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.