Stimulatory effect of the intestinal peptide PHI on glycogenolysis and gluconeogenesis in isolated rat hepatocytes.
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ABSTRACT:
SUBMITTER: Feliu JE
PROVIDER: S-EPMC1152344 | biostudies-other | 1983 Sep
REPOSITORIES: biostudies-other
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Project description:Cyclic AMP-responsive element binding protein, hepatocyte specific (CREBH), is a liver-enriched, endoplasmic reticulum-tethered transcription factor known to regulate the hepatic acute-phase response and lipid homeostasis. In this study, we demonstrate that CREBH functions as a circadian transcriptional regulator that plays major roles in maintaining glucose homeostasis. The proteolytic cleavage and posttranslational acetylation modification of CREBH are regulated by the circadian clock. Functionally, CREBH is required in order to maintain circadian homeostasis of hepatic glycogen storage and blood glucose levels. CREBH regulates the rhythmic expression of the genes encoding the rate-limiting enzymes for glycogenolysis and gluconeogenesis, including liver glycogen phosphorylase (PYGL), phosphoenolpyruvate carboxykinase 1 (PCK1), and the glucose-6-phosphatase catalytic subunit (G6PC). CREBH interacts with peroxisome proliferator-activated receptor ? (PPAR?) to synergize its transcriptional activities in hepatic gluconeogenesis. The acetylation of CREBH at lysine residue 294 controls CREBH-PPAR? interaction and synergy in regulating hepatic glucose metabolism in mice. CREBH deficiency leads to reduced blood glucose levels but increases hepatic glycogen levels during the daytime or upon fasting. In summary, our studies revealed that CREBH functions as a key metabolic regulator that controls glucose homeostasis across the circadian cycle or under metabolic stress.
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Project description:Gluconeogenesis was studied in hepatocytes isolated from phenobarbital-pretreated rats fasted for 24 h. In closed vial incubations, glucose production from lactate (20 mmol/l) and pyruvate (2 mmol/l), alanine (20 mmol/l) or glutamine (20 mmol/l) was suppressed by about 30-45%, although glycerol metabolism was not affected. In hepatocytes perifused with lactate and pyruvate (ratio 10:1), glucose production was inhibited by 50%, even at low gluconeogenic flux. From the determination of gluconeogenic intermediates at several steady states of gluconeogenic flux, we have found a single relationship between phosphoenolpyruvate and the rate of glucose production (Jglucose), and two different curves between cytosolic oxaloacetate and Jglucose in controls and in phenobarbital-pretreated hepatocytes. By using 3-mercaptopicolinate to determine the flux control coefficient of phosphoenolpyruvate carboxykinase we found that phenobarbital pretreatment led to an increase in this coefficient from 0.3 (controls) to 0.8 (phenobarbital group). These observations were confirmed by the finding that the activity of phosphoenolpyruvate carboxykinase was decreased by 50% after phenobarbital treatment. Hence we conclude that the inhibitory effect of phenobarbital on gluconeogenesis is due, at least partly, to a decrease in the flux through phosphoenolpyruvate carboxykinase.
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Project description:ObjectivesProliferation of hepatocytes in vitro can be stimulated by growth factors such as epidermal growth factor (EGF), but the role of vasoactive intestinal peptide (VIP) remains unclear. We have investigated the effect of VIP on maintenance and proliferation of human hepatocytes.Materials and methodsHuman hepatocytes were isolated from liver specimens obtained from patients undergoing liver surgery. Treatment with VIP or EGF was started 24 h after plating and continued for 3 or 5 d. DNA replication was investigated by Bromodeoxyuridine (BrdU) incorporation and cell viability detected by MTT assay. Cell lysate was analysed by western blotting and RT-PCR. Urea and albumin secretion into the culture supernatants were measured.ResultsVIP increased DNA replication in hepatocytes in a dose-dependant manner, with a peak response at day 3 of treatment. VIP treatment was associated with an increase in mRNA expression of antigen identified by monoclonal antibody Ki-67 (MKI-67) and Histone Cluster 3 (H3) genes. Western blotting analysis showed that VIP can induce a PKA/B-Raf dependant phosphorylation of extracellular signal-regulated kinases (ERK). Although EGF can maintain hepatocyte functions up to day 5, no marked efffect was found with VIP.ConclusionsVIP induces proliferation of human hepatocytes with little or no effect on hepatocyte differentiation. Further investigation of the role of VIP is required to determine if it may ultimately support therapeutic approaches of liver disease.
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