Raising plasma fatty acid concentration induces increased biogenesis of mitochondria in skeletal muscle

Abstract

A number of studies have reported that a high-fat diet induces increases in mitochondrial fatty acid oxidation enzymes in muscle. In contrast, in two recent studies raising plasma free fatty acids (FFA) resulted in a decrease in mitochondria. In this work, we reevaluated the effects of raising FFA on muscle mitochondrial biogenesis and capacity for fat oxidation. Rats were fed a high-fat diet and given daily injections of heparin to raise FFA. This treatment induced an increase in mitochondrial biogenesis in muscle, as evidenced by increases in mitochondrial enzymes of the fatty acid oxidation pathway, citrate cycle, and respiratory chain, with an increase in the capacity to oxidize fat, as well as an increase in mitochondrial DNA copy number. Raising FFA also resulted in an increase in binding of peroxisome proliferator-activated receptor (PPAR) delta to the PPAR response element on the carnitine palmitoyltransferase 1 promoter. We interpret our results as evidence that raising FFA induces an increase in mitochondrial biogenesis in muscle by activating PPARdelta.

Fig. 1.

Raising plasma FFA induces increased expression of genes encoding FAO enzymes in skeletal muscle. (a) Representative Western blots and average protein values of three enzymes used as markers for the mitochondrial β-oxidation pathway in epitrochlearis muscles of rats fed either a chow diet (control) or a high-fat diet (n = 7 per group). The rats on the high-fat diet were given daily heparin injections to raise FFA levels (↑FFA). MCAD, medium-chain acyl-CoA dehydrogenase; LCAD, long-chain acyl-CoA dehydrogenase; VLCAD, very long-chain acyl-CoA dehydrogenase. (b) Real time PCR analysis of mCPT-1 mRNA levels in triceps muscles of control and ↑FFA groups (n = 6 per group). (c) Palmitate oxidation by triceps muscles homogenates of the control and ↑FFA groups of rats (n = 7 per group). ∗, P < 0.05, significantly different from control.

Fig. 2.

Increased expression of a range of mitochondrial enzymes in response to raising plasma FFA levels. Representative Western blots and average protein values for mitochondrial enzymes used as markers for the citrate cycle [citrate synthase (CS)] and respiratory chain [NADH-ubiquinone oxidoreductase (NUO), succinate-ubiquinone oxidoreductase 70-kDa subunit (SUO), cytochrome c (Cyt c), COXI, COXIV], and ATP synthase subunit α (ATP-syn) in epitrochlearis muscles are shown. n = 7 per group. ∗, P < 0.05, significantly different from controls.

Fig. 3.

Effect of raising FFA on expression of PGC-1α, PPARα, and PPARδ. Real-time PCR analysis of mRNA levels in triceps muscles is shown. Values are means ± SEM for six muscles per group.

Fig. 4.

Mitochondrial DNA copy number was evaluated in triceps muscles of rats with raised FFA and control animals by determining the ratios of COX1 DNA to 18S DNA, and of cytochrome b (Cyt b) to 18S DNA. The COX1 and cytochrome b genes are in the mitochondrial genome, and the 18S gene is in the nuclear genome. Values are means ± SE for four rats with ↑FFA and five controls. ∗, P < 0.05.

Fig. 5.

PPARδ binding to the mCPT promoter is increased in epitrochlearis muscles of rats in response to raising plasma FFA. Values are means ± SE for six samples per group (two epitrochlearis muscles per sample). ∗, P < 0.002, significantly different from control.