mTORC1 activity is not sufficient for steatosis.

<p>Normal chow-fed, 20-week old male <i>Tsc1</i>+/+ and <i>Tsc1</i>−/− male mice were fasted overnight and sacrificed. Liver tissues were processed for histologic and biochemical analyses. A) Liver histology (H&E) and Oil Red “O” staining showing hepatic morphology and lipid content. Magnification 400X. B) Quantification of liver triglyceride content using TG assay kit (Roche Diagnostics, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018075#s2" target="_blank">Methods</a>). C) Hepatocyte cell size was deduced based on the average number of hepatocytes per high-power field from 10 randomly selected fields. *, p<0.01 compared to <i>Tsc1</i>+/+. D) Expression of genes involved in lipogenesis (SREBP1), adipogenesis (PPARg), lipolysis (ATGL) and gluconeogenesis (PEPCK) were determined by quantitative RT-PCR. *, p<0.05 compared to <i>Tsc1</i>+/+. For all graphs, values represent mean ±SEM.</p

Metabolic response to rapamycin following HFD.

<p>Six-week old, wild-type mice were randomly assigned to one of 5 groups with n = 5 in each group (see text). At the end of 6 weeks, mice were fasted overnight and sacrificed. Shown are the results of body and liver weights, fasting serum glucose and insulin, plasma and hepatic triglyceride levels for each group. Values represent mean ±SEM. * associated with HFD indicates p<0.05 with respect to NCD group. * associated with HFD-rapamycin group indicates p<0.05 with respect to NCD-rapamycin group. NCD, normal chow diet; HFD, high-fat diet; Rapa, rapamycin.</p

Histologic and biochemical effects of rapamycin on HFD-induced steatosis.

<p>A) Examples of histology (H&E) and Oil Red “O” staining of the livers procured from animals described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018075#pone-0018075-g004" target="_blank">Figure 4</a>. NCD, normal chow diet; HFD, high-fat diet; Rapa, rapamycin. Magnification, 400X. B) Western blots of representative liver lysates from each of the four groups shown in (A) highlighting the effects of chronic rapamycin on Akt(Ser473) phosphorylation. The average ratios of band intensities (Image J) between phospho- and total-Akt are summarized in the graph (n of 5 per group).</p

Primer sequences used in qRT-PCR.

<p>Primer sequences used in qRT-PCR.</p

Akt induces steatosis in the Tsc1−/− livers.

<p>A) Contrasting effects of <i>Pten</i>- and <i>Tsc1</i>-loss on Akt signaling in the liver. Immunoblot analyses of liver lysates from fasted 20 wk-old mice using indicated antibodies to highlight Akt and mTORC1 signaling. B) Effects of Akt on <i>Tsc1</i>−/− livers. <i>Tsc1</i>−/− mice were injected through the tail-vein with adenovirus (10⁷ PFUs) encoding genes for Myr-Akt1 or β–galactosidase control. After 96 hours, mice were fasted overnight and sacrificed for H&E histology and Oil Red “O” staining of the livers. Magnification, 400X. C) Expression of transgenes (HA-tagged Myr-Akt1 or β–gal) and components of the Akt/mTORC1 pathway in the <i>Tsc1</i>−/− livers following adenovirus injections. C, control for Tsc1 expression. Note up-regulation of Akt without significant alteration to mTORC1 signaling in the Myr-Akt1-treated liver.</p

Effects of rapamycin on Pten−/− livers.

<p>Hepatocyte-specific deletion of <i>Pten</i> was generated by crossing <i>Pten^fl/fl</i> with <i>Cre^Alb</i> mice. At 12 weeks of age, <i>Pten</i>−/− mice were randomly assigned to treatments with rapamycin (2 mg/kg IP daily, M-F) or DMSO as vehicle control (C) for 2 weeks and then sacrificed. A) Representative Western blot showing the effects of <i>Pten</i> loss (−/−) and rapamycin (Rapa) in the liver with respect to Akt and mTORC1 signaling. Liver lysates were subjected to immunoblot analyses with the indicated antibodies. B) Liver histology (H&E) and Oil Red “O” staining of <i>Pten</i>+/+ and <i>Pten</i>−/− mice treated with rapamycin or vehicle control. C) Quantification of liver triglyceride content of the corresponding groups. Values represent mean ±SEM. *, p<0.05 compared to <i>Pten</i>+/+ group.</p

Hepatic mRNA expression of metabolic genes.

<p>Relative expression of genes involved in hepatic lipogenesis (SREBP1, ACLY, FASN), lipolysis (ATGL), gluconeogenesis (PEPCK), glycolysis (GK), mitochondrial respiration (PGC1α) and triglyceride secretion (ApoB, Mttp) were determined by RT-PCR analyses of RNA extracted from liver samples derived from experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018075#pone-0018075-g004" target="_blank">Figures 4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018075#pone-0018075-g006" target="_blank">6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018075#pone-0018075-g008" target="_blank">8</a>. All values represent mean ±SEM. A, B) Comparison of <i>Tsc1</i>+/+ and <i>Tsc1</i>−/− mice fed normal chow (NCD) and high-fat diet (HFD) with and without rapamycin (rapa). * p<0.05 (not all significant differences are highlighted). C) <i>Pten</i>−/− mice treated with rapamycin or vehicle (dmso) compared to wild-type littermates, * p<0.05 compared to <i>Pten</i>+/+. D) Gene expression in livers of wild-type mice fed NCD or HFD with or without rapamycin treatment. *p<0.05 compared to NCD, E) Reduced FoxO1 phosphorylation in <i>Tsc1</i>−/− livers. Tissue lysates from <i>Tsc1</i>+/+ and <i>Tsc1</i>−/− livers were analyzed for the expression of the indicated proteins by immunoblot analyses. Levels of FoxO1(Ser256) phosphorylation were quantified relative to total FoxO1 expression based on densitometric analyses (Image J).</p

Liver, fat and muscle of Tsc1−/− mice show blunted response to insulin.

<p>Following an eight-hour fast, mice were injected with 0.5 U/kg of insulin or saline (control) for 10 minutes before sacrifice. A) Liver and B) white adipose tissue (WAT) and skeletal muscle tissue lysates were analyzed for the expression of indicated proteins. C. Akt response to insulin. The relative band intensities of p-Akt(Ser473) were normalized to total Akt from (A) and (B), and then the ratios between the insulin- and saline-treated animals were calculated from individual tissues tested.</p

Hepatocyte-specific deletion of Tsc1 leads to mild insulin resistance.

<p>A. Liver-specific ablation of <i>Tsc1</i>. <i>Tsc1^fl/fl</i> mice were crossed to <i>Cre^Alb</i> mice resulting in <i>Tsc1^fl/fl</i>; <i>Cre^+/+</i> (a.k.a. <i>Tsc1</i>+/+) and <i>Tsc1^fl/fl</i>; <i>Cre^Alb</i> (a.k.a. <i>Tsc1</i>−/−) littermates. Tissues from eight-week old <i>Tsc1</i>−/− and <i>Tsc1</i>+/+ animals were analyzed for the expression of Tsc1, Tsc2 and S6K by immunoblot analyses using the indicated antibodies. WAT, white adipose tissues. Note the reduced Tsc2 expression secondary to its diminished stability in the absence of Tsc1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018075#pone.0018075-Nellist1" target="_blank">[30]</a>. B. The loss of <i>Tsc1</i> in hepatocytes resulted in increased mTORC1 activity (based on the expression of phospho-S6K and phospho-S6) that was sensitive to rapamycin. <i>Tsc1</i>−/− mice were fasted and treated with or without rapamycin (2 mg/kg IP, 6 hrs). Liver lysates were analyzed by SDS-PAGE and blotted with the indicated antibodies. Note the effect of rapamycin on Akt phosphorylation in the <i>Tsc1</i>−/− liver. Actin, loading control. C. Systemic glucose tolerance (left) and insulin sensitivity (right) tests in 8-week old female (top) and male (bottom) mice. Following a 16-hr fast, glucose (1 mg/g) was given IP followed by serial blood glucose monitoring at indicated times. For insulin sensitivity test, 0.5 mU/g of insulin was injected IP after a 4-hr fast. *, p<0.05 between the <i>Tsc1</i>+/+ and <i>Tsc1</i>−/− groups. D. Fasting blood glucose and insulin levels in wild-type and mutant mice. Plasma insulin levels 30 minutes after glucose administration are also shown (filled boxes).</p

Livers with Constitutive mTORC1 Activity Resist Steatosis Independent of Feedback Suppression of Akt

<div><p>Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT and mTORC1 are key components of the insulin pathway, and play a role in promoting <i>de novo</i> lipogenesis. However, mTORC1 hyperactivity <i>per se</i> does not induce steatosis in mouse livers, but instead, protects against high-fat diet induced steatosis. Here, we investigate the <i>in vivo</i> mechanism of steatosis-resistance secondary to mTORC1 activation, with emphasis on the role of S6K1-mediated feedback inhibition of AKT. Mice with single or double deletion of <i>Tsc1</i> and/or <i>S6k1</i> in a liver-specific or whole-body manner were generated to study glucose and hepatic lipid metabolism between the ages of 6–14 weeks. Following 8 weeks of high-fat diet, the <i>Tsc1-/-;S6k1-/-</i> mice had lower body weights but higher liver TG levels compared to that of the <i>Tsc1-/-</i> mice. However, the loss of <i>S6k1</i> did not relieve feedback inhibition of Akt activity in the <i>Tsc1-/-</i> livers. To overcome Akt suppression, <i>Pten</i> was deleted in <i>Tsc1-/-</i> livers, and the resultant mice showed improved glucose tolerance compared with the <i>Tsc1-/-</i> mice. However, liver TG levels were significantly reduced in the <i>Tsc1-/-;Pten-/-</i> mice compared to the <i>Pten-/-</i> mice, which was restored with rapamycin. We found no correlation between liver TG and serum NEFA levels. Expression of lipogenic genes (<i>Srebp1c</i>, <i>Fasn</i>) were elevated in the <i>Tsc1-/-;Pten-/-</i> livers, but this was counter-balanced by an up-regulation of <i>Cpt1a</i> involved in fatty acid oxidation and the anti-oxidant protein, Nrf2. In summary, our <i>in vivo</i> models showed that mTORC1-induced resistance to steatosis was dependent on S6K1 activity, but not secondary to AKT suppression. These findings confirm that AKT and mTORC1 have opposing effects on hepatic lipid metabolism <i>in vivo</i>.</p></div