Tumour necrosis factor‐α attenuates insulin action on phosphoenolpyruvate carboxykinase gene expression and gluconeogenesis by altering the cellular localization of Foxa2 in HepG2 cells

Abstract

Circulating tumour necrosis factor‐α (TNFα) levels, which are elevated in obesity‐associated insulin resistance and diabetes, inhibit insulin signalling at several points in the signalling cascade. The liver is critical in maintaining circulating glucose levels and, in a preliminary investigation using the human hepatoma (HepG2) cell line in this study, we demonstrated the role of TNFα in the regulation of this phenomenon and determined the underlying molecular mechanisms. As the transcription factor Foxa2 has been implicated, in part, in the regulation of gluconeogenic genes, we studied the effects of TNFα and/or insulin on its cellular status in hepatocytes, followed by an assessment of its occupancy on the phosphoenolpyruvate carboxykinase (PEPCK) promoter. Preincubation of cells with TNFα, followed by insulin, significantly prevented insulin‐mediated nuclear exclusion of Foxa2 and substantially increased its nuclear concentration. Foxa2 was subsequently found to occupy its binding element on the PEPCK promoter. TNFα alone, however, did not alter the status of cellular Foxa2 or its occupancy on the PEPCK promoter. TNFα preincubation also significantly attenuated insulin‐induced inhibition of the expression of gluconeogenic enzymes and hepatic glucose production. Insulin inhibition of PEPCK expression and the preventive effect of TNFα could be partially but significantly restored in the presence of Foxa2 siRNA. Several other well‐known mediators of insulin action in the liver in general and of gluconeogenic genes in particular include Foxo1, PGC‐1 and SREBP‐1c. Our results indicate that another transcription factor, Foxa2, is at least partly responsible for the attenuating effect of TNFα on insulin action on PEPCK expression and glucose production in HepG2 cells. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7040448: TBP (uniprotkb:http://www.ebi.uniprot.org/entry/P20226), FOXA2 (uniprotkb:http://www.ebi.uniprot.org/entry/Q9Y261) and FOXA1 (uniprotkb:http://www.ebi.uniprot.org/entry/P55317) colocalize (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0403) by cosedimentation (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0027)

Molecular mechanisms of action of styrene toxicity in blood plasma and liver

“…In the present study, the higher level of the plasma SOD and protein carbonyl along with the hepatic G6Pase, GP, and PEPCK explored that these vital enzymes of gluconeogenesis are susceptible to styrene toxicity and become less accessible to insulin actions. Studies revealed that inflammatory cytokines are involved in lessening the insulin action on PEPCK along with up‐regulation of PEPCK gene which is responsible for hepatic gluconeogenesis . Beside this, higher doses of sildenafil reduced glycogenesis which ultimately lessened blood glucose level.…”

“…Studies revealed that inflammatory cytokines are involved in lessening the insulin action on PEPCK along with up-regulation of PEPCK gene which is responsible for hepatic gluconeogenesis. 52,55 Beside this, higher doses of sildenafil reduced glycogenesis which ultimately lessened blood glucose level. It has high effect on GP and lower effect on PEPCK showing antioxidant properties and no mimicking potentially.…”

IL-1β induces ER stress in a JNK dependent manner that determines cell death in human pancreatic epithelial MIA PaCa-2 cells

“…Real-Time PCR for quantitation was done for BiP and CHOP using SYBR green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions (ABI 7500, Applied Biosystems, CA) using gene specific primers (For BiP forward: 5 0 -CATCACGCCGTCCTATGTCG-3 0 and reverse: 5 0 -CG TCAAAGACCGTGTTCTCG-3 0 ; For CHOP forward: 5 0 -GCGCATGAAGGAGAAAGAAC-3 0 and reverse: 5 0 -CC AATTGTTCATGCTTGGTG-3 0 ). Reactions were performed with cDNAs from three independent experiments and the expression of each transcript was quantified by the relative standard curve method and normalized to those of 18S rRNA as described by Pandey et al from our laboratory [43]. The transcript value for the control obtained after normalization was arbitrarily assigned a value of 1.…”

Gene Expression of the Tumour Suppressor LKB1 Is Mediated by Sp1, NF-Y and FOXO Transcription Factors

“…It is unlikely that this complex is formed by another FOXO member, since none of the used cell lines expressed FOXO1 or FOXO6 (data not shown). Since HepG2 cells express the liver specific transcription factor FOXA2, also known as HNF-3β [33], it can be assumed that the observed protein-DNA complex may contain this factor. After ectopic expression of FOXA2 in “444” cells, known to lack this factor endogenously, complex formation with similar mobility could be discerned (lane 9), indicating that also other forkhead box transcription factors can bind to this element in a tissue specific manner.…”