Glucose transporter 8 (GLUT8) mediates fructose-induced de novo lipogenesis and macrosteatosis

Abstract

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world, and it is thought to be the hepatic manifestation of the metabolic syndrome. Excess dietary fructose causes both metabolic syndrome and NAFLD in rodents and humans, but the pathogenic mechanisms of fructose-induced metabolic syndrome and NAFLD are poorly understood. GLUT8 (Slc2A8) is a facilitative glucose and fructose transporter that is highly expressed in liver, heart, and other oxidative tissues. We previously demonstrated that female mice lacking GLUT8 exhibit impaired first-pass hepatic fructose metabolism, suggesting that fructose transport into the hepatocyte, the primary site of fructose metabolism, is in part mediated by GLUT8. Here, we tested the hypothesis that GLUT8 is required for hepatocyte fructose uptake and for the development of fructose-induced NAFLD. We demonstrate that GLUT8 is a cell surface-localized transporter and that GLUT8 overexpression or GLUT8 shRNA-mediated gene silencing significantly induces and blocks radiolabeled fructose uptake in cultured hepatocytes. We further show diminished fructose uptake and de novo lipogenesis in fructose-challenged GLUT8-deficient hepatocytes. Finally, livers from long term high-fructose diet-fed GLUT8-deficient mice exhibited attenuated fructose-induced hepatic triglyceride and cholesterol accumulation without changes in hepatocyte insulin-stimulated Akt phosphorylation. GLUT8 is thus essential for hepatocyte fructose transport and fructose-induced macrosteatosis. Fructose delivery across the hepatocyte membrane is thus a proximal, modifiable disease mechanism that may be exploited to prevent NAFLD.

Keywords: Diabetes; Fructose Metabolism; Glucose Transport; Hepatocyte; Metabolic Syndrome.

FIGURE 1.

Glucose transporter mRNA abundance in liver and enterocytes. qRT-PCR quantification of GLUT2, GLUT5, GLUT8, and GLUT9 in primary mouse hepatocytes (A) and in isolated mouse enterocytes (B). p < 0.01 for all interactions by one-way analysis of variance and Tukey's honest significant difference post hoc correction. Data in A and B are from n = 7 wild-type mice, each run in triplicate. C, GLUT quantification in human liver tissue using RNA-seq data from the Illumina BodyMap project (version 2.0) as described under “Experimental Procedures.” Data are expressed as reads per kilobase per million mapped reads (RPKM).

FIGURE 2.

GLUT8 is a rate-limiting, cell-surface hepatic fructose transporter. A, immunofluorescence confocal microscopy in frozen liver sections derived from chow-fed, wild-type mice. Upper panels: low-power (left) and high-power (right) views of Na⁺/K⁺ ATPase α1 subunit (green), GLUT8 (red), and DNA (blue). Lower panels: left, confocal images following pre-immune primary serum and fluorescently labeled secondary antiserum, (right) transferrin receptor localization with GLUT8. B, immunofluorescence confocal microscopy in HepG2 cultures in standard growth medium (4.5 g/liter glucose). Green, Na⁺/K⁺ ATPase α1 subunit. Red, GLUT8. Blue, DNA. Scale bars, 10 μm. C, fructose uptake in primary mouse hepatocytes (middle panel) or in HepG2 cultures (right panel) transfected with adenovirus encoding green-fluorescent protein (AdGFP) or GLUT8 (AdGLUT8). **, p ≤ 0.01 versus AdGFP (n = 4–6). GLUT8 overexpression was confirmed by GLUT8 and GAPDH immunoblotting in primary hepatocytes transfected in parallel (left panel). D, fructose uptake in HepG2 cells transfected with lentivirus encoding scrambled (Scr.) or human GLUT8-directed shRNA. *, p < 0.05 versus scrambled (n = 4–6).

FIGURE 3.

GLUT8 required for fructose-induced de novo lipogenesis. A, qRT-PCR of GLUT1, GLUT2, GLUT5, and GLUT9 in WT and GLUT8KO liver after 24 weeks HFrD (n = 4–11). B, GLUT1, GLUT2, GLUT5, and GLUT9 immunoblots in WT and GLUT8KO liver after 24 weeks of HFrD. C, decreased [¹⁴C]fructose uptake in cultured primary hepatocytes from chow-fed WT and GLUT8KO mice (n = 10 per group). D and E, decreased FA and triacylglycerol (TAG) synthesis from radiolabeled fructose in cultured hepatocytes from chow-fed WT and GLUT8KO mice (n = 4 per group). F–H, relative mRNA expression of fructose-inducible hepatocyte genes, carbohydrate response element binding protein (ChREBP), glycerol-3-phosphate acyltransferase (GPAT), acetyl coenzyme A carboxylase-1 (ACC1), analyzed by real-time qRT-PCR in the presence or absence of 5 mm fructose (24 h; n = 3–9 per group). **, p < 0.01 and ***, p < 0.001 versus WT controls. ###, p < 0.001 versus WT treated with 5 mm fructose.

FIGURE 4.

Oxidative predilection without impaired proximal insulin signaling in GLUT8KO hepatocytes. Oxygen consumption (A), caloric expenditure (B), and respiratory exchange ratio (C) in chow- and HFrD-fed WT and GLUT8KO mice (n = 7–9). *, p < 0.05 versus WT on equivalent diet. ***, p < 0.001 versus WT on equivalent diet. D, palmitate oxidation rates in cultured primary WT and GLUT8KO hepatocytes (n = 5). **, p ≤ 0.01 versus WT. E, immunoblot analysis of AKT phosphorylation (Ser-473) and GAPDH after insulin stimulation (500 nm, 5 min) in isolated hepatocytes from WT and GLUT8KO mice.

FIGURE 5.

Protection from fructose-induced hepatic steatosis in GLUT8KO mice. A, gross WT and GLUT8KO livers after 10-day (10d) chow or HFrD. B, oil Red-O staining in chow- and HFrD-fed GLUT8KO and WT liver. Scale bars, 100 μm. Shown are oil Red-O staining density (C) and droplet diameters (D) in chow- and HFrD WT and GLUT8KO liver (n = 8–12 random fields from four to eight mice per group). ***, p < 0.001 versus chow-fed WT. ## and ###, < 0.01 and < 0.001 versus WT HFrD after post hoc correction, respectively.

FIGURE 6.

Fasting plasma (A–C) and hepatic (D–F) lipids in chow- and HFrD-fed (4 weeks) WT and GLUT8KO mice. n = 4–9 mice per group. **, p ≤ 0.01 and ***, p < 0.001 versus chow-fed WT. #, ##, and ###, p < 0.05, p < 0.01, and p < 0.001 versus WT HFrD after post hoc correction, respectively.