Hepatocyte-specific knockdown of PPP2R5C leads to increased glucose uptake and improved glucose tolerance.

Abstract

<p><b>(A-C)</b> Increased glucose uptake/tolerance in mice upon liver-specific knockdown of PPP2R5C. 8–10 week CL57BL/6 male mice tail-injected with adeno-associated virus containing miRNA targeting PPP2R5C (“PPP2R5C KD”) or a scrambled control miRNA (“Control KD”). 7 weeks after knockdown mice were starved for 16 hours (“Fasting”) or starved and then given normal chow diet for 6 hours (“Refed”) prior to sacrificing. Although blood glucose levels are not altered (A), serum insulin levels are significantly reduced (B) compared to controls. (C) Glucose tolerance test performed 4 weeks after virus injection shows significantly improved tolerance in knockdown mice (2g glucose/kg body weight injected intraperitoneally, n = 12). <b>(D)</b> Insulin signaling, detected via AKT and GSK3 beta phosphorylation, does not drop in all feeding regimes in PPP2R5C HepKD livers compared to controls, despite PPP2R5C HepKD serum insulin levels being lower (see panel B). <b>(E)</b> Insulin sensitivity is increased after PPP2R5C knockdown in liver. Control C57BL/6 mice and PPP2R5C HepKD mice were virus-injected as in Fig 2. Four weeks later, mice were fasted for 6 hours, then insulin was injected 1IU/kg and 10 minutes later mice were sacrified and liver samples were taken. <b>(F-G)</b> Glucose consumption and lactate production are increased in Hepa 1–6 cells upon PPP2R5C knockdown. Hepa 1–6 cells infected by adenovirus carrying shRNA targeting all mouse PPP2R5C isoforms (PPP2R5C KD) or a negative-control scramble shRNA (Control KD). After 48h, glucose consumption (F) and lactate production (G) were measured in the medium for 24 hours, and normalized to total cell protein. (n = 3) <b>(H)</b> Glycolytic flux measured as ECAR (extracellular acidification rate) using the glycolysis stress kit from Seahorse Bioscience on the extracellular flux analyser XF96. After addition of glucose to control or PPP2R5C knockdown Hepa 1–6 cells, oligomycin is added to inhibit respiration, thereby boosting glycolytic flux. 2-deoxy-glucose is added to compete with glucose and shut down glycolysis (n = 9). <b>(I)</b> Acute glucose uptake is increased in Hepa 1–6 cells upon PPP2R5C knockdown. Stably transfected Hepa1-6 cell-lines carrying two independent, inducible shRNAs (PPP2R5C KD1 and KD2) were induced with 30 μg/ml cumate for 3 days, starved overnight in serum-free DMEM, and uptake of fluorescent 2-deoxy-glucose analog 2-NBDG was quantified by FACS. (n = 3) Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001, †p-value<10⁻⁴ by Wilcoxon signed-rank test (C) or student t-test (B, F-I).</p