Dihydromyricetin Prevents Obesity-induced Skeletal Muscle Fiber Type Transformation Via FLCN-FNIP1/AMPK Pathway

Diabetes has become a major global public health problems,the incidence of diabetes is particularly severe in China,which was reported that the prevalence of diabetes was 11.6%,and the pre-diabetes was as high as 50.1% in adults.Skeletal muscle is the predominant site of insulin-mediated glucose disposal and fatty acids oxidation,and plays a key role in preventing and curing obesity,insulin resistance,and type 2 diabetes.Obesity is the most important risk factor for skeletal muscle insulin resistance.Further,elevated non-esterified fatty acid(NEFA)play an important role in obesity-induced insulin resistance in skeletal muscle,but the exact mechanism how NEFA induced skeletal muscle insulin resistance remains unclear.Skeletal muscle possesses a remarkable plasticity,composed of heterogeneous specialized myofibers with distinct contractile and metabolic properties,which can be broadly divided into slow-twitch(type I)and fast-twitch fibers(type II).Moreover,the fiber type responds to environmental and physiological challenges by changing its phenotype in terms of size,composition,and metabolic properties.Recent studies demonstrated that insulin sensitivity is positive correlated to slow-twitch fiber proportions.In addition,skeletal muscle fiber switch to fast-twitch fiber type,thereby resulting in slow-twitch fiber proportion decrease in obesity.Thus,we speculated that elevated non-esterified fatty acid induced fiber type switch to fast-twitch fibers,thereby decreasing slow-twitch fiber proportions and ultimately resulting in insulin resistance.AMPK-PGC-1? signaling pathway plays a key role in skeletal muscle fiber type transformation.AMP-activated protein kinase(AMPK)is a sensor of cellular metabolic and energy homeostasis in eukaryotic cells.Change in the energy charge of the skeletal muscle leads to the activation of AMPK,which induces expression of its downstream transcriptional regulator,peroxisome proliferator-activated receptor-? coactivator-1?(PGC-1?),thereby directly activating the genetic programs of slow-twitch-fiber specification,oxidative metabolism,and mitochondrial biogenesis.In contrast,reduced AMPK activity is accompanied by a decline in the fraction of slow-twitch fibers in various insulin-resistance states,such as obesity,metabolic syndrome,and type 2 diabetes.Folliculin(FLCN)and folliculin-interacting protein 1(FNIP1)may regulate skeletal muscle-fiber-type specification through the AMPK/PGC-1? pathway.FLCN,the tumor suppressor protein has been reported to interact with the AMPK signaling pathway by binding to FNIP1 and act as a negative regulator of AMPK.Loss of FLCN or FNIP1 resulted in AMPK activation in skeletal muscle and a shift from fast-twitch to slow-twitch fibers,associated with increased PGC-1? expression and mitochondrial functions,which were blocked by PGC-1? knock-out.However,although the negative 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 remain unclear.Dihydromyricetin(DHM),which is extracted from Rattan tea,is a flavonoid with a long history as a medicinal and edible plant for the treatment of liver diseases and alcohol poisoning in China.We previously demonstrated that DHM improved non-alcoholic fatty liver disease,enhanced endurance exercise capacity,and stimulated myokin(irisin)secretion.Importantly,we verified that DHM could improve high-fat-induced skeletal muscle insulin resistance by autophagy induction via the AMPK signaling pathway.Together,we speculated that dihydromyricetin prevents obesity-induced slow-twitch fiber reduction,thereby ameliorating insulin resistance via FLCN/FNIP1/AMPK pathway.In the current study,high-fat-diet-induced(HFD)obese mice,ob/ob mutant mice,and palmitate-treated mouse C2C12 myotubes were used.Further,we used western blot,PCR,Seahorse XF96 Metabolic Flux Analysis,MultiPhoto Laser Scanning Microscopy,metachromatic ATPase staining,immunofluorescent staining,plasmids transfection and other methods to study the relations among non-esterified fatty acid,skeletal muscle compositions,energy metabolism and insulin sensitivity.Besides,we also tested the preventive effects of DHM on obesity-induced the changes of skeletal muscle fiber type,energy metabolism and insulin sensitivity and further demonstrated the role of FLCN/FNIP1/AMPK pathway in DHM preventing obesity-induced slow-twitch fiber reduction and insulin resistance.The main results and conclusions:1.In HFD-fed and ob/ob mice,while slow-twitch fiber proportion and insulin sensitivity were reductions,which were inhibited by DHM treatment.Meanwhile,slow-twitch fiber is negative correlated to insulin resistance.The results indicated that obesity-induced insulin resistance is mediated by decreaed slow-twitch fiber proportion.2.In HFD-fed and ob/ob mice,serum NEFA levels were significantly increase.Moreover,elevated NEFA levels were negatively correlated with slow-twitch fiber proportion and insulin sensitivity.Based on these results,we speculated that elevation of plasma NEFA-induced insulin resistance might be mediated by slow-twitch fibers decrease in obesity.Interestingly,the ameliorative effect of DHM on NEFA was not significant.However,DHM could prevent improve obesity-induced slow-twitch fibers decrease and insulin resistance.Thus,we speculated that DHM directly inhibited obesity-induced slow-twitch decrease,thereby improving insulin resistance independent of NEFA levels.3.Palmitate,one of the most elevated plasma NEFA in obesity,was used to induce insulin resistance in multiple cell lines.Palmitate induced C2C12 myotubes insulin resistance and protein expression of MyH7 decrease,the predominant MyHC isoform in slow-twitch fibers,which were prevented by DHM treatment.4.In vivo and in vitro,the expressions of FLCN,FNIP1 increased,concomitant with reductions in the phosphorylation status of AMPK and gene levels of PGC-1? in obesity and palmitate treatment,while DHM could inhibit the effects of obesity and palmitate.FLCN siRNA reversed the decrease in Myh7 protein expression,AMPK activity,p-Akt and p-IRS-1 expression,and insulin-stimulated glucose uptake induced by palmitate.Furthermore,the preventive effects of DHM on the palmitate-induced decrease in slow-twitch fibers,AMPK activation,p-Akt and p-IRS-1 expression,and glucose uptake were blocked by FLCN over-expression.The results indicated that FLCN/FNIP1/AMPK pathway play an important role in DHM preventing obesity-induced fiber type transformation and insulin resistance.Overall,our results uncovered a potential mechanism that NEFA-induced insulin resistance mediated by inhibited slow-twitch fiber decrease via FLCN-FNIP1-AMPK pathway in obesity.Moreover,DHM attenuates obesity-induced slow-twitch fibers decrease and insulin resistance through FNIP1-FLCN/AMPK signaling pathway.