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. 2016 Oct 13;36(21):2715-2727.
doi: 10.1128/MCB.00138-16. Print 2016 Nov 1.

Hepatic Overexpression of CD36 Improves Glycogen Homeostasis and Attenuates High-Fat Diet-Induced Hepatic Steatosis and Insulin Resistance

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Free PMC article

Hepatic Overexpression of CD36 Improves Glycogen Homeostasis and Attenuates High-Fat Diet-Induced Hepatic Steatosis and Insulin Resistance

Wojciech G Garbacz et al. Mol Cell Biol. .
Free PMC article

Abstract

The common complications in obesity and type 2 diabetes include hepatic steatosis and disruption of glucose-glycogen homeostasis, leading to hyperglycemia. Fatty acid translocase (FAT/CD36), whose expression is inducible in obesity, is known for its function in fatty acid uptake. Previous work by us and others suggested that CD36 plays an important role in hepatic lipid homeostasis, but the results have been conflicting and the mechanisms were not well understood. In this study, by using CD36-overexpressing transgenic (CD36Tg) mice, we uncovered a surprising function of CD36 in regulating glycogen homeostasis. Overexpression of CD36 promoted glycogen synthesis, and as a result, CD36Tg mice were protected from fasting hypoglycemia. When challenged with a high-fat diet (HFD), CD36Tg mice showed unexpected attenuation of hepatic steatosis, increased very low-density lipoprotein (VLDL) secretion, and improved glucose tolerance and insulin sensitivity. The HFD-fed CD36Tg mice also showed decreased levels of proinflammatory hepatic prostaglandins and 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictive and proinflammatory arachidonic acid metabolite. We propose that CD36 functions as a protective metabolic sensor in the liver under lipid overload and metabolic stress. CD36 may be explored as a valuable therapeutic target for the management of metabolic syndrome.

Figures

FIG 1
FIG 1
Creation of CD36Tg mice that overexpress CD36 in the liver. (A) Tet-off transgenic system to express CD36 in the liver. The open X indicates the silence of transgenic CD36 expression. (B and C) The mRNA expression of endogenous and transgenic CD36 (B) and transgenic CD36 (C) was detected by Northern blotting and real-time PCR, respectively. The Northern blotting probe detected both endogenous and transgenic CD36, whereas the real-time PCR specifically detected transgenic CD36. (D) Protein expression of the transgene and its silencing by Dox were measured by Western blotting. (E) Immunohistochemical staining of CD36. (F) Uptake of the fluorescent fatty acid analogue BODIPY-C16 by hepatocytes isolated from WT mice and CD36Tg mice treated or not treated with Dox. (G) Fluorometric quantification of BODIPY-C16 uptake. All the mice were maintained on a chow diet. *, P < 0.05; **, P < 0.01. The data are presented as means and SEM.
FIG 2
FIG 2
Overexpression of CD36 attenuated the fasting-induced steatosis, hypoglycemia, and depletion of hepatic glycogen. (A to D) Mice maintained on a chow diet were subjected to 6 h and 16 h of fasting before measurement of liver triglyceride levels (A), blood glucose (B), liver glycogen levels (C), and liver PAS staining (D). n = 4 for all groups. (E) Insulin- and glucose-stimulated glycogen synthesis rates in primary mouse hepatocytes. (F) Incorporation of [3H]glucose into hepatic glycogen during the euglycemic-hyperinsulinemic clamp. WT, n = 7; CD36Tg, n = 6. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, statistically not significant. The data are presented as means and SEM.
FIG 3
FIG 3
CD36Tg mice were protected from HFD-induced hepatic steatosis. (A) Gross appearance (top) and histology (bottom) (H&E and Oil Red O [ORO] staining) of livers of mice maintained on a chow diet (left) and mice that had been fed an HFD for 19 weeks (right). (B) Hepatic triglyceride levels in mice maintained on a chow diet and mice that had been fed an HFD for 19 weeks in the absence or presence of Dox. n = 5. (C) Expression of liver CD36 as measured by Western blotting. Densitometric quantifications of the blots are shown below. (D) The hepatic expression of genes involved in lipogenesis and fatty acid oxidation in HFD-fed mice was measured by real-time PCR. n = 5. (E) VLDL-triglyceride (TG) secretion rate in HFD-fed WT, CD36Tg, and CD36Tg-plus-Dox mice. n = 4. (F) The serum levels of ApoB100 and ApoB48 were measured by Western blotting. The serum samples were run on two parallel gels; one was used for Coomassie blue staining as a loading control, and the second gel was used for ApoB100/48 Western blotting. The densitometric intensity was normalized to the Coomassie blue staining. The densitometric scores for individual lanes are shown. On the right is shown statistical analysis of the densitometric quantification. *, P < 0.05; **, P < 0.01; n.s., not statistically significant. The data are presented as means and SEM.
FIG 4
FIG 4
CD36 overexpression improved glucose homeostasis and increased oxygen consumption. All the mice were fed an HFD for 19 weeks. (A to C) HFD-responsive dynamics of body weight gain (A), fat mass gain as measured by magnetic resonance imaging (MRI) (B), and food intake (C) in WT mice and CD36Tg mice treated or not treated with Dox. n = 7 to 9. (D) Oxygen consumption throughout the light-dark cycle. n = 4. (E to G) Metabolic cage analysis of RER (E), locomotive movement (F), and heat generated (G). n = 4. (H and I) GTT (H) and ITT (I) in WT mice and CD36Tg mice treated or not with Dox. The area under the curve (AUC) was used to quantify the GTT results. n = 5. * and #, P < 0.05; ** and ##, P < 0.01; n.s., not statistically significant. The asterisks and hash tags are comparisons between WT and CD36Tg and between CD36Tg and CD36Tg-plus-Dox mice, respectively. The data are presented as means and SEM.
FIG 5
FIG 5
Hepatic overexpression of CD36 attenuated HFD-induced insulin resistance. The mice are the same as those described in the legend to Fig. 4. (A to C) Euglycemic-hyperinsulinemic clamp measurements of glucose infusion rate (A) endogenous glucose production (B), and glucose disposal rate (C) in HFD-fed WT mice and CD36Tg mice. n = 6 or 7. (D) Basal and insulin-stimulated Akt phosphorylation in liver and skeletal muscle as measured by Western blotting. Shown on the right are densitometric quantifications of the blots. When necessary, mice were injected i.p. with insulin (0.75 U/kg) 17 min before tissue harvesting. (E) Hepatic mRNA expression of gluconeogenic genes. n = 6. (F to I) Triglyceride level (F), expression of genes involved in lipogenesis and fatty acid oxidation (G), expression of the glucose transporter GLUT4 and LPL (H), and glycogen content (I) in skeletal muscle. n = 6. (J) WAT mRNA expression of genes indicative of adipocyte remodeling and metabolism. n = 6. *, P < 0.05; **, P < 0.01; n.s., statistically not significant. The data are presented as means and SEM.
FIG 6
FIG 6
Molecular mechanism for promoting glycogen synthesis by CD36. WT, CD36Tg, and CD36Tg-plus-Dox mice maintained on a chow diet were subjected to 6 h and 16 h of fasting. (A) The hepatic mRNA expression of glycogenin, GS, and glycogen phosphorylase genes was measured by real-time PCR. (B) The protein expression of GP, total and Ser641-phosphorylated GS, total and Ser9-phosphorylated GSK3β, and total and phosphorylated AMPKα was measured by Western blotting. (C) Densitometric quantification of GP and GS-P/GS expression. (D) mRNA expression of PP1 catalytic subunits (PPP1Cs). (E) Protein expression of PPP1Cγ as measured by Western blotting. Shown on the right are densitometric quantifications of the blots. (F) mRNA expression of PPP1R3B, PPP1R3C, and PPP1R3G regulatory glycogen targeting subunits of PP1. n = 4 for all groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., statistically not significant. The data are presented as means and SEM.
FIG 7
FIG 7
Overexpression of CD36 affected arachidonic acid metabolism and decreased hepatic levels of prostaglandins and 20-HETE. All the mice were fed an HFD for 19 weeks, and the CD36Tg mice were treated or not treated with Dox. (A) Hepatic prostaglandin levels. (B) Hepatic arachidonic acid levels. (C) Hepatic expression of Cox-1 and Cox-2. (D) Hepatic 20-HETE level. (E) Hepatic expression of 20-HETE-producing Cyp4a/Cyp4f enzymes. n = 6 for all groups. *, P < 0.05; **, P < 0.01; n.s., not statistically significant. The data are presented as means and SEM.

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