Pleiotropic actions of IP6K1 mediate hepatic metabolic dysfunction to promote nonalcoholic fatty liver disease and steatohepatitis

Abstract

Objective: Obesity and insulin resistance greatly increase the risk of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). We have previously discovered that whole-body and adipocyte-specific Ip6k1deletion protects mice from high-fat-diet-induced obesity and insulin resistance due to improved adipocyte thermogenesis and insulin signaling. Here, we aimed to determine the impact of hepatocyte-specific and whole-body Ip6k1 deletion (HKO and Ip6k1-KO or KO) on liver metabolism and NAFLD/NASH.

Methods: Body weight and composition; energy expenditure; glycemic profiles; and serum and liver metabolic, inflammatory, fibrotic and toxicity parameters were assessed in mice fed Western and high-fructose diet (HFrD) (WD: 40% kcal fat, 1.25% cholesterol, no added choline and HFrD: 60% kcal fructose). Mitochondrial oxidative capacity was evaluated in isolated hepatocytes. RNA-Seq was performed in liver samples. Livers from human NASH patients were analyzed by immunoblotting and mass spectrometry.

Results: HKO mice displayed increased hepatocyte mitochondrial oxidative capacity and improved insulin sensitivity but were not resistant to body weight gain. Improved hepatocyte metabolism partially protected HKO mice from NAFLD/NASH. In contrast, enhanced whole-body metabolism and reduced body fat accumulation significantly protected whole-body Ip6k1-KO mice from NAFLD/NASH. Mitochondrial oxidative pathways were upregulated, whereas gluconeogenic and fibrogenic pathways were downregulated in Ip6k1-KO livers. Furthermore, IP6K1 was upregulated in human NASH livers and interacted with the enzyme O-GlcNAcase that reduces protein O-GlcNAcylation. Protein O-GlcNAcylation was found to be reduced in Ip6k1-KO and HKO mouse livers.

Conclusion: Pleiotropic actions of IP6K1 in the liver and other metabolic tissues mediate hepatic metabolic dysfunction and NAFLD/NASH, and thus IP6K1 deletion may be a potential treatment target for this disease.

Keywords: Hyperglycemia; IP6K1; Liver; Metabolism; NAFLD/NASH; O-GlcNAcylation.

Graphical abstract

Figure 1

Hepatocyte-specific Ip6k1 deletion (HKO) protected mice from Western diet-induced hyperglycemia, hepatic steatosis, and fibrosis. A. Ip6k1 was deleted in the liver but not in the EWAT, IWAT, or skeletal muscle of HKO mice. Data obtained from young, chow-fed mice. B. HKO mice displayed improved glucose disposal in an insulin tolerance test (ITT). C. Glucose-induced peak blood glucose levels were reduced, and its disposal was increased in HKO mice (glucose tolerance test, GTT). D. HKO mice displayed reduced peak blood glucose levels and increased disposal of glucose in a pyruvate tolerance test (PTT). E. Glucagon injection increased blood glucose levels to a lesser extent in HKO mice (glucagon tolerance test, GgTT). F. Liver weight was reduced in HKO mice. G–H. Steatosis and TAG were reduced in HKO mouse livers. Representative H&E staining images of 8 mice (n = 8). I–J. Serum TAG, AST, and ALT levels were reduced in HKO mouse livers. K–L. HKO mice displayed reduced fibrosis compared with Lox mice. Images are examples from different mice. For comparison of densitometry, the mean value of Lox was set as 1 (n = 10 per cohort). Number of mice (n) used in each experiment are presented as individual datapoints. WD-fed male mice were used unless otherwise indicated. Data are presented as mean ± SEM within dot plots. For multiple comparisons, two-way ANOVA with the Holm-Šidák multiple comparison test was used, and for two independent data sets, two-tailed unpaired Student's t-test was used. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 2

Hepatocyte-specific Ip6k1 (HKO) deletion increased mitochondrial oxidative capacity of hepatocytes but did not alter body weight or energy expenditure in mice. A–C. Hepatic levels of the mitochondrial oxidative phosphorylation proteins (NDUFB8, SDHB, UQCRC2, MTCO1, and ATP5A for complexes I–V, respectively, and UCP2) were augmented in HKO mice. D–E. Hepatocytes isolated from chow-fed HKO mice displayed increased OCR in vitro due to mitochondrial uncoupling (n = 5 experimental replicates). F. UCP2 protein level was increased in HKO hepatocytes used in Figure 2D. G–H. Body weight of Lox and HKO mice were similar at the beginning and end of WD diet feeding and intraabdominal adipose tissue stores in WD-fed mice appeared similar. I. Whole body energy expenditure (EE) was similar in Lox and HKO mice. Number of mice (n) used in each experiment are presented as individual datapoints. WD-fed male mice were used unless otherwise indicated. Data are presented as mean ± SEM within dot plots. Hepatocytes were isolated from young, chow-fed male mice. Two-tailed unpaired Student's t-test. ∗P < 0.05, ∗∗P < 0.01.

Figure 3

Whole-body deletion of Ip6k1 (KO) reduced body weight and robustly protected mice from Western diet-induced hyperglycemia, hepatic steatosis, liver injury, and fibrosis. A–D.Ip6k1-KO males displayed reduced body and fat weight and had darker, normal-appearing livers compared with the pale enlarged livers of WT mice. NMR analysis showed a reduction in percent body fat but unaltered lean and fluid masses in the knockouts. E–G.Ip6k1-KO male mice displayed reduced weight and fat accumulation in the liver. H. Macro- and micro-steatosis, hepatocyte hypertrophy, and inflammatory foci were reduced in Ip6k1-KO mice. NAFLD features were quantified from the H&E slides (n = 4 mice per cohort). I. The lipogenic ACC and FAS proteins were reduced in Ip6k1-KO livers. J. TLR4 (normalized to GAPDH in Fig. 3I) and stimulatory phosphorylation of JNK and NFκB were reduced in Ip6k1-KO mouse livers. K–L. Serum TAG and LDL were reduced in Ip6k1-KO mice. M–N.Ip6k1-KO mouse livers were robustly protected from fibrosis. For comparison, the mean value of WT was set as 1 (n = 6 per cohort). Number of mice (n) used in each experiment are presented as individual datapoints. WD-fed male mice were evaluated. Data are presented as mean ± SEM within dot plots. For multiple comparisons, two-way ANOVA with the Holm-Šidák multiple comparison test was used, and for two independent data sets, two-tailed unpaired Student's t-test was used. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 4

IP6K1 regulates various target proteins and pathways that modulate liver metabolism and fibrosis. A. Col1a1, α-SMA, and desmin were reduced in Ip6k1-KO mouse livers. For ImageJ analysis of Col1a1, all three Col1a1 bands were compared between WT and Ip6k1-KO samples. B. Integrin β1 and stimulatory phosphorylation of FAK (Y397) were reduced in Ip6k1-KO mouse livers. C.Ip6k1 depletion (siRNA) reduced α-SMA protein levels in TGFβ1-treated LX-2 cells. D. TNP treatment reduced mRNA expression of Acta2 in TGFβ1-treated LX-2 cells. E. TNP treatment impaired TGFβ1-induced mRNA expression of Col1a1 in LX-2 cells. F–G. IP6K1 but not IP6K2 was upregulated in the livers of human NASH and alcoholic cirrhosis patients. α-SMA was used as a marker of profibrogenic stellate cell activation in NASH and cirrhosis. ImageJ quantification was used to compare the expression of proteins in normal vs NASH and cirrhosis livers. H–I. Levels of protein O-GlcNAcylation were increased in human NASH and cirrhosis livers. For quantification, ImageJ analysis was performed on the most prominent O-GlcNAcylated bands (indicated by arrows) using β-actin as a control. J–M. Protein O-GlcNAcylation was reduced in WD-fed Ip6k1-KO and HKO mice compared with control mouse livers. For quantification, ImageJ analysis was performed on two O-GlcNAcylated bands that were the most prominent (indicated by arrows). N. Glucose-induced protein O-GlcNAcylation was reduced in the chow-fed HKO mouse liver. O. Model showing pleiotropic mechanisms by which IP6K1 promotes the development of NAFLD/NASH. Chronic unhealthy diets stimulate IP6K1-mediated inactivation of AMPK and activation of protein O-GlcNAcylation and integrin signaling in the liver. Consequently, mitochondrial oxidative capacity and insulin signaling are impaired, whereas HSCs are activated to a fibrogenic phenotype. IP6K1 also facilitates adipose tissue dysfunction, which causes inappropriate fatty acid release with subsequent uptake in the liver contributing to lipotoxic injury. The net effect is a phenotype of NASH and liver fibrosis. Solid and dotted red arrows denote current and previous discoveries from our group. Back arrows represent information obtained from the literature. Each experiment is presented as individual datapoints. Data are presented as mean ± SEM in dot plots. For multiple comparisons, two-way ANOVA with the Holm-Šidák multiple comparison test was used, and for two independent data sets, two-tailed unpaired Student's t-test was used. ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

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