KLF15 Is a Molecular Link between Endoplasmic Reticulum Stress and Insulin Resistance

Obesity places major demands on the protein folding capacity of the endoplasmic reticulum (ER), resulting in ER stress, a condition that promotes hepatic insulin resistance and steatosis. Here we identify the transcription factor, Kruppel-like factor 15 (KLF15), as an essential mediator of ER stress-induced insulin resistance in the liver. Mice with a targeted deletion of KLF15 exhibit increased hepatic ER stress, inflammation, and JNK activation compared to WT mice; however, KLF15-/- mice are protected against hepatic insulin resistance and fatty liver under high-fat feeding conditions and in response to pharmacological induction of ER stress. The mammalian target of rapamycin complex 1 (mTORC1), a key regulator of cellular energy homeostasis, has been shown to cooperate with ER stress signaling pathways to promote hepatic insulin resistance and lipid accumulation. We find that the uncoupling of ER stress and insulin resistance in KLF15-/- liver is associated with the maintenance of a low energy state characterized by decreased mTORC1 activity, increased AMPK phosphorylation and PGC-1α expression and activation of autophagy, an intracellular degradation process that enhances hepatic insulin sensitivity. Furthermore, in primary hepatocytes, KLF15 deficiency markedly inhibits activation of mTORC1 by amino acids and insulin, suggesting a mechanism by which KLF15 controls mTORC1-mediated insulin resistance. This study establishes KLF15 as an important molecular link between ER stress and insulin action

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

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

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

Metabolic responses to high-fat feeding in KLF15-deficient versus control mice.

<p>Male mice were placed on a high-fat diet (HFD; 60% kcal from fat) at 3-4 months of age. <b>A</b>. <b>Body composition</b>. Body weight (n=11-12) and whole-body fat mass (n=5-6) in wild-type (WT) and <i>KLF15</i>^<i>-/-</i> (KO) mice were measured immediately prior to (chow) and 13 weeks after the start of HFD. Right panels indicate change in body weight and fat mass during 13 week HFD period. <b>B</b>. <b>Metabolic cage analysis</b>. Assessment of food intake, physical activity, O₂ consumption and CO₂ production immediately prior to (chow) and 12 weeks after the start of HFD (n=6). <b>C</b>. <b>Hyperinsulinemic-euglycemic clamp study</b>. Clamp procedure was performed after 14 weeks of HFD. Top (left to right): basal plasma insulin levels after an overnight fast, basal plasma glucose levels after an overnight fast, glucose infusion rate during the insulin clamp. Bottom (left to right): basal hepatic glucose production (HGP) after an overnight fast (prior to insulin stimulation), hepatic glucose production during the insulin clamp, and insulin-stimulated whole-body glucose turnover (peripheral glucose uptake; n=5; results are representative of two individual experiments). <b>D</b>. <b>Hepatic lipid content and lipid-related gene expression in WT versus <i>KLF15</i>^<i>-/-</i> mice</b>. After 14 weeks of HFD, liver tissue from clamped mice was collected and flash frozen. To assess hepatic lipid content, a triglyceride assay was performed using a commercially available kit (left; n=5-7). To measure lipid-related gene expression, total RNA was isolated from liver tissue, reverse transcribed and amplified by QPCR using primers against the genes shown (right; n=3). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077851#pone-0077851-g001" target="_blank">Figure 1</a> statistical comparisons were made using Student’s t test for unpaired samples. Values = mean ± SEM; *p<0.05; **p<0.01 compared to WT control.</p