Knocking down PRLR expression decreases leucine deprivation–enhanced insulin sensitivity in vitro and in vivo. A and B: Male C57BL/6J mice were fed a control [(+) leu] or (−) leu diet for 7 days, and HepG2 cells were incubated in (+) leu or
(−) leu medium for 12 h. C: HepG2 cells were treated with PRLR siRNA (+ PRLR RNAi) or control reagent (− PRLR RNAi) for 36 h prior to being cultured
in (−) leu or (+) leu media for 12 h, followed by stimulation with 100 nmol/L insulin for 20 min. D–F: Mice were infected with scrambled vector (− Ad-shPRLR) and fed a (+) leu diet (indicated by white circles and solid line),
infected with scrambled vector (− Ad-shPRLR) and fed a (−) leu diet (indicated by black circles and solid line), or infected
with Ad-shRNA against PRLR (+ Ad-shPRLR) and fed a (−) leu diet (indicated by black triangles and dashed line). GTTs and ITTs
were performed at day 3 in D and E, and insulin signaling in liver was examined before (− Ins) and after (+ Ins) 2 units/kg insulin stimulation for 3 min in
these mice on day 5 in F. The mean ± SEM values shown are representative of at least three independent in vitro experiments or at least two independent
in vivo experiments, with the number of mice included in each group in each experiments indicated (n = 5–7). Statistical significance was calculated using the two-tailed Student t test or one-way ANOVA followed by the SNK test for the effects of (−) leu vs. (+) leu treatment (*P < 0.05) in A–F, Ad-shPRLR vs. the control group under (−) leu treatment (#P < 0.05) in C–F. A: Prlr mRNA. B: PRLR protein (top, Western blot; bottom, quantitative measurements of PRLR protein relative to actin). C and F: p-IR (tyr1150/1151), p-Akt (ser473), and PRLR protein (left, Western blot; right, quantitative measurements of p-IR and p-Akt protein relative to total IR and Akt, respectively). D: GTT. E: ITT. t, total.