Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: J Endocrinol. 2023 Feb 3;256(3):e220073. doi: 10.1530/JOE-22-0073

SerpinA3N deficiency attenuates steatosis and enhances insulin signaling in male mice

Melanie Tran 1, Md Golam Mostofa 1, Michael Picard 1, Jianguo Wu 1, Li Wang 2, Dong-Ju Shin 1
PMCID: PMC9930402  NIHMSID: NIHMS1867739  PMID: 36625462

Abstract

Aberrant hepatic lipid metabolism is the major cause of non-alcoholic fatty liver disease (NAFLD) and is associated with insulin resistance and type 2 diabetes. Serine (or cysteine) peptidase inhibitor, clade A, member 3N (SerpinA3N) is highly expressed in the liver, however its functional role in regulating NAFLD and associated metabolic disorders are not known. Male wildtype (WT) and hepatocyte Serpina3N knockout (HKO) mice were fed a control diet (CD), methionine and choline deficient (MCD) diet or high fat high sucrose (HFHS) diet to induce NAFLD and markers of lipid metabolism and glucose homeostasis were assessed. SerpinA3N protein was markedly induced in mice with fatty livers. Hepatic deletion of SerpinA3N attenuated steatosis which correlated with altered lipid metabolism genes, increased fatty acid oxidation activity and enhanced insulin signaling in mice with NAFLD. Additionally, SerpinA3N HKO mice had reduced epididymal white adipose tissue (eWAT) mass, leptin and insulin levels, improved glucose tolerance and enhanced insulin sensitivity which was associated with elevated insulin-like growth factor binding protein-1 (IGFBP1) and activation of the leptin receptor (LEPR)-STAT3 signaling pathway. Our findings provide a novel insight for the functional role for SerpinA3N in regulating NAFLD and glucose homeostasis.

Keywords: fatty liver, glucose metabolism, insulin signaling, leptin, fatty acid oxidation

Introduction

Non-alcoholic fatty liver disease (NAFLD) is a multi-system disease, widely regarded as the hepatic manifestation of the metabolic syndrome. Hepatic glucotoxicity and lipotoxicity are closely interrelated and contribute to the deterioration of insulin sensitivity, providing the pathophysiological mechanism underlying the development of NAFLD (Byrne and Targher, 2015, Friedman et al., 2018). A wealth of data have demonstrated a metabolic crosstalk between adipose tissue and liver which is known to promote lipogenesis and insulin resistance underscoring the importance of tissue cross talk in metabolic dysregulation of NAFLD (Qureshi and Abrams, 2007, Smith and Kahn, 2016). Although extensive research has been conducted in the field, the treatment options for NAFLD are not very effective.

Serine (or cysteine) peptidase inhibitor, clade A, member 3N (SerpinA3N) is a member of the serpin superfamily of protease inhibitors. The main function of serpins is to inhibit protease activity, although some have non-inhibitory effects and function instead as chaperones or hormone transporters in the circulation. In mice, the SerpinA3 gene has undergone extensive duplication and diversification resulting in a family of 13 closely related inhibitors, of which SerpinA3N is the closest murine orthologue of the human SerpinA3 (Horvath et al., 2005, Aslam and Yuan, 2020). SerpinA3N is synthesized by a range of cells including hepatocytes, bronchial epithelial cells, and neuronal cells. Importantly SerpinA3N is released in circulation in response to inflammation and is also known as an acute phase protein (Aslam and Yuan, 2020). Several studies have recently reported that SerpinA3N can regulate a wide range of biological processes (Haile et al., 2015, Tjondrokoesoemo et al., 2015, Vicuña et al., 2015, Gueugneau et al., 2018). In brief, SerpinA3N has been shown to induce neuroprotection in both in vitro and in vivo mouse models of neurodegenerative disease (Haile et al., 2015). Increased SerpinA3N expression was also demonstrated in muscle atrophy models mediated by glucocorticoid suggesting that elevated SerpinA3N levels can promote disease progression (Tjondrokoesoemo et al., 2015). More recently, Sergi et al. reported that SerpinA3N expression in the hypothalamus was strongly upregulated by a high fat diet (HFD) and in response to a leptin challenge (Sergi et al., 2018). The immunofluorescence staining showed increased SerpinA3N protein expression which was reported as early as one week of high fat diet feeding, consistent with early responses to HFD in the hypothalamus seen in other studies (Waise et al., 2015, Dalby et al., 2018). Additionally, recent human studies have also reported that SerpinA3 levels were highly elevated in colon and liver cancers (Cao et al., 2018, Ko et al., 2019). Our own recent study showed that SerpinA3N was highly induced in response to acetaminophen (APAP) overdose in mice (Tran et al., 2021). Moreover, hepatic deletion of SerpinA3N in mice diminished liver injury and suppressed inflammation (Tran et al., 2021) suggesting a potential role for SerpinA3N in modulating APAP-induced liver injury. However, no further studies to date have identified a role for SerpinA3N in NAFLD and its associated metabolic disorders.

In the present study, we identified a functional role for SerpinA3N in regulating NAFLD and glucose homeostasis. Hepatic deletion of SerpinA3N attenuated diet-induced steatosis and enhanced insulin signaling which was associated with activation of the LEPR-STAT3 signaling pathway. Taken together, hepatocyte-SerpinA3N deficiency might function as a protective factor against NAFLD and associated metabolic disorders.

Materials and Methods

Animal models

All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Connecticut, and animals received humane care in agreement with the approved protocols. Age-matched male mice (8–10 weeks of age) were used for all experimental procedures and were maintained on a 12 h light–dark cycle in a temperature (22°C) and humidity controlled (45–55%) environment with ad libitum access to food and water. SerpinA3N floxed mice on a C57BL6/J:129S background (stock #027511) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and were crossed with Albumin-Cre mice on a C57BL6/J background (Jackson Laboratory) to generate hepatocyte-specific SerpinA3N knockout (SerpinA3N HKO) mice (Tran et al., 2021). SerpinA3N HKO mice and their wildtype (WT) littermates (n = 6–12 mice per group) were fed a Control Diet (CD) (Harlan Teklad, TD.94149, 16.8 %kcal protein, 61.3 %kcal carbohydrate, 21.9 %kcal fat plus L-methionine [8.2 g/kg] and choline [1.4 g/kg]), methionine and choline deficient (MCD) diet (Harlan Teklad, TD.90262, 15.9 %kcal protein, 62.3 %kcal carbohydrate, 21.8 %kcal fat), or high fat high sucrose (HFHS) diet (Harland Teklad, TD.08811, 14.8% protein, 40.6% carbohydrate, 44.6% kcal fat) for 4 or 12 weeks to induce NAFLD. After 4 or 12 weeks of feeding, mice were fasted and euthanized, and blood and tissues were collected.

Metabolic function tests

Body weights were measured weekly in mice fed the HFHS diet for 12 weeks (Tabbi-Anneni et al., 2010). Plasma glucose, insulin and c-peptide levels were measured calorimetrically (Cayman Chemical, Ann Arbor, MI, USA) or using an ELISA (Crystal Chem, Elk Grove Village, IL, USA) after a 4 hour fast. Homeostatic model assessment for insulin resistance (HOMA-IR) was calculated according to the following formula: HOMA-IR = [fasting insulin concentration (mU/L) × fasting glucose concentration (mmol/L)] / 22.5 (Wallace et al., 2004). To perform a glucose tolerance test (GTT) and insulin tolerance test (ITT), mice were fasted for 6 and 4 hours during the light cycle, respectively, and then injected intraperitoneally (i.p) with glucose (2 mg/g body weight, D-glucose, Sigma Aldrich, St Louis, MO, USA) or insulin (0.5 – 0.65 mU/g body weight, Sigma Aldrich). Blood glucose levels were measured using a glucometer before (0) and after glucose or insulin injection at 5, 10, 15, 30, 60, 90 and 120 minutes. The glucose area under curve (AUC) for the GTT and ITT was calculated using GraphPad Prism software. To assess insulin signaling in liver and white adipose tissue (WAT), mice were fasted for 4 h, and then injected i.p with PBS or insulin (0.5 – 0.65 mU/g body weight), and blood and tissues were collected 10 minutes later for analysis.

Primary mouse hepatocyte isolation and cell culture

Primary mouse hepatocytes were isolated from 12 week-old WT and SerpinA3N HKO mice by a two-step collagenase perfusion method as previously described (Li et al., 2010). Oleic acid (400 μM, Sigma Aldrich) was added to the culture media for 16 h to establish an in vitro model of lipid accumulation in primary hepatocytes. For insulin signaling experiments, hepatocytes were incubated in minimum essential media (MEM, Gibco, Waltham, MA, USA) containing 0.1 % FBS for 6 h. 1 nM of insulin was added at timepoints 0, 5, 10, 15, 30 and 60 minutes after the 6 h fasting period, then cell lysates were collected for immunoblotting analyses.

Biochemical analysis

Fatty acid oxidation (FAO) enzyme activity was measured in livers of HFHS fed mice using a calorimetric assay kit from Biomedical Research Service Center (#E-141, Buffalo, NY, USA). In brief, liver samples were homogenized in 1× cell lysis solution and protein concentration of the samples were assessed with a Bradford assay and normalized to 1 mg/ml. 50 μl FAO assay solution or Control solution was added to 10μl of the liver homogenate. The plate was incubated at 37°C for 60 min and then the plate was read on a plate reader at 492 nm. Liver and plasma triglyceride (TG, Thermo Fisher Scientific, Waltham, MA, USA), alanine transaminase (ALT, Thermo Fisher Scientific), aspartate aminotransferase (AST, Thermo Fisher Scientific), SerpinA3N (LSBio, Seattle, WA, USA), IGFBP1 and IGFBP3 levels (R&D Systems Inc., Minneapolis, MN, USA) were measured according to the manufacturer’s instructions.

Immunoblotting analysis

Mouse tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer containing a cocktail of phosphatase and protease inhibitors (Thermo Fisher Scientific). Equal amounts of protein (30 μg) were pooled in each group (n = 5–6/group) or processed individually for insulin signaling experiments (n = 3–4 per group). The specific primary antibodies used were SERPINA3N (AF4709, R&D Systems Inc.), PPARα (sc-398394, Santa Cruz Biotechnology Inc., Dallas, TX), SREBP1 (sc-365513, Santa Cruz Biotechnology Inc.), Tyr1162/Tyr1163 p-IRβ (44-804G, Thermo Fisher Scientific), IRβ (sc-57342, Santa Cruz Biotechnology Inc.), Ser473 p-AKT (4060, Cell Signaling Technology, Danvers, MA), AKT (9272, Cell Signaling Technology), Thr202/Tyr204 p-ERK1/2 (4377, Cell Signaling Technology), Ser9 p-GSK3β (5558, Cell Signaling Technology), Tyr705 p-STAT3 (9145, Cell Signaling Technology), STAT3 (12640, Cell Signaling Technology), GAPDH (sc-365062, Santa Cruz Biotechnology Inc.), TUBULIN (sc-5286, Santa Cruz Biotechnology Inc.) and β-ACTIN (AM4302, Thermo Fisher Scientific). The membranes were incubated in the corresponding HRP conjugated rabbit anti-goat (HAF017, R&D Systems Inc.) goat anti-rabbit (170-6515, Bio-Rad) or goat anti-mouse (170-6516, Bio-Rad) secondary antibody for 1 hour at room temperature. Immunodetection was determined using the Pierce ECL detection system (Thermo Fisher Scientific). Images were obtained using the Image Quant LAS digital analyzer (GE Healthcare Life Sciences, Chicago, IL, USA) and immunoblots were quantified using ImageJ software.

Gene expression analysis by q-PCR

Total RNA was extracted from mouse tissues, reverse transcribed and then RT-PCR was performed as described previously (Tran et al.; 2021). A RT2 Profiler PCR array assay for Mouse Fatty Liver (PAMM-157Z, Qiagen, Germantown, MD, USA) was used for pathway-focused gene expression analysis according to the manufacturer’s instructions. The results were uploaded onto Data Analysis Center on the Qiagen website for subsequent analysis. The qPCR primer sequence used in the study are listed in Supplementary Table S1.

Histology

A piece of liver, epididymal white adipose tissue (eWAT) and pancreas were fixed in 10% formalin overnight, processed and embedded in paraffin wax. Approximately 4μm thick sections of tissues were deparaffinized and then stained with hematoxylin and eosin (H&E) or used for immunohistochemistry. Pancreatic sections were incubated with insulin (sc-8033, Santa Cruz Biotechnology Inc.) or glucagon (sc-514592, Santa Cruz Biotechnology Inc.) antibody overnight at 4°C and then incubated with horseradish peroxidase–conjugated secondary antibody. Immunoreactivities were detected with 3, 3′ diaminobenzidine tetrahydrochloride substrate (Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. Slides were examined under the Zeiss light microscope and images were taken at x20 magnification.

Statistics

Data are presented as mean ± SEM. A Student’s unpaired t-test was used to determine differences between two groups. Differences between multiple groups were compared using a one-way ANOVA with Newman-Keuls multiple comparisons test or a two-way ANOVA. Statistical significance is indicated by * p<0.05, ** p<0.01 and *** p<0.001.

Results

SerpinA3N protein is markedly upregulated in fatty livers of humans and mice

To assess the expression level of SerpinA3N, protein and RNA were extracted from mouse tissues. SerpinA3N mRNA was abundantly expressed in liver and eWAT, moderately expressed in pancreas and BAT (Supp Fig. S1A), while mRNA levels were very low in muscle, intestine, brain, heart, and kidney (Supp Fig. S1A). Therefore, hepatocyte-specific Serpina3N knockout mice were generated by crossing SerpinA3N floxed mice with the Albumin-Cre mice to delete SerpinA3N specifically in hepatocytes (SerpinA3N-HKO). mRNA and protein expression confirmed ablation of SerpinA3N in liver of HKO mice compared with WT mice (Supp Fig. S1B–C), while mRNA (Supp Fig. S1B) and protein (Supp Fig. S1C) levels of SerpinA3N in intestine, eWAT, pancreas, heart and muscle were similar between WT and HKO mice.

To determine whether SerpinA3N has a regulatory role in NAFLD, mice were fed the HFHS diet for 12 weeks. H&E staining showed increased lipid droplets, an indicator of fatty liver, in WT mice with HFHS feeding compared with WT CD-fed mice (Fig. 1A). Additionally, HFHS fed mice had elevated SERPINA3N protein expression in the liver compared with CD fed mice (Fig. 1B&C) and to a lesser extent in WAT of WT-HFHS diet compared with WT-CD (Fig. 1C). Consistent with increased protein expression, SerpinA3N mRNA was also elevated in livers of HFHS fed mice compared with CD mice (Fig 1D). Interestingly, SerpinA3N mRNA was upregulated in eWAT and muscle of HFHS fed mice compared with CD mice, while levels in pancreas and BAT remained similar between CD and HFHS (Fig. 1D). SerpinA3N is a known secretory protein, so circulating plasma levels of SerpinA3N were measured (Fig. 1E). Plasma SerpinA3N levels were markedly upregulated by 3 to 5-fold in mice fed the MCD and HFHS, respectively when compared with CD mice (Fig. 1E). Taken together, SERPINA3N protein expression was upregulated in fatty livers of mice, indicating a potential role for SERPINA3N protein in the pathogenesis of NAFLD.

FIG. 1.

FIG. 1.

Open in a new tab

SERPINA3N protein is markedly upregulated in mice with NAFLD

(A) Representative H&E images of livers of mice fed the CD and HFHS diet, scale bar = 100 μM (B) Immunoblot and quantification of SERPINA3N expression in livers of WT mice fed CD and HFHS diet (n = 5/group) (C) SERPINA3N protein expression in liver, pancreas, WAT, BAT and muscle of WT mice fed the CD and HFHS diet (D) SerpinA3N mRNA expression in liver, pancreas, WAT, BAT and muscle of WT mice fed CD and HFHS diet. RNA was pooled (n = 6/group) and processed in triplicate (E) Circulating SERPINA3N levels were measured in plasma of mice fed CD (n = 5), MCD (n = 5) and HFHS (n = 6) diet. Data are represented as mean ± SEM. A Students t-test was used to determine differences between Control vs. Fatty livers and CD vs. HFHS. For multiple groups, differences were compared using a one-way ANOVA followed by Newman-Keuls multiple comparisons test. * p<0.05 and ** p<0.01 indicate statistical significance. Abbreviations: BAT, brown adipose tissue; CD, control diet; HFHS, high fat high sucrose; MCD, methionine and choline deficient; WAT, white adipose tissue; WT, wildtype.

Hepatocyte SerpinA3N knockout mice demonstrate improved glucose homeostasis under physiological conditions

The control of glucose homeostasis is a contributing factor for development of NAFLD, therefore we assessed the effects of hepatocyte SerpinA3N deficiency in mice under physiological conditions. Body weights (Supp Fig. S1D), and absolute and relative liver weights (Supp Fig. S1E) respectively, were not altered between WT and HKO mice. Interestingly, absolute and relative eWAT weights in SerpinA3N HKO mice were reduced compared with WT mice (Fig. 2A). Consistent with the reduction in eWAT mass in SerpinA3N HKO mice, plasma leptin levels were reduced compared with their WT counterparts (Fig. 2B). On the other hand, plasma TG, ALT and AST levels were similar between WT and HKO mice (Supp Fig. S2A–B).

FIG. 2.

FIG. 2.

Open in a new tab

Hepatocyte SerpinA3N-deficiency improves glucose tolerance and enhances insulin signaling under physiological conditions

(A) WAT weights relative to body weights in WT and SerpinA3N HKO mice (n = 9–10/group). Plasma (B) leptin (n = 5–6/group) (C) glucose (n = 4–5/group) (D) and insulin levels (n = 4–5/group) (E) calculation of HOMA-IR index (n = 4–5/group) (F) Glucose tolerance test (2 mg/g body weight) and glucose AUC (n = 9–10/group) (G) Insulin tolerance test (0.5 mU/g body weight) and glucose AUC (n = 7–10/group) (H-I) Assessment of insulin signaling pathway in livers of WT and HKO mice. Phosphorylation of AKT was quantified and normalized to total AKT levels using ImageJ software analysis and was calculated based on the relative fold change from n = 3–4 individual mice per group. Data are represented as mean ± SEM. Differences were compared using a student’s t-test or a two-way ANOVA for treatment and genotype; if interaction was significant then a student’s t-test was used to determine differences between groups. Statistical significance is indicated by * p<0.05 and ** p<0.05 vs. WT, ## p<0.01 vs. PBS WT, †† p<0.05 vs. PBS HKO. Abbreviations: AUC, area under the curve; HKO, hepatocyte knockout; HOMA-IR, homeostatic model assessment for insulin resistance; WAT, white adipose tissue; WT, wildtype.

Despite similar 4 h fasting glucose levels (Fig. 2C), a 70% reduction in fasting insulin levels were observed in HKO mice compared with WT mice (Fig. 2D). Similarly, HOMA-IR, a measure of insulin resistance, was also lower in HKO mice (Fig. 2E). However, no differences were observed in plasma c-peptide levels between WT and HKO mice (Supp Fig. S2C).

Glucose tolerance and insulin sensitivity were evaluated by performing a GTT and ITT in fasted mice. Compared with WT mice, HKO mice had improved glucose tolerance, as demonstrated by the lower glucose AUC (Fig. 2F), however insulin sensitivity, as indicated by an ITT, was similar between WT and HKO mice (Fig. 2G).

To determine whether the improved glucose tolerance was associated with enhanced insulin signaling in the liver, we monitored for insulin receptor (IR) and downstream AKT activation in mice that had been fasted for 4 hours and treated with insulin by i.p injection. Hepatocyte-SerpinA3N deficiency did not promote IR activation (Fig. 2F), but strongly promoted insulin-induced AKT phosphorylation in HKO mice (Fig. 2F). Overall, our results demonstrate hepatocyte SerpinA3N-deficiency reduced insulin secretion and enhanced insulin-induced AKT phosphorylation under physiological conditions.

Hepatocyte SerpinA3N-deficiency attenuates steatosis by increasing fatty acid oxidation under conditions of diet-induced obesity

Obesity exacerbates the development of insulin resistance and promotes NAFLD development, therefore we fed mice the HFHS diet for 12 weeks and the MCD diet for 4 weeks. Body weights before and during the 12 weeks of feeding were similar between WT and HKO mice (Fig. 3A), but MCD fed mice had lower body weights after 4 weeks of feeding (Supp Fig. S3A). SerpinA3N HKO mice displayed lower relative liver weights after feeding with the HFHS diet (Fig. 3B, left panel) and the MCD diet (Supp Fig. S3B), and had reduced relative WAT mass (Fig. 3B, right panel). The decreased fat mass in male HKO mice fed the HFHS diet correlated with smaller adipocyte cell size, as shown by H&E staining (Fig. 3C) and consequently, lower circulating plasma leptin levels (Fig. 3D). Hepatic steatosis, demonstrated by H&E staining for lipid droplets, was evident after 12 weeks of HFHS feeding in WT mice compared with WT CD-fed mice, and similarly after 4 weeks of MCD feeding (Fig. 3C). On the contrary, hepatocyte-SerpinA3N deficiency in mice had attenuated lipid accumulation (Fig. 3C). However, liver injury as assessed by ALT levels, were not altered between WT and HKO mice (Fig. 3E). Similarly, SerpinA3N deficiency in mice fed the MCD also had similar ALT levels compared with their WT counterparts (Supp Fig. S3C).

FIG. 3.

FIG. 3.

Open in a new tab

Hepatocyte SerpinA3N-deficiency attenuates diet-induced hepatic steatosis

(A) Weekly body weight measurements during high fat feeding in WT and SerpinA3N HKO mice (n = 11/group) (left panel) and body weight at time of sacrifice (right panel) (B) Liver and WAT weights relative to body weights in WT and HKO mice (n = 5–6/group) (C) Representative images of H&E staining in livers of mice fed the CD, MCD and HFHS diet, as well as WAT of HFHS fed mice, scale bar = 100μm (D) Plasma leptin (n = 5–6/group) (E) Plasma ALT (n = 6–11/group). Data are represented as mean ± SEM. A Students t-test was used to determine differences between groups. Statistical significance is indicated by * p<0.05 vs. WT. Abbreviations: ALT, alanine transaminase; CD, control diet; HFHS, high fat high sucrose; MCD, methionine and choline deficient; HKO, hepatocyte knockout; WAT, white adipose tissue; WT, wildtype.

The smaller liver size and attenuated steatosis in HKO mice were associated with reduced plasma TG (Fig. 4A and Supp Fig. S3D) and liver TG content (Fig. 4A). Liver β-hydroxybutyrate levels were also found to be reduced in HKO mice (Fig. 4B), but plasma β-hydroxybutyrate levels were similar between WT and HKO mice (Fig. 4B). Accumulation of triglycerides in the cytoplasm of hepatocytes represents the hallmark of NAFLD and occurs as an abnormal balance between lipid acquisition and disposal, therefore we measured genes associated with lipid metabolism in high fat fed mice. HKO mice had elevated Mttp levels and reduced lipid synthesis and transport gene (Srebpf, Fasn and Cd36) mRNA levels compared with WT mice (Fig. 4C). At the protein level, precursor SREBP1 (pSREBP1) was not altered between WT and HKO mice (Fig. 4D) but mature SREBP1 (mSREBP1) was diminished in HKO mice (Fig. 4D). Additionally, protein and mRNA expression of Pparα, a transcription factor involved in fatty acid oxidation (FAO), were significantly upregulated in HKO mice compared with WT mice (Fig. 4C–D). Moreover, FAO enzyme activity was increased in SerpinA3N HKO mice compared with WT mice (Fig. 4E). Consistently, mRNA levels of Fgf21, a PPARα target gene, was increased in SerpinA3N HKO mice compared with WT mice, while no differences were reported in Pdk4 expression (Fig. 4H). Our results demonstrate that the attenuated steatosis observed in SerpinA3N HKO mice may be a result of diminished lipid accumulation and enhanced fatty acid oxidation enzyme activity.

FIG. 4.

FIG. 4.

Open in a new tab

Hepatocyte SerpinA3N deficiency enhances fatty acid oxidation activity in mice with NAFLD

TG content in (A) plasma (n = 10–11/group) and livers (n = 5–6/group) of mice fed the HFHS diet (B) Liver and plasma β-HB levels (n = 4–6/group) (C) qPCR analysis of genes associated with lipid transport (Cd36 and Mttp), lipogenesis (Fasn, Srebpf), cell size (Plin2) and fatty acid oxidation (Pparα) in livers of HFHS fed mice (n = 6/group) (D) Protein expression of PPARα and SREBP1 in livers of HFHS fed mice. Quantification was performed using ImageJ software and was normalized to the relative loading control. Liver lysates (n = 5–6/group) were pooled and processed in duplicate (E) Fatty acid oxidation enzyme activity was measured in livers of HFHS fed mice (n = 5/group) (F) qPCR analysis of Pparα target genes. RNA was pooled (n=5–6/group) and processed in triplicate. Data are represented as mean ± SEM. A Students t-test was used to determine differences between WT and HKO. Statistical significance is indicated by * p<0.05 and ** p<0.01. Abbreviations: β-HB, β-hydroxybutyrate; HFHS, high fat high sucrose; HKO, hepatocyte knockout; TG, triglyceride; WT, wildtype.

Hepatocyte SerpinA3N-deficiency improves glucose tolerance and insulin signaling under conditions of diet-induced obesity

Glucose homeostasis was assessed to determine whether high fat feeding contributes to impaired insulin signaling and glucotoxicity. 4-h fasting plasma glucose levels were lower in HKO mice compared with WT mice (Fig. 5A, left panel). Additionally, fasting insulin levels were lower, albeit not significant, in HKO mice (Fig. 5A, middle panel). Consistent with the lower fasting glucose and insulin levels, HOMA-IR was significantly diminished in HKO (Fig. 5A, right panel). To determine whether the reduced insulin level in HKO was a result of reduced insulin clearance, we measured c-peptide levels. We found that HKO mice had lower c-peptide levels compared with WT mice (Fig. 5B), suggesting greater insulin clearance in mice deficient for SerpinA3N. To evaluate whether there are any changes in pancreatic morphology, H&E staining was performed in the pancreas of WT and SerpinA3N HKO mice fed the HFHS diet for 12 weeks. Histologically, islet morphology was not different between WT and HKO mice (Supp Fig. S4A). Immunohistochemistry staining for insulin and glucagon expression also showed no differences between WT and HKO mice (Supp Fig. S4B), suggesting that β-cell mass and circulating insulin levels from the pancreas in SerpinA3N HKO mice may be adequate to maintain glucose homeostasis. Additionally, the expression of liver gluconeogenic genes, G6Pase and Pck1, were not altered between WT and SerpinA3N HKO mice (Fig 5C).

FIG. 5.

FIG. 5.

Open in a new tab

Hepatocyte SerpinA3N-deficiency improves glucose tolerance and enhances insulin signaling under diet-induced obesity

(A) Plasma glucose and insulin levels and calculation of HOMA-IR (n = 5–6/group) (B) Plasma c-peptide levels (n = 5 per group) (C) qPCR analysis of genes associated gluconeogenesis (G6Pase, Pck1). RNA was pooled (n=5/group) and processed in triplicate (D) Glucose tolerance test (2 mg/g body weight) and glucose AUC (E) Insulin tolerance test (0.65 mU/g body weight) and glucose AUC (n = 10–11/group) (F-G) Assessment of insulin signaling pathway (IR and AKT activation) in isolated hepatocytes treated with oleic acid (400 μm, 16 h) and in livers of HFHS fed mice. Phosphorylation of IRβ and AKT was quantified and normalized to total IRβ and AKT levels respectively, using ImageJ software analysis and was calculated based on the fold change from n = 3–4 individual mice per group. Data are represented as mean ± SEM. Differences were compared using a two-way ANOVA for treatment and genoptype; if interaction was significant then a student’s t-test was used to determine differences between groups. Statistical significance is indicated by * p<0.05 and ** p<0.01 vs. WT, # p<0.05 and ## p<0.01 vs. PBS WT, †† p<0.05 vs. PBS HKO. Abbreviations: AUC, area under the curve; HFHS, high fat high sucrose; HOMA-IR, homeostatic model assessment for insulin resistance.

Under conditions of diet-induced obesity and insulin resistance, SerpinA3N HKO mice developed improved glucose tolerance (Fig. 5D), but insulin sensitivity remained similar between WT and HKO mice (Fig. 5E). Our results demonstrate that hepatocyte SerpinA3N deficiency in mice improves glucose tolerance without changes in insulin tolerance under conditions of diet-induced obesity and insulin resistance.

Hepatocyte-SerpinA3N deficiency promotes activation of the insulin signaling pathway

Insulin-mediated activation of the insulin receptor is widely recognized as the major basis for linking insulin resistance to NAFLD. To determine whether enhanced insulin sensitivity in HKO mice correlated with increased hepatic insulin signaling, we monitored for IR activation and downstream AKT signaling in primary hepatocytes isolated from WT and HKO mice and treated with oleic acid to mimic in vivo conditions of fatty liver. Hepatocytes were treated with 1 nM of insulin accordingly at various timepoints. Insulin-induced IR and AKT activation were observed in hepatocytes of HKO mice compared with hepatocytes from WT mice (Fig. 5F) suggesting activation of insulin signaling pathway in hepatocytes of SerpinA3N KO mice. To reaffirm the enhanced insulin signaling in SerpinA3N HKO mice, we assessed in vivo IR and AKT activation. Insulin-induced IR and AKT activation were strongly induced in livers of HKO mice compared with WT mice (Fig. 5G), demonstrating enhanced insulin signaling even in the context of obesity and insulin resistance. Overall, our results demonstrate that SerpinA3N deficiency in mice activates the insulin signaling pathway to control glucose homeostasis.

SerpinA3N knockout mice demonstrate enhanced WAT insulin signaling which is associated with reduced lipid metabolism and inflammation

Our results demonstrate that high fat feeding induced SerpinA3N expression in eWAT (Fig. 1C–D), therefore we monitored for changes in insulin signaling in eWAT to determine whether the changes observed with insulin sensitivity were attributed to enhanced peripheral insulin sensitivity. Insulin-induced IR and AKT phosphorylation in eWAT was significantly induced in SerpinA3N-HKO mice compared with their WT counterparts (Fig. 6A). Our results demonstrate that enhanced insulin signaling was observed in the eWAT of HKO mice suggesting enhanced peripheral insulin sensitivity. Furthermore, the reduced adipose tissue mass and improved insulin signaling in HKO mice correlated with attenuated lipid metabolism gene expression (Acc, Scd1, Fasn) (Fig. 6B) as well as reduced expression of genes in inflammatory responses (Il-6, Il1β, Tnfα, Mcp1) (Fig. 6C). Taken together, our results point towards hepatic SerpinA3N-deficiency improving liver glucose homeostasis and enhancing peripheral insulin signaling even in the context of obesity and systemic insulin resistance.

FIG. 6.

FIG. 6.

Open in a new tab

Activation of white adipose tissue insulin signaling in SerpinA3N HKO mice is associated with reduced genes associated with lipid metabolism and inflammation

(A) Assessment of insulin signaling pathway (IR and AKT activation) in eWAT of HFHS fed mice. Densitometry was determined using ImageJ software analysis and was calculated based on fold change from n =3–4 individual mice per group. qPCR analysis of genes involved in (B) Lipid metabolism (Acc, Scd1, Fasn and Srebp1f) and (C) Inflammation (Il-6, Il1β, Tnfα and Mcp1) in livers of male mice fed the high fat high sucrose (HFHS) (n = 6/group). Data are represented as mean ± SEM. A Students t-test was used to determine differences between WT and HKO mice. For multiple groups, differences were compared using a two-way ANOVA for treatment and genoytpe; if interaction was significant then a student’s t-test was used to determine differences between groups. Statistical significance is indicated by * p<0.05 and ** p<0.01 vs. WT, # p<0.05 vs. PBS WT, †† p<0.01 vs. PBS HKO. Abbreviations: HFHS, high fat high sucrose; HKO, hepatocyte knockout; eWAT, epididymal white adipose tissue.

Hepatocyte SerpinA3N-deficiency activates the LEPR-STAT3 signaling pathway

To comprehensively understand the role of SerpinA3N in NAFLD and the associated downstream mechanisms for the attenuated steatosis and enhanced insulin sensitivity in SerpinA3N HKO mice, we performed a PCR gene array for fatty liver metabolism using liver RNA samples from high fat fed WT and HKO mice. A significant upregulation of leptin receptor (Lepr) and insulin-like growth factor binding protein 1 (Igfbp1) genes were observed in HKO mice compared with WT mice (Fig. 7A). IGFBP1 is synthesized by the liver and its levels are inversely proportional to insulin, and is a surrogate marker for insulin sensitivity (Bae, Song and Im, 2013). qPCR analysis further confirmed that hepatic Igfbp1 levels were 4-fold higher in HKO mice compared with WT mice (Fig. 7B). Other Igfbps, including Igfbp2-5 but not Igfbp6 were also significantly elevated in HKO mice (Fig. 7B). Consistent with the induction of Igfbp1 gene expression, circulating plasma levels of IGFBP1 were also significantly elevated in HKO mice fed the HFHS (Fig. 7C) and MCD diet (Fig. 7D) compared with their WT counterparts. However, no significant changes were observed with circulating IGFBP3 levels (Fig. 7E).

FIG. 7.

FIG. 7.

Open in a new tab

Hepatocyte SerpinA3N-deficiency activates LEPR-STAT3 signaling pathway.

(A) PCR array profile. Total RNA from livers of male WT and SerpinA3N HKO mice fed the HFHS diet were pooled (n = 6/group) and subjected to Fatty Liver Metabolism Pathway Finder PCR Array analysis. The fold changes of individual genes were represented in a heat map where upregulated genes are denoted in red and downregulated genes are denoted in green (B) Plasma IGFBP1 (left panel) and IGFBP3 (right panel) levels in male and female mice fed the HFHS diet (n = 8–11/group) (C) Plasma IGFBP1 levels in male (left panel) and female (right panel) mice fed the MCD diet (n = 4–5/group). qPCR analysis of Igfbp1-6, Igf1, IgfII and Igfr expression in HFHS livers of (D) male and (E) female mice. RNA was pooled (n = 5–6/group) and processed in triplicate. (F) Lepr and (G) Socs3 mRNA expression in HFHS livers of male WT and SerpinA3N HKO mice. RNA was pooled (n = 6/group) and processed in triplicate. Immunoblotting for STAT3 activation in (H) livers of HFHS fed mice and in (I) hepatocytes of WT and HKO mice treated with oleic acid (0.4mM) and stimulated with 1 nM insulin at the indicated times. Data are represented as mean ± SEM. A Students t-test was used to determine differences between WT and HKO mice. For multiple groups, differences were compared using a two-way ANOVA for group and gender; if interaction was significant then a student’s t-test was used to determine differences between groups. Statistical significance is indicated by * p<0.05 and ** p<0.01 vs. WT, ## p<0.05 vs. PBS WT and †† p<0.01 vs. PBS HKO. Abbreviations: HKO, hepatocyte knockout; HFHS, high fat high sucrose; MCD, methionine and choline deficient; WT, wildtype.

Follow up confirmation of Lepr mRNA showed a 3-fold increase in male HKO mice compared with WT mice (Fig. 7F). Activation of STAT3 via LEPR results in increased transcription and expression of Socs3, which acts as a feedback inhibitor molecule to attenuate subsequent leptin signaling. Indeed, our results showed a 40% reduction in the transcriptional expression of Socs3 in livers of HKO mice compared with WT mice (Fig. 7G), and STAT3 phosphorylation was markedly induced in SerpinA3N HKO mice fed the HFHS diet (Fig. 7H). Similarly, this was also observed in primary hepatocytes of HKO mice and treated with oleic acid, without any changes in response to insulin (Fig. 7I). Collectively, our data suggest that hepatocyte-SerpinA3N deficiency activates the LEPR-STAT3 pathway which is associated with reduced lipogenesis and enhanced fatty acid oxidation enzyme activity to attenuate hepatic steatosis.

Discussion

NAFLD is a multi-system disease occurring with concomitant insulin resistance and impaired metabolic processing. In this study, hepatic deletion of SerpinA3N attenuated diet-induced steatosis by reducing expression of genes associated with lipid accumulation, increased fatty acid oxidation measured by enzyme activity, and enhanced insulin signaling in livers of mice with NAFLD. Moreover, SerpinA3N HKO mice had improved glucose tolerance and diminished fat deposition in eWAT which may be related to reduced WAT lipid metabolism and inflammatory genes. This was associated with elevated IGFBP1 levels and activation of the LEPR-STAT3 signaling pathway. Our results provide novel insight into a functional role for SerpinA3N in regulating hepatic steatosis and insulin signaling.

It is interesting to note that the improved glucose tolerance and enhanced insulin signaling in the SerpinA3N HKO mice did not lead to increased whole body insulin sensitivity as demonstrated by the ITT. It is possible that a higher dose of insulin may be required to observe significant changes. Further studies using the hyperinsulinemic euglycemic clamp procedure, the gold standard method for assessing insulin sensitivity (Kim JJ, 2009), would shed new insights into the systemic and peripheral insulin sensitivity regulated by SerpinA3N.

Insulin has pleiotropic roles in the liver. Insulin acts via the IR-PI3K/AKT pathway to suppress hepatic glucose production to prevent glucose toxicity (Saltiel and Kahn, 2001). On the other hand, insulin also activates the transcription factor SREBP-1c, which enhances transcription of genes required for fatty acid and triglyceride synthesis (Saltiel and Kahn, 2001). In insulin-resistant states, PI3K/AKT signaling is attenuated and insulin fails to suppress hepatic gluconeogenesis, while insulin-induced lipogenesis remains intact. This phenomenon of “selective insulin resistance” is not well established, but may occur downstream of AKT and de novo lipogenesis could also occur through insulin-independent pathways (Brown and Goldstein, 2008). Our results demonstrate that the endogenous fasting insulin levels were lower in SerpinA3N HKO mice compared with WT mice, which was consistent with increased insulin sensitivity. The fatty acid oxidation enzyme activity was increased, which was associated with reduced hepatic lipid accumulation. Moreover, in response to exogenous insulin, hepatic insulin and eWAT signaling was enhanced in our SerpinA3N HKO mice, suggesting that HKO mice were more sensitive to insulin under conditions of diet-induced obesity.

IGFBPs play a key role in insulin and IGF signaling and are regulated by several proteases. IGFBP-1 is synthesized by the liver and its levels are inversely proportional to insulin, and correlate highly with insulin sensitivity (Kotronen et al., 2008, Rajwani et al., 2012, Bae et al., 2013). Our results demonstrate that SerpinA3N deficiency increased IGFBP1 levels which may be associated with enhanced insulin sensitivity. Furthermore, constitutive overexpression of IGFBP1 has been reported to inhibit adipocyte expansion (Rajkumar et al., 1999) and improve glucose tolerance and insulin sensitivity (Rajwani et al., 2012), which was similar to our observations where HKO mice had reduced WAT mass and cell size and improved glucose homeostasis. It is not clear how IGFBP1 enhances insulin sensitivity but studies have shown that IGFBP1 contains an Arg-Gly-Asp (RGD) integrin recognition sequence which can bind to cell surfaces (Jones et al., 1993). In vitro mechanistic studies reported by Haywood et al. showed that IGFBP-1 directly enhances insulin sensitivity through the RGD domain and that an RGD synthetic peptide enhances insulin secretion (Haywood et al., 2017). It may be possible that SerpinA3N can also target these IGFBPs to inhibit their function and reduce their activity, thus future studies are required to define the mechanistic basis of this interaction to ascertain whether this explains the current phenotype observed in our HKO mice.

Leptin promotes fat oxidation via leptin signaling and modulates insulin signaling (Hackl et al., 2019, Martínez-Uña et al., 2020). Experimental studies have reported that mice with lack of functional leptin (ob/ob) or its receptor (db/db), developed hepatic steatosis, while leptin replacement therapy in mice or re-expression of leptin receptor in the livers of leptin receptor-deficient Zucker diabetic fatty (ZDF) rats reversed this effect (Montague et al., 1997, Clément et al., 1998, Lee et al., 2001, Wang et al., 2014). Moreover, the pathogenesis of obesity is mediated by central leptin resistance (de Luca et al., 2005, Myers et al., 2010) while hepatic leptin resistance contributes to hepatic lipid accumulation (Fishman et al., 2007). Accordingly, our results demonstrate that circulating leptin levels were higher in WT mice with functional SerpinA3N, compared with their HKO counterparts (Fig. 2B and 3E), suggestive of hepatic leptin resistance. A study has also previously reported elevated SerpinA3N expression in the hypothalamus following a leptin challenge (Sergi et al., 2018). In our study, SerpinA3N-deficient mice had enhanced leptin signaling which resulted in reduced TG content (Fig. 4A), suggesting that HKO mice displayed enhanced leptin sensitivity and therefore contributing to the anti-steatotic effect. Our findings were in accordance with a recent study reporting that high fat fed mice with ablated hepatic leptin signaling had increased lipid accumulation in the liver (Huynh et al., 2010). Furthermore, overexpression of leptin receptor also reduced lipid synthesis and promoted fatty acid oxidation resulting in amelioration of hepatic steatosis in high fat fed mice, (Yoshino et al., 2014) which were similar to what we reported in our study.

Leptin binds to the leptin receptor, and initiates activation of STAT3 to attenuate hepatic steatosis in mice under conditions of diet induced obesity. Several studies have reported that mice with STAT3-deficiency have augmented fatty livers induced by various nutritional challenges, (Inoue et al., 2004, Horiguchi et al., 2008, Kroy et al., 2010) while overexpression of constitutively active STAT3 ameliorated high-fat diet-induced hepatic steatosis via inhibition of Srebp1c (Inoue et al., 2004, Kinoshita et al., 2008). This suggests that STAT3 activation in hepatocytes by leptin may ameliorate fatty livers. Our findings demonstrate the induction of leptin receptor in SerpinA3N-deficient mice triggered STAT3 phosphorylation, which is associated with suppressed lipid accumulation and stimulated fatty acid oxidation via STAT3 target gene, PPARα (Lee et al., 2002, Roglans et al., 2007, Wang et al., 2018) to attenuate hepatic steatosis. Importantly, additional studies are required to elucidate the mechanism of how SerpinA3N regulates leptin signaling.

Insulin resistance and ectopic fat accumulation interconnect NAFLD with other components of the metabolic syndrome. While numerous pathways are involved in this process, chronic inflammation has been implicated as driver of disease progression (Lefere and Tacke F, 2019). We found SerpinA3N protein was highly upregulated in adipose tissue of high fat fed mice compared with chow fed mice. Interestingly, high fat fed HKO mice had reduced epididymal fat mass associated with smaller adipocyte cell size, attributed to reduced levels of genes associated with lipid metabolism and inflammation, despite SerpinA3N being deleted only in livers of knockout mice. The molecular mechanism associated with this is not known as no studies to date have investigated the role of SerpinA3N in adipocyte metabolism. Serpina3 expression is mainly found in the liver, brain and adipose tissue (Zhang et al., 2017). One study showed that the expression level of the human orthologue, SERPINA3, was higher in obese individuals than in lean individuals, which suggest that SerpinA3 plays a critical role in obesity (Choi et al., 2020). Another study identified SerpinA3C, a member of the SerpinA3 clade, was markedly increased in the course of LTL-3L1 differentiation but SerpinA3N did not markedly change. Additionally, both SerpinA3N and SerpinA3C were detected in the medium from transfected cells indicating that they were secreted from cells (Choi et al., 2020). Furthermore, SerpinA3C deficiency in mice resulted in impaired metabolic phenotype which was associated with inflammation and apoptosis of white adipose tissue (Li et al., 2022). Mechanistically, SerpinA3C inhibited Cathepsin G activity which degrades integrin α5 (Li et al., 2022). However, additional studies are required to further clarify the role of SerpinA3N in adipogenesis. Aside from liver lipid metabolism, adipose tissue dysfunction is crucial in the pathogenesis of NAFLD (Cimini et al., 2017, Duval et al., 2010), and additional studies are required to determine the compensatory mechanism that may be involved in the protective phenotype observed in our SerpinA3N HKO mice. Furthermore, as SerpinA3N is secreted into circulation in response to nutritional challenges, future studies should consider using whole body knockout mice to firmly establish the specific role in driving NAFLD, since multiple tissues can contribute to NAFLD progression.

Here, we report for the first time that SerpinA3N may play a role in coordinating leptin and insulin to control lipid accumulation and insulin resistance. Taken together, hepatic deletion of SerpinA3N may function as a protective factor against NAFLD and associated metabolic disorders by alleviating dyslipidemia and improving glucose homeostasis. Our study suggests that SerpinA3N may act as a metabolic regulator in the setting of obesity and has a potential role in NAFLD.

Supplementary Material

01
02

Funding

M.T. was supported by an American Heart Association postdoctoral fellowship (18POST33960183). This work was supported by the National Institutes of Health grant (R01AA026322 to D-J.S).

Abbreviations

ALT

alanine transaminase

AST

aspartate aminotransferase

AUC

area under curve

BAT

brown adipose tissue

CD

control diet

FAO

fatty acid oxidation

GTT

glucose tolerance test

HFD

high fat diet

HFHS

high fat high sucrose

HOMA-IR

homeostatic model assessment for insulin resistance

ITT

insulin tolerance test

MCD

methionine and choline deficient

NAFLD

non-alcoholic fatty liver disease

PVDF

polyvinylidene difluoride

RIPA

radioimmunoprecipitation assay buffer

WAT

white adipose tissue

WT

wildtype

Footnotes

Conflict of interest

The authors have declared that no conflict of interest exists.

References

  1. Aslam MS & Yuan L. 2020. Serpina3n: Potential drug and challenges, mini review. Journal of Drug Targeting 28 368–378. [DOI] [PubMed] [Google Scholar]
  2. Bae JH, Song DK & Im SS. 2013. Regulation of IGFBP-1 in Metabolic Diseases. Journal of lifestyle medicine 3 73–79. [PMC free article] [PubMed] [Google Scholar]
  3. Brown MS and Goldstein JL. 2008. Selective versus total insulin resistance: a pathogenic paradox. Cell Metabolism 7 95–96. [DOI] [PubMed] [Google Scholar]
  4. Byrne CD & Targher G. 2015. NAFLD: a multisystem disease. Journal of Hepatology 62 S47–S64. [DOI] [PubMed] [Google Scholar]
  5. Cao LL, Pei XF, Qiao X, Yu J, Ye H, Xi CL, Wang PY & Gong ZL. 2018. SERPINA3 Silencing Inhibits the Migration, Invasion, and Liver Metastasis of Colon Cancer Cells. Digestive Diseases and Sciences 63 2301–2319. [DOI] [PubMed] [Google Scholar]
  6. Choi Y, Choi H, Toon BK, Lee H, Seok JW, Kim HJ & Kim JW. 2020. Serpina3c Regulates Adipogenesis by Modulating Insulin Growth Factor 1 and Integrin Signaling. iScience 23 100961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cimini FA, Barchetta I, Carotti S, Bertoccini L, Baroni MG, Vespasiani-Gentilucci U, Cavallo MG, Morini S. 2017. Relationship between adipose tissue dysfunction, vitamin D deficiency and the pathogenesis of non-alcholic fatty liver disease. World Journal of Gastroenterology 23 3407–3417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clément K, Vaisse C, Lahlou N, Cabrol S, Pelloux V, Cassuto D, Gourmelen M, Dina C, Chambaz J, Lacorte JM, et al. 1998. A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 392 398–401. [DOI] [PubMed] [Google Scholar]
  9. Dalby MJ, Aviello G, Ross AW, Walker AW, Barrett P & Morgan PJ. 2018. Diet induced obesity is independent of metabolic endotoxemia and TLR4 signalling, but markedly increases hypothalamic expression of the acute phase protein, SerpinA3N. Scientific Reports 8 15648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duval C, Thissen U, Keshtkar S, Accart B, Steinstra R, Boekschoten MV, Roskams T, Kersten S, Muller M. 2010. Adipose tissue dysfunction signals progression of hepatic steatosis towards nonalcoholic steatohepatitis in C57BL/6 mice Diabetes 59 3181–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fishman S, Muzumdar RH, Atzmon G, Ma X, Yang X, Einstein FH & Barzilai N. 2007. Resistance to leptin action is the major determinant of hepatic triglyceride accumulation in vivo. The FASEB Journal 21 53–60. [DOI] [PubMed] [Google Scholar]
  12. Friedman SL, Neuschwander-Tetri BA, Rinella M & Sanyal AJ. 2018. Mechanisms of NAFLD development and therapeutic strategies. Nature Medicine 24 908–922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gueugneau M, d’Hose D, Barbé C, de Barsy M, Lause P, Maiter D, Bindels LB, Delzenne NM, Schaeffer L, Gangloff YG et al. 2018. Increased Serpina3n release into circulation during glucocorticoid-mediated muscle atrophy. Journal of Cachexia, Sarcopenia and Muscle 9 929–946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hackl MT, Fürnsinn C, Schuh CM, Krssak M, Carli F, Guerra S, Freudenthaler A, Baumgartner-Parzer S, Helbich TH, Luger A et al. 2019. Brain leptin reduces liver lipids by increasing hepatic triglyceride secretion and lowering lipogenesis. Nature Communications 10 1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haile Y, Carmine-Simmen K, Olechowski C, Kerr B, Bleackley RC & Giuliani F. 2015. Granzyme B-inhibitor serpina3n induces neuroprotection in vitro and in vivo. Journal of Neuroinflammation 12 157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haywood NJ, Cordell PA, Tang KY, Makova N, Yuldasheva NY, Imrie H, Viswambharan H, Bruns AF, Cubbon RM, Kearney MT et al. 2017. Insulin-like growth factor binding protein 1 could improve glucose regulation and insulin sensitivity through its RGD domain. Diabetes 66 287–299. [DOI] [PubMed] [Google Scholar]
  17. Horiguchi N, Wang L, Mukhopadhyay P, Park O, Jeong W Il, Lafdil F, Osei-Hyiaman D, Moh A, Fy XY, Pacher P, et al. 2008. Cell Type-Dependent Pro- and Anti-Inflammatory Role of Signal Transducer and Activator of Transcription 3 in Alcoholic Liver Injury. Gastroenterology 134 1148–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Horvath AJ, Irving JA, Rossjohn J, Law RH, Bottomley SP, Quinsey NS, Pike RN, Couglin PB, & Whisstock J. 2005. The murine orthologue of human antichymotrypsin: A structural paradigm for clade A3 serpins. Journal of Biological Chemistry 280 43168–78 [DOI] [PubMed] [Google Scholar]
  19. Huynh FK, Levi J, Denroche HC, Gray SL, Voshol PJ, Neumann UH, Speck M, Chua SC, Covey SD & Kieffer TJ. 2010. Disruption of hepatic leptin signaling protects mice from age- and diet-related glucose intolerance. Diabetes 59 3032–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Inoue H, Ogawa W, Ozaki M, Haga S, Matsumoto M, Furukawa K, Hashimoto N, Kido Y, Mori T, Sakaue H, et al. 2004. Role of STAT-3 in regulation of hepatic gluconeogenic genes and carbohydrate metabolism in vivo. Nature Medicine 10 168–74. [DOI] [PubMed] [Google Scholar]
  21. Jones JI, Gockerman A, Busby WH, Wright G & Clemmons DR. 1993. Insulin-like growth factor binding protein 1 stimulates cell migration and binds to the α5β1 integrin by means of its Arg-Gly-Asp sequence. Proceedings of the National Academy of Sciences of the United States of America 90 10553–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kim JJ. 2009. Hyperinsulinemic-euglycemic clamp to assess insulin sensitivity in vivo. Methods Molecular Biology. 560 221–38. [DOI] [PubMed] [Google Scholar]
  23. Kinoshita S, Ogawa W, Okamoto Y, Takashima M, Inoue H, Matsuki Y, Watanabe E, Hiramatsu R & Kasuga M. 2008. Role of hepatic STAT3 in the regulation of lipid metabolism. Kobe Journal of Medical Sciences 54 E200–8. [PubMed] [Google Scholar]
  24. Ko E, Kim JS, Bae JW, Kim J, Park SG & Jung G. 2019. SERPINA3 is a key modulator of HNRNP-K transcriptional activity against oxidative stress in HCC. Redox Biology 24 101217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kotronen A, Lewitt M, Hall K, Brismar K & Yki-Järvinen H. 2008. Insulin-like growth factor binding protein 1 as a novel specific marker of hepatic insulin sensitivity. Journal of Clinical Endocrinology and Metabolism 93 4867–4872. [DOI] [PubMed] [Google Scholar]
  26. Kroy DC, Beraza N, Tschaharganeh DF, Sander LE, Erschfeld S, Giebeler A, Liedtke C, Wasmuth HE, Trautwein C & Streetz KL. 2010. Lack of interleukin-6/glycoprotein 130/signal transducers and activators of transcription-3 signaling in hepatocytes predisposes to liver steatosis and injury in mice. Hepatology 51 463–73. [DOI] [PubMed] [Google Scholar]
  27. Lee Y, Wang MY, Kakuma T, Wang ZW, Babcock E, McCorkle K, Higa M, Zhou YT & Unger RH. 2001. Liporegulation in Diet-induced Obesity: The antisteatotic role of hyperleptinemia. Journal of Biological Chemistry 276 5629–35. [DOI] [PubMed] [Google Scholar]
  28. Lee Y, Yu X, Gonzales F, Mangelsdorf DJ, Wang MY, Richardson C, Witters LA & Unger RH. 2002. PPARα is necessary for the lipopenic action of hyperleptinemia on white adipose and liver tissue. Proceedings of the National Academy of Sciences of the United States of America 99 11848–11853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lefere S & Tacke F. 2019. Macrophages in obesity and non-alcoholic fatty liver disease: Crosstalk with metabolism. JHEP Reports 1 30–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Li BY, Guo YY, Xiao G, Guo L, & Tang QQ. 2022. SERPINA3C ameliorates adipose tissue inflammation through the Cathepsin G/Integrin/AKT pathway. Molecular Metabolism 61 101500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Li WC, Ralphs KL & Tosh D. Isolation and culture of adult mouse hepatocytes. Methods in molecular biology. 185–197. [DOI] [PubMed] [Google Scholar]
  32. de Luca C, Kowalski TW, Zhang Y, Elmquist JK, Lee C, Kilimann MW, Ludwig T, Liu SM & Chua SC. 2005. Complete rescue of obesity, diabetes, and infertility in db/db mice by neuron-specific LEPR-B transgenes. Journal of Clinical Investigation 115 3484–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Marroquí L, Gonzalez A, Nẽco P, Caballero-Garrido E, Vieira E, Ripoll C, Nadal A & Quesada I. 2012. Role of leptin in the pancreatic β-cell: Effects and signaling pathways. Journal of Molecular Endocrinology 49 R9–R17. [DOI] [PubMed] [Google Scholar]
  34. Montague CT, Farooqi IS, Whitehead JP, Soos MA, Rau H, Wareham NJ, Sewter CP, Digby JE, Mohammed SN, Hurst A, et al. 1997. Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature 387 903–8. [DOI] [PubMed] [Google Scholar]
  35. Muoio DM & Dohm GL 2002. Peripheral metabolic actions of leptin. Best Practice and Research: Clinical Endocrinology and Metabolism 16 653–666. [DOI] [PubMed] [Google Scholar]
  36. Myers MG, Leibel RL, Seeley RJ & Schwartz MW. 2010. Obesity and leptin resistance: Distinguishing cause from effect. Trends in Endocrinology and Metabolism 21 643–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Qureshi K and Abrams GA. 2007. Metabolic liver disease of obesity and role of adipose tissue in the pathogenesis of nonalcoholic fatty liver disease. World Journal of Gastroenterology 13 3540–3553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rajkumar K, Modric T & Murphy LJ. 1999. Impaired adipogenesis in insulin-like growth factor binding protein-1 transgenic mice. Journal of Endocrinology 162 457–465. [DOI] [PubMed] [Google Scholar]
  39. Rajwani A, Ezzat V, Smith J, Yuldasheva NY, Duncan ER, Gage M, Cubbon RM, Kahn MB, Imrie H, Abbas A, et al. 2012. Increasing circulating IGFBP1 levels improves insulin sensitivity, promotes nitric oxide production, lowers blood pressure, and protects against atherosclerosis. Diabetes 61 915–924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Roglans N, Vilà L, Farré M, Alegret M, Sánchez RM, Vázquez-Carrera M & Laguna JC. 2007. Impairment of hepatic STAT-3 activation and reduction of PPARα activity in fructose-fed rats. Hepatology 45 778–788. [DOI] [PubMed] [Google Scholar]
  41. Saltiel AR & Khan CR. 2001. Insulin signaling and the regulation of glucose and lipid metabolism. Nature 414 799–806. [DOI] [PubMed] [Google Scholar]
  42. Sergi D, Campbell FM, Grant C, Morris AC, Bachmair EM, Koch C, McLean FH, Muller A, Hoggard N, de Roos B, et al. 2018. SerpinA3N is a novel hypothalamic gene upregulated by a high-fat diet and leptin in mice Genes and Nutrition 13 28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Smith U & Kahn BB. 2016. Adipose tissue regulates insulin sensitivity: role of adipogenesis, de novo lipogenesis and novel lipids. Journal of Internal Medicine 280 465–475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tabbi-Anneni I, Cooksey R, Gunda V, Liu S, Mueller A, Song G, McClain DA, Wang L. 2010. Overexpression of nuclear receptor SHP in adipose tissues affects diet-induced obesity and adaptive thermogenesis. American Journal of Physiology: Endocrinology And Metabolism 298 E961–E970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tjondrokoesoemo A, Schips T, Kanisicak O, Sargent MA & Molkentin JD. 2015. Genetic overexpression of serpina3n attenuates muscular dystrophy in mice. Human Molecular Genetics 25 1192–1202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tran M, Lee SM, Shin DJ & Wang L. 2017. Loss of miR-141/200c ameliorates hepatic steatosis and inflammation by reprogramming multiple signaling pathways in NASH. JCI Insight 2 e96094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tran M, Wu J, Wang L & Shin DJ. 2021. A potential role for SerpinA3N in acetaminophen-induced hepatotoxicity. Molecular Pharmacology 99 277–285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vicuña L, Strochlic DE, Latremoliere A, Bali KK, Simonetti M, Husainie D, Prokosch S, Riva P, Griffin RS, Njoo C et al. 2015. The serine protease inhibitor SerpinA3N attenuates neuropathic pain by inhibiting T cell-derived leukocyte elastase. Nature Medicine 21 518–523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Waise TMZ, Toshinai K, Naznin F, NamKoong C, Md Moin AS, Sakoda H & Nakazato M. 2015. One-day high-fat diet induces inflammation in the nodose ganglion and hypothalamus of mice. Biochemical and Biophysical Research Communications 464 1157–1162. [DOI] [PubMed] [Google Scholar]
  50. Wallace TM, Levy JC & Matthews DR. 2004. Use and abuse of HOMA modeling. Diabetes Care 27 1478–95. [DOI] [PubMed] [Google Scholar]
  51. Wang B, Chandrasekera P & Pippin J. 2014. Leptin- and Leptin Receptor-Deficient Rodent Models: Relevance for Human Type 2 Diabetes. Current Diabetes Reviews 10 131–145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wang T, Fahrmann JF, Lee H, Li YJ, Tripathi SC, Yue C, Zhang C, Lifshitz V, Song J, Yuan Y et al. 2018. JAK/STAT3-Regulated Fatty Acid β-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance. Cell Metabolism 27 136–150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yoshino S, Satoh T, Yamada M, Hashimoto K, Tomaru T, Katano-Toki A, Kakizaki S, Okada S, Shimizu H, Ozawa A et al. 2014. Protection against high-fat diet-induced obesity in Helz2-deficient male mice due to enhanced expression of hepatic leptin receptor. Endocrinology 155 3459–72. [DOI] [PubMed] [Google Scholar]
  54. Zhang Y, He J, Xu M, Lou D, Tso P, Li Z & Li X. 2017. Effect of ApoA4 on SERPINA3 mediated by nuclear receptors NR4A1 and NR1D1 in hepatocytes. Biochemical and Biophysical Research Communications 487 327–332. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS1867739-supplement-01.docx (15.8KB, docx)
02
NIHMS1867739-supplement-02.pdf (317.5KB, pdf)

RESOURCES