Study On The Mechanism Of Alternate-day Fasting Improving Vascular Endothelial Function In Type 2 Diabetic Mic

Objective1.To discuss how the TCM dietary theory links to the regulatory role of dietary intervention in weight management,glucose metabolism and insulin sensitivity in type 2 diabetic and control mice.2.To determine the mechanisms of how alternate day fasting(ADF)improves vascular endothelial function in type 2 diabetic mice.3.To test the hypothesis that ADF protects against diabetes-associated endothelial dysfunction,at least partly through modulating adipokine profiles.Methods1.Animal models:Type 2 diabetic mice(Leprdb)and the m Leprdb control mice were purchased from the Jackson laboratory.Leprdb mice exhibit morbid obesity,chronic hyperglycemia,polyphagia,polydipsia,and polyuria.Heterozygous control mice(m Leprdb)had normal body weight,blood glucose and insulin.Control mice(m Leprdb)and diabetic mice(Leprdb)were treated with 12-weeks of ADF.Glucose metabolism,endothelial function,and adipokine profile were assessed.2.Metabolic parameters:Blood glucose was measured with glucose meter.ELISA kits were used to measure the serum levels of fasting insulin and for the calculation of HOMA-IR.Insulin tolerance test was performed.Blood cholesterol was determined using kit.3.Functional assessment of small mesenteric arteries(SMA):SMAs were mounted on a Myograph 610M to determine acetylcholine(ACh)-and sodium nitroprusside(SNP)-induced vasodilation,as well as phenylephrine(PE)-induced vasoconstriction.4.Circulating levels and mesenteric adipose tissue(MAT)expression of adipokines:Circulating adiponectin,resistin,and leptin were measured by ELISA.mRNA levels of Adipoq,Retn,and Lep were determined by qRT-PCR.5.Supplementation of adiponectin by adenovirus and supplementation of recombinant resistin:Adenovirus expression adiponectin(Ad-APN)or β-galactosidase(Ad-βgal)were produced and purified for i.v.injection.Recombinant resistin supplementation was also achieved via i.p.injection.6.Western Blotting for protein expression:Western blot experiments were performed using 10 mg of MAT or 4-6 SMA branches using anti-nitrotyrosine primary antibody,anti-tubulin primary antibody,and horseradish peroxidase-conjugated secondary antibody.7.Data Analysis:All data are expressed as mean ± SEM.For ITT and vasomotor responses at different doses,two-way repeated ANOVA was used to determine how responses were affected by two factors(ADF × time as repeated measures for ITT,ADF × dose as vasomotor response repeated measures),followed by a post-hoc Tukey’s multiple comparison test.For additional data obtained from four groups(m Leprdb,m Leprdb+ADF,Leprdb,Leprdb+ADF),one-way ANOVA was used to compare groups defined by one factor,followed by post hoc Fisher’s least significant difference test for multiple comparisons.P<0.05 was considered as statistically significant.Results1.The effect of ADF on body weight,abdominal circumference and blood lipid:the weight and abdominal circumference of Leprdb diabetic mice were higher than those of m Leprdb control mice.In m Leprdb control mice,12 weeks of ADF reduced body weight and abdominal circumference.In Leprdb mice,ADF did not significantly reduce body weight and abdominal circumference.Leprdb diabetic mice had higher fasting total cholesterol than m Leprdb control mice.12 weeks of ADF had no significant effect on lipids.2.The effect of ADF on blood glucose,insulin and insulin resistance(HOMA-IR):Leprdb diabetic mice had higher fasting blood glucose,fasting insulin and HOMA-IR than m Leprdb control mice.In m Leprdb control mice,12 weeks of ADF significantly reduced insulin.In Leprdb mice,ADF significantly decreased blood glucose and HOMA-IR without affecting insulin levels.3.Effect of ADF on mesenteric arteriole(SMA)vascular function:Compared with m Leprdb control mice,SMA acetylcholine(ACh)-induced endothelium-dependent vasodilation in Leprdb diabetic mice was impaired.Incubation with a nitric oxide synthase inhibitor(L-NAME)largely attenuates ADF-induced improvement in endothelial function in diabetic mice,suggesting that the protective effect of ADF on endothelial relaxation is primarily regulated by increased nitric oxide.Sodium nitroprusside(SNP)-induced endothelium-independent vasodilation and phenylephrine(PE)-induced vasoconstriction were not significantly impaired in Leprdb diabetic mice.ADF also had no further effect on SNP-induced endothelium-independent vasodilation or PE-induced vasoconstriction in m Leprdb control mice and Leprdb diabetic mice.4.Effects of ADF on circulating levels of adipokines and adipose tissue expression levels:Leprdb mice had lower levels of adiponectin in serum compared to m Leprdb control mice.ADF increases serum adiponectin in Leprdb diabetic mice to levels in m Leprdb control mice.However,ADF did not affect serum adiponectin levels in m Leprdb mice.Serum resistin in Leprdb mice was lower than m Leprdb control mice.ADF increases m resistin in Leprdb control mice and Leprdb diabetic mice.As expected,Leprdb mice showed a significant increase in serum leptin compared with m Leprdb control mice,a result of hyperleptinemia due to the absence of leptin receptors.ADF decreased serum leptin in m Leprdb control mice but further increased serum leptin in Leprdb mice.In mesenteric adipose tissue(MAT),there were similar trends as seen for the circulating adipokines.5.ADF protects vascular endothelial function in diabetic mice by regulating adipokine:adenovirus-mediated adiponectin supplementation improves endothelial function in Leprdb mice,further supporting the role of adiponectin in diabetes-associated endothelial dysfunction protective effect.Recombinant resistin had no significant effect on endothelial function in Leprdb mice.6.Effect of ADF on protein expression of oxidative stress marker nitrotyrosine in SMA and MAT:Protein tyrosine nitration is one of the indicators of oxidative stress.Nitrotyrosine protein levels were elevated in SMA and MAT in Leprdb mice compared with m Leprdb control mice.ADF reduced the nitrotyrosine protein in SMA of Leprdb mice,but had no significant effect on the nitrotyrosine protein in MAT,indicating that the anti-nitrotyrosine damage of ADF mainly acts on blood vessels.ADF did not affect the levels of SMA and MAT nitrotyrosine proteins in m Leprdb control mice,indicating that ADF would not further affect oxidative stress indicators at physiologically normal levels.Conclusion1.12 weeks of ADF significantly improved blood glucose metabolism and insulin resistance in type 2 diabetic mice,but had no significant effect on body weight,visceral adiposity,and blood lipids.In non-obese non-diabetic control mice,ADF exhibited reduced body weight and a modest further increase in glucose metabolism and insulin sensitivity.2.In type 2 diabetic mice,ADF exerted a significant protective effect on vascular endothelium,and had no further effect on smooth muscle relaxation and contraction.Meanwhile,in non-obese non-diabetic control mice,ADF showed a modest further enhancement of vascular endothelial function and no further effect on smooth muscle relaxation and contraction.3.ADF affected both circulating adipokines and adipokines expression in adipose tissue,mainly manifested as increased circulating adiponectin,resistin and leptin.In control mice,ADF had no effect on circulating adiponectin,but increased circulating resistin and decreased circulating leptin.4.Through the mechanism study of adenovirus-mediated supplementation of adiponectin and recombinant resistin,we further confirmed that the mechanism of ADF’s vascular endothelial protection is at least partly achieved by enhancing circulating adiponectin,while resistin and leptin are not may be involved.In addition to adipokines,oxidative stress products directly affect vascular endothelial function.ADF reduced markers of oxidative stress in resistance arteries but not in adipose tissue.5.In summary,ADF can improve blood sugar metabolism and vascular endothelial function damage in type 2 diabetes.Its vascular endothelial protective effect is related to the improvement of blood glucose metabolism,the regulation of adipokines profile,and the reduction of vascular oxidative stress indicators.ADF also had positive effects on glucose metabolism and endothelial function in non-obese non-diabetic control mice.Therefore,ADF has the potential to be a very effective and promising major lifestyle intervention modality in the treatment of diabetes and its associated vascular endothelial dysfunction,as well as lifestyle regulation in non-obese non-diabetic populations.6.Through theoretical discussion and experimental research,this paper establishes the scientific mechanism of ADF as one of the dietary regulation methods to effectively improve diabetes and vascular complications,as well as blood sugar and blood vessel health care in healthy people.It provides an extremely critical and groundbreaking scientific basis for TCM dietary regulation,especially dieting,as an indispensable part in TCM syndrome differentiation and treatment of diabetes and TCM health care.