Vitamin K2 Alleviates Insulin Resistance In Skeletal Muscle By Improving Mitochondrial Function In SIRT1 Dependent Manner

BackgroundWhile the etiology of type 2 diabetes mellitus（T2DM）is poorly defined,chronic consumption of a high-fat diet（HFD）is a major contributor to whole-body glucose intolerance and insulin resistance（IR）by increasing mitochondrial dysfunction.Recent studies demonstrated the therapeutic potential of micronutrients for the prevention and/or management of many chronic diseases including diabetes.Vitamin K（VK）is an essential micronutrient known for its beneficial role in blood coagulation.Recently,however,coagulation-unrelated functions of VK,such as improving insulin sensitivity,modulating glucose metabolism,and reducing the risk of T2DM,have attracted scientific attention.A study found that VK2 supplementation increased insulin sensitivity in healthy men and IR patients.However,the signaling mechanisms of VK2 in the beneficial effect on insulin sensitivity and IR remain unclear.Evidence from prospective observational studies and clinical trials has shown T2DM risk reduction with VK2 supplementation and demonstrated that VK2 has a better effect than VK1 on T2DM.Hence,VK2 has a more significant effect on IR than VK1,and exploring the effects of VK2 has profound significance.However,there has so far been no mechanistic study describing in detail the molecular mechanism underlying the beneficial role of VK2 in IR.Research has found that VK1 has a positive effect on lowering fasting glucose and enhancing insulin sensitivity in T2DM by regulating the SIRT1 signaling pathway.We hypothesized that VK1 and VK2 have the same naphthoquinone ring and a similar aliphatic chain structure;thus,as a natural and non-toxic micronutrient,VK2’s amelioration of IR may also be attributed to activation of the SIRT1 signaling pathway.Sirtuin 1（SIRT1）is a conserved protein NAD⁺-dependent deacylase,and its function is intrinsically linked to cellular metabolism.Over the past two decades,accumulating evidence has indicated that SIRT1not only is an important energy status sensor but also protects cells against metabolic stresses.Furthermore,SIRT1 plays a crucial role in reducing oxidative stress and improving mitochondrial function,resulting in amelioration of IR in insulin-sensitive tissues like skeletal muscle and adipose tissue.ObjectiveOur study aimed to identify whether essential micronutrient VK2 protects from IR and to explore the potential molecular mechanisms involved.We hypothesized that VK2’s alleviation of IR and improvement of insulin sensitivity may be due to activation of the SIRT1 signaling pathway.To investigate this hypothesis,we have tested VK2’s regulatory mitochondrial function for protection against IR by SIRT1 in mice and cell models.Our results demonstrated that VK2 administration and treatment alleviates IR and improves mitochondrial function in a SIRT1-dependent manner in HFD-induced mice and palmitate acid（PA）-exposed C₂C₁₂ cells.Method1.C57BL/6 male mice（4 weeks old）were fed with high fat diet（HFD）for 16 weeks to establish a mouse IR model.VK2 was administrated at different dose of BW daily by oral gavage for last eight weeks.The effect of VK2 on body weight and fat mass were observed,and the degree of intraperitoneal glucose tolerance test,insulin tolerance test,fatty acid,serum biomarker levels,and pathological changes among mice fed with different diet were compared to evaluate whether VK2 could protect HFD-fed mice against IR.The effects of VK2 on skeletal muscle mitochondrial function were detected by transmission electron microscopy,ATP production,mitochondrial complexⅠandⅣenzyme activities,mitochondrial membrane potential（MMP）and expression level of mitochondrial respiratory chain complexes.Furthermore,VK2-mediated beneficial effect on enhancing the mitochondrial antioxidant capacity were evaluated by measuring SOD2enzyme activity,ac-SOD2 and SIRT3 protein expressions.Otherwise,VK2’s effects on plasma free fatty acid（FFA）concentration by GC-MS analysis.2.PA-exposed C₂C₁₂ cells were used to mimic IR in vivo.By detecting cell viability and glucose uptake in PA-exposed C₂C₁₂ cells,the optimal PA concentration and VK2intervention concentration were determined.The effect of VK2 on decreasing ROS were measured by DCFH-DA and Mitosox probe in PA-exposed C₂C₁₂ cells.Mitochondrial biogenesis and antioxidant capacity were detected by Western blot and RT-PCR.VK2’s effects on mitochondrial function were also evaluated by measuring the oxygen consumption rate and the enzymatic activity as well as expression of mitochondrial respiratory chain complex in PA-C₂C₁₂ cells.The molecular mechanism of VK2-mediated the beneficial effect on improving mitochondrial function were also evaluated.Thus,SIRT1 signaling pathway were detected by Western blot and RT-PCR in HFD-induced mice and PA-exposed cells.In order to investigate the role of SIRT1 in VK2-mediated protection on mitochondrial function,C₂C₁₂ cells were transfected or treated with SIRT1si RNA or SRT1720.The effect of VK2 on SIRT1 was further evaluated by using an AMPK si RNA and AMPK specific inhibitor Compound C in PA-C₂C₁₂ cells.3.CCK-8 used to detect the cell viability of geranylgeraniol（GGOH,side chain structure of VK2）or menadione（Mena,naphthoquinone ring of VK2）pretreatment of C₂C₁₂ cells.The effect of GGOH and Mena on mitochondrial function were investigated by mitochondrial oxygen consumption rate（OCR）and corresponding kits,and JC-1detects MMP in PA-exposed C₂C₁₂ cells.Western blot was detected by the effects of GGOH,Mena and VK2 on SIRT1 signaling protein expression.Results1.VK2 supplementation inhibits insulin resistance by increasing insulin sensitivity,improving mitochondrial function and oxidative fibers in HFD-fed miceOur results showed that the HFD group and HFD+OO group gained more body weight and fat mass than the respective control diet groups;however,these effects were attenuated by VK2 supplementation in HFD mice.We also found that VK2supplementation significantly inhibited the IR induced by the HFD via decreasing the fasting insulin levels,improving glucose tolerance and area under the curve,and improving insulin sensitivity in the K_itt,insulin tolerance test,and AUC,as well as enhancing glucose homeostasis model assessment of insulin resistance（HOMA-IR）compared to the non-VK2-supplemented mice.We identified soleus muscle composition in HFD-fed mice after 8 weeks of treatment with VK2 by nicotinamide adenine dinucleotide tetrazolium reductase（NADH-TR）and hematoxylin-eosin（HE）staining.HE staining revealed that there were non-obvious pathological changes in the muscle tissue of HFD-fed mice.The results showed that the number of oxidative fibers was decreased and the number of glycolytic fibers was increased as well as the ratio of oxidative and glycolytic fibers was decreased in the control group compared with the HFD and HFD+OO groups.Based on these results,VK2 improved obesity-induced IR through increasing insulin sensitivity and oxidative fibers.HFD feeding elicited aggravated mitochondrial dysfunction in skeletal muscle,as seen by the decreased ATP content,activity of the manganese superoxide dismutase enzyme,and increased levels of malonaldehyde（MDA）,H₂O₂ and mitochondrial reactive oxygen species（mt-ROS）in HFD-fed mice.In HFD-fed mice,mitochondrial complex I and IV activities,mt-DNA contents and expression levels of genes encoding proteins involved in mitochondria biogenesis were also reduced;however,these were attenuated by VK2 administration.Ultrastructural analysis by transmission electron microscopy revealed hyperproliferation of swollen mitochondria with disordered cristae in the HFD and HFD+OO groups compared with control group.In addition,there were significant differences in the total numbers and average areas of the mitochondria among all groups.However,after supplementation of VK2 in HFD mice,the shape and distribution of the mitochondria were apparently improved.These results clearly indicated that VK2improves mitochondrial function in HFD-fed mice.2.VK2 enhances mitochondrial function in a SIRT1-dependent manner in PA-exposed C₂C₁₂ cellsOur findings revealed that VK2 treatment also inhibited the PA-induced increase in ROS and mt-ROS in C₂C₁₂ cells.In PA-exposed C₂C₁₂ cells,mitochondrial complex I and IV activities were significantly decreased,which was notably attenuated by VK2.Analysis of mitochondrial respiration capacity in C₂C₁₂ cells showed that PA treatment alone significantly reduced the mitochondrial respiration oxygen consumption rate（OCR）,ATP production,and spare respiratory capacity in mitochondria,which were all markedly rescued by VK2 treatment.Western blot revealed that VK2 supplementation or treatment caused an increase in the protein expression of SIRT1,phospho-adenosine monophosphate-activated protein kinase（p-AMPK）,peroxisome proliferator-activated receptor-c coactivator-1 alpha（PGC-1a）,and peroxisome proliferators activated receptor a（PPARa）in both HFD-fed mice and PA-exposed C₂C₁₂ cells.The downstream targets of PGC-1a,nuclear respiratory factor 1（Nrf1）,and mitochondrial transcription factor A（TFAM）,which regulate mitochondrial content,were likewise significantly upregulated by VK2 administration and treatment groups.In our study,Western blot analysis revealed that HFD feeding and PA exposure increased the acetylation of SOD2 and decreased SIRT3 protein expression which was suppressed by VK2 administration and treatment via SIRT1.These results indicate that the SIRT1 signaling pathway may play an important role in VK2’s improvement of mitochondrial function.Western blot showed that the inhibitive effects of VK2 against PA-exposed reductions in AMPK phosphorylation and decreases in PGC-1a,PPARa,Nrf1,and TFAM expression were abrogated in C₂C₁₂ cells transfected with SIRT1 si RNA.The effect of VK2 on SIRT1 was further evaluated by using an AMPK inhibitor and AMPK si RNA transfection in PA-exposed C₂C₁₂ cells.VK2-triggered SIRT1 activation was not inhibited in the presence of Compound C（a potent AMPK inhibitor）or AMPK si RNA.Concomitantly,pretreatment with SIRT1specific agonist SRT1720 notably improved MMP and complex activities in PA-exposed C₂C₁₂ cells.Western blot revealed that VK2 and SIRT1 specific agonist SRT1720 had similar effect on improving the SIRT1 signaling pathway.However,the beneficial effect of VK2 on increasing SIRT1 protein expression was abrogated in SRT1720-pretreated cells.The results showed that VK2 improves mitochondrial function in a SIRT1-dependent manner in PA-exposed C₂C₁₂ cells.3.Side chain structure of VK2 has beneficial effect on mitochondrial function via SIRT1The results showed that VK2 and GGOH improved MMP,mitochondrial complex I and IV activities compared to Mena treatment in PA-exposed C₂C₁₂ cells.Meanwhile,mitochondrial respiratory capacity was also detected by OCR,ATP production,and spare respiratory capacity.The also results revealed that VK2 and GGOH pretreatment both significantly improved mitochondrial respiratory capacity in PA-exposed C₂C₁₂ cells.Western blot revealed that VK2 and GGOH pretreatment also inhibited PA-induced decreases in SIRT1,PGC-1a,PPARa,Nrf1,TFAM and SIRT3 expression.However,Mena did not increase the relative expression of SIRT1 compared to the VK2 and GGOH treatment group.These results indicated that VK2 and GGOH both improved mitochondrial function via SIRT1.ConclusionOur research for the first time proposed a potential mechanism underlying the amelioration of IR by VK2 mediated through SIRT1.Given the intense clinical interest in developing novel preventive and therapeutic strategies that can mitigate both insulin resistance and the consequences of mitochondrial dysfunction,our findings that VK2 has both of these functions indicate that it could have significant potential for development as a novel pharmacological agent to improve IR.