Effects of diet manipulation and 1,25 (OH)₂ vitamin D supplementation on NF-κB activation (panel A) and TNF-alpha levels (panel B) in the mouse gastrocnemious.

<p>(A) Representative western blotting analysis for the expression of NF-κB p65 subunit. Protein expression was analyzed by Western blot on cytosol and nucleus homogenates of gastrocnemious from animals fed a control diet or HFHS diet with or without vitamin D supplementation (7 μg/kg, 3 times per week). Densitometric analysis of the bands is expressed as relative optical density (O.D.) corrected for the GADPH (cytosol) or histone (nucleus) contents, and normalized using the related control band. NF-κB p65 subunit translocation was expressed as nucleus/cytosol ratio normalized using the related control band. (B) TNF-alpha levels were measured by ELISA in the mouse gastrocnemious homogenates. The data are expressed by medians and interquartile range) of 5–6 randomly selected animals per group. Statistical analysis was performed with Kruskal–Wallis test with Dunn’s post hoc test. *p<0.05 <i>vs</i> Control; #p<0.05 <i>vs</i> HFHS.</p

Effects of vitamin D on insulin resistance and myosteatosis in diet-induced obese mice

<div><p>Epidemiological studies pointed out to a strong association between vitamin D deficiency and type 2 diabetes prevalence. However, the role of vitamin D supplementation in the skeletal muscle, a tissue that play a crucial role in the maintenance of glucose homeostasis, has been scarcely investigated so far. On this basis, this study aimed to evaluate the effect of vitamin D supplementation in a murine model of diet-induced insulin resistance with particular attention to the effects evoked on the skeletal muscle. Male C57BL/6J mice (n = 40) were fed with a control or a High Fat-High Sugar (HFHS) diet for 4 months. Subsets of animals were treated for 2 months with vitamin D (7 μg·kg-1, i.p. three times/week). HFHS diet induced body weight increase, hyperglycemia and impaired glucose tolerance. HFHS animals showed an impaired insulin signaling and a marked fat accumulation in the skeletal muscle. Vitamin D reduced body weight and improved systemic glucose tolerance. In addition, vitamin D restored the impaired muscle insulin signaling and reverted myosteatosis evoked by the diet. These effects were associated to decreased activation of NF-κB and lower levels of TNF-alpha. Consistently, a significantly decreased activation of the SCAP/SREBP lipogenic pathway and lower levels of CML protein adducts and RAGE expression were observed in skeletal muscle of animals treated with vitamin D.</p><p>Collectively, these data indicate that vitamin D-induced selective inhibition of signaling pathways (including NF-κB, SCAP/SREBP and CML/RAGE cascades) within the skeletal muscle significantly contributed to the beneficial effects of vitamin D supplementation against diet-induced metabolic derangements.</p></div

Effects of diet manipulation and 1,25 (OH)₂ vitamin D supplementation on insulin signaling transduction in the mouse gastrocnemious.

<p>The expression of total IRS-1 protein and its Ser³⁰⁷ phosphorylation (panel A), total Akt protein and its Ser⁴⁷³ phosphorylation (panel B), and total GSK-3β protein and its Ser⁹ phosphorylation (panel C) were analyzed by Western blot on gastrocnemious homogenates of animals fed a control diet or HFHS diet, with or without vitamin D supplementation (7 μg/kg, 3 times per week). Densitometric analysis of the bands is expressed as relative optical density (O.D.) and normalized using the related control band. The data are expressed by medians and interquartile range of 5–8 randomly selected animals per group. Statistical analysis was performed with Kruskal–Wallis test with Dunn’s post hoc test. *p<0.05, **p<0.01 <i>vs</i> Control; #p<0.05, ##p<0.01 <i>vs</i> HFHS.</p

Effects of diet manipulation and 1,25 (OH)₂ vitamin D supplementation on lipid accumulation in the mouse gastrocnemious.

<p>Representative photomicrographs (10x, 20x, 40x magnification) of Oil Red staining on gastrocnemious sections from animals fed a control diet or HFHS diet with or without vitamin D supplementation (7 μg/kg, 3 times per week).</p

Effects of diet manipulation and 1,25 (OH)₂ vitamin D supplementation on oral glucose tolerance test and HOMA index measured at the end of the experimental protocol (week 16) in fasted animals.

<p>Panel A. Oral glucose tolerance test <b>(</b>OGTT). Values are means ± S.E.M. of 6–10 animals per group. Statistical analysis was performed with Two-way analysis of variance with Bonferroni's post-hoc test. *p<0.05, ***p<0.001 <i>vs</i> Control; ^#p<0.05, ^##p<0.01, ^###p<0.001 <i>vs</i> HFHS. Panel B. HOMA index. Data are expressed by medians and interquartile range of 4–6 animals randomly selected per group. Statistical analysis was performed with Kruskal–Wallis test with Dunn’s post hoc test. *p<0.05 <i>vs</i> Control; ^#p<0.05 <i>vs</i> HFHS.</p

Effects of diet manipulation and 1,25 (OH)₂ vitamin D supplementation on SCAP/SREBP pathway activation in the mouse gastrocnemious.

<p>(A) Representative Western blotting analysis for the expression of SCAP and active/inactive SREBP1c. Protein expression was evaluated on gastrocnemious homogenates of animals fed a control diet or HFHS diet with or without vitamin D supplementation (7 μg/kg, 3 times per week). (B) Densitometric analysis of the bands is expressed as relative optical density (O.D.), corrected for the GADPH contents, and normalized using the related control band. The data are means ± S.E.M. of 6–8 randomly selected animals per group. Statistical analysis was performed by One-way analysis of variance with Bonferroni's post-hoc test. **p<0.01, ***p<0.001 <i>vs</i> Control; ^##p<0.01, ^###p<0.001 <i>vs</i> HFHS.</p

Effects of diet manipulation and 1,25 (OH)₂ vitamin D supplementation on mice body weight.

<p>Values are means ± S.E.M. of 6–14 animals per group. Statistical analysis was performed with Two-way analysis of variance with Bonferroni's post-hoc test. ^###p<0.001 <i>vs</i> HFHS. p<0.001 HFHS groups <i>vs</i> Control groups from week 1, symbol are not reported in the figure.</p

Effects of diet manipulation and 1,25 (OH)₂ vitamin D supplementation on levels of CML protein adducts and RAGE expression in the mouse gastrocnemious.

<p>(A) Representative Western blotting analysis for CML-modified proteins and RAGE expression. Protein expression was evaluated on gastrocnemious homogenates of animals fed a control diet or HFHS diet with or without vitamin D supplementation (7 μg/kg, 3 times per week). (B) Densitometric analysis of the bands is expressed as relative optical density (O.D.), corrected for the GADPH contents, and normalized using the related control band. The data aremeans ± S.E.M. of 6–9 randomly selected animals per group. Statistical analysis was performed by One-way analysis of variance with Bonferroni's post-hoc test. *p<0.05, ***p<0.001 <i>vs</i> Control; #p<0.05 <i>vs</i> HFHS.</p

Effects of diet manipulation and 1,25 (OH)₂ vitamin D supplementation on serum 25-OH vitamin D level.

<p>Levels of 25-OH vitamin D were measured by ELISA in the serum of 4–6 randomly selected animals per group. Data are expressed by medians and interquartile range. Statistical analysis was performed with Kruskal–Wallis test with Dunn’s post hoc test.</p

Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis

<div><p>Non-Alcoholic Fatty Liver Disease (NAFLD) is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH), fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs) released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1β. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.</p></div