Effect of puerarin in promoting fatty acid oxidation by increasing mitochondrial oxidative capacity and biogenesis in skeletal muscle in diabetic rats

Abstract

Background: Type 2 diabetes is characterized by dyslipidemia and the accumulation of lipids in non-adipose tissue, including skeletal muscle. Puerarin, which is a natural isoflavonoid isolated from the root of the plant Pueraria lobata, has been shown to have antidiabetic activity. However, the lipid-reducing effect of puerarin, in particular in skeletal muscle, has not yet been addressed.

Methods: We examined the effect of puerarin on mitochondrial function and the oxidation of fatty acids in the skeletal muscle of high-fat diet/streptozotocin-induced diabetic rats.

Results: Puerarin effectively alleviated dyslipidemia and decreased the accumulation of intramyocellular lipids by upregulating the expression of a range of genes involved in mitochondrial biogenesis, oxidative phosphorylation, the detoxification of reactive oxygen species, and the oxidation of fatty acids in the muscle of diabetic rats. Also, the effect of puerarin on mitochondrial biogenesis might partially involve the function of the μ-opioid receptor. In addition, puerarin decreased the trafficking of fatty acid translocase/CD36 to the plasma membrane to reduce the uptake of fatty acids by myocytes. In vitro studies confirmed that puerarin acted directly on muscle cells to promote the oxidation of fatty acids in insulin-resistant myotubes treated with palmitate.

Conclusions: Puerarin improved the performance of mitochondria in muscle and promoted the oxidation of fatty acids, which thus prevented the accumulation of intramyocellular lipids in diabetic rats. Our findings will be beneficial both for elucidating the mechanism of the antidiabetic activity of puerarin and for promoting the therapeutic potential of puerarin in the treatment of diabetes.

Fig. 1. Puerarin prevented the body weight gain and alleviated hyperlipidemia in diabetic rats

a A schematic representation of experimental process. b The body weight gain measurement. c Serum TG, FFA, and TC levels. d Serum SOD activity and MDA content. Data were presented as mean ± s.d., n = 8. ^*P < 0.05, ^**P < 0.01, vs. NC group; ^#P < 0.05, ^##P < 0.01 vs. DM group. NC normal control, DM diabetes mellitus, Pue puerarin

Fig. 2. Puerarin decreased IMCLs accumulation in diabetic rats

Representative blot and quantification of (a) total CD36 protein in muscle lysates, and (b) membrane CD36 in muscle sarcolemma. β subunit of insulin receptor (IR) was performed to confirm PM localization and normalization. c Western blot analyses and quantification of CPT-1b, p-AMPK (Thr172)/AMPK and p-ACC (Ser79)/ACC in muscle lysates. d Relative mRNA expressions of LCAD, ACOX1, PPAR-δ were analyzed by real-time RT-PCR and normalized to β-actin. e Representative TEM micrographs showed significant IMCLs accumulation (arrows) in diabetic rats and with much reduced IMCLs in puerarin-treated animals. Scale bar: 1 μm. Data were normalized to NC and presented as mean ± s.d., n = 5. ^**P < 0.01, vs. NC group; ^#P < 0.05, ^##P < 0.01 vs. DM group

Fig. 3. Puerarin restored mitochondrial function in the muscle of diabetic rats

a Representative western blots and quantification of mitochondrial proteins used as markers for OXPHOS complexes (I–IV). b Real-time RT-PCR analysis of mRNA levels of mitochondrial proteins used as markers for electron transport chain (MTCO1, COIV) and TAC (CS) and normalized to β-actin. c Representative western blots and quantification of p-p66Shc(Ser36)/Shc, UCP2, SIRT3 and SOD2. d The mRNA levels of UCP2, UCP3, and SIRT3 were analyzed by real-time RT-PCR and normalized to β-actin. e Representative TEM images of mitochondrial ultrastructure from muscle. Scale bar: 0.2 μm. Data were normalized to NC and presented as mean ± s.d., n = 5. ^*P < 0.05, ^**P < 0.01, vs. NC group; ^#P < 0.05, ^##P < 0.01 vs. DM group

Fig. 4. Puerarin increased mitochondrial biogenesis in muscle of diabetic rats

a, e Representative blots and quantification of SIRT1 and PGC-1α. b Relative mRNA levels of SIRT1, PGC-1α, and TFAM analyzed by real-time RT-PCR and normalized to β-actin. c The mtDNA quantity evaluated as the ratios of 16S rRNA to β-actin determined by real-time PCR. d, f Mitochondrial density assessed by TEM in muscle of rats. Scale bar: 2 μm. Data were normalized to NC and presented as mean ± s.d., n = 5. (a–c) *P < 0.05, **P < 0.01, vs. NC group; ^#P < 0.05, ^##P < 0.01 vs. DM group. (e) ^★ P < 0.01, vs. DM + Pue group. Nal naloxone

Fig. 5. Puerarin promoted the oxidation of fatty acids in palmitate-induced insulin-resistant myotubes

a Insulin-stimulated phosphorylation of Akt (Ser473) determined by western blot. Cells were incubated with 100 nM insulin during the last 20 min of treatment. b Western blot analyses and quantification of total and membrane CD36 in myotubes. IR-β was performed to confirm PM localization and normalization. c Relative mRNA expressions of ACSL1, LCAD, and ACOX1 analyzed by real-time RT-PCR and normalized to β-actin. d Western blot analyses and quantification of CPT-1b and p-ACC (Ser79)/ACC. e FFA content in cells normalized by respective protein content. f Representative electron micrographs showed the deposition of lipid droplets (arrowheads) in palmitate-treated myotubes but few lipids accumulation in cells pretreated with puerarin. Scale bar: 0.5 μm. All data were from three independent experiments and presented as mean ± s.d. *P < 0.05, **P < 0.01, vs. BSA group; ^#P < 0.05, ^##P < 0.01 vs. PA group. PA palmitate