Perilipin 2 improves insulin sensitivity in skeletal muscle despite elevated intramuscular lipid levels

Abstract

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)-coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation-related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet-induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance.

FIG. 1.

PLIN2 knockdown lowers intracellular neutral lipid accumulation. A: C2C12 myotubes were incubated overnight with 80 µmol/L BSA complexed to 200 µmol/L oleate (OA), palmitate (PA), octanoate (OctA), or solely BSA as a control. PLIN2 and TAG levels are expressed as fold change relative to the control treatment. *P < 0.05 compared with the control treatment. B: PLIN2 protein expression in TA muscle of C57Bl6 mice fed an HFD or normal chow diet for 8 weeks. C and D: C2C12 myotubes were transfected with PLIN2 siRNA (P2) or a scrambled control (S) and incubated overnight with 200 µmol/L BSA-coupled oleate, palmitate, or solely 80 µmol/L BSA. Subsequently, cells were harvested to measure PLIN2 protein expression and TAG levels. #P < 0.05 vs. control (FA effect) and *P < 0.05 vs. scrambled control with the corresponding FA treatment. Data are expressed as mean ± SEM. n = 3–4. D and E: Electron microscopy pictures. D scale bars represent 2 microns. E: The oleate-treatment condition (increased LD size upon PLIN2 knockdown). Scale bars represent 0.5 micron.

FIG. 2.

Gene expression profiles of PLIN2 knockdown in C2C12 myotubes. Selected pathways identified by gene score resampling in ErmineJ. Only pathways that were significantly up- or downregulated (P < 0.05) are shown. The enrichment score reflects the degree to which a gene set is overrepresented at the top (upregulated, positive score) or bottom (downregulated, negative score) of the ranked gene list and is corrected for gene set size. Black bars, control condition; gray bars, oleate; white bars, palmitate treatment. Microarray data have been submitted to Gene Expression Omnibus (GSE38590).

FIG. 3.

Altered lipid metabolism and oxidative capacity in PLIN2 knockdown cells. A: Protein expression of the OXPHOS complexes I, II, III, and V in cell cultures transfected with either PLIN2 siRNA (P2) or scrambled control siRNA (S), expressed as arbitrary units (AU). n = 4 for each treatment condition. OA, oleate; PA, palmitate. Analysis of 3-h ¹⁴C-FA oxidation rates in PLIN2 knockdown and negative control cells: total oxidation (sum of ¹⁴C-CO₂ and ¹⁴C ASMs) (B), complete oxidation to CO₂ (C), and incomplete oxidation to ASMs (D). n = 6 per treatment condition. ¹⁴C-palmitate incorporation into total neutral lipids (E), TAG (F), phospholipids (G), and DAG (H). Values were normalized to protein levels. Incorporation into TAG, DAG, and phospholipids is expressed relative to the control condition. I: Insulin-stimulated deoxyglucose uptake. J: Insulin-stimulated pAkt phosphorylation. *P < 0.05. Error bars represent SEM.

FIG. 4.

PLIN2 overexpression protects against palmitate-induced insulin resistance in C2C12 myotubes. A: Western blot demonstrating efficiency of PLIN2 overexpression. B: TAG levels. Incorporation into neutral lipids (C), TAG (D), DAG (E), and phospholipids (PL) (F). Incorporation into TAG, DAG, and PL is expressed relative to the control condition. G: Insulin-stimulated deoxyglucose uptake. EV, empty vector; PA, palmitate (400 μmol/L); PL, phospholipids; C, control; DPM, disintegrations per minute. *P < 0.05. Error bars represent SEM.

FIG. 5.

Gene electrotransfer–mediated overexpression of PLIN2 in rat TA muscle. A: Representative Western blot demonstrating overexpression of PLIN2 in the left TA muscle. EV, empty vector. SR-actin, sarcomeric actin B: Lipid accumulation (red, Oil-Red-O) and PLIN2 expression (green) (overlap of PLIN2 and lipids appears yellow, but depends on the place of the cross-cut through the intracellular LDs) in TA muscle sections of the right (control, empty vector) and left (PLIN2 vector) leg of rats fed an HFD. Cell membranes are stained in blue. Representative pictures are shown. The image on the right bottom panel is a magnification of a selection of the picture above. C: Ultrastructure (transmission electron microscopy [TEM]) of muscle samples from sham- and PLIN2-electroporated muscle. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

Overexpression of PLIN2 in TA muscle of rats on an HFD improves insulin sensitivity of the muscle. Skeletal muscle DAG (A) and ceramide (B) levels in PLIN2- or empty vector (EV)–electroporated muscle of rats on an HFD. C: ³H-labeled deoxyglucose uptake in control and PLIN2-electroporated TA muscle of rats on an HFD (n = 11). Error bars represent SEM. *P < 0.05.