Targeting Hepatic Glutaminase 1 Ameliorates Non-alcoholic Steatohepatitis by Restoring Very-Low-Density Lipoprotein Triglyceride Assembly

Abstract

Non-alcoholic steatohepatitis (NASH) is characterized by the accumulation of hepatic fat in an inflammatory/fibrotic background. Herein, we show that the hepatic high-activity glutaminase 1 isoform (GLS1) is overexpressed in NASH. Importantly, GLS1 inhibition reduces lipid content in choline and/or methionine deprivation-induced steatotic mouse primary hepatocytes, in human hepatocyte cell lines, and in NASH mouse livers. We suggest that under these circumstances, defective glutamine fueling of anaplerotic mitochondrial metabolism and concomitant reduction of oxidative stress promotes a reprogramming of serine metabolism, wherein serine is shifted from the generation of the antioxidant glutathione and channeled to provide one-carbon units to regenerate the methionine cycle. The restored methionine cycle can induce phosphatidylcholine synthesis from the phosphatidylethanolamine N-methyltransferase-mediated and CDP-choline pathways as well as by base-exchange reactions between phospholipids, thereby restoring hepatic phosphatidylcholine content and very-low-density lipoprotein export. Overall, we provide evidence that hepatic GLS1 targeting is a valuable therapeutic approach in NASH.

Keywords: GLS1; GLS2; NAFLD; NASH; TCA cycle; VLDL; folate cycle; glutaminase; methionine cycle; phospholipids.

Figure 1.. Glutaminase 1 (GLS1) is overexpressed in clinical non-alcoholic steatohepatitis (NASH).

A. Serum levels of glutamine and the product of glutamine catabolism, glutamate, in a large cohort of patients diagnosed with NASH (n= 131) relative to a control group of healthy subjects (n= 90). A table showing the main serum biochemical parameters relative to these patients is shown. B. Liver immunohistochemical staining and respective quantification for the isoform 1 of glutaminase (GLS1), and inset zoom, the isoform 2 of glutaminase (GLS2) and glutamine synthetase in another cohort of NASH patients (n= 13) versus a control group of healthy subjects (n= 13). Scale bar corresponds to 100 μm. V-venous region; P-portal region. C. mRNA levels of GLS1 in a cohort of NASH patients (n=16) against a control group of age- and body-weight matched healthy controls (n=5). Data is shown as average ± SEM and Student’s t-test was used to compare groups. *p<0.05 and ****p<0.0001 against the control group are shown (See also Table S1).

Figure 2.. Glutaminase 1 (GLS1) is overexpressed in a mouse model of non-alcoholic steatohepatitis (NASH) of choline deficient and 0.1% methionine diet (0.1% MCDD)-fed rodents.

A. Hepatic Glutaminase 1 (GLS1), with higher magnification zoom shown in inset, and Glutaminase 2 (GLS2) immunostaining and respective quantifications. Scale bar corresponds to 100 μm. V-venous region; P-portal region; B. Hepatic GLS1 and GLS2 protein levels by Western blot analysis. Glyceraldehyde-3-phosphate (GAPDH) was used as a loading control; and C. Hepatic Gls1 and Gls2 mRNA levels in mice fed a choline deficient and 0.1% methionine diet (0.1% MCDD) against a standard chow diet (SC diet) during 2, 4 and 6 weeks. Data is shown as average ± SEM and Student’s t-test was used to compare groups. *p<0.05, **p<0.01 and ***p<0.001 are shown versus age- and gender-matched animals maintained on SC diet. D. Representative consecutive slides staining for GLS1, F4/80 and Sirius red staining in liver biopsies of animals maintained for four weeks or six weeks on 0.1% MCDD (fibrosis areas highlighted with white dashed line and GLS1 with yellow dashed line). E. Immunofluorescence double co-staining for GLS1 and albumin, a marker of hepatocytes, and alpha smooth muscle actin (aSMA), a marker of hepatic stellate cells (HSC) in 0.1% MCDD-fed mice for 4 weeks. (See also Supplemental Figure 1, Supplemental Figure 2, Supplemental Figure 3 and Supplemental Figure 4).

Figure 3.. Targeting Glutaminase 1 (GLS1) in vitro and in vivo resolves the accumulation of hepatic triglycerides and non-alcoholic steatohepatitis (NASH).

A. Western blot analysis of total protein levels of Glutaminase 1 (GLS1) and Glutaminase 2 (GLS2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control; B. Representative BODIPY staining micrographs and respective quantification. Scale bar corresponds to 100 μm; and C. Mass-spectrometry analysis of different triglyceride (TG) isoforms in mouse isolated hepatocytes treated for 48 h with methionine- and choline-deficient media (MCD) after treatment with siRNA against Gls1 (siGls1) or unrelated control (siCtrl). At least triplicates were used for each experimental condition. Data is shown as average ± SEM and Student’s t-test was used to compare between the groups. *p<0.05 and **p<0.01 versus MCD + siCtrl and ^##p<0.01 versus mcd + siCtrl are shown. D. GLS1, GLS2 and glutamine synthetase levels quantified by Immunohistochemistry and representative micrographs of Sudan Red staining and respective quantification in animals maintained on a choline deficient and 0.1% methionine diet (0.1% MCDD). From weeks two to four of diet, two different experimental groups were treated either with siCtrl or siGls1. Scale bar corresponds to 100 μm. At least n=5 were used for each experimental group. Data is shown as average ± SEM and one-way ANOVA followed by Bonferroni post-test was used to compare between multiple groups. *p<0.05, **p<0.01 and ***p<0.001 versus SC diet and ^#p<0.05 versus 0.1%MCDD + siCtrl are shown (See also Supplemental Figure 4, Supplemental Figure 5, Supplemental Figure 6 and Supplemental Table2).

Figure 4.. Targeting Glutaminase 1 (GLS1) in vitro and in vivo restores very-low-density lipoproteins (VLDL) triglyceride export after choline and methionine deprivation.

A. Representative BODIPY staining micrographs and respective quantification in mouse isolated hepatocytes treated with control media (Ctrl) or methionine- and choline -deficient media (MCD) for 24 h after overnight treatment with siRNA against Gls1 (siGls1) or unrelated control (siCtrl). In some experimental conditions lomitapide was added at 600 nM for 24 hours. Scale bar corresponds to 100 μm. At least triplicates were used for each experimental condition. Data is shown as average ± SEM and Student’s t-test was used to compare between groups. *p<0.05 and **p<0.01 versus Ctrl + siCtrl and ^##p<0.01 versus MCD + siCtrl are shown. B. Liver phosphatidylcholine (Ptd-Cho), phosphatidylethanolamine (Ptd-Et) and phosphatidylserine (Ptd-Ser) hepatic levels and C. Serum very-low-density lipoprotein (VLDL) phospholipids and lipid content in mice fed either a standard chow (SC) diet or a diet deficient in choline with 0.1% methionine (0.1% MCDD) for four weeks. From weeks two to four of diet, two different experimental groups were treated either with siCtrl or siGls1. Animals were administered poloxamer 407 (P407) and serum VLDL isolated and analyzed at six hours after P407 administration. At least n=5 were used for each experimental group. Data is shown as average ± SEM and one-way ANOVA followed by Bonferroni post-test was used to compare between multiple groups. **p<0.01, ***p<0.001 and ****p<0.0001 versus SC diet and ^#p<0.05 and ^##p<0.01 versus 0.1%MCDD + siCtrl are shown (See also Supplemental Figure 7).

Figure 5.. Targeting Glutaminase 1 (GLS1) in vitro and in vivo reduces oxidative stress.

A. Total and mitochondrial reactive oxygen species (ROS) levels in mouse isolated hepatocytes treated with control media (Ctrl) or methionine and choline deficient media (MCD) for 48 h after overnight treatment with siRNA against Gls1 (siGls1) or unrelated control (siCtrl). At least triplicates were used for each experimental condition. Data is shown as average ± SEM and one-way ANOVA followed by Bonferroni post-test was used to compare between multiple groups. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 versus Ctrl media as well as ^###p<0.001 versus MCD media + siCtrl are shown. B. Oxidized glutathione (GSSG) and reduced glutathione (GSH) ratio and incorporation of ¹³C carbons from U-¹³C(glutamine) carbons into 5-¹³C(GSH) in mice fed either a standard chow diet (SC) or a diet deficient in choline with 0.1% methionine (0.1% MCDD) for four weeks. From weeks two to four of diet, two different experimental groups were treated either with siCtrl or siGls1. At least n=5 were used for each experimental group. Data is shown as average ± SEM and one-way ANOVA followed by Bonferroni post-test was used to compare between multiple groups. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 versus SC diet and ^#p<0.05 and ^##p<0.01 versus 0.1%MCDD+siCtrl are shown. C. Oxidized and reduced glutathione ratio (GSSG/GSH) in animals maintained on a choline deficient and high fat diet (CD-HFD) for 6 weeks. From week 3 to 6 of diet, two different experimental groups were treated either with siCtrl or siGls1 (CD-HFD + siCtrl or CD-HFD + siGls1). At least n=5 were used for each experimental group. Data is shown as average ± SEM and Student’s t-test was used to compare between groups. *p<0.05 versus CD-HFD + siCtrl is shown. D. Malondialdehyde (MDA) levels as a measurement of lipid peroxidation; E. Citrate and incorporation of U-¹³C(glutamine) carbons on 1-¹³C(citrate) levels; F. Fatty acid oxidation (FAO) rate quantified from the incorporation of ¹⁴C-palmitate into CO₂ and in acid-soluble metabolites (ASM); and G. Mitochondrial Oxygen Consumption Rate (OCR) in different states of the respiration (State 2, State 3, State 4o, State 3u) in mice fed either a standard chow diet (SC) or a diet deficient in choline with 0.1% methionine (0.1% MCDD) for four weeks. From weeks two to four of diet, two different experimental groups were treated either with siCtrl or siGls1. At least n=5 were used for each experimental group. Data is shown as average ± SEM and one-way ANOVA followed by Bonferroni post-test was used to compare between multiple groups. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 versus SC diet and ^#p<0.05 and ^##p<0.01 versus 0.1%MCDD+siCtrl are shown (See also Supplemental Figure 3).

Figure 6.. GLS1-mediated reduction of oxidative stress is associated with increased phospholipid synthesis and the activation of folate and methionine cycles.

A. Differential expression of mRNA levels from genes significantly different involved either in glutathione (GSH) synthesis through the transsulfuration pathway, and the folates and methionine cycles in mice on a 0.1% MCDD and with Gls1 silencing (siGls1) versus control silencing (siCtrl) for four weeks. (Bhmt = betaine-homocysteine S-methyltransferase; Cbs = cysthationine-beta synthase; Cth = cysthathionine gamma-lyase; Gclc = glutamate-cysteine ligase, catalytic subunit; Gclm = glutamate-cysteine ligase, modifier subunit; Gls1 = glutaminase 1; GNMT = glycine N-methyltransferase; GSS = glutathione synthetase; Mat1a = methionine adenosyltransferase 1A; Mat2a = methionine adenosyltransferase 2A; Mat2b = methionine adenosyltransferase 2B; Ms = methionine synthetase; Mthfr = methylenetetrahydrofolate reductase; Mthfs = synthetase; Sahh = S-adenosyl-homocysteinase). (DMG = dimethyglycine; MTHF = L-methylfolate; SAMe = S-adenosylmethionine; SAH = S-adenosylhomocysteine; THF = tetrahydrofolate). B. Differential expression of mRNA levels from genes significantly different involved in phospholipid biosynthesis in mice on a 0.1%MCDD with siGls1 versus siCtrl for four weeks. (Cept1 = choline/ethanolamine phosphotransferase 1; Chk = choline kinase; Chpt1 = choline phosphotransferase 1; Etnk2 = ethanolamine kinase 2; Pcyt1a = phosphate cytidylyltransferase 1, choline; Pcyt2 = phosphate cytidylyltransferase 2, ethanolamine; Pemt = phosphatidylethanolamine methyltransferase; Pisd = phosphatidylserine decarboxylase; Ptdss1 = phosphatidylserine synthase 1; Ptdss2 = phosphatidylserine synthase 2). At least n=5 were used for each experimental group. Student’s t-test was used to compare the two groups and significance was set to p<0.05 (See also Supplemental Table 3).

Figure 7.. Flowchart of Glutaminase 1 (GLS1)-inhibition resolution of non-alcoholic steatohepatitis (NASH).

Deprivation of methionine and choline induces NASH through the inhibition of very-low-density lipoprotein (VLDL) assembly/export. Under these circumstances, excessive glutamine catabolism mediated by GLS1 in NASH accounts for increased levels of glutamate that can be further deaminated to a-ketoglutarate in order to sustain augmented tricarboxylic acids (TCA) cycle anaplerosis and augmented fatty acid oxidation (FAO). Hepatic anaplerotic pathways are energetically backed by elevated oxidative metabolism in the liver through oxidative phosphorylation (OXPHOS). On the other hand, GLS1 inhibition under methionine and choline deprivation is associated with decreased TCA cycle activity, associated diminishment of mitochondrial OXPHOS, and reduced production of reactive oxygen species (ROS). Under these circumstances, serine demand for glutathione (GSH) synthesis is diminished and in alternative serine is channeled to phophatidylcholine synthesis, leading to the formation of lipid-enriched VLDL particles and decreased liver steatosis in a metabolic reprogramming where folates and methionine cycles are relevant. Overall, GLS1 inhibition is suggested as a novel therapeutic approach during NASH management.