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PGC-1 gene expression is down-regulated by Akt- mediated phosphorylation and nuclear exclusion of FoxO1 in insulin-stimulated skeletal muscle

Robert J. Southgate^*, Clinton R. Bruce, Andrew L. Carey^*, Gregory R. Steinberg, Ken Walder, Robert Monks^¶, Matthew J. Watt^*, John A. Hawley, Morris J. Birnbaum^¶ and Mark A. Febbraio^*,1

^* Cellular and Molecular Metabolism Laboratory and
Exercise Metabolism Group, RMIT University, Bundoora;
St. Vincent’s Institute and Department of Medicine, The University of Melbourne, Fitzroy, Vic;
Metabolic Research Unit, School of Health Sciences, Deakin University, Waurn Ponds, Australia; and
^¶ Howard Hughes Medical Institute, The Cox Institute, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

¹Correspondence: Cellular and Molecular Metabolism Laboratory, RMIT University, P.O. Box 71, Bundoora 3083, Victoria, Australia. E-mail: mark.febbraio{at}rmit.edu.au

SPECIFIC AIMS

There are multiple binding domains on the promoter region of the peroxisome proliferator activator receptor coactivator-1 (PGC-1) gene, including a trio of insulin-responsive elements that are activated by the forkhead box class-O (FoxO1) winged helix transcription factor, which is regulated by acute transforming retrovirus thymoma (Akt). In healthy skeletal muscle, insulin phosphorylates Akt, a process that can be impaired in the skeletal muscle of patients with type 2 diabetes. Surprisingly, no studies have examined Akt-mediated regulation FoxO1 and PGC-1 by insulin in skeletal muscle, but this is warranted given the limited but conflicting data with respect to insulin regulation of PGC-1 in skeletal muscle. In the present study we examined the effect of insulin on Akt and FoxO1 phosphorylation and PGC-1 mRNA expression in skeletal muscle from healthy (CON) and insulin-resistant (TYPE 2) humans. To confirm our in vivo data, we next performed experiments in vitro in human primary muscle cells treated with either vehicle (VEH) or palmitic acid (PALM) to render them insulin resistant before subjecting them to insulin stimulation. Finally, to confirm that aberrant FoxO1 function was Akt dependent, we performed experiments in muscles from Akt2 –/– and wild-type (WT) mice. We hypothesized that in healthy, but not insulin-resistant, skeletal muscle insulin stimulation would decrease PGC-1 gene transcription via Akt-dependent phosphorylation and nuclear exclusion of FoxO1.

PRINCIPAL FINDINGS

1. Insulin phosphorylates FoxO1 at Ser²⁵⁶ and Thr²⁴ and results in reduced nuclear abundance of FoxO1 protein expression in healthy but not insulin-resistant muscle
Basal phosphorylation of FoxO1 (Ser²⁵⁶) residue was higher in CON than in TYPE 2, and the same was true of basal FoxO1 (Ser²⁵⁶) phosphorylation in VEH compared with PALM myotubes. Insulin did not increase phosphorylation at this residue in either group in vivo. However, in vitro, insulin increased FoxO1 (Ser²⁵⁶) phosphorylation in VEH- but not PALM-treated cells. Basal phosphorylation of FoxO1 (Thr²⁴) residue was not different when comparing groups in the basal state in vivo or in vitro. However, while insulin stimulation increased FoxO1 (Thr²⁴) phosphorylation in CON, it did not do so in TYPE 2 and a similar pattern of phosphorylation of FoxO1 (Thr²⁴) was observed when comparing VEH- with PALM-treated myotubes. While insulin treatment decreased (P<0.05) the nuclear abundance of FoxO1 total protein in VEH, it did not do so in PALM-treated cells (Fig. 1 ).

View larger version (17K): [in this window] [in a new window]   Figure 1. Basal and insulin-stimulated nuclear abundance of total FoxO1 protein in human primary skeletal muscle myotubes treated with vehicle (VEH) or palmitate (PALM). *Difference (P<0.05) compared with VEH. Difference (P<0.05) compared with BASAL. Data are mean ± SE. Phospho-FOXO1 (Ser256) expressed as a ratio of total FoxO1.

2. Insulin suppresses PGC-1 and downsteam genes in healthy but not insulin-resistant muscle
We observed, in vivo, that insulin decreased (P<0.05) the mRNA expression of PGC1-, COX4, and NRF-1 in CON. In contrast, insulin did not repress the expression of any of the genes measured in TYPE 2. We also observed in vitro that insulin decreased the mRNA abundance of PGC-1 in VEH-treated myotubes, but not in PALM-treated cells. These data are consistent with our findings that insulin decreased FoxO1 total protein in the nucleus in healthy but not insulin-resistant muscle, as FoxO1 is a major transcription factor for PGC-1 (Fig. 2 ).

View larger version (21K): [in this window] [in a new window]   Figure 2. Insulin-stimulated (post-clamp relative to pre-clamp=1) mRNA expression of protein peroxisome proliferator-activated receptor coactivator-1 (PGC-1), nuclear encoded cytochrome oxidase (COX4), forkhead box class-O (FoxO1), mitochondrial transcription factor A (tFAM), presenilins-associated rhomboid-like protein (PSARL), and nuclear respiratory factor-1 (NRF-1) in patients with type 2 diabetes (TYPE 2) and healthy control subjects (CON) (A). Insulin-stimulated (post-insulin relative to pre-insulin=1) mRNA expression of PGC-1 in primary human skeletal muscle myotubes treated with vehicle (VEH INSULIN) or palmitate (PALM INSULIN) (B). *Difference (P<0.05) compared with pre-clamp or pre-insulin. Data are mean ± SE. *Difference (P<0.05) compared with all other conditions. Data are mean ± SE.

3. The effect of insulin on FoxO1 phosphorylation, nuclear extrusion and PGC-1 mRNA abundance are regulated by Akt
To determine whether the regulation of FoxO1 was Akt dependent, we next treated Akt2 –/– and wild-type mice with or without insulin. Insulin phosphorylated Akt and FoxO1 (Thr²⁴, Ser²⁵⁶), resulting in reduced nuclear expression of FoxO1 total protein in wild-type but not Akt2 –/– skeletal muscle.

CONCLUSIONS

The regulation of PGC-1 by insulin in skeletal muscle is not fully clear. Here we show that in skeletal muscle biopsy specimens from healthy humans and cultured human skeletal myotubes, insulin phosphorylates Akt (Ser⁴⁷³) and FoxO1 (Thr²⁴, Ser²⁵⁶), leading to reduced nuclear abundance of FoxO1 total protein. This is associated with an insulin-mediated repression of the mRNA expression PGC-1 and downstream genes associated with oxidative phosphorylation. In contrast, in muscle taken from insulin-resistant humans or in palmitate-treated insulin-resistant myotubes, neither Akt nor FoxO1 were phosphorylated by insulin, resulting in a failure for nuclear exclusion of FoxO1 total protein, and an inability for insulin to repress the mRNA expression of PGC-1 and downstream genes. To determine whether the regulation of FoxO1 was Akt dependent, we treated Akt2 –/– and wild-type mice with or without insulin. Insulin phosphorylated Akt and FoxO1, resulting in a reduced nuclear expression of FoxO1 total protein in wild-type but not Akt2 –/– skeletal muscle. We conclude that insulin decreases the expression of genes involved in oxidative metabolism in healthy but not insulin-resistant muscle due to a decrease in FoxO1 phosphorylation and nuclear exclusion secondary to reduced Akt activity. Our data provide new insight into the regulation of PGC-1 in healthy and insulin resistance human skeletal muscle and suggest that Akt and FoxO1 signaling are critical in their regulation of this process.

View larger version (35K): [in this window] [in a new window]   Figure 3. Proposed schematic control of PGC1- in skeletal muscle. Three promoter regions are calcium (Ca2+) and protein kinase A (PKA) -responsive. Upon contraction Ca2+ activates calcineurin (CnA) and calmodulin kinases (CamK). CnA activates nuclear factor of activated T cells (NFAT) and myocyte enhancer factor 2 (MEF2), which act as transcription factors for PGC1-. CamK phosphorylates histone dyacetylases (HDAC), which inhibit the MEF2-responsive element on PGC1-. Increased adrenaline can activate cyclic AMP (cAMP) to phosphorylate cAMP-responsive element binding protein (CREB), which activates CRE on PGC1-. There is also trio of insulin-responsive elements on the promoter region. These are activated by the forkhead box class-O (FoxO1) winged helix transcription factor, previously known as forkhead homologue of rhabdomyosarcoma (FKHR). When insulin binds to its receptor and activates PI3K, resulting in Akt phosphorylation, FoxO1 is phosphorylated and extruded from the nucleus. This process is impaired in insulin resistance.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3993fje;

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