Dramatic and widespread changes in insulin signaling molecules in CPEB knockout mice.

<p>(A) Western blot analysis and quantification of IRS1, IRS2, PTEN, PDK1, phospho-Stat3 (S727), total Stat3, Socs3, and tubulin as a loading control from WT and CPEB knockout liver, fat, and muscle. (B) Quantitative RT-PCR analysis of mRNAs encoding IRS1, IRS2, PTEN, PDK1, Stat3, and Socs3 mRNAs from WT and <i>Cpeb1</i> KO liver. Data are represented as mean +/− SEM. At least 3 animals per group were used for the experiment. Asterisks refer to statistical significance at the p<0.05 (*) or p<0.01 (**) levels (Student's t test).</p

CPEB controls the synthesis of Stat3 and PTEN.

<p>(A and B) 3′ UTR sequences of Stat3 and PTEN from human, mouse and cow. The nucleotides in bold represent putative CPEs. (C and D) Western blots of Stat3 and PTEN following CPEB depletion in HepG2 cells. In panels C-H, tubulin served as a negative or input control. (E and F) Quasi-quantitative RT-PCR for Stat3 and PTEN RNAs following CPEB depletion. (G and H) HepG2 cells depleted of CPEB were pulse labeled with ³⁵S-methionine for 15 min followed by Stat3, PTEN and tubulin (as a control) immunoprecipitation and SDS-PAGE analysis. These same proteins were also analyzed by western blots. (I) Representation of Renilla and firefly luciferase RNAs that were electroporated into HepG2 cells. Renilla luciferase RNA, which contained an irrelevant 3′ UTR, served as a normalization control. Firefly luciferase contained the Stat3 or PTEN 3′ UTRs as noted in panels A and B; in some cases, the CPEs in these 3′ UTRs were mutated. (J and K) The firefly and Renilla RNAs noted above were electroporated into HepG2 cells, some of which were depleted of CPEB. Firefly luciferase was normalized to the Renilla luciferase transfection control; luciferase activity derived from all RNAs was then made relative to the control shRNA. The Stat3 and PTEN data were analyzed with ANOVA; p values were 0.009 and 0.005, respectively. The asterisk refers to statistical significance (p<0.05). Data are represented as mean +/− SEM. The firefly and Renilla luciferase RNAs were also analyzed for relative stability by quasi-quantitative RT-PCR; all the RNAs had similar stabilities. At least 3 animals were used for each experiment.</p

CPEB mediates insulin signaling in the liver.

<p>(A) Western blot and quantification of total and phospho-Akt (serine 473 and threonine 308) from liver of WT and <i>Cpeb1</i> KO mice, some of which were injected with insulin. The pAkt (Ser473) and pAkt (Thr308) data were analyzed with ANOVA with (p<0.05, *; p<0.01, **). Data are represented as mean +/− SEM. In this and all panels, at least 3 animals per group were used for the experiment. (B,C) Phospho-Akt (threonine 308) in CPEB KO fat and muscle, respectively. Analysis as in panel A. (D) Examination of insulin signaling molecules in WT and CPEB KO liver. Analysis as in panel A.</p

Proposed model for CPEB-dependent regulation of the insulin-signaling ribonome regulation.

<p>The dark gray boxes refer to mRNAs that contain conserved CPEs and can potentially be regulated by CPEB. The light gray boxes refer to mRNAs that contain conserved CPEs but because they are not co-immunoprecipitated with CPEB, are probably not regulated by this protein in the current settings. The striped boxes refer to mRNAs that contain conserved CPEs and are regulated by CPEB. Insulin mRNA does not contain a CPE.</p

<i>Cpeb1</i> KO mice are insulin-resistant.

<p>(A) WT and KO mice were fed a normal chow diet and then examined for glucose tolerance test, serum insulin levels, and insulin tolerance. ANOVA values are as indicated in the figure. (B) Measurements for lean mass, fat mass, and total body mass of WT and <i>Cpeb1</i> KO mice fed a high fat diet. (C) Animals fed a high fat diet were subjected to euglycemic clamp analysis that determined glucose infusion rate (GIR), glucose turnover, whole body glycolysis, glycogen synthesis, hepatic glucose production (HGP), and liver insulin action (the ratio of basal to clamped HGP). The HGP data were analyzed with ANOVA with a value of 0.006. The asterisks in this panel as well as panel D refer to statistical significance (p<0.05). (D) Following the euglycemic clamp, liver proteins from WT and KO animals were probed on western blots for total and phospho-Akt (S473 and T308). The pAkt (Ser473) and pAkt (Thr308) data were analyzed with ANOVA with suggestive values of 0.01999 and 0.08335 values respectively. Data are represented as mean +/− SEM. At least 3 animals per group were used for the Western blots and at least 6 animals per group were used to measure the physiological parameters.</p

Protection against ER stress-induced hepatic lipid accumulation and insulin resistance <i>KLF15</i>^<i>-/-</i> liver.

<p><b>A</b>. <b>Hepatic lipid accumulation in response to tunicamycin treatment in WT versus <i>KLF15</i>^<i>-/-</i> mice</b>. 5-month-old male WT and <i>KLF15</i>^<i>-/-</i> mice on a standard chow diet with free access to food were i.p. injected with vehicle or 3mg/kg tunicamycin (Tm). 24h post injection, mice were sacrificed and liver tissue removed and flash frozen. Liver tissues were subjected to triglyceride assay with a commercially available kit (left; n=7). Oil Red O staining (right) was performed on frozen liver sections for detection of neutral lipid. Representative samples are pictured. <b>B</b>. <b>Effect of acute ER stress on AKT activity in WT versus <i>KLF15</i>^<i>-/-</i> primary hepatocytes</b>. Hepatocytes were isolated from standard chow-fed 2.5-month-old male WT and <i>KLF15</i>^<i>-/-</i> mice and treated for 10 minutes with vehicle, 100nM insulin or 100nM insulin preceded by 20h treatment with 2µg/ml tunicamycin (Tm). Lysates were subjected to immunoblotting with antibodies against total and phospho-AKT (Ser473, Thr308). Three individual experiments were performed in triplicate; each lane indicates a technical replicate. Quantitation graph shown next to immunoblot (white bar = WT, black bar = KLF15^-/-). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077851#pone-0077851-g004" target="_blank">Figure 4</a> statistical analysis was performed using Student’s t-test for unpaired samples. Values = mean ± SEM. *p<0.05.</p

Regulation of the unfolded protein response by KLF15.

<p><b>A</b>. <b>UPR activity in WT versus <i>KLF15</i>^<i>-/-</i> liver after high-fat feeding</b>. 2-month-old WT and <i>KLF15</i>^<i>-/-</i> male mice received HFD (60% kcal from fat) for 8 weeks, then were fasted for 5h, i.p. injected with 10mU/g insulin and sacrificed 10 minutes post-injection. Liver lysates were subjected to immunoblotting (n=3). <b>B</b>. <b>Western analysis of UPR activity in WT versus <i>KLF15</i>^<i>-/-</i> primary hepatocytes</b>. Hepatocytes were isolated from standard chow-fed 4-month-old male WT and <i>KLF15</i>^<i>-/-</i> mice. <b>C</b>. <b>Effect of KLF15 overexpression on UPR activity</b>. Western analysis of hepatocytes isolated from chow-fed 4-month-old WT male mice infected with empty vector (EV) or <i>KLF15</i>-expressing adenovirus. Cells were treated for 6h with vehicle (DMSO) or 5µg/ml tunicamycin (Tm) before harvest. <b>D</b>. <b>Induction of KLF15 in response to acute ER stress</b>. AML-12 hepatocytes were treated for 7h hours with vehicle or 5µg/ml tunicamycin (Tm; top panel) or for 5h with vehicle or 1µM thapsigargin (Tg; bottom panel). Total RNA was isolated from harvested cells, reverse transcribed and subjected to QPCR. <b>E</b>. <b>Ultrastructural examination of ER morphology</b>. WT and <i>KLF15</i>^<i>-/-</i> female mice (n=3) received HFD (60% kcal from fat) for 4 weeks starting at age 3 months. Mice were fasted for 21h before sacrifice and liver tissue was removed and prepared for transmission electron microscopy analysis as described in Materials and Methods. Left: Representative electron micrographs (original magnification = 20,500x) of liver sections. Lower pictures show an enlarged portion of the field above. Scale bar = 0.5 μm. Right: Quantitation of ER lumen diameter. Values = average lumen diameter/cell representative of 9 cells/group. In each cell, lumen diameter measurements were taken along the length of each of 10 ER cisternae. For <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077851#pone-0077851-g002" target="_blank">Figure 2B, C and D</a>, two individual experiments were performed in triplicate; each lane (in B, C) indicates a technical replicate. For <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077851#pone-0077851-g002" target="_blank">Figure 2A</a>, each lane represents one mouse. Values = mean ± SEM; *p<0.05; **p<0.01 (Student’s t test for unpaired samples).</p

Uncoupling of ER stress and insulin resistance in mice with a liver-specific knockdown of KLF15.

<p><b>A</b>. <b>Effect of liver-specific knockdown of KLF15 on the hepatic UPR</b>. C57BL/6 male mice received a tail vein injection of 1.5 x 10¹¹ viral particles of either shControl or sh<i>KLF15</i> adenovirus under chow feeding conditions at 13 weeks of age or after 5 weeks of high-fat feeding at 14 weeks of age. Chow-fed mice (Day 4 post-injection) and HFD-fed mice (Day 6 post-injection) were fasted for 4h followed by i.p. injection of 10mU/g insulin and were sacrificed 10 minutes post-insulin injection. Liver lysates were subjected to Western analysis with the indicated antibodies. Each lane represents one mouse (n=3). Quantitation graphs are shown below blots. <b>B</b>. <b>Glucose tolerance tests</b>. C57BL/6 male mice received a tail vein injection of 1.5 x 10¹¹ viral particles of shControl or sh<i>KLF15</i> adenovirus under chow feeding conditions at 10 weeks of age or after 5 weeks of high-fat feeding at 14 weeks of age. Four days after tail vein injection, mice were injected i.p. with 1g glucose/kg body weight after a 16h fast. Tail vein blood samples were assessed for glucose concentration immediately before i.p. injection (Time 0) and at 15, 30, 60 and 120 minutes post-injection. Blood glucose concentrations during the GTT and corresponding area under the curve calculations for glucose values are shown for chow- and HFD-fed mice. n=6-7 mice/group for both studies. <b>C</b>. <b>Liver specificity of KLF15 knockdown</b>. Quantitative PCR evaluation of <i>KLF15</i> mRNA levels in liver, quadriceps muscle, white adipose and heart tissues isolated from chow-fed mice that were tail vein-injected with shControl (C) or sh<i>KLF15</i> (K) adenovirus. n=3. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077851#pone-0077851-g003" target="_blank">Figure 3</a> statistical comparisons were made using Student’s t test for unpaired samples or, for glucose values during GTT, analysis of variance for repeated measures with a Bonferroni post hoc test. Values = mean ± SEM; *p<0.05; **p<0.01 compared to WT control.</p

KLF15 regulation of autophagy.

<p><b>A</b>. <b>Autophagy marker expression in WT and <i>KLF15</i>^<i>-/-</i> primary hepatocytes and liver tissue</b>. (Top) Hepatocytes were isolated from standard chow-fed 4-month-old male WT and <i>KLF15</i>^<i>-/-</i> mice. Protein lysates were subjected to immunoblotting with the antibodies shown. (Bottom) 2-month-old WT and <i>KLF15</i>^<i>-/-</i> male mice were placed on a HFD (60% kcal from fat) for 8 weeks. Mice were fasted for 5h, then i.p. injected with 10mU/g insulin and sacrificed 10 minutes post-injection. Lysates from flash frozen livers were subjected to immunoblotting with the antibodies shown (n=3). Quantitation graphs are shown next to immunoblots (white bar = WT, black bar = KLF15^-/-). <b>B</b>. <b>Ultrastructural examination of autophagic vesicles in liver tissue from HFD-fed WT and <i>KLF15</i>^<i>-/-</i> mice</b>. WT and <i>KLF15</i>^<i>-/-</i> female mice (n=3) received HFD (60% kcal from fat) for 4 weeks starting at age 3 months. Mice were fasted for 21h before sacrifice and liver tissue was removed and prepared for transmission electron microscopy (TEM) analysis as described in Materials and Methods. (Top) Electron micrographs (original magnification = 43,000x) of liver sections showing clustering of autophagic vesicles (indicated by red arrows) in <i>KLF15</i>^<i>-/-</i> versus WT hepatocytes. Scale bar = 200nm. (Bottom) Quantitation of autophagic vesicles in WT and <i>KLF15</i>^<i>-/-</i> liver. TEM was used to visualize autophagic vesicles in intact hepatocytes. Values = number of autophagic vesicles per cell calculated from 15 hepatocytes per group. <b>C</b>. <b>Chloroquine treatment of primary hepatocytes</b>. Hepatocytes were isolated from 3.5-month old male WT and <i>KLF15</i>^<i>-/-</i> mice and treated with vehicle or 50µM chloroquine (CQ) for 2h prior to harvest. Protein lysates were subjected to immunoblotting with an antibody against LC3B. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077851#pone-0077851-g006" target="_blank">Figure 6</a> primary hepatocyte experiments were performed at least twice in triplicate; each lane indicates a technical replicate. For <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077851#pone-0077851-g006" target="_blank">Figure 6A</a> liver blot, each lane represents one mouse. Statistical analysis was performed using Student’s t-test for unpaired samples. Values = mean ± SEM. *p<0.05; **p<0.01.</p

Potential mechanisms underlying the uncoupling of ER stress and insulin resistance in KLF15-deficient liver.

<p><b>A</b>. <b>Inflammatory marker expression</b>. Total RNA was extracted from hepatocytes isolated from chow-fed 4-month-old male WT and <i>KLF15</i>^<i>-/-</i> mice (top) or from liver tissues isolated from WT and <i>KLF15</i>^<i>-/-</i> male mice that received HFD (60% kcal from fat) for 8 weeks starting at age 2 months, were fasted for 5h, i.p. injected with 10mU/g insulin and sacrificed 10 minutes post-injection (n=3; bottom). First strand cDNA was subjected to QPCR using primers against <i>TNFα</i> and <i>MCP-1</i>. <b>B</b>. <b>Activation of hepatic JNK, AKT and markers of energy availability</b>. Western analysis of protein lysates prepared from (left) hepatocytes that were isolated from chow-fed 3.5-month-old male WT and <i>KLF15</i>^<i>-/-</i> mice and harvested 10 minutes after addition of saline or 100nM insulin, or from (right) liver tissues described in (A). <b>C</b>. <b>Proximal insulin signaling pathway activity</b>. Western analysis of IRS-1 and IRS-2 immunoprecipitated from protein lysates of liver tissues from WT and <i>KLF15</i>^<i>-/-</i> male mice that received HFD (60% kcal from fat) for 8 weeks starting at age 2 months, were fasted for 5h, i.p. injected with saline or 10mU/g insulin and sacrificed 10 minutes post-injection (n=3). <b>D</b>. <b>Effect of KLF15 overexpression on mTORC1 activity</b>. Hepatocytes were isolated from standard chow-fed 2.5-month-old female WT mice and infected with EV or <i>KLF15</i>-containing adenovirus. Protein lysates were subjected to immunoblotting with the antibodies shown. <b>E</b>. <b>Effect of KLF15 deficiency on insulin and amino acid-mediated activation of mTORC1</b>. Hepatocytes were isolated from 3.5 month-old male WT and <i>KLF15</i>^<i>-/-</i> mice. Following an overnight incubation in serum-free Williams <i>E medium</i> (without L-glutamine), cells were harvested after a 45-minute treatment with PBS or (top) 10mM L-glutamine, (middle) 10mM L-leucine or after a 10-minute treatment with PBS or 100nM insulin (bottom). Protein lysates were subjected to immunoblotting with the antibodies shown. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077851#pone-0077851-g005" target="_blank">Figure 5</a> primary hepatocyte experiments were performed twice in triplicate; each lane (in B, D and E) indicates a technical replicate. For <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077851#pone-0077851-g005" target="_blank">Figure 5</a> liver blots, each lane represents one mouse. Statistical analysis was performed using Student’s t-test for unpaired samples. Values = mean ± SEM. *p<0.05; **p<0.01.</p