Vitamin D3 mitigates myopathy and metabolic dysfunction in rats with metabolic syndrome: the potential role of dipeptidyl peptidase-4

Abstract

Metabolic syndrome is associated with vitamin D3 deficiency. This work aims to examine the efficacy of vitamin D3 in inhibiting MetS-induced myopathy and to determine whether the beneficial effects of vitamin D3 are mediated by the inhibition of dipeptidyl peptidase-4 (DPP-4). An in silico study investigated the potential effectiveness of vitamin D3 on the inhibition of the DPP-4 enzyme. An in vitro assay of the DPP-4 inhibitory effect of vitamin D3 was performed. In vivo and over 12 weeks, both diet (with 3% salt) and drinking water (with 10% fructose) were utilized to induce MetS. In the seventh week, rats received either vitamin D3, vildagliptin, a combination of both, or vehicles. Serum lipids, adipokines, glycemic indices, and glucagon-like peptide-1 (GLP-1), muscular glucose transporter type-4 (GLUT-4) content, DPP-4, adenosine monophosphate kinase (AMPK) activities, and Sudan Black B-stained lipids were assessed. Muscular reactive oxygen species (ROS), caspase-3, and desmin immunostaining were used to determine myopathy. MetS-induced metabolic dysfunction was ameliorated by vitamin D3, which also reduced intramuscular glycogen and lipid accumulation. This is demonstrated by the attenuation of MetS-induced myopathy by vitamin D3, decreased oxidative stress, increased desmin immuno-expression, and caspase-3 activity. Our in silico data demonstrated that vitamin D3 is capable of inhibiting DPP-4, which is further supported by biochemical findings. Vitamin D3 increased serum GLP-1, muscular AMPK activity, and GLUT-4 content, whereas the levels of muscular ROS were decreased in MetS. Vildagliptin and its combination with vitamin D3 yielded comparable results. It is suggested that the DPP-4 inhibitory potential of vitamin D3 is responsible for the amelioration of MetS-induced metabolic changes and myopathy.

Keywords: DPP-4 inhibitors; Metabolic syndrome; Myopathy; Skeletal muscles; Vildagliptin; Vitamin D3.

Fig. 1

The binding subsites of the various classes of dipeptidyl peptidase-4 (DPP-4) inhibitors into the DPP-4 active site (a). Docking poses of vitamin D3 or its metabolites with DPP-4 (PDB ID:2RGU) are shown, where panels b and c show 2D and 3D docking poses of cholecalciferol (green), panels d and e show 2D and 3D docking poses of calcifediol (cyan). In contrast, panels f and g show 2D and 3D docking poses of calcitriol (purple)

Fig. 2

3D binding interaction pattern of vitamin D3 (green, panel a) and calcitriol (purple, panel b) in the binding site of DPP-4 (PBD: 2RGU). The binding orientation of these compounds is similar to that of the co-crystalized inhibitor (BI 1356, linagliptin) shown in orange

Fig. 3

Flexible alignment of cholecalciferol (green), calcifediol (cyan), and calcitriol (purple) with the co-crystalized inhibitor (BI 1356, linagliptin) (orange) of DDP-4 (PDB ID:2RGU) (a) and the vildagliptin (yellow) (b)

Fig. 4

Effect of 6 weeks of treatment with vitamin D3 (10 µg/kg/day, gavage), vildagliptin (Vilda,10 mg/kg/day, gavage), or combination on metabolic syndrome (MetS)-induced changes in systolic blood pressure (a), diastolic blood pressure (b), and mean arterial pressure (c). MetS was induced by feeding rats 3% salt and 10% fructose for 12 weeks. Values are presented as mean ± SEM (n = 6/group). One-way ANOVA followed by Tukey’s test for multiple comparisons was used for analysis, ^a vs control and ^b vs MetS at p < 0.05

Fig. 5

Effect of 6 weeks of treatment with vitamin D3 (10 µg/kg/day, gavage), vildagliptin (Vilda,10 mg/kg/day, gavage), or combination on metabolic syndrome (MetS)-induced anthropometrics alterations as expressed by body weight (BW, a), waist circumference (WC, b), body mass index (BMI, c), and visceral adipose tissue weight/bodyweight (VATW/BW, d). They also demonstrated their effect influence on MetS-induced disturbances of lipid profile, as manifested by serum levels of triglycerides (TG, e), total cholesterol (TC, f), high-density lipoprotein-cholesterol (HDL-C, g), low-density lipoprotein-cholesterol (LDL-C, h), and atherogenic index (i). MetS was induced by feeding rats 3% salt and 10% fructose for 12 weeks. Values are presented as mean ± SEM (n = 6/group). One-way ANOVA, followed by Tukey’s test for multiple comparisons, were used for analysis, ^a vs control, ^b vs MetS, and.^c vs vildagliptin at p < 0.05

Fig. 6

The effect of 6 weeks of treatment with vitamin D3 (10 µg/kg/day, gavage), vildagliptin (Vilda, 10 mg/kg/day, gavage), or combination on metabolic syndrome (MetS)-induced impairment of glycemic control demonstrated by the area under glycemic curve of oral glucose tolerance test (AUC of OGTT, a), fasting insulin (b), HOMA-IR (c), and HbA1C (d). Additionally, they demonstrated their effect on MetS-induced changes in serum uric acid (e) and serum adipokines (adiponectin, f, and leptin, g) as well as vitamin D3 deficiency (1,25(OH)₂D3, h). MetS was induced by feeding rats 3% salt and 10% fructose for 12 weeks. Values are presented as mean ± SEM (n = 6/group). One-way ANOVA, followed by Tukey’s test for multiple comparisons, was used for analysis, ^a vs control, ^b vs MetS, and.^c vs vildagliptin at p < 0.05

Fig. 7

The effect of 6 weeks treatment with vitamin D3 (10 µg/kg/day, gavage), vildagliptin (Vilda,10 mg/kg/day, gavage), or combination on metabolic syndrome (MetS)-induced glycogen accumulation (a). MetS was induced by feeding rats 3% salt and 10% fructose for 12 weeks. Values are presented as mean ± SEM (n = 6/group). One-way ANOVA, followed by Tukey’s test for multiple comparisons, were used for analysis, ^a vs control, ^b vs MetS, and.^c vs vildagliptin at p < 0.05. Panel b represents photomicrographs of Sudan Black staining for total lipids of the longitudinal sections from the soleus muscle (n = 3/group). Scale bar 50 µm × 400

Fig. 8

The effect of 6 weeks of treatment with vitamin D3 (10 µg/kg/day, gavage), vildagliptin (Vilda,10 mg/kg/day, gavage), or combination on metabolic syndrome (MetS)-induced changes in soleus muscle DPP-4 activity (a), serum GLP-1 (b), soleus muscle AMPK activity (c), and soleus muscle GLUT-4 (d). MetS was induced by feeding rats 3% salt and 10% fructose for 12 weeks. Values are presented as mean ± SEM (n = 6/group). One-way ANOVA, followed by Tukey’s test for multiple comparisons, was used for analysis, ^a vs control, ^b vs MetS, and.^c vs vildagliptin at p < 0.05

Fig. 9

The effect of 6 weeks treatment with vitamin D3 (10 µg/kg/day, gavage), vildagliptin (Vilda, 10 mg/kg/day, gavage), or combination on metabolic syndrome (MetS)-induced myopathy expressed as soleus muscle advanced glycation end products (AGEs, a), reactive oxygen species (ROS, b), nicotinamide adenine dinucleotide phosphate oxidase (NADPH Oxidase, c), and caspase-3 activity (d). MetS was induced by feeding rats 3% salt and 10% fructose for 12 weeks. Values are presented as mean ± SEM (n = 6/group). One-way ANOVA, followed by Tukey’s test for multiple comparisons, was used for analysis, ^a vs control, ^b vs MetS, and.^c vs vildagliptin at p < 0.05. Panel e represents photomicrographs of immunohistochemical staining of desmin in the longitudinal section of rat soleus muscle sections from study groups (n = 3/group) (Scale bar 50 µm × 400)