Growth Hormone Control of Hepatic Lipid Metabolism

Page 1 of 31 Diabetes

Growth hormone control of hepatic lipid metabolism

Zhongbo Liu1, Jose Cordoba-Chacon2,3, Rhonda D Kineman2,3, Bruce N Cronstein4, Radhika Muzumdar5, Zhenwei Gong5, Haim Werner6, and Shoshana Yakar1

1. David B. Kriser Dental Center, Department of Basic Science and Craniofacial Biology New York University College of Dentistry New York, NY 10010-4086 2. Jesse Brown VA Medical Center, Research and Development, Chicago IL 60612 3. Department of Medicine, Section of Endocrinology, Diabetes and Metabolism, University of Illinois Chicago, IL 60612 4. Department of Medicine, New York University School of Medicine, New York 10016 5. Division of Endocrinology and Diabetes, Children’s Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine 6. Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Israel

Running title: Liver GH resistance

Key words: Growth hormone receptor (GHR), insulin-like growth factor-1 (IGF-1), liver, steatosis, lipid metabolism, de novo lipogenesis, oxidative stress, inflammation

Correspondence: Shoshana Yakar, Ph.D. Associate Professor New York University College of Dentistry David B. Kriser Dental Center Department of Basic Science and Craniofacial Biology 345 East 24th Street New York, New York 10010-4086 Tel : 212-998-9721 [email protected]

Diabetes Publish Ahead of Print, published online September 27, 2016 Diabetes Page 2 of 31

Abstract In humans low levels of growth hormone (GH) and its mediator, insulin-like growth factor-1 (IGF-1), associate with hepatic lipid accumulation. In mice, congenital liver-specific ablation of the GH receptor (GHR) results in reductions in circulating IGF-1 and hepatic steatosis, associated with systemic insulin-resistance. Due to the intricate relationship between GH and IGF-1, the relative contribution of each hormone to the development of hepatic steatosis is unclear. Our goal was to dissect the mechanisms by which hepatic GH resistance leads to steatosis and overall insulin resistance, independent of IGF-1. We have generated a combined mouse model with liver-specific ablation of GHR in which we restored liver IGF-1 expression via hepatic IGF-1 transgene. We found that liver- GHR ablation leads to increases in lipid uptake, de novo lipogenesis, hyperinsulinemia and hyperglycemia accompanied with severe insulin resistance, and increased body adiposity and serum lipids. Restoration of IGF-1 improved overall insulin sensitivity, lipid profile in serum, reduced body adiposity, but was insufficient to protect against steatosis-induced hepatic inflammation or oxidative stress. We conclude that the impaired metabolism in states of GH resistance results from direct actions of GH on lipid uptake and de novo lipogenesis, while its actions on extrahepatic tissues are mediated by IGF-1.

Page 3 of 31 Diabetes

INTRODUCTION According to Browning et al [1] non-alcoholic fatty liver disease (NAFLD) affects almost one third of the adult population in North America. Low levels of growth hormone (GH) in the general population associate with NAFLD [2]. Multi single nucleotide polymorphism (SNP) analyses of genome-wide association study (GWAS) have revealed 18 SNPs in the GH pathway that relate to the development and progression of NAFLD [3]. Also, patients with GH receptor (GHR) loss of function (Laron’s syndrome) exhibit NAFLD [4]. Likewise, cessation of GH treatment in GH deficient (GHD) children after achieving adult height leads to development of NAFLD and dyslipidemia in 29% of patients surveyed 10 years after therapy [2]. Importantly, reductions in circulating GH [2, 5] or its mediator, the insulin-like growth factor-1 (IGF-1) [6-8], associate with NAFLD even after adjusting to body mass index (BMI) [9]. Obese patients manifest GH resistance and can show declines in GH [10, 11], that may be as low as those observed in GHD subjects [12, 13]. GH therapy reduces fat in young men with abdominal obesity [14], patients with primary GHD [5, 15, 16], and hepatosteatosis in patients with HIV lipodystrophy [17]. On the other hand, humans treated with the GHR antagonist (pegvisomant and somatostatin analogs) to control for endogenous GH levels [18], show increased hepatic triglyceride content.

In rodents, diet-induced fatty liver associates with reduced circulating GH [19, 20] and reduced signal transducer and activator of transcription-5 (STAT5) phosphorylation in response to GH stimulation [21]. Liver-specific deletion of GHR in mice results in a marked decrease in serum IGF-1 levels, significant increases in fat mass and serum lipids, and severe hepatic steatosis, associated with systemic insulin resistance [22]. A similar metabolic phenotype is observed in mice with liver-specific disruption of the GHR mediators, Janus kinase-2 (JAK2) [23], or STAT5 [24]. GH treatment in mice reduced diet induced hepatosteatosis [21, 25] and overexpression of a GHR antagonist increased hepatic steatosis [26].

Clearly, clinical and experimental data indicate that reductions in circulating GH levels or hepatic response to GH are a typical component of NAFLD. Based on the above evidence, a clinical trial is currently underway to test the effectiveness of GH treatment in reducing hepatic lipid content in NAFLD patients (NCT02217345).

Addressing the molecular mechanisms by which GH regulates hepatic lipid metabolism is challenging; models with ablation of GHR action in liver, show significantly reduced serum IGF-1 levels accompanied with high GH levels, that affect carbohydrate and lipid metabolism in extrahepatic tissues. Thus, the direct roles of hepatic GH in lipid and carbohydrate metabolism are not known. We hypothesized that hepatic GHR regulates lipid metabolism independent of IGF-1, and that reductions Diabetes Page 4 of 31 in hepatic GHR signaling are not merely a consequence of NAFLD but a contributor to hepatic lipid accumulation in NAFLD. To address our hypothesis we generated a mouse model with hepatic ablation of the GHR (Li-GHRKO) and restored IGF-1 gene expression via hepatic IGF-1 transgene (HIT). The Li-GHRKO-HIT mice show normal levels of serum IGF-1 and exhibit hepatic GH resistance.

METHODS Animals: The generations of hepatic igf-1 transgenic (HIT) mice [27] and the floxed ghr mice [28] were previously described. Gene inactivation of the ghr in liver was achieved by the cre/lox-P system, as described by us previously [29]. All mice were in the C57BL/6J genetic background. Weaned mice were allocated randomly into cages separated according to their sex. Mice were housed 2–5 animals/cage in a facility with 12-h light:dark cycles and free access to food/water. The different analyses were performed in male mice at the indicated ages.

All animal procedures were approved by the Institutional Animal Care and Use Committee of the NYU School of Medicine (assurance number A3435-01, USDA licensed No. 465).

Serum hormones: Serum/plasma were collected via orbital bleeding immediately after euthanasia with CO2 between 8 and 10 AM. Hormones were measured by ELISA; GH (Millipore Corporation, Cat# EZRMGH-45K), IGF-1 (ALPCO, CAT# 22-IG1MS-E01), leptin (Millipore Corporation, Cat# EZRL-83K), insulin (Mercodia Inc NC9440604), IGFBP-3 (Thermo Fisher, CAT# EMIGFBP3), and cytokines (Meso Scale Discovery, Cat# K152A0H-1), serum IGFBP-1 (CELLAPLICATION INC Cat#CL0383) and IGFBP-2 (INNOVATIVE RESEARCH INC. Cat#IRKTAH5375). Serum ALS levels were determined by Western immunoblotting (R&D, CAT# AF1436).

Serum and tissue lipids: free fatty acids (FFAs) were measured using calorimetric assay (Roche, Cat#11383175001). Liver triglyceride (TG) content was assessed following chloroform-methanol extraction and quantified using the TG reagent (PointeScientific, Cat# T7531). Serum samples were sent to AntechDiagnostics Inc, New Hyde Park, New York for cholesterol measurements.

Tissue fatty acids (FA) composition: were determined by gas chromatography/mass spectrometry (GC/MS) after lipid extraction, as described previously [30].

Liver glycogen content: was measured using colorimetric assay (Cayman Chemical Glycogen kit NC0294986).

Page 5 of 31 Diabetes

Liver enzymes: Serum samples were sent to Antech Diagnostics Inc, New Hyde Park, New York for measurements of aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (Alk-Phos).

Blood glucose measurements, insulin, glucose, and pyruvate tolerance tests: mice were injected with 0.5U/kg insulin, 2mg/g glucose, or 2g/kg sodium-pyruvate (Sigma cat# S8636) for tolerance tests. Blood glucose levels were measured using a glucometer (Elite; Bayer, Mishawaka, IN). Insulin tolerance was measured in fed mice, glucose tolerance in 8 hours fasted mice and pyruvate tolerance in 15 hours fasted mice.

Lipid peroxidation: Thiobarbituric Acid Reactive Substances (TBARS) were measured using commercial kit (Cayman, CAT# 10009055).

Protein oxidation: Carbonylated proteins were detected using OxiSelect Protein Carbonyl Spectrophotometric assay (STA-315, OxiSelect).

Gene expression: RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) or RNeasy Plus (Cat# 74134, Qiagen), reverse-transcribed (Cat# 18080-051, Life technologies), and subjected to real- time PCR using SYBR master mix (Life Technologies/Applied Biosystems, NY, USA Cat#4385612). Transcript levels were corrected to 18S. The primer sequences are presented in Supplemental table 2.

Western immunoblot assay: Proteins were extracted using CHAPS buffer (1.25% CHAPS, Thermo Scientific Cat#28300) with protease inhibitor cocktail [Roche, Cat# 04693132001]), separated on 4– 20% SDS-PAGE (LifeTechnologies, Cat# NP0335), and transferred to nitrocellulose membranes (Bio- Rad, Cat# 170-4158). Antibodies: Anti-F4/80 (AbDSerotec MCA497R), Anti-IR (SantaCruz, Cat# sc- 711), actin (CellSignaling, Cat# 4970) and secondary antibodies (CellSignaling, Cat# 7074).

Histology: Tissues were fixed in 10% zinc formalin and then processed for paraffin sectioning (5-m sections) and stained with hematoxylin and eosin. Anti-F4/80 (AbD Serotec MCA497R) was used to assess macrophage infiltration in the liver.

Statistical Analysis: Data are presented as means ± SEM. Differences between groups were tested using 1-way ANOVA and post hoc Tukey’s test, significance accepted at p < 0.05. Diabetes Page 6 of 31

RESULTS Restoration of hepatic IGF-1 in the Li-GHRKO mice To restore IGF-1 in the liver-specific GHRKO mice, we crossed the Li-GHRKO mice with the hepatic IGF-1 transgenic (HIT) mice [27] (Figure 1A), where the rat IGF-1 transgene expressed specifically in liver under the transthyretin promoter.

Ghr gene ablation in liver resulted in ~95% reductions in serum IGF-1 in Li-GHRKO mice (Figure 1B), while expression of the Igf-1 transgene in the HIT mice increased serum IGF-1 levels by ~2-fold. Crossing of the Li-GHRKO and HIT mice normalized serum IGF-1 levels (in Li-GHRKO-HIT). Serum GH levels increased 5-fold in the Li-GHRKO, while restoration of liver IGF-1 (Li-GHRKO-HIT) normalized GH levels (Figure C).

The acid labile subunit (ALS) is a surrogate marker of GHR action in liver. Accordingly, ALS protein levels in serum were undetectable in both Li-GHRKO and the Li-GHRKO-HIT mice (Figure 1D). Serum IGF-1 binding protein-3 (IGFBP-3) levels reduced significantly in Li-GHRKO and Li-GHRKO- HIT mice (Figure 1E), likely due to blunted expression of ALS (that stabilizes the IGF-1/IGFBP3 complex in serum) and increased susceptibility of unbound IGFBP-3 to degradation.

IGF-1 improves systemic glucose/lipid homeostasis and body composition in the Li-GHRKO- HIT mice. Li-GHRKO mice show increased blood glucose, serum insulin levels, and show severely impaired ITT (Figure 2). Restoration of hepatic Igf-1 (Li-GHRKO-HIT mice) normalized the fed and fasting blood glucose (Figure 2A), insulin (Figure 2B), and ITT (Figure 2D). In accordance with a recent study [31] showing that elevated IGFBP-1 improves whole body insulin sensitivity and glucose tolerance, we found that in the Li-GHRKO-HIT mice serum IGFBP-1 increased 5folds (Figure 2E), suggesting that it may play a role in the improvement of overall insulin sensitivity. A previous study using the Li-GHRKO mice [32] showed that IGFBP-1 levels increased by 2.5 fold despite elevated levels of its inhibitor insulin. This may be in part due to hepatic insulin resistance, progressive hepatic steatosis [33], or alternatively a compensatory response to significant reductions in IGFBP-3. Our results differ from those previously reported [22], in which administration of rhIGF-1 via osmotic pump into the Li- GHRKO mice did not correct the metabolic phenotype. We have previously shown that in the absence of ALS the half-life of rhIGF-1 is very short and it is rapidly cleared from circulation [34]. Thus, infusing rhIGF-1 via osmotic pumps for relatively short time (4 weeks) to Li-GHRKO mice with already severe hepatic steatosis is inefficient.

In view of the improved whole body insulin sensitivity (suggesting improved muscle insulin sensitivity), Page 7 of 31 Diabetes

we performed intraperitoneal pyruvate tolerance test (iPTT) to examine whether hepatic insulin sensitivity was also improved in Li-GHRKO-HIT mice. We found significant improvement in iPTT in Li- GHRKO-HIT as compared to Li-GHRKO mice, suggesting that part of whole body improvement in insulin sensitivity was also due to decreased hepatic glucose production (HGP) (Figure 2F).

Lipid profile in serum revealed significant increases in TG and cholesterol (Figure 3A), and serum free fatty acid (FFA) levels (Figure 3B) in the Li-GHRKO mice, which were normalized in Li-GHRKO- HIT mice, suggesting that increases in hepatic-derived IGF-1 can override the impaired systemic glucose and lipid metabolism resulting from GHR resistance in the liver.

Insulin resistance in the Li-GHRKO mice associated with changes in body composition. Body weight was not affected in Li-GHRKO or the Li-GHRKO-HIT mice (Supplemental figure 1A). However, we detected ~2-fold increase in the weights of several fat pads in Li-GHRKO (Figure 3C), which correlated with increases in serum leptin levels (Figure 3D). With age (at 52 weeks), control mice showed increases in body adiposity, resulting in a difference of only 25% between Li-GHRKO and control mice. Hepatic-derived Igf-1 in Li-GHRKO-HIT mice brought body adiposity (Figure 3C) and serum leptin (Figure 3D) to normal levels. A Previous study has demonstrated that leptin regulates IGFBP-2, which in turn improves glucose metabolism and hepatic insulin sensitivity by suppressing HGP and gene expression of enzymes involved in gluconeogenesis and fatty acid synthesis [34]. We found that serum IGFBP-2 levels increased over 10fold in the Li-GHRKO-HIT mice (Figure 3E), which may explain in part the improvement in overall insulin sensitivity in those mice.

IGF-1 is insufficient to restore lipid metabolism in the livers of GH resistant mice. Relative liver weight in the Li-GHRKO mice increased at all ages, and reduced in the Li-GHRKO-HIT mice (Figure 4A). Li-GHRKO mice exhibited steatotic livers by histology (Figure 4B), and showed ~6 fold increase in liver triglyceride (TG) content (Figure 4C) and fatty acid (FA) content detected by gas chromatography/mass spectrometry (GC/MS) (Figure 4D, Supplement table 1). Hepatic steatosis persisted even after restoration of liver Igf-1 (Li-GHRKO-HIT mice), although to a lesser degree (Figure 4B).

In accordance with previous findings with mutated GHR and liver-specific STAT5KO [35] mice showing a negative regulation of the fatty acid translocase CD36 by STAT5, we found significantly increased CD36 expression in both Li-GHRKO and the Li-GHRKO-HIT mice (Figure 5A). Likewise, the fatty acid transport proteins-2 and 5 (fatp2, 5) and the low-density lipoprotein receptor (LDLR) that regulates cholesterol metabolism, increased in the Li-GHRKO and Li-GHRKO-HIT mice (Figure 5A), suggesting increased hepatic lipid uptake in the absence of GHR. Further, we found that liver content Diabetes Page 8 of 31 of linoleic and docosahexaenoic acids, which are not synthesized de novo and must be uptaken by hepatocytes, are increased in Li-GHRKO mice (Supplement table 1). Although, the levels of hepatic linoleic acid were reduced in Li-GHRKO-HIT when compared to Li-GHRKO mice, they were not normalized. In an attempt to decrease GH-mediated lipolysis in fat we deleted the GHR in fat of the Li- GHRKO-HIT mice using the adiponectin-driven cre transgenic mice. We show that despite normalization of serum GH, IGF-1, and insulin, as well as inactivation of the GHR in fat, hepatic lipid content in the Li/Fat-GHRKO-HIT mice was not normalized (Supplement figure 1B). Overall, the data suggest that GHR in liver controls lipid uptake independent of IGF-1 or insulin.

Gene expression of stearoyl-CoA desaturase (SCD1), the rate-limiting enzyme that converts the palmitate (16:0) and stearate (18:0) to palmitoleate (16:1n7) and oleate (18:1n9), respectively, increased significantly in both Li-GHRKO and the Li-GHRKO-HIT mice, suggesting an increase in de novo lipogenesis (DNL) (Figure 5B). In accordance with previous publications [36, 37], we show that the SCD-1 index (the ratio of 18:1/18:0 by GC/MS) increased 3 fold in the Li-GHRKO mice (Figure 5C, Supplement table 1), suggesting an increased DNL. Li-GHRKO-HIT showed a significant decrease in SCD-1 index as compared to the Li-GHRKO mice, but this was still significantly higher than controls, suggesting that hepatic IGF-1 improved but did not resolve DNL in the absence of GHR. Increased DNL correlated with increased gene expression of the sterol regulatory element- binding protein-1 (SREBP1c), acetyl-CoA carboxylase (ACAC), and fatty acid synthase (Fas), (Figure 5B) in Li-GHRKO and the Li-GHRKO-HIT mice. Finally, Carnitine palmitoyltransferase 1 (CPT1), and peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (Pgc1α) increased in Li-GHRKO and the Li-GHRKO-HIT mice, suggesting increased fatty acid oxidation.

Compromised hepatic lipid metabolism in Li-GHRKO and the Li-GHRKO-HIT mice was associated with impaired glucose metabolism (Figure 5D). Gene expression of the glucose transporter-2 (GLUT2) and glucokinase (GCK) increased in Li-GHRKO mice indicating enhanced glucose flux. Pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PCK1/PEPCK), and the glucose 6 phosphatase (G6PC), which are critical enzymes for gluconeogenesis, were also elevated in livers of Li-GHRKO. Despite significant improvement in iPTT in Li-GHRKO-HIT mice, expression of theses genes was still elevated. Expression of genes involved in glycogen synthesis was highly variable (supplement Figure 1C). However, we found significant increases in hepatic glycogen content in Li- GHRKO mice (Figure 5E), which is in agreement with studies in humans showing that in states of hyperinsulinemia with hyperglycemia (Li-GHRKO mice), hepatic glycogen synthesis is maximized [38]. Improvement in insulin sensitivity associated with decreased glycogen content in the livers of the Li-GHRKO-HIT mice (Figure 5E).

Page 9 of 31 Diabetes

IGF-1 modulates oxidative stress, but is insufficient to resolve liver inflammation in GH resistant mice. Liver expression of glutathione peroxidase (GPx), super oxide dismutase (SOD), catalase 2 (cat2), and the nuclear factor erythroid 2-related factor-2 (Nrf2), increased significantly in Li-GHRKO and Li- GHRKO-HIT mice (Figure 6A). Protein carbonylation (oxidatively damaged proteins) in the liver did not differ between the groups (Figure 6B). However, Li-GHRKO mice showed significantly increased levels of oxidized lipids in liver (Figure 6C) and serum (Figure 6D) that were normalized in the Li- GHRKO-HIT mice. Since adult hepatocytes express very low levels of the IGF-1 receptor (IGF-1R), we postulated that IGF-1 elicited its effects on lipid peroxidation via its actions on hepatic stellate cells or via the insulin receptor (IR) on hepatocytes. We found that IR levels increased significantly in livers of Li-GHRKO-HIT as compared to Li-GHRKO mice (Supplement Figure 1D), suggesting that hepatic-derived IGF-1 may act locally via the IR to reduce oxidative stress in liver. Hepatic steatosis in the Li-GHRKO mice associated with liver injury as indicated by elevated levels of aspartate transaminase (AST) and alanine transaminase (ALT) (Figure 6E) and elevated lipid peroxidation (Figure 6C) that were normalized with hepatic-IGF-1. Note that muscle protein carbonylation (Supplement Figure 1E) and the levels of oxidized lipids (Supplement Figure 1F) reduced in Li- GHRKO-HIT mice and may contribute to overall improved insulin sensitivity.

Livers of Li-GHRKO and Li-GHRKO-HIT mice showed significantly more F4/80 positive cells (Figure 7A), increased F4/80 protein levels (Figure 7B), increases f4/80 expression (Figure 7C) and the monocyte chemoattractant protein-1 (mcp1), both are markers of macrophages. Expression of the inflammatory cytokines tumor necrosis factor α (TNFα), interleukin-1 beta (IL-1β), IL-6, and IL-18 increased in livers of Li-GHRKO and the Li-GHRKO-HIT mice (Figure 7C). The pathogenesis of NAFLD associates with increased toll-like receptor (TLR) and its adaptor protein MyD88 [39], which both increased in livers of Li-GHRKO and Li-GHRKO-HIT mice. Lastly, the chemokine RANTES (CCL5), implicated in the progression of hepatic inflammation [40] also increased in livers of Li- GHRKO and Li-GHRKO-HIT mice. Note that infiltration of T-cells was not evident histologically or by CD4/CD8 gene expression. Serum levels of IL-6 and TNFα did not reflect the gene expression patterns (Figure 7D). IL-6 levels increased only in serum of the HIT mice, while serum RRRαRwas similar between the groups, suggesting that GH-resistance-induced steatosis in the adult mouse results in inflammatory response that at young adulthood is localized to the liver. Diabetes Page 10 of 31

DISCUSSION We have created a new mouse model to dissect the effects of GHR signaling in liver metabolism where we excluded the possible contribution of concomitant reductions in IGF-1 to the overall phenotype. Our study clearly indicates that restoration of IGF-1 in the context of hepatic GH resistance normalized whole body insulin sensitivity and serum lipid profile in the adult mouse (16-24 weeks), a phenotype that persisted to 52 weeks of age. However, IGF-1 did not resolve hepatic steatosis, as enhanced hepatic lipid uptake and DNL were still evident. Importantly, IGF-1 was insufficient for protecting against steatosis-induced hepatic inflammation.

Li-GHRKO and the LID mice, with high levels of GH show insulin resistance [41]. Hepatic production of IGF-1 (Li-GHRKO-HIT) rescued overall insulin sensitivity likely via normalization of serum GH levels and improved muscle insulin responsiveness. Improvement in overall insulin sensitivity in the Li- GHRKO-HIT mice associated also with normalized iPTT, suggesting partial improvement in hepatic insulin sensitivity. IGFBP-1, a surrogate marker of insulin resistance was elevated in the Li-GHRKO- HIT mice and may also contribute to the improvement in whole body insulin sensitivity. Li-GHRKO-HIT mice also showed improved lipid profile in serum and normalized body adiposity that correlated with normalized serum leptin levels and over 10fold increase in IGFBP-2 levels that were previously shown to enhance insulin sensitivity [34]. At this point, suffice it to say that improvement in whole body insulin sensitivity together with normalized leptin levels in the Li-GHRKO-HIT mice most likely involves central (neuroendocrine) regulation, which controls body composition via leptin dependent and independent mechanisms.

The contribution of GH to body adiposity is documented in both humans and mice. Laron’s syndrome patients [42], GH deficient subjects [43], and the GHRKO mice exhibit increases in body adiposity [44], while patients with acromegaly [45] and the bGH transgenic mice are lean [46]. Together with the documented lipolytic effects of GH, it is suggested that GH-mediated lipolysis leads to reductions in fat mass. Nonetheless, the Li-GHRKO and the LID mouse models, which show high levels of GH, exhibit increased body adiposity, suggesting a more complex regulatory mechanism that may involve central regulatory pathways in the hypothalamus. Our studies indicate that the insulin resistance in LID [41] and Li-GHRKO mice contributes to the enhanced body adiposity. When serum IGF-1 was restored (Li- GHRKO-HIT), insulin sensitivity improved and resulted in decreased body adiposity.

Despite overall improved body composition and insulin responsiveness in the Li-GHRKO-HIT mice, hepatic insulin resistance was not completely resolved. Previous reports have shown that augmented HGP (gluconeogenesis) in states of insulin resistance [31], associate with increased expression of PC Page 11 of 31 Diabetes

and PEPCK, as seen in Li-GHRKO and Li-GHRKO-HIT mice, although in the latter iPTT (which indicates improved liver responsiveness to insulin) improved, and the insulin resistance was insufficient to cause hyperglycemia. We also found increases in glycogen content in livers of the Li- GHRKO that manifest systemic insulin resistance, while glycogen content in livers of Li-GHRKO-HIT mice was comparable to normal, suggesting an improved (but likely unresolved) insulin sensitivity in the latter.

In cases of GH resistance, the increase in liver TG content may be due to secondary effects of GH- mediated lipolysis in white adipose tissue. However, ablation of JAK2 both in liver and adipose tissue, resulted in significant reductions in hepatic TG levels as compared to JAK2L mice [23], but was still ~2.5 fold greater than controls. Ablation of CD36 in the JAK2L mice (DKO-JAK2/CD36) improved hepatic TG content [5] as compared to JAK2L, but was still ~20 fold higher than control mice. We found increases in CD36 gene expression in Li-GHRKO and Li-GHRKO-HIT mice that correlated with hepatic increases in the levels of linoleic and docosahexaenoic acids, which are not synthesized de novo, suggesting increased lipid uptake. Hepatic IGF-1 significantly reduced the levels of these acids (Li-GHRKO-HIT) but not to normal levels. Further, ablation of GHR in both liver and adipose tissue (Liver/fat-GHRKO-HIT mice) din not resolved hepatic steatosis, despite normal levels of blood glucose and insulin, again suggesting that an increase in white adipose tissue lipolysis is not the only cause for hepatic lipid accumulation in states of GH resistance.

Based on the theory of Brown and Goldstein [47] fatty liver can become resistant to insulin-mediated suppression of HGP, but remains sensitive to insulin-mediated stimulation of lipogenesis. In the Li- GHRKO-HIT mice, despite normal insulin levels and iPTT, hepatic steatosis remained, suggesting that lipid synthesis in livers of these mice is independent of insulin signaling and most likely relates to ablation of the GHR. Our data is also supported by by Vanter et al who showed that the rate of hepatic TG production depends on the rate of fatty acid uptake and independent of hepatic insulin signaling [48], and with clinical data showing that low levels of circulating IGF-1 may have a role in the development of NAFLD, independent of insulin resistance. Accordingly, we note that the high levels of insulin found in the LID mice did not lead to hepatic steatosis [15, 41], suggesting that increased hepatic lipid synthesis in the Li-GHRKO mice relates directly to the ablation of GHR action in liver. Consequently, we detected increases in SCD1 gene expression and SCD-1 index in the Li-GHRKO and the Li-GHRKO-HIT mice, suggesting that hepatic GHR regulates liver DNL independent of IGF-1. Notably, in the Li-GHRKO-HIT mice SCD-1 index was reduced but not normalized, suggesting that GHR signaling directly suppresses hepatic DNL. These data are in agreement with previous study showing a 2-fold increase in de novo lipogenesis in GH resistant mice [49]. Overall it is postulated that both lipid uptake and DNL occur in states of hepatic GH resistance independent of IGF-1. Diabetes Page 12 of 31

Excess lipid storage in the liver leads to enhanced β-oxidation of FFAs, which is regulated by CPT-1. Li-GHRKO and the Li-GHRKO-HIT mice show increases in CPT-1 gene expression, suggesting increases in β-oxidation. Increased β-oxidation results in over production of reactive oxygen species (ROS) eventually leading to mitochondrial dysfunction. Oxidation of lipids by extra-mitochondrial oxidation systems, such as microsomal and peroxisomal oxidation, also leads to increased ROS production [40]. Accumulation of ROS and nitrogen species is a hallmark of NAFLD [26]. We found increases in lipids peroxidation in the Li-GHRKO mice in serum and liver, which was restored to normal by hepatic IGF-1 (Li-GHRKO-HIT mice). Despite improvement in lipid peroxidation, an increase in oxidative stress may still be occurring in the Li-GHRKO-HIT liver since we found significant increases in SOD and catalase in both Li-GHRKO and the Li-GHRKO-HIT mice.

NAFLD associates with low-grade inflammation, which often proceeds to chronic inflammation [7]. Kupffer cells are liver resident macrophages that show high levels of the cell surface marker F4/80. Activated Kupffer cells produce pro-inflammatory cytokines such as IL-1β, and TNFα and secrete chemokines to recruit blood-derived monocytes/macrophages into the liver and play key roles in the initiation and progression of liver inflammation [50]. Indeed, we found increases in F4/80 and MCP-1 in both Li-GHRKO and the Li-GHRKO-HIT mice, which associated with increased expression of MyD88, RANTES (CCL5), IL-6, IL-1β, and TNFα. Serum levels of IL-1β, and TNFα at 16-24 weeks did not differ from controls, but during advanced adulthood (1 and 2 years) they both increased in Li- GHRKO and the Li-GHRKO-HIT mice (data not shown).

In summary, we have shown that hepatic GH resistance associates with overall impaired lipid metabolism, insulin resistance, and increased body adiposity. Hepatic lipid accumulation in states of chronic GH resistance results from increased lipid uptake and increased DNL, indicating that GH directly regulates these processes in the liver. The impaired lipid metabolism, along with severe hepatic insulin resistance leads to overall increased oxidative stress and initiates inflammation (Figure 8). Restoration of hepatic IGF-1 (Li-GHRKO-HIT) resolved overall insulin resistance and partially alleviated hepatic oxidative stress, but did not resolve hepatic steatosis or inflammation. Page 13 of 31 Diabetes

REFERENCES

1. Browning JD, Szczepaniak LS, Dobbins R, Nuremberg P, Horton JD, Cohen JC, Grundy SM, Hobbs HH: Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology 2004, 40(6):1387-1395. 2. Xu L, Xu C, Yu C, Miao M, Zhang X, Zhu Z, Ding X, Li Y: Association between serum growth hormone levels and nonalcoholic fatty liver disease: a cross-sectional study. PLoS One 2012, 7(8):e44136. 3. Chen QR, Braun R, Hu Y, Yan C, Brunt EM, Meerzaman D, Sanyal AJ, Buetow K: Multi-SNP analysis of GWAS data identifies pathways associated with nonalcoholic fatty liver disease. PLoS One 2013, 8(7):e65982. 4. Laron Z, Ginsberg S, Webb M: Nonalcoholic fatty liver in patients with Laron syndrome and GH gene deletion - preliminary report. Growth Horm IGF Res 2008, 18(5):434-438. 5. Hazlehurst JM, Tomlinson JW: Non-alcoholic fatty liver disease in common endocrine disorders. Eur J Endocrinol 2013, 169(2):R27-37. 6. Arturi F, Succurro E, Procopio C, Pedace E, Mannino GC, Lugara M, Procopio T, Andreozzi F, Sciacqua A, Hribal ML et al: Nonalcoholic fatty liver disease is associated with low circulating levels of insulin-like growth factor-I. J Clin Endocrinol Metab 2011, 96(10):E1640-1644. 7. Fusco A, Miele L, D'Uonnolo A, Forgione A, Riccardi L, Cefalo C, Barini A, Bianchi A, Giampietro A, Cimino V et al: Nonalcoholic fatty liver disease is associated with increased GHBP and reduced GH/IGF-I levels. Clin Endocrinol (Oxf) 2012, 77(4):531- 536. 8. Sumida Y, Yonei Y, Tanaka S, Mori K, Kanemasa K, Imai S, Taketani H, Hara T, Seko Y, Ishiba H et al: Lower levels of insulin-like growth factor-1 standard deviation score are associated with histological severity of non-alcoholic fatty liver disease. Hepatol Res 2015, 45(7):771-781. 9. Volzke H, Nauck M, Rettig R, Dorr M, Higham C, Brabant G, Wallaschofski H: Association between hepatic steatosis and serum IGF1 and IGFBP-3 levels in a population-based sample. Eur J Endocrinol 2009, 161(5):705-713. 10. Huang L, Steyn FJ, Tan HY, Xie TY, Veldhuis JD, Ngo ST, Chen C: The decline in pulsatile GH secretion throughout early adulthood in mice is exacerbated by dietary-induced weight gain. Endocrinology 2012, 153(9):4380-4388. 11. Iranmanesh A, Lizarralde G, Veldhuis JD: Age and relative adiposity are specific negative determinants of the frequency and amplitude of growth hormone (GH) secretory bursts and the half-life of endogenous GH in healthy men. J Clin Endocrinol Metab 1991, 73(5):1081-1088. 12. Dichtel LE, Yuen KC, Bredella MA, Gerweck AV, Russell BM, Riccio AD, Gurel MH, Sluss PM, Biller BM, Miller KK: Overweight/Obese adults with pituitary disorders require lower peak growth hormone cutoff values on glucagon stimulation testing to avoid overdiagnosis of growth hormone deficiency. J Clin Endocrinol Metab 2014, 99(12):4712-4719. 13. Shalet SM, Toogood A, Rahim A, Brennan BM: The diagnosis of growth hormone deficiency in children and adults. Endocr Rev 1998, 19(2):203-223. Diabetes Page 14 of 31

14. Bredella MA, Gerweck AV, Lin E, Landa MG, Torriani M, Schoenfeld DA, Hemphill LC, Miller KK: Effects of GH on body composition and cardiovascular risk markers in young men with abdominal obesity. J Clin Endocrinol Metab 2013, 98(9):3864-3872. 15. Nishizawa H, Iguchi G, Murawaki A, Fukuoka H, Hayashi Y, Kaji H, Yamamoto M, Suda K, Takahashi M, Seo Y et al: Nonalcoholic fatty liver disease in adult hypopituitary patients with GH deficiency and the impact of GH replacement therapy. Eur J Endocrinol 2012, 167(1):67-74. 16. Takahashi Y, Iida K, Takahashi K, Yoshioka S, Fukuoka H, Takeno R, Imanaka M, Nishizawa H, Takahashi M, Seo Y et al: Growth hormone reverses nonalcoholic steatohepatitis in a patient with adult growth hormone deficiency. Gastroenterology 2007, 132(3):938- 943. 17. Schwarz JM, Mulligan K, Lee J, Lo JC, Wen M, Noor MA, Grunfeld C, Schambelan M: Effects of recombinant human growth hormone on hepatic lipid and carbohydrate metabolism in HIV-infected patients with fat accumulation. J Clin Endocrinol Metab 2002, 87(2):942. 18. Madsen M, Krusenstjerna-Hafstrom T, Moller L, Christensen B, Vendelbo MH, Pedersen SB, Frystyk J, Jessen N, Hansen TK, Stodkilde-Jorgensen H et al: Fat content in liver and skeletal muscle changes in a reciprocal manner in patients with acromegaly during combination therapy with a somatostatin analog and a GH receptor antagonist: a randomized clinical trial. J Clin Endocrinol Metab 2012, 97(4):1227-1235. 19. Steyn FJ, Xie TY, Huang L, Ngo ST, Veldhuis JD, Waters MJ, Chen C: Increased adiposity and insulin correlates with the progressive suppression of pulsatile GH secretion during weight gain. J Endocrinol 2013, 218(2):233-244. 20. Luque RM, Kineman RD: Impact of obesity on the growth hormone axis: evidence for a direct inhibitory effect of hyperinsulinemia on pituitary function. Endocrinology 2006, 147(6):2754-2763. 21. Qin Y, Tian YP: Preventive effects of chronic exogenous growth hormone levels on diet-induced hepatic steatosis in rats. Lipids Health Dis 2010, 9:78. 22. Fan Y, Menon RK, Cohen P, Hwang D, Clemens T, DiGirolamo DJ, Kopchick JJ, Le Roith D, Trucco M, Sperling MA: Liver-specific deletion of the growth hormone receptor reveals essential role of growth hormone signaling in hepatic lipid metabolism. J Biol Chem 2009, 284(30):19937-19944. 23. Nordstrom SM, Tran JL, Sos BC, Wagner KU, Weiss EJ: Disruption of JAK2 in adipocytes impairs lipolysis and improves fatty liver in mice with elevated GH. Mol Endocrinol 2013, 27(8):1333-1342. 24. Cui Y, Hosui A, Sun R, Shen K, Gavrilova O, Chen W, Cam MC, Gao B, Robinson GW, Hennighausen L: Loss of signal transducer and activator of transcription 5 leads to hepatosteatosis and impaired liver regeneration. Hepatology 2007, 46(2):504-513. 25. List EO, Palmer AJ, Berryman DE, Bower B, Kelder B, Kopchick JJ: Growth hormone improves body composition, fasting blood glucose, glucose tolerance and liver triacylglycerol in a mouse model of diet-induced obesity and type 2 diabetes. Diabetologia 2009, 52(8):1647-1655. 26. Yang T, Householder LA, Lubbers ER, List EO, Troike K, Vesel C, Duran-Ortiz S, Kopchick JJ, Berryman DE: Growth hormone receptor antagonist transgenic mice are protected from hyperinsulinemia and glucose intolerance despite obesity when placed on a HF diet. Endocrinology 2015, 156(2):555-564. 27. Wu Y, Sun H, Basta-Pljakic J, Cardoso L, Kennedy OD, Jasper H, Domene H, Karabatas L, Guida C, Schaffler MB et al: Serum IGF-1 is insufficient to restore skeletal size in the Page 15 of 31 Diabetes

total absence of the growth hormone receptor. J Bone Miner Res 2013, 28(7):1575- 1586. 28. Wu Y, Wang C, Sun H, LeRoith D, Yakar S: High-efficient FLPo deleter mice in C57BL/6J background. PLoS One 2009, 4(11):e8054. 29. Yakar S, Liu JL, Stannard B, Butler A, Accili D, Sauer B, LeRoith D: Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc Natl Acad Sci U S A 1999, 96(13):7324-7329. 30. Kineman RD, Majumdar N, Subbaiah PV, Cordoba-Chacon J: Hepatic PPARgamma is not essential for the rapid development of steatosis following loss of hepatic GH signaling, in adult male mice. Endocrinology 2016:en20152077. 31. Rajwani A, Ezzat V, Smith J, Yuldasheva NY, Duncan ER, Gage M, Cubbon RM, Kahn MB, Imrie H, Abbas A et al: Increasing circulating IGFBP1 levels improves insulin sensitivity, promotes nitric oxide production, lowers blood pressure, and protects against atherosclerosis. Diabetes 2012, 61(4):915-924. 32. List EO, Berryman DE, Funk K, Jara A, Kelder B, Wang F, Stout MB, Zhi X, Sun L, White TA et al: Liver-specific GH receptor gene-disrupted (LiGHRKO) mice have decreased endocrine IGF-I, increased local IGF-I, and altered body size, body composition, and adipokine profiles. Endocrinology 2014, 155(5):1793-1805. 33. Li HH, Doiron K, Patterson AD, Gonzalez FJ, Fornace AJ, Jr.: Identification of serum insulin-like growth factor binding protein 1 as diagnostic biomarker for early-stage alcohol-induced liver disease. J Transl Med 2013, 11:266. 34. Hedbacker K, Birsoy K, Wysocki RW, Asilmaz E, Ahima RS, Farooqi IS, Friedman JM: Antidiabetic effects of IGFBP2, a leptin-regulated gene. Cell Metab 2010, 11(1):11-22. 35. Barclay JL, Nelson CN, Ishikawa M, Murray LA, Kerr LM, McPhee TR, Powell EE, Waters MJ: GH-dependent STAT5 signaling plays an important role in hepatic lipid metabolism. Endocrinology 2011, 152(1):181-192. 36. Lee JJ, Lambert JE, Hovhannisyan Y, Ramos-Roman MA, Trombold JR, Wagner DA, Parks EJ: Palmitoleic acid is elevated in fatty liver disease and reflects hepatic lipogenesis. Am J Clin Nutr 2015, 101(1):34-43. 37. Silbernagel G, Kovarova M, Cegan A, Machann J, Schick F, Lehmann R, Haring HU, Stefan N, Schleicher E, Fritsche A et al: High hepatic SCD1 activity is associated with low liver fat content in healthy subjects under a lipogenic diet. J Clin Endocrinol Metab 2012, 97(12):E2288-2292. 38. Petersen KF, Laurent D, Rothman DL, Cline GW, Shulman GI: Mechanism by which glucose and insulin inhibit net hepatic glycogenolysis in humans. J Clin Invest 1998, 101(6):1203-1209. 39. Miura K, Ohnishi H: Role of gut microbiota and Toll-like receptors in nonalcoholic fatty liver disease. World J Gastroenterol 2014, 20(23):7381-7391. 40. Bonekamp NA, Volkl A, Fahimi HD, Schrader M: Reactive oxygen species and peroxisomes: struggling for balance. Biofactors 2009, 35(4):346-355. 41. Yakar S, Setser J, Zhao H, Stannard B, Haluzik M, Glatt V, Bouxsein ML, Kopchick JJ, LeRoith D: Inhibition of growth hormone action improves insulin sensitivity in liver IGF-1- deficient mice. J Clin Invest 2004, 113(1):96-105. 42. Laron Z: Lessons from 50 Years of Study of Laron Syndrome. Endocr Pract 2015, 21(12):1395-1402. 43. Maison P, Griffin S, Nicoue-Beglah M, Haddad N, Balkau B, Chanson P: Impact of growth hormone (GH) treatment on cardiovascular risk factors in GH-deficient adults: a Metaanalysis of Blinded, Randomized, Placebo-Controlled Trials. J Clin Endocrinol Metab 2004, 89(5):2192-2199. Diabetes Page 16 of 31

44. Masternak MM, Bartke A, Wang F, Spong A, Gesing A, Fang Y, Salmon AB, Hughes LF, Liberati T, Boparai R et al: Metabolic effects of intra-abdominal fat in GHRKO mice. Aging Cell 2012, 11(1):73-81. 45. Freda PU, Shen W, Heymsfield SB, Reyes-Vidal CM, Geer EB, Bruce JN, Gallagher D: Lower visceral and subcutaneous but higher intermuscular adipose tissue depots in patients with growth hormone and insulin-like growth factor I excess due to acromegaly. J Clin Endocrinol Metab 2008, 93(6):2334-2343. 46. Benencia F, Harshman S, Duran-Ortiz S, Lubbers ER, List EO, Householder L, Al-Naeeli M, Liang X, Welch L, Kopchick JJ et al: Male bovine GH transgenic mice have decreased adiposity with an adipose depot-specific increase in immune cell populations. Endocrinology 2015, 156(5):1794-1803. 47. Li S, Brown MS, Goldstein JL: Bifurcation of insulin signaling pathway in rat liver: mTORC1 required for stimulation of lipogenesis, but not inhibition of gluconeogenesis. Proc Natl Acad Sci U S A 2010, 107(8):3441-3446. 48. Vatner DF, Majumdar SK, Kumashiro N, Petersen MC, Rahimi Y, Gattu AK, Bears M, Camporez JP, Cline GW, Jurczak MJ et al: Insulin-independent regulation of hepatic triglyceride synthesis by fatty acids. Proc Natl Acad Sci U S A 2015, 112(4):1143-1148. 49. Cordoba-Chacon J, Majumdar N, List EO, Diaz-Ruiz A, Frank SJ, Manzano A, Bartrons R, Puchowicz M, Kopchick JJ, Kineman RD: Growth Hormone Inhibits Hepatic De Novo Lipogenesis in Adult Mice. Diabetes 2015, 64(9):3093-3103. 50. Lanthier N: Targeting Kupffer cells in non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: Why and how? World J Hepatol 2015, 7(19):2184-2188.

Page 17 of 31 Diabetes

FIGURE LEGENDS

Figure 1: Restoration of hepatic IGF-1 gene expression in Li-GHRKO mice. (A) HIT were crossed with Li-GHRKO mice to yield the following groups; Control mice harbor the floxed ghr gene, hepatic IGF-1 transgenic (HIT) mice harbored the rat IGF-1 transgene and the floxed ghr gene, Li-GHRKO harbor the floxed ghr gene and the albumin-promoter-derived Cre transgene, and the Li-GHRKO-HIT mice harbor the floxed ghr gene, the albumin-promoter-derived Cre transgene, and the HIT. (B) Serum IGF-1 levels in male mice at 16 weeks of age. (C) Serum GH levels in male mice at 8-16 weeks of age. (D) Serum ALS levels of 16 weeks old male mice. (E) Serum IGF-BP3 in male mice at 16 weeks of age. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT.

Figure 2: Hepatic-derived IGF-1 improves systemic glucose homeostasis in the Li-GHRKO mice. (A) Blood glucose levels and B) serum insulin levels were determined in male mice at 16 weeks of age. (C) Intraperitoneal glucose tolerance test and (D) Intraperitoneal insulin tolerance test performed in 16-20 weeks old male mice in the fed state. (E) Serum IGFBP-1 levels determined in male mice at 16-24 weeks of age. (F) Intraperitoneal pyruvate tolerance test performed in 16-20 weeks old male mice. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT.

Figure 3: Hepatic-derived IGF-1 improves systemic lipid homeostasis and body composition in the Li-GHRKO mice. (A) Serum triglyceride (TG) and cholesterol in males at 16 weeks of age. (B) Serum free fatty acids (FFA) in the fed state in males at 16 weeks of age. (C) Relative gonadal fat pad wet-weight to body weight at the indicated ages, and (D) Serum leptin levels in males at 16 weeks of age. (E) Serum IGFBP-2 levels in males at 16-24 weeks of age. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT.

Figure 4: Hepatic igf-1 transgene does not resolve hepatic steatosis in the Li-GHRKO mice. (A) Relative liver wet-weight to body weight was followed in several age groups as indicated. (B) Hematoxylin/Eosin staining of liver sections from 16 weeks old control, Li-GHRKO, HIT and Li- GHRKO-HIT mice. (C) Hepatic triglycerides (TG) content. (D) Hepatic fatty acids (FA) content in 16 weeks old male mice measured by GC/MS. Data presented as mean ± SEM, N indicates sample size, Diabetes Page 18 of 31 significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO- HIT versus HIT. Figure 5: Hepatic igf-1 transgene does not resolve hepatic lipid metabolism in the Li-GHRKO mice. (A,B) Liver gene expression of key players in lipid metabolism was determined by real time PCR (n=8 per each genotype). (C) SCD-1 index determined by the ratio of hepatic stearic and oleic acid concentrations determined by GC/MS. (D) Hepatic gene expression of key players in glycolysis (GLUT2, glucokinase-GCK, liver pyruvate kinase-PKLr), and gluconeogenesis (pyruvate carboxylase- Pc, phosphoenolpyruvate carboxykinase-Pck1, glucose 6 phosphatase-G6pc. Liver RNA extracted from 16-24 weeks old male mice and processed for real-time PCR assay. (E) Hepatic glycogen content assayed in livers of 16 weeks old male mice. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li- GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT.

Figure 6: Hepatic-IGF-1 modulates oxidative stress in liver and serum. (A) Liver gene expression of enzymes involved in oxidative stress response in males at 16 weeks measured by real time PCR. (B) Protein carbonylation was determined using a spectrophotometric assay of liver protein extracts from male mice at 24 weeks of age. (C) Lipid peroxidation measured using TBARS assay in liver, and (D) serum from male mice at 24 weeks of age. (E) Serum levels of aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (Alk-Phos) in males at 16 weeks of age. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT.

Figure 7: Hepatic-IGF-1 modulates oxidative stress in liver and serum, but is insufficient to resolve liver inflammation in the Li-GHRKO mice. (A) Liver sections from 16 weeks old mice immunostained with anti-F4/80 antibody, arrows indicate F4/80 positive cells. (B) F4/80 protein levels assessed by western immunoblotting in 16 weeks old mice. (C) Liver gene expression of inflammatory markers (n=6 per each genotype). (D) Serum levels of IL-6 and TNFα at 16-24 weeks of age. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT.

is delivered to tissues via circulation. IGF-1 feeds back negatively on GH secretion. (B) Ablation of GHR in liver (Li-GHRKO) results in significant reductions in liver IGF-1 production and elevations in GH secretion. Increased GH levels contribute to overall increased insulin resistance (reflected by high levels of serum insulin and elevated blood glucose) and increased body adiposity (evident by increased circulating leptin levels). Li-GHRKO mice show severe hepatic steatosis, resulting from increased de novo lipogenesis and increased lipid uptake. The impaired lipid metabolism in the Li- GHRKO mice associates with protein and lipid oxidation in liver, as well as inflammation in the liver. (C) Restoration of hepatic IGF-1 in the Li-GHRKO-HIT mice normalizes serum IGF-1 and GH secretion. This associates with reduction in body adiposity and serum leptin, normalization of serum lipids, blood glucose and serum insulin levels. Hepatic steatosis in Li-GHRKO-HIT mice reduced as compared to Li-GHRKO mice, but was not resolved. Expression levels of genes involved in de novo lipogenesis and lipid uptake were increased to levels comparable to those in Li-GHRKO mice. Liver inflammation was high and did not resolve with hepatic IGF-1. Lastly, (D) the HIT mice express the IGF-1 transgene in liver and exhibit 2fold increase in serum IGF-1. HIT mice show normal levels of serum GH, and insulin during young adulthood.

Diabetes Page 20 of 31

FOOTNOTE PAGE:

Abbreviations: Growth hormone (GH), insulin-like growth factor-1 (IGF-1), non-alcoholic fatty liver disease (NAFLD), GH receptor (GHR), hepatic IGF-1 transgene (HIT), nucleotide polymorphism (SNP), genome-wide association study (GWAS), IGF-binding protein 3 (IGFBP-3), Janus kinase-2 (JAK2), signal transducer and activator of transcription 5 (STAT5), free fatty acids (FFAs), triglyceride (TG), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (Alk-Phos), Thiobarbituric Acid Reactive Substances (TBARS), acid labile subunit (ALS), gas chromatography/mass spectrometry (GC/MS), fatty acid transport proteins-2 and 5 (fatp2, 5), low- density lipoprotein receptor (LDLR), stearoyl-CoA desaturase (SCD1), de novo lipogenesis (DNL), sterol regulatory element-binding protein-1 (SREBP1c), acetyl-CoA carboxylase (ACAC or ACC), and fatty acid synthase (Fas), Carnitine palmitoyltransferase 1 (CPT1), and peroxisome proliferator- activated receptor-gamma co-activator 1 alpha (Pgc1α), glucose transporter-2 (GLUT2), glucokinase (GCK), pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PCK1/PEPCK), glucose 6 phosphatase (G6PC), reactive oxygen species (ROS), glutathione peroxidase (GPx), super oxide dismutase (SOD), catalase 2 (cat2), nuclear factor erythroid 2-related factor-2 (Nrf2), IGF-1 receptor (IGF-1R), insulin receptor (IR), monocyte chemoattractant protein-1 (mcp1), tumor necrosis factor α (TNFα), interleukin (IL), toll-like receptor (TLR).

All authors concur with the submission. The material submitted for publication has not been previously reported and is not under consideration for publication elsewhere.

Author Contributions Z.L. conducted the experiments, JCC and RDK measured fatty acid composition by GC/MS, B.N.C. and H.L conducted liver histology and serum lipid profile, R.M. and Z.G conducted liver protein carbonylation assay, H.W helped in experimental design and discussion, and S.Y designed the experiments and wrote the paper.

Acknowledgments We thank Dr. Papasani V. Subbaiah, Section of Endocrinology, Diabetes and Metabolism, University of Illinois at Chicago, for providing technical expertise in setting up the GC/MS for analysis of fatty acid composition.

Dr. Yakar is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors concur with the submission. The material submitted for publication has not been previously reported and is not under consideration for publication elsewhere. None of the authors have COI. Financial support received from National Institutes of Health Grant DK100246 to SY, Bi-national Science Foundation Grant 2013282 to SY and HW, VA Merit Award BX001114 to RDK, AR056672, AR068593, and UL1TR001445 to BNC.

Funding: Financial support received from National Institutes of Health Grant DK100246 to SY, and Bi-national Science Foundation Grant 2013282 to SY and HW. RDK supported by VA Merit Award BX001114. BC supported by AR056672, AR068593, UL1TR001445 from National Institutes of Health.

FigurePage 21 of 31 1 Diabetes

B A Li- 900 Serum IGF-1, ng/ml HIT GHRKO 800 X 700 cef 600 P<0.0001 flx/flx flx/flx Ghr HIT Alb-Cre Ghr 500 df 400 300 Li- Li- N= Control N= HIT 200 ade N=9 GHRKO GHRKO- N=7 1 1 3 9

0 Control HIT Li-GHRKO Li-GHRKO- flx/flx HIT Ghr Ghrflx/flx Alb-Cre Alb-Cre flx/flx flx/flx HIT Ghr Ghr HIT ALS in serum D Control Li-GHRKO HIT Li-GHRKO-HIT

IGF-ALS

C Serum GH, ng/ml E Serum IGFBP3, ng/ml 35 ade 350 30 300 P<0.0001 25 P<0.0001 250 20 200 c 15 150 ade 10 d 100 bdf 5 50 N=18 N=15 N=30 N=9 N=10 N=10 N=10 N=10 0 0 Control HIT Li-GHRKO Li-GHRKO-HIT Control HIT Li-GHRKO Li-GHRKO- HIT

Figure 1: Restoration of hepatic IGF-1 gene expression in Li-GHRKO mice. (A) HIT were crossed with Li-GHRKO mice to yield the following groups; Control mice harbor the floxed ghr gene, hepatic IGF-1 transgenic (HIT) mice harbored the rat IGF-1 transgene and the floxed ghr gene, Li-GHRKO harbor the floxed ghr gene and the albumin-promoter-derived Cre transgene, and the Li-GHRKO- HIT mice harbor the floxed ghr gene, the albumin-promoter-derived Cre transgene, and the HIT. (B) Serum IGF-1 levels in male mice at 16 weeks of age. (C) Serum GH levels in male mice at 8-16 weeks of age. (D) Serum ALS levels of 16 weeks old male mice. (E) Serum IGF-BP3 in male mice at 16 weeks of age. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT. Figure 2 Diabetes Page 22 of 31 A B Fed state Fasting state 250 4.5 Fed state Fasting state ade P<0.05 4 ade

200 l

3.5 m l P<0.05 / P<0.05 g /d

n 3 g

150 , n m

ade li 2.5 , u e

s 2 ins o 100 c N=

ade N= m

u 1.5 l u 9 r 9 g

N N N N N N N= N S 1 N= N= N= =1 =3 =3 =2 =2 =6 50 =6 N= 8 N= N 6 0 2 8 2

11 15 11 =

0.5 8 9 8

0 0 Control HIT Li- Li- Control HIT Li- Li- Control HIT Li- Li- Control HIT Li- Li- GHRKO GHRKO- GHRKO GHRKO- GHRKO GHRKO- GHRKO GHRKO- HIT HIT HIT HIT C 450 400 D 120 P<0.05 350 100 ad ad ad 300 80 250 , % of basal ,

200 60

150 glucose

Glucose, mg/dlGlucose, Control n=22 40 100 Li-GHRKO n=28 Control n=3 Li-GHRKO-HIT n=6 Li-GHRKO n=7 Blood Blood 20 50 HIT n=6 Li-GHRKO-HIT n=6

0 0 0 15 30 60 120 0 15 30 60 Time (Min) F Time, min after insulin injection E 300

7000 P<0.0001 250 6000 bdf

l l m /d / 200 g 5000 g p

m ,

1 e - d d

4000 s

P 150 o

a c bd u l

GFB 3000 I

G 100 bd bd m

u 2000 r Control N=5 e

S 50 Li-GHRKO N=8 1000 Li-GHRKO-HIT N=5 N=16 N=17 N=19 N=16 0 0 0 10 20 30 60 90 120 Time following pyruvate injection, min

Figure 2: Hepatic-derived IGF-1 improves systemic glucose homeostasis in the Li-GHRKO mice. (A) Blood glucose levels and B) serum insulin levels were determined in male mice at 16 weeks of age. (C) Intraperitoneal glucose tolerance test and (D) Intraperitoneal insulin tolerance test performed in 16-20 weeks old male mice in the fed state. (E) Serum IGFBP-1 levels determined in male mice at 16-24 weeks of age. (F) Intraperitoneal pyruvate tolerance test performed in 16-20 weeks old male mice. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO- HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT. FigurPage 23 ofe 31 3 Diabetes

2.5 P<0.0001 A 250 Control n=5 P<0.001 B HIT n=5 ade ade 2

200 Li-GHRKO n=5 Li-GHRKO-HIT n=5 df mM

, 1.5 150 a A F F 1

100 m u r Se N N= N= 50 0.5 N =2 =4 9 8 1

0 0 TG, Cholesterol, Control HIT Li- Li- mg/dl mmol/l GHRKO GHRKO- C 4.00 Control HIT ade HIT P<0.002 D y Li-GHRKO d 3.50 o Li-GHRKO-HIT ade ade 14 f b 3.00 ade P<0.0001 o 12 %

2.50 l t t 10 h h g /m g i i 2.00 8 ng we we d 1.50 d d n, 6 fat pti al

1.00 Le 4 ad N=7 N=5 N= N= N= N=8 N=9 N= N= N= N= N=

n d 1 1 1 1 2 1 2 1 2 4 0 0 6 8 4 4 0 0.50 Go

N=11 N=10 N=17 N=9 0.00 0 16 24 52 Control HIT Li-GHRKO Li-GHRKO-HIT Age, weeks

30000 E P<0.0001 bdf

10000

/m 2000 1800 ng 1600 2, - 1400 P

B 1200 F 1000 IG

800 600 um

r 400 e 200 S N=16 N=17 N=19 N=16 0 Control HIT Li-GHRKO Li-GHRKO-HIT

Figure 3: Hepatic-derived IGF-1 improves systemic lipid homeostasis and body composition in the Li-GHRKO mice. (A) Serum triglyceride (TG) and cholesterol in males at 16 weeks of age. (B) Serum free fatty acids (FFA) in the fed state in males at 16 weeks of age. (C) Relative gonadal fat pad wet- weight to body weight at the indicated ages, and (D) Serum leptin levels in males at 16 weeks of age. (E) Serum IGFBP-2 levels in males at 16-24 weeks of age. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li- GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT. Figure 4 Diabetes Page 24 of 31

9.00 A Control

t HIT P<0.0001 ade B Control HIT h 8.00 g

i Li-GHRKO e 7.00 Li-GHRKO-HIT w ade ade y

d 6.00 o

b bd df f 5.00 d o

% 4.00 t,

h Li-GHRKO Li-GHRKO-HIT g i 3.00 e w

r 2.00 N=7 N=5 N= N= N= N=8 N=9 N= N= N= N= N= e v i 1 1 1 1 2 1 2 1 L 4 0 0 6 8 4 4 0 1.00

0.00 16 24 52 Age, weeks

e 14 v

45 li P<0.0001 C D ade

P<0.0001 g 40 ade / 12 g

35 , er 10 ) v i S

l 30

M 8 /

m 25 bdf a bdf r GC 6 20 g y b

15 ( 4 mg/

10 FA 2 G, 5 N=5 N=5 N=6 N=6 T N=4 N=5 N=6 N=6 0 0 Total Control HIT Li-GHRKO Li-GHRKO- Control HIT Li-GHRKO Li-GHRKO-HIT HIT Figure 4: Hepatic igf-1 transgene does not resolve hepatic steatosis in the Li-GHRKO mice. (A) Relative liver wet-weight to body weight was followed in several age groups as indicated. (B) Hematoxylin/Eosin staining of liver sections from 16 weeks old control, Li-GHRKO, HIT and Li- GHRKO-HIT mice. (C) Hepatic triglycerides (TG) content. (D) Hepatic fatty acids (FA) content in 16 weeks old male mice measured by GC/MS. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO- HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li- GHRKO-HIT versus HIT. FigurPage 25 ofe 31 5 Diabetes A Lipid uptake B Lipid synthesis Lipid oxidation P<0.002 Control n=8 30 P<0.0001 6 P<0.002 HIT n=8 8 ae Li-GHRKO n=8 ae bf 14 ae 25 5 Li-GHRKO-HIT n=8 6 45 ae 7 bf r bf bf 12

e 40

v 20 4 b 5 6 li 35 ae

, 10

e ae 5

g 30 4 ae bf n 15 3 ae f 8 ae a bf 25 a 4 h ae bf

c 3 ncrease

i 20 6

ld 10 2 3 d o

F 15 2 Fol 4 2 5 1 10 1 5 2 1 0 0 0 0 0 0 CD36 Fatp2 Fatp5 LDLR SCD1 Fas2 SREBP1c ACC PPARg CPT1 PGC1a

C Control n=6 Glycolysis Gluconeogenesis D HIT n=6 SCD-1 index (18:1/18:0) Li-GHRKO n=6 7.00 4.50 b 4.5 Li-GHRKO-HIT n=6 ade 4.00 b 4 6.00 ae bf d 3.5 ae b 3.50 bf

5.00 3 3.00 ase ase e e 4.00 r 2.5 r 2.50 c c n n i 2 i 3.00 2.00 d d l bdf l o 1.5 o 1.50 F 1 F 2.00 1.00 0.5 1.00 N=5 N=5 N=6 N=6 0.50 0 Control HIT Li-GHRKO Li-GHRKO- 0.00 0.00

HIT GLUT2 GCK PKLr PC PCK1 G6PC P<0.003 P<0.02 P=0.1 P<0.004 P<0.02 P=0.1

140 E P=0.046 ae ) 120 r e v i l 100 g m /

g 80 u (

n e

g 60 co y l 40 G

r e N= N= v N= N= i 20 L 1 1 1 1 0 0 0 0

0 Control HIT Li-GHRKO Li-GHRKO- HIT

Figure 5: Hepatic igf-1 transgene does not resolve hepatic lipid metabolism in the Li-GHRKO mice. (A,B) Liver gene expression of key players in lipid metabolism was determined by real time PCR (n=8 per each genotype). (C) SCD-1 index determined by the ratio of hepatic stearic and oleic acid concentrations determined by GC/MS. (D) Hepatic gene expression of key players in glycolysis (GLUT2, glucokinase-GCK, liver pyruvate kinase-PKLr), and gluconeogenesis (pyruvate carboxylase-Pc, phosphoenolpyruvate carboxykinase-Pck1, glucose 6 phosphatase-G6pc. Liver RNA extracted from 16-24 weeks old male mice and processed for real-time PCR assay. (E) Hepatic glycogen content assayed in livers of 16 weeks old male mice. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li- GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT. Figure 6 Diabetes Page 26 of 31

ae Gene expression in liver A 7.0 bf 6.0

ae 5.0 ae bf bf a 4.0 bf crease Control n=5 n i 3.0

d HIT n=5 l 2.0

Fo Li-GHRKO n=6 1.0 Li-GHRKO-HIT n=6 0.0 CAT2 GPX SOD Nrf2 P<0.0001 P<0.0001 P<0.0001 P<0.005 C Liver-protein carbonylation B 140 Liver-Lipid peroxidation

4.5 P=0.27 120 ade ins P=0.004 e

t 4

o 100 )

r 3.5 ) g p

M m d 3 80 / u l e (

o 2.5 A lat 60 m

y 2 D n n ( M o 1.5 40 N N N N b N N N N r = = 1 = = a = = = = 8 8 8 8 20 5 6 6 6 C 0.5 0 0 Control Control HIT Li- Li- HIT Li-GHRKO Li-GHRKO- GHRKO GHRKO- HIT HIT E D Serum-Lipid peroxidation 50 250 ade ade ade P=0.006 Control 40 200 HIT

Li-GHRKO

uM 30 Li-GHRKO-HIT

150 l / A U D 20 I M 100

10 N N N N = = = = 50 6 5 6 6 N N N N N N N N N N N N ======5 5 5 5 5 5 5 5 5 5 5 0 5 Control HIT Li- Li- 0 GHRKO GHRKO- AST ALT Alk Phos HIT

A B Control Li-GHRKO Control HIT Li-GHRKO Li-GHRKO-HIT F4/80 b-actin

ve 1.8 b ati

l 1.6 P<0.002 ) re ts i 1.4

n ty e i u

s 1.2 as

Li-GHRKO-HIT HIT n rb

re 1 A te c

, l n i in

0.8 o d d tr l 0.6 n o an F o b

0.4 n

i N=6 N=6 N=6 N=6 to

te 0.2

ro 0 p ( Control HIT Li-GHRKO Li-GHRKO -HIT Gene expression in liver P<0.01 Control 4.5 C 6 Control P<0.05 HIT HIT b ae 4.0 b Li-GHRKO 5 bf Li-GHRKO Li-GHRKO-HIT bf 3.5 e f

Li-GHRKO-HIT bf e e s s 4 bf 3.0 ae a a ae ae ae re re 2.5 c c 3 n n i i 2.0

f e f d d e l l 2 1.5 Fo Fo 1.0 1 0.5 0 0.0 F4/80 MCP1 IL1b IL6 IL18 TNFa CD4 CD8 Myd88II Myd88V RANTES

D 500 IL-6, pg/ml 25 TNF-α, pg/ml 400 20

300 15

200 10 N N N N N N N N = = = 100 5 = = = = = 16 26 17 23 16 26 17 23

0 0 Control HIT Li-GHRKO Li-GHRKO-HIT Control HIT Li-GHRKO Li-GHRKO-HIT

Figure 7: Hepatic-IGF-1 modulates oxidative stress in liver and serum, but is insufficient to resolve liver inflammation in the Li-GHRKO mice. (A) Liver sections from 16 weeks old mice immunostained with anti-F4/80 antibody, arrows indicate F4/80 positive cells. (B) F4/80 protein levels assessed by western immunoblotting in 16 weeks old mice. (C) Liver gene expression of inflammatory markers (n=6 per each genotype). (D) Serum levels of IL-6 and TNFa at 16-24 weeks of age. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li- GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li- GHRKO-HIT versus HIT. Figure 8 Diabetes Page 28 of 31

A Control B Li-GHRKO C Li-GHRKO-HIT D HIT

Pituitary GH GH GH GH

- GHR GHR Igf-1 gene expression Liver Steatosis HIT Steatosis Lipid uptake Lipid uptake HIT De novo-lipogenesis De novo-lipogenesis Lipid peroxidation Lipid peroxidation Inflammation Inflammation Circulation Glucose Glucose Insulin IGF-1 Insulin IGF-1 IGF-1 IGF-1

FFA/TG FFA/TG ~ Insulin resistance resistance Insulin

Fat Leptin Leptin

Figure 8: IGF-1-independent effects of GHR in liver involve mainly de novo lipogenesis. (A) In control animals GH, secreted from the pituitary, promotes liver production and secretion of IGF-1 that is delivered to tissues via circulation. IGF-1 feeds back negatively on GH secretion. (B) Ablation of GHR in liver (Li-GHRKO) results in significant reductions in liver IGF-1 production and elevations in GH secretion. Increased GH levels contribute to overall increased insulin resistance (reflected by high levels of serum insulin and elevated blood glucose) and increased body adiposity (evident by increased circulating leptin levels). Li- GHRKO mice show severe hepatic steatosis, resulting from increased de novo lipogenesis and increased lipid uptake. The impaired lipid metabolism in the Li-GHRKO mice associates with protein and lipid oxidation in liver, as well as inflammation in the liver. (C) Restoration of hepatic IGF-1 in the Li-GHRKO-HIT mice normalizes serum IGF-1 and GH secretion. This associates with reduction in body adiposity and serum leptin, normalization of serum lipids, blood glucose and serum insulin levels. Hepatic steatosis in Li-GHRKO-HIT mice reduced as compared to Li-GHRKO mice, but was not resolved. Expression levels of genes involved in de novo lipogenesis and lipid uptake were increased to levels comparable to those in Li- GHRKO mice. Liver inflammation was high and did not resolve with hepatic IGF-1. Lastly, (D) the HIT mice express the IGF-1 transgene in liver and exhibit 2fold increase in serum IGF-1. HIT mice show normal levels of serum GH, and insulin during young adulthood. Page 29 of 31 Diabetes Supplement figure 1 A Control B 40.00 HIT 45 * P<0.0001 ade Li-GHRKO 40

35.00 g r

, Li-GHRKO-HIT t e 35 h 30.00 v i ig l 30 we

25.00 y 25 d bdf 20.00 Bo

/gram 20 15.00

mg 15

10.00 , 10 N=7 N= N=5 N= N= N=8 N=9 N= N= N= N= N= TG 1 1 1 1 5.00 2 1 2 1 4 0 0 6 8 4 4 0

0.00 0 16 24 52 Control HIT Li- Li- Age, weeks Li/Fat GHRKO GHRKO- GHRKO- N=4 N=5 N=6 HIT HIT N=6 N=4 D C Glycogen metabolism Control HIT Li-GHRKO Li-GHRKO-HIT 4.00 IR

3.50 f Liver b e -Actin 3.00 e ae

1.6 ase 2.50 e e

r d c 2.00 1.4 n

i P=0.003

d 1.2 l 1.50 o F 1.00 1 Control n=6 HIT n=6 0.50 0.8 Li-GHRKO n=6 Li-GHRKO-HIT n=6 0.00 0.6 GYS2 PYGL GSK3a GSK3b 0.4 P<0.02 P<0.02 P<0.02 P<0.02 Fold increase 0.2 to control, Arb Arb units) control, to

(protein band intensity intensity (protein band relative IR E Muscle-protein carbonylation 10 14 Muscle-Lipid peroxidation

ade s 12 P=0.23

n P<0.0001

i 8 10

prote 6 M) 8 m u d / ( l

e o

A 6

at 4 l m y n N N N ( n 4 MD N N N N N = = = = bo = = = = 2 10 10 10 9

2 6 5 6 6 ar C 0 0 Liver- Liver- Control Liver- Liver- Control HIT HIT GHRKO GHRKO-HIT GHRKO GHRKO-HIT

Supplement Figure 1: (A) Body weight at the indicated ages. (B) Hepatic triglycerides (TG) content measured in control, HIT, Li-GHRKO, Li-GHRKO-HIT, and mice with liver and fat ablation of the GHR that express HIT (Li/fat-GHRKO-HIT). (C) Liver expression of genes involved in glycogen metabolism, detected by real-time PCR (GYS2-liver glycogen synthase, PYGL-liver glycogen phosphorylase, GSK3- glycogen synthase kinase-3 a, b). (D) Insulin receptor (IR) levels in liver extracts detected by Western immunoblotting. (E) Muscle protein carbonylation and (F) lipid peroxidation in 16-24 week old male mice. Data presented as mean ± SEM, N indicates sample size, significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT.

Diabetes Page 30 of 31

SUPPLEMENTARY TABLE 1 Table 1: FA composition in liver extracts from 16 weeks old male mice as determined by GC/MS

FA, mg /g liver Control (n=5) HIT (n=5) Li-GHRKO (n=6) Li-GHRKO-HIT (n=6) Total FA 3.5303±0.237 3.2722±0.196 10.2059±1.453ae 4.7894±0.227bdf Palmitic acid (16:0) 0.8547±0.047 0.7833±0.054 2.4535±0.306 ae 1.2026±0.094 bdf Palmitoleic acid (16:1, n-7) 0.0347±0.004 0.0308±0.007 0.1781±0.033 ae 0.0570±0.008 bdf Stearic acid (18:0) 0.6187±0.027 0.5972±0.031 0.7060±0.041 0.6543±0.060 Oleic acid (18:1, n-9) 0.3499±0.052 0.3266±0.034 2.4254±0.468 ae 0.8110±0.098 bdf Vaccenic acid (18:1, n-7) 0.0510±0.002 0.0543±0.005 0.2207±0.037 ae 0.0822±0.006 bdf Linoleic acid (18:2, n-6) 0.8738±0.103 0.7084±0.056 3.1283±0.527 ae 1.2069±0.092 bdf γ-linoleic acid (18:3, n-6) 0±0 0±0 0.1416±0.025 0.0495±0.002 bdf Arachidonic acid (20:4, n-6) 0.3845±0.011 0.3668±0.017 0.3890±0.032 0.3213±0.022 Docosahexaenoic acid (22:6, n-3) 0.3630±0.013 0.4048±0.032 0.5633±0.049 ae 0.4047±0.022 bdf Data presented as mean ± SEM, tested by 2way ANOVA significance accepted at p<0.05, (a) Control versus Li-GHRKO, (b) Control versus Li-GHRKO-HIT, (c) Control versus HIT, (d) Li-GHRKO versus Li-GHRKO-HIT, (e) Li-GHRKO versus HIT, (f) Li-GHRKO-HIT versus HIT.

Page 31 of 31 Diabetes

SUPPLEMENTARY TABLE 2 Table 2:Primers used for real time Q-PCR Gene Sense primer Anti-sense primer Reference 18S 5`- CGGCTACCACATCCAAGGAA-3’ 5`-GCTGGAATTACCGCGGCT -3’ IGF system MsIgf1 5’-GGACCAGAGACCCTTTGCGGGG-3’ 5’-GGCTGCTTTTGTAGGCTTCAGTGG-3’ RnIgf1 5’-CACAGACGGGCATTGTGGAT-3’ 5’-CCGGATGGAACGAGCTGACTT-3’ Igfbp3 5’-ATTCCAAGTTCCATCCACTC-3’ 5’-AGGAGAAGTTCTGGGTGTCT-3’ als 5’-CCTGCAGAATCTCTACCATCT-3’ 5’-CAAACTGAGTGAAGCCAGAC-3’ Glycolysis Glut2 5’-TGCTGGCCTCAGCTTTATTC-3’ 5’-TTTCTTTGCCCTGACTTCCTC-3’ Gck 5’-AAACTACCCCTGGGCTTCAC-3’ 5’-ATTGCCACCACATCCATCTC-3’ Pklr-liver 5’-AGGAGTCTTCCCCTTGCTCTAC-3’ 5’-GGAGAGGCGTTTCAGGATATG-3’ Gluconeogenesis Pc 5’-GATGACCTCACAGCCAAGCA-3’ 5’-GGGTACCTCTGTGTCCAAAGGA-3’ Pck1 5’-TGGGGTGTTTGTAGGAGCAG-3’ 5’-CCAGGTATTTGCCGAAGTTG-3’ G6pc 5’-TCGAGGAAAGAAAAAGCCAAC-3’ 5’-CAATGCCTGACAAGACTCCA-3’ Glycogen GYS2 5’-CCAGCTTGACAAGTTCGACA-3’ 5’-ATCAGGCTTCCTCTTCAGCA-3’ metabolism PYGL 5’-GCGACTACTACTTCGCCCTTG-3’ 5’-GAGGTAATACACCCTCTTGGGA-3’ Gsk-3a 5’-GAGATCATCAAGGTACTAGGA-3’ 5’-AGATTTGAACACCTTTGTCCA-3’ Gsk-3b 5’-AACTTTACCACTCAAGAACTGTCA-3’ 5’-AGCATTAGTATCTGAGGCTGC-3’ Lipid metabolism ACAC 5’- GCCATTGGTATTGGGGCTTAC-3’ 5’- CCCGACCAAGGACTTTGTTG-3’ CPT1 5’-TTTGGGCCGGTTGCTGATGACGGC-3’ 5’-GCGGTGTGAGTCTGTCTCAGGGCT-3’ FAS 5’-GGCATCATTGGGCACTCCTT-3’ 5’-GCTGCAAGCACAGCCTCTCT-3’ PGC1a 5’-CGGGATGATGGAGACAGCTATGGT-3’ 5’-TGAGTTGGTATCTAGGTCTGCATA-3’ PPARg 5’-TTCCGAAGAACCATCCGATTG-3’ 5’-TGGCATTGTGAGACATCCCCAC-3’ CD36 5’-TGGAGCTGTTATTGGTGCAG-3’ 5’-TGGGTTTTGCACATCAAAGA-3’ Fatp2 5’-CTTCGGGAACCACAGGTCTTC-3’ 5’-CATAGCAAGGCCTGTCCCATAC-3’ Fatp5 5’-TTCGAAAGAACCAACCCTTCCT-3’ 5’-GCGTCGTACATTCGCAACAA-3’ SREBP1c 5’- GGAGCCATGGATTGCACATT-3’ 5’- GCTTCCAGAGAGGAGGCCAG-3’ SCD1 5’-CCGAAGTCCACGCTCGAT-3’ 5’-TGGAGATCTCTTGGAGCATGTG-3’ Oxidative stress Gpx 5’-GTGGTGCTCGGTTTCCCGTGC-3’ 5’-CCCGCCACCAGGTCGGACGTA-3’ Cat2 5’-GCAAGTTCCATTACAAGACC-3’ 5’-CATAATCCGGATCTTCCTGA-3’ SOD 5’-CTGGCCAAGGGAGATGTTACA-3’ 5’-GTCACGCTTGATAGCCTCCAG-3’ Nrf2 5’-CTCAGCATGATGGACTTGGAG-3’ 5’-CACTTCTCGACTTACTCCAAGAT-3’ Inflammation F4/80 5’-GAAGGCTCCCAAGGATATGGA-3’ 5’-TGCTTGGCATTGCTGTATCTG-3’ MCP1 5’-CTTCTGGGCCTGCTGTTCA-3’ 5’-GAGTAGCAGCAGGTGAGTGGG-3’ IL1 5’-AAATACCTGTGGCCTTGGGC-3’ 5’-CTTGGGATCCACACTCTCCAG-3’ IL6 5’-TCTGCAAGAGACTTCCATCC-3’ 5’-TTAGCCACTCCTTCTGTGAC-3’ IL18 5’-TCTTCTGCAACCTCCAGCATC-3’ 5’-GACATGGCAGCCATTGTTCC-3’ TNFa 5’-TTCTGTCTACTGAACTTCGGGGTGATCG-3’ 5’-GTATGAGATAGCAAATCGGCTGACGGTG-3’ CD4 5’-TCTGGCAACCTGACTCTGAC-3’ 5’-TCATCACCACCAGGTTCACT-3’ CD8 5’-GCTCAGTCATCAGCAACTCG-3’ 5’-ATCACAGGCGAAGTCCAATC-3’ MyD88-II 5’-ACTCGCAGTTTGTTGGATG-3’ 5’-CCACCTGTAAAGGCTTCTCG-3’ MyD88-V 5’-CATGGTGGTGGTTGTTTCTG-3’ 5’-CTTGGTGCAAGGGTTGGTAT-3’ RANTES 5’- CATATGGCTCGGACACCA -3’ 5’- ACACACTTGGCGGTTCCT -3’