Adult-Onset Hepatocyte GH Resistance Promotes NASH in Male Mice, Without Severe Systemic Metabolic Dysfunction

Abstract

Nonalcoholic fatty liver disease (NAFLD), which includes nonalcoholic steatohepatitis (NASH), is associated with reduced GH input/signaling, and GH therapy is effective in the reduction/resolution of NAFLD/NASH in selected patient populations. Our laboratory has focused on isolating the direct vs indirect effects of GH in preventing NAFLD/NASH. We reported that chow-fed, adult-onset, hepatocyte-specific, GH receptor knockdown (aHepGHRkd) mice rapidly (within 7 days) develop steatosis associated with increased hepatic de novo lipogenesis (DNL), independent of changes in systemic metabolic function. In this study, we report that 6 months after induction of aHepGHRkd early signs of NASH develop, which include hepatocyte ballooning, inflammation, signs of mild fibrosis, and elevated plasma alanine aminotransferase. These changes occur in the presence of enhanced systemic lipid utilization, without evidence of white adipose tissue lipolysis, indicating that the liver injury that develops after aHepGHRkd is due to hepatocyte-specific loss of GH signaling and not due to secondary defects in systemic metabolic function. Specifically, enhanced hepatic DNL is sustained with age in aHepGHRkd mice, associated with increased hepatic markers of lipid uptake/re-esterification. Because hepatic DNL is a hallmark of NAFLD/NASH, these studies suggest that enhancing hepatocyte GH signaling could represent an effective therapeutic target to reduce DNL and treat NASH.

Figure 1.

aHepGHRkd rapidly (1 wk) leads to steatosis, which progresses to NASH (27 and 55 wk after AAV8 treatment) in chow-fed mice. Hepatic (A) Ghr, (B) Igf1, and (C) Socs2 gene expression. (D) Body weight (BW), (E) liver weight, and (F) TG content. (G) Representative images of hematoxylin and eosin–stained liver sections (scale bars, 50 μm; 20× magnification). Hepatic (H) Tnfα and (I) Tgfβ1 expression. (J) Plasma ALT levels. Open columns represent control mice; filled columns represent aHepGHRkd mice. Data are represented as means ± SEM of four different age groups, that is, 1, 9, 27, and 55 wk after administration of AAV8 vectors. Gene expression is represented as mRNA copy number normalized with a normalization factor (NF) calculated from the expression of three housekeeping genes, Ppia, β-actin, and Hprt. n = 4 to 10 mice per group. Asterisks indicate significant difference between control and aHepGHRkd mice within age group: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2.

aHepGHRkd mice (27 wk after AAV8 treatment) show early signs of fibrosis and oxidative stress. Hepatic (A) Col1a1 and (B) αSma expression. Hepatic (C) MDA levels and (D) ratio of phosphorylated NF-κB/NF-κB. (E) Western blot of phosphorylated NF-κB/NF-κB. Open columns indicate control mice (C); filled columns indicate aHepGHRkd mice (Kd). Data are represented as means ± SEM. Gene expression is shown as mRNA copy number divided by a normalization factor (NF) calculated from the expression levels of three housekeeping genes, Ppia, β-actin, and Hprt. n = 9 to 10 mice per group. Asterisks indicate significant difference between control and aHepGHRkd mice within age group: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

aHepGHRkd mice (9 to 32 wk after AAV8 treatment) show elevated plasma GH and insulin levels, but normal glucose and insulin tolerance tests and circulating NEFA. Basal levels (4 h after food removal at 0700 h) of (A) plasma GH, (B) plasma insulin, and (C) blood glucose from mice 9, 27, or 32 wk after aHepGHRkd induction. (D) Plasma GH levels in lateral vein blood samples obtained prior to (1100 h) and after BODIPY-C16 injections (at 1200, 1400, and 1600 h). (E) Glucose tolerance tests (2 g/kg, IP). (F) Insulin tolerance tests (1.5 U/kg, IP). (G and H) Plasma NEFA levels in (G) trunk blood after 4-h food withdrawal starting at 0700 h and from (H) lateral tail vein blood obtained at just prior to lights out (1700 h, pre-fast), after overnight fasting (0900 h, fasted), and after 6-h refeeding with a chow diet (1500 h, refed). Open columns and filled circles with solid lines indicate control mice (C); filled columns and open squares with discontinuous line indicate aHepGHRkd mice (Kd). Data are represented as means ± SEM of groups of mice used at different times after administration of AAV8 vectors. n = 7 to 11 mice per group. Asterisks indicate significant difference between control and aHepGHRkd mice within age group: *P < 0.05; ***P < 0.001. Different letters in (H) indicate significant difference (P < 0.05) between the fed state within group.

Figure 4.

aHepGHRkd mice (32 wk after AAV8 treatment) did not show evidence of increased WAT lipolysis. Ex vivo lipolysis of (A) urogenital fat (UG-fat) and (B) subcutaneous fat (inguinal, SC-fat). Ratio of pHSL/total HSL in (C) UG-fat and (D) SC-fat, with representative Western blots included above graphs. Open columns indicate control mice (C); filled columns indicate aHepGHRkd mice (Kd). Data are represented as means ± SEM. n = 7 mice per group. Asterisks indicate significant difference between vehicle (Veh)-treated and isoproterenol (1µM Iso)-stimulated WAT explants within group: ***P < 0.001; ****P < 0.001.

Figure 5.

aHepGHRkd mice show fat depot–selective reduction in weight associated with increased systemic lipid utilization and tissue-specific FA uptake. (A) Fat depot weight of 1- to 55-wk aHepGHRkd mice. (B) Respiratory quotient (RQ) of aHepGHRkd mice 27 wk after AAV injection. Night period (1900 to 0700 h) is indicated by gray background. (C) Average of day and night RQ. (D) Percentage of BODIPY-C16 uptake in different tissues of 32-wk aHepGHRkd mice, where controls were set at 100%. Open columns and filled circles with solid line indicate control mice; filled columns and open squares with discontinuous line indicate aHepGHRkd mice. Data are represented as means ± SEM. n = 5 to 7 mice per group. Asterisks indicate significant difference between control and aHepGHRkd: *P < 0.05; **P < 0.01; ***P < 0.001. Letters indicate significant different between day and night values within group: b, P < 0.01; c, P < 0.001. BAT, brown adipose tissue; GAS, gastrocnemius; HRT, heart; LIV, liver; nm, not measured; PR, perirenal/retroperitoneal fat; SC, subcutaneous fat; SOL, soleus; UG, urogenital fat.

Figure 6.

aHepGHRkd mice (1 to 55 wk after AAV8 treatment) show increased expression of genes important for DNL and lipid uptake/esterification. Hepatic (A) Gck, (B) Khk, (C) Acc1, (D) Fasn, (E) Scd1, (F) Pparγ, (G) Cd36, and (H) Mogat1 gene expression. Open columns indicate control mice; filled columns indicate aHepGHRkd mice. Data are represented as means ± SEM of four different age groups, that is, 1, 9, 27, and 55 wk after administration of AAV8 vectors. Gene expression is represented as mRNA copy number divided by a normalization factor (NF) calculated from the expression level of three housekeeping genes, Ppia, β-actin, and Hprt. n = 3 to 10 mice per group. Asterisks indicate significant difference between control and aHepGHRkd within group: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 7.

aHepGHRkd mice (1 to 55 wk after AAV8 treatment) show changes in hepatic FA composition that are indicative of enhanced DNL. Absolute levels of hepatic (A) 16:0, (B) 16:1(n-6), (C) 18:0, (D) 18:1(n-9), and (E) 18:2(n-6). Hepatic (F) SCD index [16:1(n-7)/16:0], (G) SCD index [18:1(n-9)/18:0], and (H) DNL index [16:0/18:2 (n-6)]. Relative hepatic levels (individual FA percentage of total FA) of (I) 16:0, (J) 16:1(n-7), (K) 18:0, (L) 18:1(n-9), and (M) 18:2(n-6). As illustrated in the inset diagram, FAs can be generated de novo from glucose oxidation that is incorporated in the tricarboxylic cycle (TCA cycle). The newly synthetized saturated FAs (16:0 and 18:0) are then desaturated by SCD and subsequently esterified in TGs. SCD activity is closely related with DNL activity in hepatocytes. FAs can also come from extrahepatic sources, where 18:2(n-6), an FA that cannot be synthetized in animal cells (essential FA), is used as an indicator of extrahepatic FA uptake. Therefore, an increase in the ratio of 16:0/18:2(n-6) is also used as an indicator of DNL. Open columns indicate control mice; filled columns indicate aHepGHRkd mice. Data are represented as means ± SEM of four different age groups, that is, 1, 9, 27, and 55 wk after administration of AAV8 vectors. Individual FAs were quantified using an internal standard (13,16,19–docosatrienoic acid; 22:3). n = 4 to 9 mice per group. Asterisks indicate significant difference between control and aHepGHRkd within group: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.