Cardiac specific deletion of KLF15 alters lipid profile.

<p>Metabolomic analysis of long chain acylcarnitines in cardiac tissue from control (MHC-Cre) vs. KLF15-cKO with and without 48 hour fast, (n = 5), *P<0.05 by one-way analysis of variance (ANOVA) with the Tukey post hoc test.</p

Short-chain diet rescues the KLF15-dependent attenuation of cardiac function in response to fasting.

<p>(A) qPCR analysis of expression of transporter genes in MHC-Cre vs. KLF15-cKO under fed vs. 48 hour fasting conditions. *P<0.05 vs. Cre Fed, **P<0.05 vs. CKO Fed, # P<0.05 vs. Cre Fast. Values normalized to <i>Ppib</i>. (B) <i>Slc25a20</i> expression (qPCR) in MHC-Cre vs. KLF15-cKO under fed vs. 48 hour fasting conditions. *P<0.05 vs. Cre Fed, **P<0.05 vs. CKO Fed, # P<0.05 vs. Cre Fast. Values normalized to <i>Ppib</i>. (C) Western blot analysis of CACT levels in MHC-Cre vs KLF15-cKO under fed and 48 hour fasting conditions. α-tubulin used as loading control. (D) Quantification of data in C (n = 3 per group). Two-tailed Student's <i>t</i>-test for unpaired data was used. *P<0.05. (E) Left ventricular fractional shortening from echocardiography performed in control (MHC-Cre) vs. KLF15-cKO under fed vs. 48 hours fasting conditions following 10 weeks of short-chain fatty acid diet, (n = 10). (F) Representative echocardiography image from MHC-Cre vs. KLF15-cKO following 48 hours fasting and 10 weeks of short-chain fatty acid diet. (G) Tabular representation of echocardiography data in MHC-Cre vs. KLF15-cKO under fed vs. 48 hour fasting conditions following 10 weeks of short-chain fatty acid diet.</p

Cardiac KLF15 is required for the heart’s functional adaptation in response to fasting.

<p>(A) Left ventricular fractional shortening from echocardiography performed in control (MHC-Cre) vs KLF15-cKO under fed vs. 48 hours fasting conditions, (n = 5), *P<0.05 vs. MHC-Cre Fast. (B) Representative echocardiography image from MHC-Cre vs. KLF15-cKO following a 48 hour fast. (C) Tabular representation of echocardiography data in MHC-Cre vs. KLF15-cKO under fed vs. 48 hour fasting conditions.</p

Systemic KLF15 is required for the heart’s functional adaptation in response to fasting.

<p>(A) Left ventricular fractional shortening from echocardiography performed in wild-type (WT) vs. systemic <i>Klf15</i>-null (<i>Klf15-/-</i>) under fed vs. 48 hours fasting conditions, (n = 5), *P,0.05 vs. WT Fast. (B) Representative echocardiography image from WT vs. <i>Klf15-/-</i> following a 48 hour fast. (C) Tabular representation of echocardiography data in WT vs. <i>Klf15-/-</i> under fed vs. 48 hour fasting conditions.</p

Cardiac specific deletion of KLF15 alters tissue and plasma levels of free fatty acids and triglycerides.

<p>Cardiac FFA (A) and TG (B) levels in control (MHC-Cre) vs. KF15-cKO following 48 hours fasting, (n = 5), *P<0.05 vs. Cre Fed, **P<0.05 vs. CKO Fed, # P<0.05 vs. Cre Fast. Plasma FFA (C) and TG (D) levels in control (MHC-Cre) vs. KLF15-cKO following 48 hours fasting, (n = 5), *P<0.05 vs. Cre Fed, **P<0.05 vs. CKO Fed, # P<0.05 vs. Cre Fast.</p

Change in long chain acylcarnitine profiles after loss of cardiac KLF15 expression.

<p>Change in long chain acylcarnitine profiles after loss of cardiac KLF15 expression.</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

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

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