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. 2009 Nov;15(11):1307-11.
doi: 10.1038/nm.2049. Epub 2009 Oct 18.

Foxo1 integrates insulin signaling with mitochondrial function in the liver

Zhiyong Cheng  1 , Shaodong Guo, Kyle Copps, Xiaochen Dong, Ramya Kollipara, Joseph T Rodgers, Ronald A Depinho, Pere Puigserver, Morris F White
Affiliations

Foxo1 integrates insulin signaling with mitochondrial function in the liver

Zhiyong Cheng et al. Nat Med. 2009 Nov.

Abstract

Type 2 diabetes is a complex disease that is marked by the dysfunction of glucose and lipid metabolism. Hepatic insulin resistance is especially pathogenic in type 2 diabetes, as it dysregulates fasting and postprandial glucose tolerance and promotes systemic dyslipidemia and nonalcoholic fatty liver disease. Mitochondrial dysfunction is closely associated with insulin resistance and might contribute to the progression of diabetes. Here we used previously generated mice with hepatic insulin resistance owing to the deletion of the genes encoding insulin receptor substrate-1 (Irs-1) and Irs-2 (referred to here as double-knockout (DKO) mice) to establish the molecular link between dysregulated insulin action and mitochondrial function. The expression of several forkhead box O1 (Foxo1) target genes increased in the DKO liver, including heme oxygenase-1 (Hmox1), which disrupts complex III and IV of the respiratory chain and lowers the NAD(+)/NADH ratio and ATP production. Although peroxisome proliferator-activated receptor-gamma coactivator-1alpha (Ppargc-1alpha) was also upregulated in DKO liver, it was acetylated and failed to promote compensatory mitochondrial biogenesis or function. Deletion of hepatic Foxo1 in DKO liver normalized the expression of Hmox1 and the NAD(+)/NADH ratio, reduced Ppargc-1alpha acetylation and restored mitochondrial oxidative metabolism and biogenesis. Thus, Foxo1 integrates insulin signaling with mitochondrial function, and inhibition of Foxo1 can improve hepatic metabolism during insulin resistance and the metabolic syndrome.

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Figures

Figure 1
Figure 1
Mitochondrial morphology and biogenesis in the insulin-resistant livers. (a–c) Electron microscopy study of mitochondrial biogenesis and morphology in the livers. Scale bar, 2 μm. Cntr refers to the control (double floxed Irs1 and Irs2) liver. (d,e) Mitochondrial number (d) and area (e) in control, DKO and TKO liver. (f,g) Mitochondrial number (f) and area (g) in control, db/db, db/db-FKO and ob/ob liver. (h–j) Top, immunoblotting detection of expression of mitochondrial fusion and fission proteins in control, DKO, TKO, db/db, db/db-FKO and ob/ob liver. Bottom, quantification of band density normalized to porin. Mfn1, mitofusin-1; Mfn2, mitofusin-2; Dnm1l, dynamin-1–like; Fis1, fission-1; Dctn2, dynactin-2. The results in bar graphs are presented as means ± s.d. (n = 5). *P < 0.05; **P < 0.01.
Figure 2
Figure 2
Evaluation of mitochondrial function. (a–e) RCR (a), APR (b), ATP content (c), ETC activity (d) and NAD/NADH ratios (e) in the indicated groups of mice. The mice used in panels a–e were 8 weeks old, insulin resistant and hyperglycemic. (f–h) ETC activity (f), APR (g) and membrane potential (h) of the mitochondria from 4-week-old DKO mice that were insulin resistant but normoglycemic. All values are means ± s.d., n = 4; *P < 0.05; **P < 0.01.
Figure 3
Figure 3
Ppargc-1α inactivation in liver. (a) Acetylation of Ppargc-1α (acPpargc-1α) and expression of downstream target proteins NRF-1 and Tfam in control, DKO and TKO liver, as determined by immunoprecipitation assay. (b) Acetylation of Ppargc-1α and expression of downstream target proteins NRF-1 and Tfam in control, db/db and db/db-FKO liver, as determined by immunoprecipitation assay. (c) Acetylation of Ppargc-1α and expression of downstream target proteins NRF-1 and Tfam in control and ob/ob liver, as determined by immunoprecipitation assay. (d) The expression of wild-type Ppargc-1α, R13-Ppargc-1α and the acetylation levels in DKO liver after adenovirus infection, as determined by immunoprecipitation assay. The bar graphs in panels a–d are density quantification of the related bands normalized to porin. (e) Electron microscopy assay of liver sections from DKO mice infected with adenoviruses expressing GFP, wild-type Ppargc-1α and R13-Ppargc-1α. Scale bar, 500 nm. (f,g) Mitochondrial (Mito) number (f) and area (g) in DKO liver infected with adenoviruses expressing GFP, wild-type Ppargc-1α and R13-Ppargc-1α. All values in bar graphs are means ± s.d., n = 4; *P < 0.05; **P < 0.01.
Figure 4
Figure 4
Foxo1 regulates electron transport chain via Hmox1. (a) The expression pattern of protein markers of complexes I–V (mt-Nd6, Sdha, Uqcrc1, mt-Co1 and Atp5a1, respectively) in control, DKO and TKO liver. (b) The expression pattern of protein markers of complexes I–V (mt-Nd6, Sdha, Uqcrc1, mt-Co1 and Atp5a1, respectively) in control, db/db, db/db-FKO and ob/ob liver. (c) Heme content measurement. (d) Expression profile of Hmox1 protein. (e) The effect of siRNA on Hmox1 expression. The bar graphs in panels a, b, d and e are density quantification of the related bands normalized to porin. (f–h) Effects of Hmox1 suppression on mitochondrial functions. White bar, primary hepatocytes from the control mice transfected with nonspecific siRNA (−); Gray bars, primary hepatocytes from DKO, db/db and ob/ob mice transfected with nonspecific siRNA (−) and Hmox1 siRNA (+), respectively. (i) ChIP assay of the molecular interaction between Foxo1 and Hmox1 promoter. Left, immunoprecipitates subjected to immunoblot assay with Foxo1-specific antibody; whole tissue lysate was used as input control, and normal rabbit IgG was used as a negative control. Right, PCR and gel analysis using a pair of primers flanking the insulin response element (IRE) sequence in the Hmox1 promoter. Double floxed Irs1 and Irs2 (dflox) and triple floxed Irs1, Irs2 and Foxo1 (tflox) livers were used as the control of DKO and TKO livers, respectively. All values are the means ± s.d., n = 4; *P < 0.05; **P < 0.01. (j) A schematic model showing Foxo1 activation deregulates mitochondria under insulin resistance. Sirt1, sirtuin-1.
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