Effects Of DHA/EPA On Skeletal Muscle Glucose Metabolism And Bone Formation In Diabetes

N-3 polyunsaturated fatty acid(PUFA)has attracted wide attention due to its biological activities.It has been pointed out that n-3 PUFA plays an important role in resisting obesity,improving dyslipidemia,alleviating inflammation,and delaying aging,which makes contributions to functional food development.According to the World Health Organization(WHO),there are more than 140 million diabetes patients in China,ranking first in the world.In addition,over 90% of diabetes are type 2diabetes(T2D),indicating that the prevention and therapy of T2 D is imperative.In previous studies,although dietary intervention with n-3 PUFA alleviated hyperglycemic phenotypes in obese or diabetic models,different results have been reported in other studies,suggesting that the ameliorative effects of n-3 PUFA on type2 diabetes are still controversial and the involved underlying mechanism remain unclear.In addition,diabetic osteoporosis,as the classic complications of diabetes,has become a major public health problem due to the increasing number of diabetes and population aging,which has increased the burden of social medical and economic systems.Although high n-3 PUFA level in blood is associated with higher bone mineral density,the effects of n-3 PUFA on bone under diabetic situation is rarely reported,and the related mechanisms is necessary to be explored.In order to fully elucidate the effect of n-3 PUFA on type 2 diabetes,db/+mice and fat1 mice with the same background were hybridized to obtain db/db/fat1 mice,which can endogenously synthesize n-3 PUFA and have diabetic phenotype in the same time.The db/db/fat1 model can eliminate the interference of interaction between feed components,lipid oxidation,and sensory differences of diets on interventional experiments.In addition,the skeletal muscle,which is responsible for 80% of glucose metabolism in whole body,has been selected as the target organ in this study to explore the potential mechanism of n-3 PUFA on hypoglycemic effects through non-targeted metabolomics.Subsequently,the CRISPR/Cas9 was used to construct a gene knock-out cell model to verify the related signaling pathway.At the same time,we focused on myokines and explored the protective effects of n-3 PUFA on bone through the “muscle-bone” axis and its underlying mechanism.The main results of this study are as follows:(1)In db/db/fat1 mice,after 13-week feeding with 10% safflower oil diet,endogenous n-3 PUFA significantly improved the impaired glucose metabolism in diabetic mice.In addition,an increase of 1.9 times in AKT phosphorylation was found in db/db/fat1 mice.Moreover,the endogenous n-3 PUFA increased 1.3 times of GLUT4 protein expression.Meanwhile,it also significantly upregulated Rab8 a and t-SNAREs expression.The Rab8 a is responsible for the traffic of GLUT4-vesicles,and the t-SNAREs are involved in the fusion of GLUT4-vesicles with cytoplasmic membrane.These results suggested that endogenous n-3 PUFA improved glucose homeostasis in diabetic mice by promoting the expression and translocation of GLUT4 in skeletal muscle.(2)In order to elucidate the direct regulatory effects of n-3 PUFA on skeletal muscle under insulin-resistance situation,C2C12 cells were induced into insulin resistance through palmitic acid,and DHA and EPA interventions were performed subsequently.The results showed that both DHA and EPA significantly increased the glucose consumption of myotubes in insulin-resistance situation,and the effect of EPA was better than that of DHA.In addition,EPA significantly increased 1.4 times of AKT phosphorylation and elevated 1.7 times of GLUT4 expression.Both DHA and EPA promoted the m RNA expressions of genes involved in GLUT4-vesicle traffic,including Rab5,Rab8 a and Rab13.Moreover,EPA significantly increased the protein and m RNA expressions of Syntaxin4 and SNAP23,while DHA significantly increased the m RNA level of Syntaxin4.Finally,immunofluorescence visually demonstrated that DHA and EPA promoted GLUT4 translocation into the cytoplasmic membrane in myoblasts under insulin stimulation.(3)Non-targeted metabolomics of cell content and cell supernatant were carried out to explore the underlying mechanism.In cell content,the glucose contents in DHA and EPA groups were increased,while glycolysis products such as pyruvate and lactic acid were also increased in EPA.In addition,TCA cycle intermediates including citric acid and ketoglutaric acid were elevated in DHA and EPA groups.In cell supernatant,glutamic acid,pyroglutamic acid and glutamine were up-regulated in the DHA group,which were negatively correlated with insulin resistance.KEGG pathway analysis showed that in intracellular and extracellular,the sphingolipid metabolism was enriched in both DHA and EPA groups,indicating S1 P levels may be increased by DHA and EPA.Furthermore,creatine,as a differential metabolite,was significantly increased in DHA and EPA groups,which may promote the expression of irisin by activating PGC1α accompanied with S1 P.Further experiments confirmed that DHA and EPA significantly up-regulated 1.3 times of FNDC5 expression(irisin encoding gene).Subsequently,the FNDC5 knock-out experiments showed that irisin played an important mediating role in promoting GLUT4 translocation and improving muscle glucose metabolism induced by DHA and EPA.(4)In order to investigate the effects of endogenous n-3 PUFA on bone and its relationship with myokines in diabetic mice,the bone density related indexes and myokines expressions in db/db/fat1 mice were detected.The results showed that endogenous n-3 PUFA significantly increased 1.3 times of FNDC5 expression,alone with significantly increased bone mineral density of femur in diabetic mice.In addition,db/db/fat1 mice had higher bone volume fraction,trabecular number,connectivity density and lower bone trabecular separation in femur compared with db/db mice,suggesting that endogenous n-3 PUFA improved bone microstructure and bone mineral density in diabetic mice.Moreover,the results of serum calcium and phosphorus showed that n-3 PUFA rescued abnormal bone metabolism in diabetic mice.Furthermore,pathological sections showed no significant difference between db/db mice and db/db/fat1 mice in the TRAP positive region in distal femur,indicating that endogenous n-3 PUFA did not affect the bone resorption in diabetic mice.Notably,OCN staining and Western Blotting of OCN showed that OCN expression was significantly increased in db/db/fat1 mice.Combined with the elevated serum ALP level,it indicated that n-3 PUFA improved bone mineral density and bone microstructure in diabetic mice by increasing bone formation.(5)The effects and potential mechanisms of n-3 PUFA on bone formation were further explored.The direct interventions of DHA and EPA on osteoblasts showed that low doses of DHA and EPA had no significant effect on osteogenic differentiation,while high doses of DHA and EPA inhibited osteogenic differentiation,suggesting that n-3 PUFA did not promote bone formation through directly effecting on osteoblasts.Moreover,“muscle-bone” indirect co-culture experiment has shown that DHA and EPA promoted osteogenic differentiation based on the “muscle-bone” axis by regulating the expression of myokines in myoblasts,especially irisin.When FNDC5-KO C2C12 cells were used as culture medium donors in “muscle-bone”co-culture system,the effects of DHA and EPA on promoting osteogenesis through the “muscle-bone” axis disappeared,which further confirming that irisin,as a myokine,plays an important mediating role in the “muscle-bone” axis regulated by DHA and EPA.In conclusion,this study showed that endogenous n-3 PUFA improved glucose homeostasis by regulating GLUT4 translocation in skeletal muscle.In cell experiments,the metabolomics was carried out,which revealed that n-3 PUFA activated the insulin signaling pathway in myotubes under insulin resistance and further accelerated GLUT4 translocation by regulating the expression of myokines.Furthermore,the key mediating role of irisin was confirmed by FNDC5-KO C2C12 cells.On the other hand,endogenous animal experiments also showed that n-3 PUFA significantly improved bone health in diabetes.Indirect co-culture models suggested that n-3 PUFA improved bone formation via “muscle-bone” axis relying on irisin.Take together,the current study demonstrated new evidences for the improvement effects of n-3 PUFA on glucose homeostasis from an endogenous perspective,and provided references for application of dietary fatty acids in the prevention and treatment of diabetes and related bone diseases.Moreover,focusing on myokines,such as irisin,our results exhibited new sights for further explorations on physiological functions of n-3 PUFA.