Nicotine in combination with a high-fat diet causes intramyocellular mitochondrial abnormalities in male mice

Abstract

Smoking is a major risk factor for diabetes, cardiovascular disease, and nonalcoholic fatty liver disease. The health risk associated with smoking can be exaggerated by obesity. We hypothesize that nicotine when combined with a high-fat diet (HFD) can also cause ectopic lipid accumulation in skeletal muscle, similar to recently observed hepatic steatosis. Adult C57BL6 male mice were fed a normal chow diet or HFD and received twice-daily ip injections of nicotine (0.75 mg/kg body weight) or saline for 10 weeks. Transmission electron microscopy of the gastrocnemius muscle revealed substantial intramyocellular lipid accumulation in close association with intramyofibrillar mitochondria along with intramyofibrillar mitochondrial swelling and vacuolization in nicotine-treated mice on an HFD compared with mice on an HFD treated with saline. These abnormalities were reversed by acipimox, an inhibitor of lipolysis. Mechanistically, the detrimental effect of nicotine plus HFD on skeletal muscle was associated with significantly increased oxidative stress, plasma free fatty acid, and muscle triglyceride levels coupled with inactivation of AMP-activated protein kinase and activation of its downstream target, acetyl-coenzyme A-carboxylase. We conclude that 1) greater oxidative stress together with inactivation of AMP-activated protein kinase mediates the effect of nicotine on skeletal muscle abnormalities in diet-induced obesity and 2) adipose tissue lipolysis is an important contributor of muscle steatosis and mitochondrial abnormalities.

Figure 1.

Panel A, Time course of body weight measured over 10 weeks of nicotine treatment in mice fed an NCD or HFD. By 10 weeks of combined treatment with nicotine and HFD, mean body weight was significantly (P < .05) reduced relative to mice fed an HFD and saline. Panel B, Average food intake in mice fed an NCD or HFD in the absence or presence of nicotine. Panel C, Plasma FFA levels in various treatment groups. Panels D and E, Addition of nicotine to HFD also caused a significant increase in muscle triglyceride levels (panel D) and muscle oxidative stress as indicated by a low GSH to GSSG ratio (panel E). Values are given as mean ± SE of 5–6 mice per group. Means with different letters are significantly (P < .05) different from each other. Means with the same letter are not different.

Figure 2.

A and B, Representative transmission electron micrographs of gastrocnemius muscle from mice fed an NCD without (A) or with (B) nicotine exhibit normal myofibrillar architecture and sarcomeric pattern with abundant normal-appearing IMF mitochondria and no IMCL accumulation. C, Appearance of muscle fiber in mice fed an HFD is similar to that seen in mice on an NCD in the absence (A) or presence (B) of nicotine. D, Representative electron micrograph shows IMCL accumulation in close association with IMF mitochondria (arrow). E, A higher-magnification view of the area marked in D showing IMCL accumulation (asterisk). F and G, Nicotine plus HFD also causes mitochondrial vacuolization (F; arrow) and mitochondrial swelling with broken cristae (G; asterisk). Scale bar, 1 μm (A–D, F, and G) and 0.4 μm (E). Data are representative of 4 mice in each group.

Figure 3.

A and B, Representative transmission electron micrographs of gastrocnemius muscles from mice fed an HFD (A) and mice receiving acipimox plus HFD (B) show normal ultrastructural myofibrillar architecture, with normal morphology of both SS and IMF mitochondria. C, As expected, nicotine plus HFD treatment shows IMF mitochondrial abnormalities, including IMCL accumulation. D, Acipimox treatment fully prevents nicotine plus HFD-induced IMCL accumulation and mitochondrial abnormalities. Scale bar, 1 μm. Data are representative of 4 mice in each group.

Figure 4.

A, Western blot analysis shows that mice fed an HFD have decreased p-AMPK and p-ACC levels compared with mice on an NCD with or without nicotine treatment. Addition of nicotine to HFD leads to complete dephosphorylation of AMPK and ACC. B, Treatment with acipimox significantly attenuates the HFD plus nicotine-induced decrease in p-AMPK and p-ACC levels.