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Inactivation of NF- κ B p65 (RelA) in Liver Improves Insulin Sensitivity and Inhibits cAMP/PKA Pathway

View Article: PubMed Central - PubMed

ABSTRACT

The transcription factor nuclear factor-κB (NF-κB) mediates inflammation and stress signals in cells. To test NF-κB in the control of hepatic insulin sensitivity, we inactivated NF-κB in the livers of C57BL/6 mice through deletion of the p65 gene, which was achieved by crossing floxed-p65 and Alb-cre mice to generate L-p65-knockout (KO) mice. KO mice did not exhibit any alterations in growth, reproduction, and body weight while on a chow diet. However, the mice on a high-fat diet (HFD) exhibited an improvement in systemic insulin sensitivity. Hepatic insulin sensitivity was enhanced as indicated by increased pyruvate tolerance, Akt phosphorylation, and decreased gene expression in hepatic gluconeogenesis. In the liver, a decrease in intracellular cAMP was observed with decreased CREB phosphorylation. Cyclic nucleotide phosphodiesterase-3B (PDE3B), a cAMP-degrading enzyme, was increased in mRNA and protein as a result of the absence of NF-κB activity. NF-κB was found to inhibit PDE3B transcription through three DNA-binding sites in the gene promoter in response to tumor necrosis factor-α. Body composition, food intake, energy expenditure, and systemic and hepatic inflammation were not significantly altered in KO mice on HFD. These data suggest that NF-κB inhibits hepatic insulin sensitivity by upregulating cAMP through suppression of PDE3B gene transcription.

No MeSH data available.


Related in: MedlinePlus

Hepatic insulin action in L-p65-KO mice. A: PTT. After 24 weeks on HFD, mice received pyruvate 2 g/kg i.p. following overnight fasting (n = 8–10). B: Insulin signaling in hepatocytes. Primary hepatocytes were prepared from 8-week-old mice and cultured in vitro. After 15 min of insulin treatment, phosphorylation of Akt at T308 and S473 was examined in cell lysate by Western blotting. C: mRNA expression of gluconeogenic genes in hepatocytes of KO liver (n = 6). D: Liver weight. Livers were collected from mice on HFD for 16 weeks and used in the weight analysis (n = 10–12). E: Liver function. ALT and AST were tested using plasma collected from mice after 4 weeks on HFD (n = 7). Data are mean ± SEM. *P < 0.05.
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Figure 3: Hepatic insulin action in L-p65-KO mice. A: PTT. After 24 weeks on HFD, mice received pyruvate 2 g/kg i.p. following overnight fasting (n = 8–10). B: Insulin signaling in hepatocytes. Primary hepatocytes were prepared from 8-week-old mice and cultured in vitro. After 15 min of insulin treatment, phosphorylation of Akt at T308 and S473 was examined in cell lysate by Western blotting. C: mRNA expression of gluconeogenic genes in hepatocytes of KO liver (n = 6). D: Liver weight. Livers were collected from mice on HFD for 16 weeks and used in the weight analysis (n = 10–12). E: Liver function. ALT and AST were tested using plasma collected from mice after 4 weeks on HFD (n = 7). Data are mean ± SEM. *P < 0.05.

Mentions: Liver insulin sensitivity was examined to understand the improved systemic insulin sensitivity in KO mice on HFD. Pyruvate tolerance was studied to determine hepatic gluconeogenesis, a process inhibited by insulin. Insulin sensitivity was inversely associated with glucose elevation in PTT. The tolerance was enhanced in L-p65-KO mice for a smaller increase in blood glucose (Fig. 3A). Phosphorylation of Akt was examined to determine insulin signaling activity in hepatocytes. Akt phosphorylation at T308 and S473 was enhanced in KO mice (Fig. 3B). Expression of gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) was examined in hepatocytes to determine the mechanism of reduced gluconeogenesis, and both were decreased in the KO liver (Fig. 3C). Liver weight was not significantly altered in KO mice on HFD (Fig. 3D), although plasma ALT was elevated in KO mice (Fig. 3E). These data suggest that hepatic insulin sensitivity is improved in L-p65-KO mice on HFD.


Inactivation of NF- κ B p65 (RelA) in Liver Improves Insulin Sensitivity and Inhibits cAMP/PKA Pathway
Hepatic insulin action in L-p65-KO mice. A: PTT. After 24 weeks on HFD, mice received pyruvate 2 g/kg i.p. following overnight fasting (n = 8–10). B: Insulin signaling in hepatocytes. Primary hepatocytes were prepared from 8-week-old mice and cultured in vitro. After 15 min of insulin treatment, phosphorylation of Akt at T308 and S473 was examined in cell lysate by Western blotting. C: mRNA expression of gluconeogenic genes in hepatocytes of KO liver (n = 6). D: Liver weight. Livers were collected from mice on HFD for 16 weeks and used in the weight analysis (n = 10–12). E: Liver function. ALT and AST were tested using plasma collected from mice after 4 weeks on HFD (n = 7). Data are mean ± SEM. *P < 0.05.
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Related In: Results  -  Collection

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Figure 3: Hepatic insulin action in L-p65-KO mice. A: PTT. After 24 weeks on HFD, mice received pyruvate 2 g/kg i.p. following overnight fasting (n = 8–10). B: Insulin signaling in hepatocytes. Primary hepatocytes were prepared from 8-week-old mice and cultured in vitro. After 15 min of insulin treatment, phosphorylation of Akt at T308 and S473 was examined in cell lysate by Western blotting. C: mRNA expression of gluconeogenic genes in hepatocytes of KO liver (n = 6). D: Liver weight. Livers were collected from mice on HFD for 16 weeks and used in the weight analysis (n = 10–12). E: Liver function. ALT and AST were tested using plasma collected from mice after 4 weeks on HFD (n = 7). Data are mean ± SEM. *P < 0.05.
Mentions: Liver insulin sensitivity was examined to understand the improved systemic insulin sensitivity in KO mice on HFD. Pyruvate tolerance was studied to determine hepatic gluconeogenesis, a process inhibited by insulin. Insulin sensitivity was inversely associated with glucose elevation in PTT. The tolerance was enhanced in L-p65-KO mice for a smaller increase in blood glucose (Fig. 3A). Phosphorylation of Akt was examined to determine insulin signaling activity in hepatocytes. Akt phosphorylation at T308 and S473 was enhanced in KO mice (Fig. 3B). Expression of gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) was examined in hepatocytes to determine the mechanism of reduced gluconeogenesis, and both were decreased in the KO liver (Fig. 3C). Liver weight was not significantly altered in KO mice on HFD (Fig. 3D), although plasma ALT was elevated in KO mice (Fig. 3E). These data suggest that hepatic insulin sensitivity is improved in L-p65-KO mice on HFD.

View Article: PubMed Central - PubMed

ABSTRACT

The transcription factor nuclear factor-&kappa;B (NF-&kappa;B) mediates inflammation and stress signals in cells. To test NF-&kappa;B in the control of hepatic insulin sensitivity, we inactivated NF-&kappa;B in the livers of C57BL/6 mice through deletion of the p65 gene, which was achieved by crossing floxed-p65 and Alb-cre mice to generate L-p65-knockout (KO) mice. KO mice did not exhibit any alterations in growth, reproduction, and body weight while on a chow diet. However, the mice on a high-fat diet (HFD) exhibited an improvement in systemic insulin sensitivity. Hepatic insulin sensitivity was enhanced as indicated by increased pyruvate tolerance, Akt phosphorylation, and decreased gene expression in hepatic gluconeogenesis. In the liver, a decrease in intracellular cAMP was observed with decreased CREB phosphorylation. Cyclic nucleotide phosphodiesterase-3B (PDE3B), a cAMP-degrading enzyme, was increased in mRNA and protein as a result of the absence of NF-&kappa;B activity. NF-&kappa;B was found to inhibit PDE3B transcription through three DNA-binding sites in the gene promoter in response to tumor necrosis factor-&alpha;. Body composition, food intake, energy expenditure, and systemic and hepatic inflammation were not significantly altered in KO mice on HFD. These data suggest that NF-&kappa;B inhibits hepatic insulin sensitivity by upregulating cAMP through suppression of PDE3B gene transcription.

No MeSH data available.


Related in: MedlinePlus