Obesity is often associated with decreases in the proportion of skeletal muscle slow-twitch fibers and insulin sensitivity. Slow-twitch fibers are rich in mitochondria and utilize fatty acid oxidative phosphorylation for energy production. In their new study, Zhou et al. (2017) explore the role of the FLCN/FNIP1/AMPK signalling pathway in obesity-induced reductions in slow-twitch fibers and insulin sensitivity in skeletal muscle using high-fat-diet-induced (HFD) obese mice, ob/ob mutant mice, and palmitate-treated C2C12 myotubes. The authors also assess the effects of dihydromyricetin (DHM) on the obesity-induced decrease in slow-twitch fibers, and the molecular mechanisms responsible for this effect.
AMP-activated protein kinase (AMPK) plays a central role in skeletal muscle oxidative metabolism and fiber-type specification. AMPK activation in skeletal muscle induces expression of its downstream transcriptional regulator PGC-1α. FLCN, responsible for Birt-Hogg Dubé syndrome, interacts with the AMPK signalling pathway by binding to folliculin-interacting protein 1 (FNIP1) (Baba et al., 2006). Folliculin (FLCN) and FNIP1 may regulate skeletal muscle-fiber-type specification through the AMPK/PGC-1α pathway (Hasumi et al., 2012). Although the interaction between FLCN/FNIP1 and AMPK appears to play an important role in skeletal muscle adaptations, its involvement in the obesity-induced decrease in slow-twitch fibers and insulin resistance remains unclear.
Exercise is commonly prescribed for obesity and metabolic diseases, including insulin resistance and diabetes, since it increases AMPK activity, promoting slow-twitch fibers and increasing the use of fatty acids in skeletal muscle (Lantier et al., 2014). The authors have previously reported that the flavonoid DHM enhanced exercise performance (Zou et al., 2014), and improved skeletal muscle insulin resistance by autophagy induction via AMPK (Shi et al., 2015). However, the ability of DHM to increase the proportion of skeletal muscle slow-twitch fibers via the AMPK signalling pathway remains unclear.
In the HFD-fed and ob/ob mice the proportions of slow-twitch fibers, insulin sensitivity (detected by the markers of insulin sensitivity, insulin-stimulated Akt and insulin receptor substrate 1 (IRS-1) phosphorylation) and oxidative metabolism in skeletal muscle were decreased compared with control mice, and this effect was prevented by DHM treatment.
Increased non-esterified fatty acids (NEFA) levels are closely associated with insulin resistance in obesity and type 2 diabetes. In line with these results, the authors found that plasma NEFA levels were significantly increased in HFD-fed and ob/ob mice compared with controls and they negatively correlated with slow-twitch-fiber proportion.
To verify the results obtained in mice, in vitro experiments with C2C12 myotubes were performed. Palmitate, one of the most elevated plasma NEFA in obesity, was used to induce insulin resistance in C2C12 myotubes and shown to decrease expression of slow-fiber specification Myh7 protein, this was inhibited by DHM.
Western blot analysis show decreased phosphorylation of AMPK in both HFD-fed and ob/ob mice, and in palmitate-treated C2C12 myotubes. The similar trends in AMPK activity and changes in slow-twitch fibers and insulin resistance suggest that AMPK might be involved in these obesity-induced changes. FNIP1 and FLCN expression levels were significantly increased in skeletal muscle in HFD-fed and ob/ob mice, and in palmitate-treated C2C12 myotubes and negatively correlated with AMPK activity. These results implicated FNIP1/FLCN in obesity-induced AMPK inactivation, and the subsequent decreases in slow-twitch fibers and insulin sensitivity in skeletal muscle.
The role of the FLCN/FNIP1/AMPK signalling pathway in obesity-induced insulin resistance and the decrease in slow-twitch fibers was further clarified using over-expression and knock-down of FNIP1 and FLCN. Transfection of C2C12 myotubes with FNIP1 resulted in a corresponding increase in FLCN levels. AMPK phosphorylation levels increased following FLCN or FNIP1 knock-down, and decreased following their over-expressions. mRNA levels of the PGC-1α encoding gene were assessed and results showed that its expression was negatively related to FNIP1/FLCN expression, and consistent with AMPK activity.
DHM ameliorated the obesity-induced decrease in slow-twitch-fiber proportion, insulin sensitivity, and AMPK activity. However, it was necessary to verify if these effects of DHM were mediated by FNIP1 and FLCN. FNIP1 and FLCN expression levels were significantly decreased following DHM administration in HFD-fed and ob/ob mice, and in palmitate-induced C2C12 models. Also, the preventive effects of DHM on the palmitate-induced decrease in slow-twitch fibers, AMPK activation, p-Akt and p-IRS-1 expression, were blocked by FLCN over-expression. These results demonstrated that the effects of DHM were mediated by the FNIP1/FLCN/AMPK signalling pathway.
Yan et al. recently reported FLCN/AMPK as a novel molecular pathway involved in regulating mitochondrial function and browning of white adipocytes, this was discussed on a previous blog. Here, the results of the study demonstrate that the FNIP1/FLCN complex might play an important role in AMPK/PGC-1α signalling in the obesity-induced decreases in slow-twitch-fibers and insulin sensitivity. Furthermore, DHM acts as a potential exercise mimetic by attenuating these obesity-induced effects via the FNIP1/FLCN/AMPK signalling pathway. These results provide new insights into the FNIP1/FLCN/AMPK signalling pathway, key in BHD research, and novel mechanisms and potential targets for treatments of insulin resistance and type 2 diabetes.
- Zhou Q, Gu Y, Lang H, Wang X, Chen K, Gong X, Zhou M, Ran L, Zhu J, & Mi M (2017). Dihydromyricetin prevents obesity-induced slow-twitch-fiber reduction partially via FLCN/FNIP1/AMPK pathway. Biochimica et biophysica acta PMID: 28363698