlation was observed between PME and other ratios in relation to body mass index, waist circumference, body fat percentage and fasting serum insulin levels in all the three groups. ConclusionsDerangements in hepatic gluconeogenesis as assessed non-invasively using 31P MRS, was observed in obese and non-obese, non-diabetic Asian Indians with NAFLD. Further research is warranted whether this investigation in NAFLD subjects could be developed as a non-invasive tool to assess those predisposed to develop hyperglycemia.

Altered fluxes responsible for reduced hepatic glucose production and gluconeogenesis by exogenous glucose in rats.

The net release of glucose from the liver, or hepatic glucose production (HGP), and apparent gluconeogenesis (GNG) are reduced by exogenous glucose. We investigated the changes in metabolic fluxes responsible. Flux through the hepatic GNG pathway was quantified by mass isotopomer distribution analysis (MIDA) from [2-13C]glycerol. Unidirectional flux across hepatic glucose-6-phosphatase (G-6-Pase), or total hepatic glucose output (THGO), and hepatic glucose cycling (HGC) were also measured by using glucuronate (GlcUA) to correct for glucose 6-phosphate (G-6-P) labeling. Infusion of glucose (15-30 mg.kg-1.min-1 iv) to 24 h-fasted rats caused two important metabolic alterations. First was a significant increase in hepatic glucose uptake and HGC: > 60% of THGO was from HGC. Second, although flux through hepatic G-6-P increased (from 15.7 to 17.7-22.7 mg.kg-1.min-1), the partitioning of G-6-P flux changed markedly [from 30-35% to 55-60% entering UDP-glucose (UDP-Glc), P < 0.01]. Total flux through the GNG pathway remained active during intravenous glucose, but increased partitioning into UDP-Glc lowered GNG flux plasma glucose by 50%. In summary, the suppression of HGP and GNG flux into glucose is not primarily due to reduced carbon flow through hepatic G-6-Pase or the hepatic GNG pathway. THGO persists, but hepatic G-6-P is derived increasingly from plasma glucose, and flow through GNG persists, but the partitioning coefficient of G-6-P into UDP-Glc doubles. These adjustments permit net HGP to fall despite increased total production of hepatic G-6-P during administration of glucose.