Fig 1: Impact of scaffolds on proteins associated with insulin signaling and glucose uptake in the epididymal fat pad, gastrocnemius muscle, and liver. (A,C,E) Representative western blots and (B,D,F) band quantification of Akt ratio for the epididymal fat pad (A,B), gastrocnemius (C,D) and liver (E,F). (G,H) Glut4 expression normalized to GPADH is depicted for the epididymal fat pad (G) and the gastrocnemius (H). Statistics were calculated by one-way ANOVA with Tukey's multiple comparisons test. * Represents p < 0.05 compared to HFD+Sh, †represents p < 0.05 compared to HFD+Sc. n = 5 mice per group.
Fig 2: Effect of treatment with high glucose and palmitate on CSE-H2S, PGC-1α, FNDC5 (irisin), and glucose uptake in C2C12 mouse myotubes. Differentiated myotubes treated with high glucose (25mM) or palmitate (0.6mM) for 24 h. Mannitol was used as an osmolarity control. (A) Western blot analysis (CSE, PGC-1α, and FNDC5) was performed on total protein extracts (n = 3 independent experiments) in mouse myotubes; (B–D) semi-quantitative analysis of the abundance ratio of protein to GAPDH. (E) RT-qPCR was performed to assess the level of the GLUT4 gene, as indicated (n = 3). (F) Glucose uptake; (G) levels of intracellular H2S; (H) cell culture medium H2S; (I) cell culture medium irisin levels. One-way ANOVA followed by SNK (Student’s Newman–Keul’s) means comparison was performed between the control and treatment groups. * p ≤ 0.05 was considered significant for a statistical test. Data are expressed as mean ± SEM.
Fig 3: Effect of H2S producing enzyme inhibitors (propargylglycine or aminooxyacetate) on CSE-H2S, PGC-1a, FNDC5 (irisin), and glucose uptake in C2C12 mouse myotubes. Differentiated myotubestreated with propargylglycine (PPG; 100 µM) oraminooxyacetate (AOA; 100 µM) or a combination of both for 6 h. (A) Western blot analysis (CSE, PGC-1a, and FNDC5) was performed on total protein extracts (n = 3 independent experiments) in mouse myotubes; (B–D) semi-quantitative analysis of the abundance ratio of protein to GAPDH. (E) RT-qPCR was performed to assess the level of the GLUT4 gene, as indicated (n = 3). (F) Glucose uptake; (G) levels of intracellular H2S; (H) cell culture medium H2S; (I) cell culture medium irisin levels. One-way ANOVA followed by SNK (Student’s Newman–Keul’s) means comparison was performed between the control and treatment groups. * p = 0.05 was considered significant for a statistical test. Data are expressed as mean ± SEM.
Fig 4: Glucose transporters, insulin receptor and adrenergic receptor ß2 expression in skeletal muscleProtein expression levels of GLUT1 (A), GLUT4 (B), ADRß2 (ADRB2; C) and insulin receptor ß (INSR; D) were measured in semitendinosus muscle from control (n = 8; 4M/4F), IUGR (n = 8; 3M/5F) and IUGR-AR (n = 7; 1M/6F) lambs. Representative images of western blots are shown for the glucose transporters and receptors and ß-Tubulin (TUBB), which was used for normalization. Means ± SEM are presented in the bar graphs and differences (P < 0.05) are indicated with different letters.
Fig 5: Endothelial NOX5 expression increased Glut4 levels in mesenteric and epididymal fat in mice fed a HFD for 10 weeks. (A,B) Glut4 mRNA levels in (A) mesenteric fat and (B) epididymal fat. (C,D) Representative Western blot and protein levels of GLUT4 in total lysates of (C) mesenteric fat and (D) epididymal fat. Control diet: control Cre (n = 12), Nox5/Cre (n = 11); high-fat diet: control Cre (n = 10), Nox5/Cre (n = 12). Values are expressed as median with confidence interval. mRNA levels are relative to Gapdh. Protein levels are relative to B-ACTIN. # p < 0.05, ## p < 0.01: diet differences (dotted lines); * p < 0.05: genotype differences (solid lines). Statistical test used: Aligned Rank ANOVA.
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