Hepatocyte-specific, PPARγ-regulated mechanisms to promote steatosis in adult mice

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   Figure 1

   Hepatocyte specificity expression of AAV8-TBGp-driven transgene. Hepatocyte-specific expression of GFP in AAV8-TBGp-EGFP-injected wild-type mice (green, A and B). GFP expression was absent in non-hepatocyte cells (yellow arrows) in sinusoids (Sn), central vein (CV), portal vein (PV), bile duct (BD) or artery (HA). TBGp-GFP was not expressed in hepatic stellate cells (HSC, desmin +, red, C) or macrophages (Mac, F4/80+, red, D). Sections were counterstained with DAPI (blue nuclei, A, B, C and D). Hepatic GFP (E) and Cre (F) expression was detected only in liver extracts of AAV8-TBGp-EGFP- and AAV8-TBGp-Cre-injected mice, respectively. To confirm the hepatocyte-specific activity of Cre recombinase, PPARγ^fl/fl mice were injected with AAV8-TBGp-Cre and expression of PPARγ was reduced in hepatic extracts but not adipose tissue (G). Ctx, cortex; eWAT, epididymal fat; Int, intestine; iWAT, inguinal fat. Asterisks indicate the difference between AAV8-TBGp-Cre-injected mice as compared to AAV8-TBGp-Null mice. *P < 0.05; ***P < 0.0001. n = 3–6 mice/group.
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   Figure 2

   Adult-onset hepatocyte-specific PPARγ knockdown (aLivPPARγkd) in chow-fed mice (A, B and C) and diet-induced obese mice (D, E and F). (A) PPARγ^fl/fl mice were injected at 10 weeks of age with 1.5 * 10¹¹ GC AAV8-TBGp-Null (C, open columns) or 1.5 * 10¹¹ GC AAV8-TBGp-Cre (Kd, shaded columns) via lateral tail vein and killed 1 or 18 weeks after. (B) Hepatic PPARγ mRNA and (C) protein expression of 1 week and 18 weeks aLivPPARγkd and their littermate control mice. Representative image of the western blot for hepatic PPARγ and Histone (as loading control). Of note, nuclear protein variability within total hepatic extracts (assessed by Histone) alters the amount of PPARγ protein detected. n = 4–5 mice/group. (D) LF-fed PPARγ^fl/fl mice were injected at 10 weeks as described above and immediately half of the mice were fed a high-fat diet (HF, 60% kcal from fat) to induce liver steatosis, whereas the rest were maintained on a low-fat diet (LF, 10% kcal from fat). Mice were killed 14 weeks after. (E) Hepatic PPARγ mRNA and (F) protein expression of 14 weeks LF- and HF-fed aLivPPARγkd and their littermate controls. Representative images of the western blot for hepatic PPARγ and Histone (as loading control). Asterisks indicate the differences between C and Kd. *P < 0.05; ***P < 0.0001. Letters indicate the differences between 1 week and 18 weeks or LF- and HF-fed mice within group. ^bP < 0.01; ^cP < 0.0001. n = 5–6 mice/group.
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   Figure 3

   Impact of aLivPPARγkd on hepatic gene expression of molecular mechanisms controlling hepatic TAG levels. (A) 1-week (top) and 18-week (bottom) chow-fed and (B) 14-week LF-fed (top) and HF-fed (bottom) aLivPPARγkd-induced regulation of hepatic gene expression. Graphs represent the natural logarithm of the relative change in the gene expression of aLivPPARγkd mice as compared to their littermates controls (set at 0, x-axis), within age (A) or diet (B). Absolute values are included in Supplementary Table 1A and B. Asterisks show significant changes between C and aLivPPARγkd within age (A) or diet (B). *P < 0.05; **P < 0.01; ***P < 0.0001. Livers were collected at 11:00 h (4 h after food removal). DNL, de novo lipogenesis; FA_ox, fatty acid oxidation; TAG_hydr, TAG hydrolysis; VLDL_syn, VLDL synthesis; TAG_syn, TAG synthesis. Selected genes of these metabolic pathways represented in Fig. 3: peroxisome proliferator-activated receptor α (PPARα), acyl-CoA synthetase long-chain family member 1 (Acsl1), carnitine palmitoyltransferase 1α (Cpt1α), hepatic nuclear factor 4 α (Hnf4α), PPARγ co-activator 1 α (Pgc1α), Cyp4a10, adipose triglyceride lipase (Atgl), hormone-sensitive lipase (Hsl), monoacylglycerol lipase (Mgll), apolipoprotein B (ApoB), microsomal triglyceride transfer protein (Mttp), sterol response element-binding protein 1c (Srebp1c), acetyl-CoA carboxylase 1 (Acc1), fatty acid synthase (Fasn), fatty acid elongase (Elovl6), stearoyl–CoA desaturase 1 (Scd1), hepatic lipase (Hl), low-density lipoprotein lipase receptor (Ldlr), very-low-density lipoprotein receptor (Vldlr), lipoprotein-related protein 1 (Lrp1), fatty acid translocase (Cd36), glycerol phosphate acyltransferase (Gpat1), monoacylglycerol acyltransferase 1 or 2 (Mogat1/2), diacylglycerolacyltransferase 1/2 (Dgat1/2).
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   Figure 4

   aLivPPARγkd reduces HF diet-induced FA levels, but has little impact on FA indices of DNL. (A) Hepatic FA levels of 16:0, palmitate; 16:1, palmitoleate; and 18:2, linoleate levels and (B) hepatic FA ratios indicative of DNL include the SCD index (16:1/16:0) and the DNL index (16:0/18:2), where control (open columns, C) and aLivPPARγkd (closed columns, Kd) mice were fed a LF or a HF diet for 14 weeks. Asterisks indicate differences between C and Kd. ***P < 0.0001. Letters indicate differences between LF- and HF-fed mice within group. ^aP < 0.05; ^cP < 0.0001. n = 4–6 mice/group.
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   Figure 5

   aLivPPARγkd reduces HF diet-induced hepatic TAG and DAG levels, whereas increasing MAG levels, independent of diet, indicative of impaired hepatic MAG pathway activity in aLivPPARγkd mice. (A) Schematic representation of acylglycerol synthesis by glycerol-3-phosphate (G3P) pathway that produces DAG by subsequent re-esterification of FA in G3P and lysophosphatidic acid (LPA) or by monoacylglycerol (MAG) pathway that produces DAG after re-esterification of FA in MAG. (B) Relative hepatic TAG, DAG and MAG levels assessed by liquid chromatography/mass spectrometry (LC/MS) in control (open columns) and aLivPPARγkd (close columns) mice. TAG, DAG and MAG are shown as relative values of LF-fed controls. Asterisks indicate differences between control and aLivPPARγkd. ***P < 0.0001. Letters indicate differences between LF- and HF-fed mice within group. ^bP < 0.01; ^cP < 0.0001. n = 5–6 mice/group.

• © 2017 Society for Endocrinology