PPP2R5C HepKD in <i>db/db</i> mouse liver improves insulin sensitivity, decreases hyperglycemia, but worsens the dyslipidemia.

<p><b>(A)</b> Hyperglycemia in <i>db/db</i> mice is decreased upon hypatocyte-specific PPP2R5C knockdown with adeno-associated virus. After 5 weeks knockdown, <i>db/db</i> mice were sacrificed under <i>ad libitum</i> feeding with normal chow diet. Blood glucose was monitored at each week, week 0 was one day before virus injection (n = 6). <b>(B)</b> Insulin tolerance test shows improved insulin sensitivity in PPP2R5C knockdown <i>db/db</i> mice at week 4 after virus injection (1.5IU/kg insulin was tail-injected after 6 hour fasting) (n = 6). <b>(C-F)</b> PPP2R5C HepKD in <i>db/db</i> mice increases body weight (C), whole body fat content (D), and liver weight (E), without changing abdominal adipose tissue weight (Abd.WAT) (F) (n = 6). Error bars: std. dev. *p-value<0.05 and **p-value<0.01 by student t-test (D-E). p-value in the (A-C) was calculated by two-way ANOVA.</p

Hepatocyte-specific knockdown of PPP2R5C leads to increased lipogenesis and lipid secretion.

<p><b>(A-C)</b> Liver-specific knockdown of PPP2R5C leads to pro-anabolic changes including increased liver lipid synthesis and secretion and reduced glycogen breakdown. As in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005561#pgen.1005561.g002" target="_blank">Fig 2</a>, 7 weeks post hepatocyte-specific knockdown, liver weight (A), glycogen (B) and triglycerides (C) were quantified. (n = 5 or 6) <b>(D-E)</b> Cellular triglyceride levels are increased in Hepa 1–6 (D) or mouse primary hepatocytes (E) upon PPP2R5C knockdown. Cells infected as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005561#pgen.1005561.g002" target="_blank">Fig 2</a>. (n = 3) (<b>F-G</b>) Liver-specific knockdown of PPP2R5C leads to elevated serum VLDL levels. 7 weeks post hepatocyte-specific knockdown, serum triglycerides were quantified either in aggregate (F), or when fractionated by FPLC to resolve lipoprotein particles of various densities (G). (n = 5 or 6) Error bars: std. dev. *p-value<0.05, **p-value<0.01 by one-way ANCOVA with liver NEFA as a covariate (C, “Random”) or student t-test (A-F).</p

PPP2R5C knockdown leads to SREBP-1 and ChREBP activation.

<p><b>(A)</b> PPP2R5C knockdown leads to upregulation of genes enriched for PPARA and SREBP-1 targets. Genes either up- or down-regulated upon PPP2R5C knockdown in mouse primary hepatocytes were analyzed using TFactS software [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005561#pgen.1005561.ref037" target="_blank">37</a>] to identify transcription factors putatively misregulated upon PPP2R5C knockdown. FDR (False Discovery Rate) rate was controlled using the Benjamini-Hochberg procedure. <b>(B-C)</b> Expression of bona-fide SREBP-1 target genes is increased upon PPP2R5C knockdown in primary hepatocytes in culture (B) or in mouse liver in vivo (C). PPP2R5C was knocked-down in mouse primary hepatocytes using adenovirus and in vivo using adeno-associated virus as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005561#pgen.1005561.g002" target="_blank">Fig 2</a>. SREBP-1 target genes quantified by Q-RT-PCR, normalized to TBP. <b>(D)</b> Upon PPP2R5C knockdown in liver, SREBP-1 protein levels are elevated. <b>(E)</b> Expression of ChREBP target genes is increased upon PPP2R5C knockdown in mouse liver in vivo. PPP2R5C was knocked-down in vivo using adeno-associated virus as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005561#pgen.1005561.g002" target="_blank">Fig 2</a>. ChREBP target genes quantified by Q-RT-PCR, normalized to TBP. <b>(F)</b> Graphical representation of the metabolic changes induced upon PPP2R5C knockdown in mouse liver. Livers with reduced PPP2R5C have increased glucose uptake, increased TAG synthesis, and increased VLDL secretion. Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001 by student t-test (B-C,E) (n = 4 for mouse primary hepatocytes, and 5 or 6 for mouse liver).</p

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

<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

PPP2R5C expression in human liver correlates with insulin resistance.

<p><b>(A)</b> Expression of PPP2R5C is elevated in livers of diabetic patients. Quantitative RT-PCR of Human PPP2R5C in liver of healthy controls (n = 40) or type 2 diabetic patients (n = 26), normalized to 18S rRNA. <b>(B)</b> Expression of PPP2R5C is elevated in non-diabetics with visceral obesity. People enrolled in the analysis were divided into 3 subgroups according to their adiposity: Lean (n = 12), Subcutaneous (“SC”) Obesity (n = 21) and Visceral (“VIS”) Obesity (n = 7). <b>(C)</b> Expression of PPP2R5C inversely correlates with insulin sensitivity. Pearson correlation analysis was done for PPP2R5C expression levels and glucose infusion rate (GIR) during hyperinsulemic-euglycemic clamp. **p-value<0.01, ***p-value<0.001 by student t-test (A-B).</p

Expression of PPP2R5C is nutritionally regulated in metabolically relevant tissues.

<p>(A-C) mRNA levels of PPP2R5C are nutritionally regulated in various mouse tissues. Quantitative RT-PCR of PPP2R5C (NCBI splice variant 4) from liver (A), abdominal white adipose tissue (B) and gastrocnemius muscle (C) of control C57BL/6 (“WT”) or diabetic leptin-receptor deficient (“db/db”) 8–10 week old male mice. Mice were first starved for 16 hours (“Fasting”) and then given normal chow diet for 6 hours (“Refed”). Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001 by student t-test (n ≥ 3).</p

PPP2R5C regulates AMPK and HIF1α.

<p><b>(A)</b> Schematic diagram of “BioID”—proximity-dependent biotinylation of proteins—to identify PPP2R5C substrates interacting transiently with the phosphatase complex. Biotin ligase (BirA) was fused to either the N-terminus (Myc-BirA-PPP2R5C) or C-terminus (PPP2R5C-BirA-HA) of PPP2R5C (NCBI variant 1). As negative controls, only BirA-HA or Myc-BirA were used. A mutation in the active center of the catalytic domain was introduced (PP2A-C D85N) as a substrate-trapping mutation. Biotinylated proteins from Hepa 1–6 cells were purified by cell lysis and streptavidin pulldown, and subsequently detected by immunoblotting. <b>(B-B’)</b> BioID identifies AMPK beta 1, HIF1<b>α</b>, STAT3 and S6K as PPP2R5C interacting proteins. Proteins interacting with PPP2R5C in vivo in Hepa 1–6 cells were biotinylated and purified as in (A), and probed by immunoblotting. LXR, PPAR alpha, SREBP-1, Actin, and Tubulin were included as negative control proteins which were not detected in the pulldowns. (Lysates shown at same exposure as biotin pulldowns.) <b>(C)</b> AMPK phosphorylation and activity increase upon PPP2R5C knockdown in Hepa 1–6 cells. PPP2R5C was knocked-down using two independent, inducible shRNAs as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005561#pgen.1005561.g002" target="_blank">Fig 2G</a>. AMPK phosphorylation increases on T172 upon PPP2R5C knockdown. AMPK activity determined via phosphorylation of two substrates, ACC1 and TBC1D1. <b>(D)</b> HIF1α phosphorylation increases upon PPP2R5C knockdown in Hepa 1–6 cells. HIF1α phosphorylation is detected as reduced mobility (up) on a 25 μM PhosTag gel (8% gel), which is abolished upon treatment of cell lysates with calf intestinal phosphatase (CIP) prior to gel electrophoresis (right panel). PPP2R5C knockdown was performed by generating stable cell lines carrying two independent inducible shRNA constructs (KD1 and KD2) to exclude off-target effects. <b>(E)</b> Expression of HIF1<b>α</b> target genes increases in primary hepatocytes upon PPP2R5C knockdown. (n = 4) Error bars: std. dev. *p-value<0.05, **p-value<0.01, ***p-value<0.001 by student t-test (E).</p