PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle

Abstract

The pathophysiology underlying mitochondrial dysfunction in insulin-resistant skeletal muscle is incompletely characterized. To further delineate this we investigated the interaction between insulin signaling, mitochondrial regulation, and function in C2C12 myotubes and in skeletal muscle. In myotubes elevated insulin and glucose disrupt insulin signaling, mitochondrial biogenesis, and mitochondrial bioenergetics. The insulin-sensitizing thiazolidinedione pioglitazone restores these perturbations in parallel with induction of the mitochondrial biogenesis regulator PGC-1alpha. Overexpression of PGC-1alpha rescues insulin signaling and mitochondrial bioenergetics, and its silencing concordantly disrupts insulin signaling and mitochondrial bioenergetics. In primary skeletal myoblasts pioglitazone also up-regulates PGC-1alpha expression and restores the insulin-resistant mitochondrial bioenergetic profile. In parallel, pioglitazone up-regulates PGC-1alpha in db/db mouse skeletal muscle. Interestingly, the small interfering RNA knockdown of the insulin receptor in C2C12 myotubes down-regulates PGC-1alpha and attenuates mitochondrial bioenergetics. Concordantly, mitochondrial bioenergetics are blunted in insulin receptor knock-out mouse-derived skeletal myoblasts. Taken together these data demonstrate that elevated glucose and insulin impairs and pioglitazone restores skeletal myotube insulin signaling, mitochondrial regulation, and bioenergetics. Pioglitazone functions in part via the induction of PGC-1alpha. Moreover, PGC-1alpha is identified as a bidirectional regulatory link integrating insulin-signaling and mitochondrial homeostasis in skeletal muscle.

FIGURE 1.

Insulin signaling and mitochondrial phenotype in response to insulin resistance and pioglitazone. Control myotubes were exposed to 25 mm glucose, and the insulin resistant myotubes were exposed to 40 mm glucose and 100 nm insulin for 48 h. A represents insulin-responsive signaling as shown by the representative Western blots (pTyr, phosphotyrosine; cont, control; IR, insulin resistance). B, transcript levels, expressed as % control, of genes encoding for mitochondrial regulatory proteins (PPARγ, peroxisome proliferator-activated receptor γ; NRF1, nuclear respiratory factor 1; Tfam, mitochondrial transcription factor A and SIRT1. C, steady-state mitochondrial electron transfer chain protein levels (COX I and III, cytochrome c oxidase subunits I and III; Cyt c, cytochrome c; ND 6, NADH dehydrogenase, subunit 6; VDAC, voltage-dependent anion channel) as well as levels of voltage-activated anion channel, SIRT1, and actin as a housekeeping protein; D, relative mitochondrial genomic copy number (% control). E shows insulin-responsive signaling comparing insulin-resistant to pioglitazone (Pio)-treated insulin-resistant myotubes. F, transcript levels, expressed as % control, of genes encoding for mitochondrial regulatory proteins. G, steady-state mitochondrial electron transfer chain protein levels comparing insulin-resistant to insulin-resistant myotubes exposed to pioglitazone. H, relative mitochondrial genomic copy number (% control). The asterisk represents a p < 0.05 versus the respective control in this and all subsequent figures (unless otherwise stated).

FIGURE 2.

Insulin resistance and pioglitazone modulation of mitochondrial biology. The mitochondrial bioenergetic profile was compared between control and insulin-resistant myotubes and to the administration of pioglitazone. Results are representative cytometric profile of MitoTracker green, which represents that relative mitochondrial size (A), and tetramethylrhodamine methyl ester (TMRM), which represents the relative mitochondrial membrane potential (% control) (B) comparing control to insulin-resistant cells. C, total cellular ATP levels. D, the relative rate of oxygen consumption (% control). E, comparing insulin resistant cells to those exposed to pioglitazone (Pio) showing a representative cytometric profile of MitoTracker green fluorescence depicting the relative mitochondrial size. F, tetramethylrhodamine methyl ester represents the relative mitochondrial membrane potential (% insulin-resistant controls). G, total cellular ATP levels. H, relative rate of oxygen consumption (% insulin-resistant controls). RFU, relative fluorescent units.

FIGURE 3.

Pioglitazone restores the mitochondrial bioenergetic profile in primary myocytes and in the db/db mouse. A, pioglitazone (Pio) up-regulates the expression of the gene encoding for PGC-1α in primary myoblasts. B, pioglitazone reverses the diminution in mitochondrial size and membrane potential evoked by insulin resistance in primary skeletal myoblasts. C, pioglitazone prevents the development of fasting hyperglycemia in db/db mice. D, pioglitazone up-regulates PGC-1α steady-state protein levels in db/db mice. The levels of PGC-1α were diminished in untreated db/db mice compared with strain-matched C57BL/6J control mice.

FIGURE 4.

PGC-1α modulates mitochondrial biology and insulin signaling. A shows murine PGC-1α promoter activation in response to pioglitazone (cont, control; Pio, pioglitazone). B, infection of PGC-1α up-regulates PGC-1α and COX I protein levels. MOI, multiplicity of infection. C, augmentation of relative rate of oxygen consumption in cells with increasing levels of PGC-1α and the relative O₂ consumption comparing GFP to PGC-1α vector at a multiplicity of infection of 100. D shows the restoration of insulin-mediated signaling (% GFP/cont without insulin) by PGC-1α overexpression (pTyr, phosphotyrosine; cont, control). E, induction of oxygen consumption by PGC1α overexpression in, insulin resistance myotubes. F, effect of PGC-1α silencing on transcript levels and on oxygen consumption (% si cont); G, the diminution of Akt threonine phosphorylation by insulin after partial silencing of PGC-1α.

FIGURE 5.

Disruption of insulin signaling and mitochondrial respiration. A, effect of knockdown of InsRβ on Akt phosphorylation (p-Akt). B, PGC-1α levels in response to knockdown of InsRβ. C, quantitative reduction of PGC-1α protein after InsRβ knockdown. D, attenuation in mitochondrial size and membrane potential in insulin receptor knockdown cells. TMRM, tetramethylrhodamine methyl ester; Mito G, MitoTracker green. E, in insulin receptor knock-out (KO) skeletal myoblasts the mitochondrial membrane potential and mitochondrial mass is diminished compared with wild type (WT) control cells.