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. 2022 Apr 26;323(1):R43–R58. doi: 10.1152/ajpregu.00006.2022

Figure 3.

Figure 3.

Downregulation of heat shock protein 72 (HSP72) (via small interfering RNA) or upregulation HSP72 (via heating) in cultured endothelial cells does not impair and enhance insulin signaling (i.e., activation of Akt), respectively, nor is c-Jun amino-terminal kinase (JNK) activity altered. HSP72 (A), phospho-JNK/JNK (B), and insulin-stimulated phospho-AktS473/Akt expression (C) in human skeletal muscle microvascular endothelial cells (hSMMECs), 48 h after 2nd transfection with scramble or HSP72 siRNA (20 nM for 6 h) (A, n = 12/condition; B, n = 12/condition; C, n = 6/condition). HSP72 (E), phospho-JNK/JNK (F), and insulin-stimulated phospho-AktS473/Akt expression (G) in hSMMECs, 24 h after sham (37°C) or heat treatment (40.5 °C) for 60 min/day, over 5 days (E, n = 10/condition; F, n = 10/condition; G, n = 5/condition). HSP72 (I), phospho-JNK/JNK (J), and insulin-stimulated phospho-AktS473/Akt expression (K) in hSMMECs, 24 h after incubation with a glucolipotoxic or vehicle milieu (I, n = 10/condition; J, n = 10/condition; K, n = 5/condition). HSP72 (M), phospho-JNK/JNK (N), and insulin-stimulated phospho-AktS473/Akt expression (O) in glucolipotoxic-treated hSMMECs, 24 h after sham (37°C) or heat treatment (40.5°C) for 60 min (M, n = 22/condition; N, n = 22/condition; O, n = 11/condition). D, H, L, and P: representative Western blot bands for proteins of interest (bands in L were obtained from the same gel but are noncontiguous, as denoted by separated boxes). Cells were incubated with insulin (100 nM) for 30 min before collection. Comparisons of HSP72 (A, E, I, M) and phospho-JNK/JNK (B, F, J, N) between conditions within each experiment were performed with unpaired two-tailed Student’s t test. Comparisons of phospho-AktS473/Akt (C, G, K, O) between conditions in each experiment were performed with two-way ANOVA and Bonferroni post hoc test. Means ± SE and individual data are reported. In A, E, I, M, *significant effect of condition (P < 0.05). In C, G, K, *significant main effect of insulin stimulation (P < 0.05). In K, #significant main effect of glucolipotoxicity (P < 0.05).