PPARγ coactivator-1α contributes to exercise-induced regulation of intramuscular lipid droplet programming in mice and humans

Abstract

Intramuscular accumulation of triacylglycerol, in the form of lipid droplets (LD), has gained widespread attention as a hallmark of metabolic disease and insulin resistance. Paradoxically, LDs also amass in muscles of highly trained endurance athletes who are exquisitely insulin sensitive. Understanding the molecular mechanisms that mediate the expansion and appropriate metabolic control of LDs in the context of habitual physical activity could lead to new therapeutic opportunities. Herein, we show that acute exercise elicits robust upregulation of a broad program of genes involved in regulating LD assembly, morphology, localization, and mobilization. Prominent among these was perilipin-5, a scaffolding protein that affects the spatial and metabolic interactions between LD and their surrounding mitochondrial reticulum. Studies in transgenic mice and primary human skeletal myocytes established a key role for the exercise-responsive transcriptional coactivator PGC-1α in coordinating intramuscular LD programming with mitochondrial remodeling. Moreover, translational studies comparing physically active versus inactive humans identified a remarkably strong association between expression of intramuscular LD genes and enhanced insulin action in exercise-trained subjects. These results reveal an intimate molecular connection between intramuscular LD biology and mitochondrial metabolism that could prove relevant to the etiology and treatment of insulin resistance and other disorders of lipid imbalance.

Fig. 1.

Intramuscular lipid droplets are physically and functionally associated with skeletal muscle mitochondria. (A–D) Electron micrographs showing presence of mitochondria and lipid droplets in white (A) versus red (B) quadriceps muscle and in the tibialis anterior muscle of standard chow (C) versus high-fat (D) diet fed animals (bar = 2 μm). (E) Accumulation and depletion of [1-¹⁴C]labeled triacylglycerol in mouse soleus and extensor digitorum longus (EDL) muscles pulsed for 1 h with 1 mM [1-¹⁴C]oleic acid in KHB buffer supplemented with 1% BSA and 0.5 mM L-carnitine followed by a 1 h chase in the absence of exogenous fatty acid. (F) Glycerolipid-derived ¹⁴CO₂ produced during the 1 h chase. (G) Oxidation of 5 mM [U-¹⁴C]glucose by isolated mouse muscle during a 1 h incubation in KHB buffer ± 100 µM etomoxir (Etx). Values are means ± SE (n = 5–6 muscles per group). *P < 0.05 versus soleus muscle; ^#P < 0.05 versus pulse or control condition.

Fig. 2.

Acute exercise and Pgc-1α overexpression stimulate transcriptional induction of the lipid droplet program in mouse skeletal muscle. Expression of genes involved in lipid droplet metabolism were measured in the TA muscles of (A) wild-type mice at rest and following 90 min of treadmill exercise and (B–E) MCK-Pgc-1α mice and NT littermates. Wild-type mice were euthanized at the indicated time points after exercise. TA muscles from NT or MCK-Pgc-1α transgenic mice were used for gene expression and protein content. (B and C) Gene expression data for key transcription factors and lipid droplet metabolism. (D and E) Representative Western blot and protein quantification of Plin5 (perilipin 5/OxPAT), Plin2 (perilipin 2/Adrp), and ATGL (adipose tissue triglyceride lipase) protein abundance. Data represent means ± SE for n = 4 mice per group. *P < 0.05 versus nonexercised or NT controls.

Fig. 3.

Pgc-1α overexpression promotes desaturation of muscle lipids. (A) Electron microscopy (bar = 1 or 2 μm as noted) and (B) measurement of intramuscular TAG and DAG. Fatty acid composition of (C) TAG and (D) DAG in TA muscles from MCK-Pgc-1α mice and NT littermates. (E and F) Fatty acid composition of muscle glycerolipids expressed as percentages of the total fatty acids present in either TAG or DAG. Data are means ± SE for n = 4 animals per group. *P < 0.05 versus NT controls.

Fig. 4.

Overexpression Pgc-1α affects lipid droplet metabolism in primary human skeletal muscle myotubes. (A) Western blot analysis of mouse Pgc-1α protein abundance in differentiation day 7 human skeletal myotubes. Cells were harvested 72 h after transduction with increasing doses (PFU/cm²) of adenoviruses expressing β-galactosidase (β-gal) or mouse Pgc-1α. (#) denotes the dose used for subsequent gene expression and pulse-chase studies. (B) mRNA expression analysis of myotubes harvested 72 h after virus treatment. Data are means ± SEM for triplicate wells from two independent experiments analyzed in triplicate and normalized to 18 s. (C) Day 4 myotubes were exposed to media containing 100 µM oleate/palmitate (1:1) bound to BSA at a 5:1 ratio for 72 h. Neutral lipids were stained using AdipoRed and visualized by fluorescent microscopy. (D) Schematic representation of the pulse-chase experimental design. L-carnitine (0.5 mM) was present only during the chase. (E) Representative radiogram of myotube lipids measured at the end of the 24 h pulse showing incorporation of [1-¹⁴C]oleate into TAG, DAG, and phospholipids (PL). Quantitation of [1-¹⁴C]oleate-labeling of (F) TAG, (G) DAG, and (H) PL during the pulse-chase experiment. Cells were harvested in 0.1% SDS lysis buffer at times 0 (immediately after the 24 h pulse), 3, 8, and 24 h during the carnitine chase. (I) ¹⁴CO₂ production and (J) glycerol release into the medium at 3 and 8 h during the carnitine chase. Data are means ± SE of two independent experiments performed in triplicate and expressed as a percentage relative to levels in cells treated with β-gal virus at time 0 (the end of the pulse period). In (F–J), data are normalized to total cellular protein per well. *P < 0.05 versus β-gal treated control cells at the same time point.

Fig. 5.

Sequestering intramuscular triacylglycerol into lipid droplets blunts expression of PPAR target genes. Human skeletal myocytes were treated on differentiation day 4 with recombinant adenovirus (5.2 × 10⁸ PFU/cm²) to express either β-galactosidase (β-gal) or mouse perilipin (Plpn1). After 24 h, cells were treated an additional 72 h with 0.1% BSA or 0.1 mM oleate/palmitate (1:1) bound to BSA at a 5:1 ratio, followed by (A) neutral lipid staining with AdipoRed, (B) measurement of glycerol in the culture medium, and (C) gene expression analyses by RT-PCR. (D) On differentiation day 5, human myocytes were pretreated 24 h with 100 µM of the lipase inhibitor diethylumbeliferyl phosphate (DEUP) or the vehicle alone (DMSO), followed by 72 h treatment with 0.1 mM oleate/palmitate (1:1) and measurement of gene expression. (E) Model showing the proposed metabolic interplay between lipid droplets and mitochondria. Data are means ± SE of at least two independent experiments performed in triplicate. Glycerol was normalized to total cellular protein, and gene expression analyses were normalized to 18 s. *P < 0.05 versus β-gal or vehicle control. ^#P < 0.05 versus BSA treatment.

Fig. 6.

Assessment of IMTG metabolism, fuel selection, and insulin action in trained compared with untrained human subjects. (A) Intramyocellular lipid content was measured by Oil Red O staining and combined with an immunofluorescence staining against slow myosin heavy chain (sMHC) to determine Type I fibers. Cells not stained for sMHC were considered to be Type II fibers. (B) mRNA levels of genes involved in lipid metabolism were measured by qRT-PCR in skeletal muscle tissue of trained compared with untrained men and expressed relative to GAPDH. (C) Representative blots and protein expression levels of lipid droplet coating (PLIN2, PLIN5) and lipolytic (ATGL, CGI58) proteins in skeletal muscles of trained compared with untrained men were measured by Western blot analysis. Insulin sensitivity expressed as insulin-stimulated change in (D) Rd (ΔRd), (E) glucose oxidation, and (F) nonoxidative glucose disposal during a euglycemic-hyperinsulinemic clamp with simultaneous infusion of glycerol. All parameters are expressed as µmol kg⁻¹ min⁻¹. Untrained subjects (n = 9) are represented by black bars and trained subjects (n = 10) by white bars. Data expressed as mean ± SE, *P < 0.05.