Itaconic acid underpins hepatocyte lipid metabolism in non-alcoholic fatty liver disease

doi:10.21203/rs.3.rs-2109910/v1

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Itaconic acid underpins hepatocyte lipid metabolism in non-alcoholic fatty liver disease

https://doi.org/10.21203/rs.3.rs-2109910/v1

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published 12 Jun, 2023

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Itaconate, the product of the decarboxylation of cis-aconitate, regulates numerous biological processes. We and others have revealed itaconate as a regulator of fatty acid beta-oxidation, generation of mitochondrial reactive oxygen species and the metabolic interplay between resident macrophages and tumors. In the present study, we show that itaconic acid is upregulated in human non-alcoholic steatohepatitis and a mouse model of non-alcoholic fatty liver disease. Mice deficient in the gene responsible for itaconate production (Immunoresponsive gene /Irg-1) have exacerbated lipid accumulation in the liver, glucose and insulin intolerance and mesenteric fat deposition. Treatment of mice with the itaconate derivative, 4-OI, reverses dyslipidemia associated with high fat diet feeding. Mechanistically, itaconate treatment of primary hepatocytes reduces lipid accumulation and increases their oxidative phosphorylation in a manner dependent upon fatty acid oxidation. We propose a model whereby macrophage-derived itaconate acts in trans upon hepatocytes to modulate the liver’s ability to metabolize fatty acids.

Non-alcoholic fatty liver disease (NAFLD) is a global health crisis in adults and children¹. Often associated with excess consumption of calories, accumulated adipose tissue and obesity, NAFLD represents a spectrum of liver disease which is closely linked with inflammation, metabolic syndrome, insulin resistance and a host of risk factors for advanced disease such as liver cirrhosis and hepatocellular carcinoma ^2-4. As the predominant site for the uptake, storage and export of lipid, the liver plays an indispensable role in the metabolism of fat. The liver is a major site for the oxidation of triglycerides, generating fatty acids that are exported to the circulation, used by various tissues in the body for energy or stored in adipose tissue³. Hepatic lipid accumulation results from an imbalance between lipid availability and removal via fatty acid oxidation or lipoprotein secretion. Enhanced lipolysis in adipose tissue can result in higher circulating fatty acid levels and hepatic lipogenesis. When excess adiposity occurs, often due to dietary or genetic factors, free fatty acids released from adipose tissue may overwhelm the liver’s capacity to oxidize or export lipids, contributing to the development of fatty liver disease and steatosis. Obesity-associated changes in blood glucose levels and insulin intolerance can result in “lipid spillover” characterized by alterations in lipid metabolism that contribute to high circulating levels of lipid and precede the development of metabolic syndromes such as Type 2 diabetes, atherosclerosis and a progression of hepatic steatosis, fatty liver disease and liver cirrhosis ^{3, 4}. A better understanding of the metabolic regulation of lipid metabolism by the liver may facilitate the development of therapeutic interventions that counteract this growing health crisis.

Regulation of lipid homeostasis and metabolism is complex, governed by a large array of hormones, metabolites and transcription factors ^{2, 3}. Among metabolites, acetyl-coenzyme A (acetyl-CoA), a product of the oxidation of pyruvate during glycolysis, beta oxidation of fatty acids or metabolism of certain amino acids, plays a central role in lipid metabolism³. In addition to its role as the entry point into the Citric Acid Cycle (TCA), acetyl-CoA also serves as a principal precursor for the synthesis of fatty acids and lipids. We hypothesized that other metabolic intermediates might exert as yet poorly defined roles in lipid metabolism. As one example, we and others showed previously that the immunometabolite itaconic acid ⁵ (itaconate) regulated beta oxidation of lipids promoting mitochondrial reactive oxygen species (ROS) and oxidative phosphorylation of macrophages ^{6, 7}. Itaconate is formed by diverting cis-aconitate away from the TCA during inflammatory macrophage activation, a decarboxylation reaction catalyzed by the enzyme aconitate decarboxylase 1 (Acod1, referred to here as immunoresponsive gene-1; Irg1) ⁸. Itaconate has been described as an anti-inflammatory immunometabolite that exerts regulatory roles in numerous pathologies including cancer, infection, sepsis, and tissue injury responses ^{7, 9-13}. Metabolism of itaconate begins with its activation to itaconyl-CoA by succinyl-CoA ligase, which has been postulated to serve as a “CoA sink” that may regulate fatty acid metabolism and other CoA-dependent pathways ¹⁴.

In the present study, we identify an important and underappreciated role for itaconic acid in lipid metabolism in the liver and the progression of NAFLD. We find that both Irg1 and itaconate accumulates in liver macrophages in a mouse model of NAFLD and human NASH livers. Mice with global or myeloid-specific deletion of Irg1 demonstrated a dramatic accumulation in adiposity, exacerbated lipid accumulation (prominently triglycerides) in the liver and exacerbated glucose and insulin intolerance. Mechanistically, we find that macrophage-derived itaconate acts in trans to alter the oxidative profile and lipid metabolism of hepatocytes. Our results reveal novel pathways whereby itaconate regulates hepatocyte responses to lipid and may help identify Irg1 and itaconate as potential targets during NAFLD.

A role for the immunometabolite itaconic acid in the beta oxidation of lipids as a source of fuel for oxidative phosphorylation and mitochondrial ROS has been reported in tumor-associated macrophages, J774.2 and zebrafish macrophage lineage cells ^{6, 7}. We asked whether Irg1 might regulate lipid homeostasis in primary bone marrow macrophages (BMM). Using unbiased metabolomics we found that Irg1^−/− mice have dysregulated lipid metabolism compared to wildtype cells. BMM from Irg1^−/− mice showed dramatic accumulation of carnitine conjugated fatty acids, along with reduced levels of many long-chain polyunsaturated fatty acids (Fig. 1A). Although the BMM in this analysis were unstimulated, we confirmed itaconate production during BMM culture using mass spectrometry (Fig. 1B) suggesting that exposure of BMM to itaconate during derivation affects their lipid metabolism.

We also detected a similar lipid dysfunction in resident peritoneal macrophages. Unstimulated peritoneal resident macrophages from Irg1^−/− mice as well as Irg1 ^fl/fl x LysM ^Cre mice had significantly elevated neutral lipid content as detected by BODIPY staining compared to cells from wildtype mice (Fig. 1C. Taken together, these results prompted us to investigate the role for Irg1 and itaconic acid in a mouse model of hyperlipidemia.

Mice fed a Western diet (WD) for 12 weeks develop hyperlipidemia in the liver, denoted by extensive lipid accumulation, elevated serum triglycerides and insulin resistance^{15, 16}. Our analysis of previously published transcriptomic data of hepatic myeloid cells in WD fed wildtype mice ¹⁷ indicated Irg1 expression was significantly upregulated in the Clec4f⁻ macrophage population that is derived from immigration of circulating macrophages following 12–36 weeks of WD feeding (Fig. 2A). Irg1 expression was detectable in resident Kupffer cells (KC), however this expression dramatically declined with WD feeding and no Irg1 expression was detectable in proliferating KCs. Robust Irg1 expression was noted in neutrophils, however this was completely independent of WD feeding suggesting the macrophage but not neutrophil expression of Irg1 was related to hyperlipidemia. Because Irg1 expression has been reported in hepatocytes during ischemia-reperfusion disease ¹⁰, we investigated further the cellular source of hepatic Irg1 and itaconic acid and interrogated the role of Irg1-catalyzed itaconic acid during WD feeding. We compared lipid pathologies between wildtype and Irg1^−/− mice bred on to identical genetic backgrounds. Quantitative PCR analysis on livers confirmed induction of Irg1 mRNA after 12 weeks of WD feeding (Fig. 2B). Molecular analysis of livers from mice bearing tissue-specific deletion of Irg1 showed that its expression was confined to the myeloid compartment since WD-induced Irg1 expression was abrogated in Irg1^fl/fl x LysM^Cre but preserved in Irg1^fl/fl x Alb^Cre mice that have the gene deleted in hepatocytes (Fig. 2B). No Irg1 expression was detected in macrophages isolated from adipose tissue (data not shown). Mass spectrometry confirmed the accumulation of itaconic acid in isolated F4/80 positive macrophages from these livers, while no itaconic acid was expressed in F4/80 negative cell fractions (Fig. 2C). Together these data demonstrate that loss of Irg1 is associated with lipid accumulation in macrophages and that WD feeding results in Irg1 and itaconate expression in hepatic macrophages, but not hepatocytes. To determine whether itaconate was upregulated in human NASH, we obtained liver biopsies from 10 non-NASH controls and 16 NASH patients. By qPCR, Irg1 mRNA expression was significantly increased in NASH as compared to non-NASH controls (Fig. 2D). Furthermore, itaconate levels were significantly upregulated in liver tissue from human NASH, as compared to non-NASH controls (Fig. 2E). The diagnosis of NASH provided to us by University of Pittsburgh was further corroborated by a significant upregulation of IL-6 mRNA expression in NASH samples (Supplemental Fig. 1A). Among other metabolites, we found succinate and glutamine were unchanged, whereas lactate, citrate and alpha-ketoglutarate were significantly upregulated in NASH, (Supplemental Figs. 1B-F).

Remarkably, Irg1^−/− mice fed WD exhibited increased lipid accumulation in the liver as compared to wildtype mice (Figs. 3A and 3B for oil red O [ORO] staining and Fig. 3C for hematoxylin and eosin [H&E] staining). Intriguingly, Irg1^−/− mice fed control chow diet also had significantly increased lipid staining as compared to control wildtype mice, suggesting an important role for Irg1 in both homeostatic and pathogenic lipid metabolism in the liver. Adipose and liver tissue from WD-fed mice but not control diet fed mice was significantly increased in weight (Fig. 3D and 3E) and Irg1^−/− mice fed either control or Western diets had significantly elevated free fatty acids in the blood (Fig. 3F) and increases in liver triglycerides (Fig. 3g). No differences in serum triglycerides or cholesterol were observed nor was there any difference in adipose triglycerides (Supplemental Fig. 2).

Having established an important role for itaconate in the regulation of lipid metabolism in the liver, we asked whether treatment of WD-fed mice with the cell-permeable derivative, 4-OI, could ameliorate dysfunctional lipid metabolism in Irg1^−/− mice. We treated mice with 4-OI for the 12 weeks of WD feeding and observed that 4-OI treatment significantly reduced liver ORO staining (Fig. 4A-4C) and mesenteric fat weight (Fig. 4D). These findings indicate that exogenous administration of itaconate can successfully reverse the severe hyperlipidemic phenotype in WD fed Irg1-/- mice.

Given the changes in liver lipid burden we assessed characteristics of metabolic syndrome in mice lacking Irg1. Irg1^−/− mice fed either control or WD develop glucose intolerance as compared to respective diet fed wildtype mice (Fig. 5A). Irg1^−/− mice on WD also had significantly elevated blood insulin levels (Fig. 5B) and insulin insensitivity (Fig. 5C). These findings were evident even though no differences were observed in body weight between wildtype and Irg1^−/− mice (Fig. 5D) and no differences were observed in the amount of food intake by the mice (Supplemental Fig. 3). Consistent with the patterns of expression we documented, Irg1 deficiency in the myeloid compartment was sufficient to yield elevated insulin resistance since Irg1^fl/fl x LysM^Cre mice exhibited similar insulin intolerance as global Irg1^−/− mice; regardless of whether mice were fed a control diet (Fig. 5E) or WD (Fig. 5F).

WD-driven metabolic disorder is associated with substantial remodeling of adipose tissue by lipid-associated macrophages (LAM). Even though we did not detect Irg1 expression in LAM during control or WD feeding, the increased mesenteric fat burden of Irg1^−/− mice prompted us to assess LAM number and architecture. We found the accumulation of lipid in the mesenteric fat and liver in mice lacking Irg1 was accompanied by a striking loss of F4/80⁺ macrophages in both these tissues. In the liver, significant reductions in the frequencies of F4/80⁺ cells were observed in both Irg1^−/− and Irg1^fl/fl x LysM^Cre mice fed the WD (Figs. 6A and 6B), and hepatic Crown like structures were severely reduced (Fig. 6C). Similarly, F4/80⁺ cells were reduced and Crown like structures were nearly absent in adipose tissue of Irg1^−/− mice fed a WD (Figs. 6D-6E). Given the important homeostatic role for hepatic and adipose macrophages, our findings suggest their striking loss in Irg1^−/− mice accompany dysregulated lipid metabolism.

To begin to reveal the mechanisms behind these phenomena, we isolated F4/80⁺ macrophages from the livers of wildtype or Irg1^−/− mice and performed targeted metabolomics. These data confirmed preferential accumulation of several long-chain fatty acids in the macrophages from Irg1^−/− mice fed the Western diet (Supplemental Fig. 4A). Moreover, several key metabolites associated with mitochondrial function including pyruvate, fumarate, citrate, and oxaloacetate were reduced in macrophages from WD fed Irg1^−/− mice compared to their wild type counterparts (Supplemental Fig. 4B). These data suggest subdued mitochondrial function in the absence of Irg1. We considered that metabolic dysfunction in hepatic macrophages alone would be unlikely to fully explain the substantial liver disease phenotype of mice lacking Irg1, but that these cells may represent sentinels of greater liver metabolic changes. Based on the lipid dysfunction we observed in BMM and liver macrophages lacking Irg1, we hypothesized that myeloid-derived itaconic acid might act in trans to modulate lipid metabolism by hepatocytes. We tested this possibility by assessing the metabolic response of hepatocytes to exogenous itaconate. Exogenous itaconate readily accumulated in hepatocytes (Supplemental Fig. 5A). Surprisingly, we saw no itaconate-induced accumulation of succinate (Supplemental Fig. 5B) in hepatocytes; nor did we detect any alterations in cis-aconitate or pyruvate levels (Supplemental Figs. 5C and 5D, respectively). Next, we assayed lipid accumulation in cultures of primary hepatocytes challenged with exogenous lipid with or without ex vivo treatment with sodium itaconate ¹⁸. Targeted lipidomics indicated that multiple triglycerides (Fig. 7A) and acyl-carnitines (Fig. 7B) were significantly downregulated in itaconate treated hepatocytes. Moreover, hepatocytes challenged with lipid showed the expected increase in BODIPY staining and itaconate exposure at either 5 mM or 10mM reduced that lipid staining (Fig. 7C). By both BODIPY staining of neutral lipids (Fig. 7C) and targeted lipidomics (Supplemental Fig. 6), significantly reduced lipid accumulation was observed in itaconate treated hepatocytes. Similarly, itaconate treatment of the human hepatocyte (HepG2) cell line caused a significant reduction in BODIPY staining, as a single agent or in combination with exogenously added lipid (Supplemental Fig. 6).

Flux analysis of similarly treated cells showed that lipid challenge of hepatocytes increased their oxygen consumption rates (OCR), and that this was accentuated by exposure to itaconate (Fig. 7D). The itaconate-mediated increase in lipid-induced OCR was completely abrogated in the presence of etomoxir, confirming the role for fatty acid oxidation as the source of lipid- and itaconate-induced increases in oxygen consumption in hepatocytes. In these experiments, itaconate treatment also caused an increase in glycolysis in hepatocytes, both as a single agent and in combination with lipid challenge (Fig. 7E). Together, these data demonstrate the ability of exogenous itaconate to enhance oxidative phosphorylation of lipids and that this increase is not simply the result of glycolytic inhibition.

Although these data strongly imply a fundamental itaconate-induced alteration in metabolic processing, multiple reports have shown itaconate capable of eliciting substantial changes in transcription. Thus, to determine whether itaconate exerted transcriptional differences in hepatocytes that could account for the hyperlipidemic phenotype, we performed RNA seq on lipid challenged hepatocytes exposed or not to itaconate. We then interrogated expression of genes in the KEGG reference pathways for fatty acid metabolism ¹⁹. Although the expression of 3 out of 35 genes (Adh7, Echs1, Eci2) in the fatty acid reference pathway showed statistical differences between untreated and itaconate treated hepatocytes, and 4 genes (Aldh1b1, Echs1, Eci2, Hadha) were significantly changed between lipid and lipid + itaconate treated hepatocytes, Gene Set Enrichment Analysis showed no significant difference in this pathway (Supplemental Fig. 7A). Moreover, despite previous reports implicating hepatic Irg1 induced by ischemia/reperfusion in the regulation of the Nrf2 antioxidant response ¹⁰ and those suggesting itaconate as a key regulator of Nrf2 activity in macrophages ²⁰, we show that itaconate exposure of hepatocytes had only a minimal effect on Nrf2 target expression ²¹. Out of 13 target genes, just one (Gsta3) showed significance upon itaconate treatment, but with less than 30% change between treatment groups, whereas 12 other genes showed no significant differences attributable to itaconate (Supplemental Fig. 7B).

We next hypothesized metabolic changes within itaconate-exposed hepatocytes might underlie enhancements in beta oxidation of lipid. As expected, itaconate treatment of hepatocytes resulted in increased levels of itaconyl CoA (Fig. 8A). Whereas lipid treatment increased all CoA species we interrogated, we saw that itaconate had no effect on the levels of succinyl CoA (Fig. 8B), malonyl CoA (Fig. 8C) or acetyl CoA (Fig. 8D). Moreover, the addition of exogenous CoA (CoASH) had no effect on the BODIPY lipid staining in lipid + itaconate treated hepatocytes (Fig. 8E). Taken together, these data argue against the CoA sink model in which itaconyl CoA might regulate fatty acid metabolism and other CoA-dependent pathways by sequestering CoA ¹⁴. We next interrogated whether itaconate could suppress mitochondrial substrate level phosphorylation (mSLP) as proposed ²². Indeed, itaconate-treated hepatocytes display a shift in their ADP:ATP ratio consistent with the loss or consumption of ATP due to conversion of itaconate to itaconyl CoA accompanied by increases in ADP (Fig. 8F). These data are consistent with a model where reduced ATP levels due to a block in mSLP lead to compensatory increases in both beta oxidation and glycolysis, as we observed (Fig. 7), to facilitate the partial control of lipid levels and restore cellular ATP levels (schematized in Fig. 8G).

Altered metabolic pathways in the context of excess caloric intake can profoundly contribute to the development of fatty liver disease or hepatic steatosis, a chronic condition emerging as a major global health crisis^{1, 4}. The liver is the principal organ involved in the homeostasis of lipid metabolism, where tightly regulated biochemical pathways regulate the storage, biosynthesis, transport, and elimination of fatty acids. Hepatic metabolism of fatty acids and triglycerides occurs primarily in hepatocytes³. Although the liver normally stores small amounts of triglycerides, in the context of excess caloric intake and disrupted lipid metabolism, triglycerides accumulate within hepatocytes, with severe impacts upon inflammation, cell death and fibrosis. Excess lipid accumulation can lead to altered lipid homeostasis that may manifest clinically as a spectrum of pathologies including obesity, type 2 diabetes, and insulin resistance. Extensive steatosis may progress to liver failure involving cirrhosis and hepatocellular carcinoma.

Mitochondrial beta-oxidation represents a principal route for the processing of most fatty acids in hepatocytes³. Our study highlights an underappreciated role for metabolites such as itaconic acid to regulate fatty acid oxidation in hepatocytes. Itaconic acid, and Irg1, the gene responsible for itaconate synthesis, was discovered as a metabolite produced mainly by macrophages upon activation with a range of stimuli, including lipopolysaccharide, interferons and other Toll-like receptor stimuli^{5, 8, 12}. Itaconate is noteworthy because it has anti-inflammatory ^{20, 23–25} and potentially pro-tumorigenic^{7, 26} properties and the understanding of its expression beyond myeloid immune cells has widened. In the liver, itaconate production by both hematopoietic cells and hepatocytes was responsible for protective responses following ischemia/reperfusion injury by stimulating the Nrf2-dependent protection of hepatocytes against oxidative stress ¹⁰. Interestingly, we did not detect Nrf2 activation in hepatocytes treated with itaconate, a discrepancy possibly attributable to opposing effects on electrophilic stress and immune modulation between unmodified itaconate used in our studies and itaconyl derivatives such as 4-OI ¹⁸. However, 4-OI potently reversed the hyperlipidemia and adiposity in high fat diet-fed Irg1-deficient mice. The finding that 4-OI did not have an effect in wildtype mice we believe highlights a poorly recognized homeostatic role for endogenous itaconate levels in the liver during lipid-related pathology.

Our data contribute to the expanding literature of myeloid-centric expression of Irg1 and itaconic acid during pathogenic processes. In the mouse model of NAFLD we employed, Irg1 expression was seemingly confined to the myeloid compartment of cells and indistinguishable lipid phenotypes were observed in mice bearing both global and myeloid cell-specific deletion of Irg1. Given the absence of Irg1 and itaconate expression by hepatocytes in the model we used, we believe that Irg1 expression by hepatocytes following ischemia-reperfusion¹⁰ likely represents an important protective response that is specific to the injury achieved in that model. During the progression of fatty liver disease, resident Kupffer cells and local hematopoietic macrophages play critical roles in liver tissue remodeling and response to injury ^{27, 28}. Hepatic macrophages can be activated by gut-derived endotoxins, fatty acids, cholesterol metabolites and molecules released from damaged hepatocytes whereby they can secrete proinflammatory cytokines, chemotactic factors, and ROS; contribute to extracellular matrix degradation and tissue remodeling and promote angiogenesis. The frequency of activated macrophages is a predictive factor in the progression of human NAFLD²⁹. Although activated macrophages also accumulate in adipose tissue and further fuel insulin resistance and hepatic lipid accumulation during NAFLD^{30, 31}, we found Irg1 and itaconate expression was essentially confined to the macrophages of the liver, consistent with their unique roles in integrating signals from the gut/diet and as drivers of the progression of NAFLD to fibrosis and advanced steatohepatitis³⁰. Although itaconate exerts protective effects against oxidative stress in hepatocytes, we believe this is unlikely to be the case within the context of hepatic macrophages, since we and others showed itaconic acid promotes lipid beta oxidation as a fuel source for oxidative phosphorylation that generates mitochondrial reactive oxygen species contributable to oxidative stress in macrophage lineage cells ^{6, 7}. Itaconate deficient macrophages have defective oxidative phosphorylation, and we cannot rule out contributions of hepatic macrophages to the lipid phenotype evident in Irg1^−/− mice. We believe it likely, however that any such effects by macrophages are likely to be dwarfed by the direct influence of itaconate upon lipid metabolism by hepatocytes, due to their proficiency in lipid processing.

Of particular importance is the upregulated expression of Irg1 and itaconate in human NASH. Moreover, we show that human hepatocytes exposed to itaconate show reductions in BODIPY lipid staining both in the presence and absence of exogenously added lipid. Taken together, we demonstrate the plausible role for itaconate in the regulation of lipid metabolism in NASH patient livers.

A role for Irg1 in metabolic disease was reported recently ³². In that study, Irg1 expression by adipocytes regulated inflammation and glucose homeostasis, although in that report, hepatic expression of Irg1 was not investigated. As the central organ for the metabolism of fatty acids, our findings of Irg1 expression in the liver in both a mouse model of NAFLD and human NASH cases, and the subsequent impacts upon hepatocyte metabolism expand considerably our knowledge of the role by itaconate in lipid metabolism. Moreover, our data showing reduced lipid accumulation by the itaconate derivative, 4-OI, provides evidence of the therapeutic potential for targeting metabolites, including itaconate, for treating metabolic disease.

Given the excessive adiposity upon Irg1 deficiency, we propose a feedback system in which IRG1 expression by hepatic macrophages emerges as an important mechanism underlying the livers’ ability to metabolize lipid. Lipid homeostasis in Kupffer cells isolated from high fat diet-fed mice is already profoundly dysregulated, with prevailing bioactive and toxic lipid metabolites³³. Our study supports the premise that Irg1 expression is induced in NAFLD as an injury response mechanism to metabolize excess lipid, although the potential for antimicrobial responses due to gut leakiness remains another possibility. In the absence of myeloid Irg1 expression, liver lipid metabolism is impaired and potentially overwhelmed, resulting in exacerbated hyperlipidemia in association with the dramatic accumulation of carnitine species of fatty acids. The conjugation of carnitine to long-chain fatty acids is a prerequisite for their subsequent transfer into the mitochondria for beta-oxidation³⁴. As the liver becomes less able to process and oxidize lipid, the accumulating lipid becomes associated with increases in adiposity and a dramatic loss of macrophages in both adipose tissue and liver, presumably due to lipid-associated toxicity. During NAFLD, adipose tissue becomes dysfunctional, characterized by low-grade inflammation and the appearance of crown-like structures comprised of F4/80⁺ macrophages surrounding dying adipocytes ^{35, 36}. In the liver, a similar structure has been described, consisting of F4/80⁺ macrophages forming a ring-like structure, generally surrounding dead or dying hepatocytes, a common feature of NASH¹⁸.

The loss of Acod1⁺ Kupffer cells (KC) accompanied by the significant upregulation of Acod1/Irg1 expression in F4/80⁺ macrophages is consistent with a model whereby resident macrophages undergo cell death and are replaced by newly recruited macrophages ^{27, 37}. Of note, studies of single cell transcriptomic analysis during NAFLD revealed Irg1 expression was upregulated in response to WD feeding in the CLEC4f⁻ macrophages recruited from the circulation but not resident KC ¹⁷. In that study, WD feeding elicited the loss of KC due to apoptosis concomitant with the dramatic appearance of Irg1⁺ macrophages from the circulation. Although recruited macrophages undergo reprogramming in the liver environment and eventually take up characteristics of resident KC³⁷, Irg1⁺ KC remain undetectable even as late as 36 weeks of WD feeding, indicating hepatic Irg1 expression in our study is predominantly macrophages of circulatory origin. Neutrophils also accumulate during NASH and contribute to hepatocyte injury ³⁸ and we cannot rule out contributions of Irg1⁺ neutrophils towards lipid metabolism in the liver. However, Irg1 expression by neutrophils is independent of Western diet feeding, neutrophil cell numbers across all cellular zones of the liver are dwarfed by those of monocytes and macrophages in the steatotic liver ¹⁷ and we show itaconate expression confined to F4/80-sorted macrophages. IRG1 was co-expressed with interferon receptor (IFNAR) and Stat1 gene expression ^{17, 27}, which potently induce IRG1 expression.

We show for the first time that itaconate significantly reduces lipid accumulation in primary hepatocytes and that this is accompanied by an increase in oxidative phosphorylation. The itaconate mediated increases in OXPHOS were abrogated in the presence of etomoxir, supporting the premise that beta-oxidation of lipids drives the increase in hepatocyte OXPHOS. Itaconate has been shown to inhibit aerobic glycolysis ^{12, 39–41}, however this was described in actively dividing cells and not for relatively quiescent cells such as cultured hepatocytes⁴² where we also did not observe accumulations or alterations in levels of succinate or other glycolytic intermediates due to itaconate treatment. Although succinate was shown to accumulate in itaconate-exposed Huh7 cells ¹⁴, we detected no change in succinate levels in primary hepatocytes that could account for the ability of itaconate to reduce lipid accumulation. Shifts away from glycolysis and towards OXPHOS could possibly explain the increases in OXPHOS we observed, however we found no such evidence; in fact, itaconate treatment caused an increase in the overall metabolic activity of hepatocytes by increasing OXPHOS as well as glycolysis in a lipid-dependent manner. Although multiple reports have shown itaconate to capable of eliciting substantial changes in transcription ^{20, 23}, despite substantial metabolic alterations during itaconate exposure, we found no evidence of significant transcriptional alterations in fatty acid oxidation or oxidative phosphorylation KEGG pathways in hepatocytes. Thus, we believe regulation of lipid oxidation by itaconate is due to metabolic, rather than transcriptional effects.

We demonstrate the ability of itaconate to promote fatty acid oxidation in hepatocytes. Mechanistically our data support the purported role of itaconate in suppressing mitochondrial substrate level phosphorylation (mSLP)²². We and others find that cellular itaconate is effectively activated to itaconyl-CoA, presumably via succinyl-CoA ligase. Within the TCA, this enzyme produces ATP while converting succinyl-CoA to succinate and free CoA. Itaconate subjugates this enzyme for the reverse reaction and in the process consumes ATP and CoA yielding ADP and itaconyl-CoA. Our supplementation of CoA showed little effect on lipids, ruling out itaconate as a CoA-sink¹⁴. Rather, we find that addition of itaconate increases ADP in hepatocytes in conjunction with the appearance of itaconyl-CoA. Thus, we propose a model where a reduction of ATP levels, driven by a loss of mSLP and subsequent consumption of ATP during activation of itaconate, leads to a compensatory increase in beta oxidation and glycolysis. This fatty acid-dependent beta oxidation, our data suggests, facilitates the partial control of lipid levels in the hepatocytes of mice fed a high fat diet and blunts metabolic disorder.

In summary, we identify an important connection between immunometabolism and lipid metabolism in the liver. Itaconate expression in human NASH and mouse models of fatty liver disease highlights the necessity of further investigation of mechanisms whereby this key immunometabolite regulates lipid oxidation by hepatocytes and potentially other cells. These findings suggest that interventions to alter itaconate levels may hold therapeutic potential to regulate metabolic disease and dyslipidemia.

The authors have declared that no conflict of interest exists.

Acknowledgements

This research was supported, in part, by the Intramural Research Program of the NIH, National Cancer Institute, CCR, CIL. RNA sequencing and initial data analysis were conducted at the Frederick National Laboratory for Cancer Research at the CCR Sequencing Facility, NCI Frederick. Human specimens were provided by the Clinical Biospecimen Repository and Processing Core of the Pittsburgh Liver Research Center (supported by National Institutes of Health grant 1P30DK120531). Acyl-CoA analysis was supported by R01GM132261 to NWS. We thank Dr. Christos Chinopoulos at Semmelweis University (Budapest, Hungary) for helpful discussions.

Mice

Wildtype C57Bl/6J mice were obtained from The Jackson Laboratory and bred at the National Cancer Institute-Frederick Cancer Research and Development Center. Irg1^−/− mice were obtained from Dr. Michael Diamond (Washington University School of Medicine, St. Louis, MO) and backcrossed 10 generations onto C57Bl/6J background. LysM^Cre or Alb^Cre mice on C57BL/6J background at NCI Frederick breeding core were crossed with Irg1^fl/fl mice (generously provided by Dr. Diamond) to generate LysM^Cre x Irg1^fl/fl and Alb^Cre x Irg1^fl/fl mice, respectively. The C57Bl/6J background of all mice was confirmed by DartMouse (DartMouse Lab, Lebanon, NH; under NIH grants 1S10OD021667-01 and 5P30CA023108). Male mice were used at 8–10 weeks of age in accordance with an approved NCI Frederick Animal Care and Use Committee protocol. The mouse model of non-alcoholic fatty liver disease was induced by feeding mice ad libitum with RD Western diet (Research Diets Inc., New Brunswick, NJ) supplemented with 10% sucrose in drinking water for 12 weeks as described ^{15, 16, 43, 44}. Some mice were treated 3x/week i.p. with 50 mg/kg 4-Octyl itaconate (Cayman Chemical, Ann Arbor, MI) in 40% cyclodextrin/PBS as described ^{18, 39} for 12 weeks.

Human tissue

Frozen liver tissue from deidentified 16 patients with non-alcoholic steatohepatitis (NASH) and 10 non-NASH controls were provided through the Clinical Biospecimen Repository and Processing Core of the Pittsburgh Liver Research Center. The non-NASH controls had diagnoses of hemangioma, focal nodular hyperplasia, hepatic adenoma, hepatocellular carcinoma and benign liver mass. Samples were homogenized in lysis buffer or 80% methanol and processed as described below for qPCR and metabolomics, respectively.

Histologic analysis/quantitation

Liver tissue sections were fixed in 4% buffered formalin and embedded in paraffin or snap frozen in OCT compound. Fixed, paraffin embedded tissue sections were incubated with antibody to F4/80 (clone BM4, eBioscience), followed by horseradish peroxidase or alkaline phosphatase conjugated secondary antibodies. Frozen tissues were stained in Oil red O (Sigma Aldrich). At least 5 non-overlapping fields per sample were imaged using Aperio ImageScope software (Leica Biosystems, Deer Park, IL) and quantitation was performed using ImageJ software using thresholding of object sizes to eliminate debris. Average ORO droplet size was calculated by dividing the sum of stained area by the total number of droplets in the same field.

Isolation of F4/80 ⁺ macrophages

Liver tissues were homogenized in 200U/mg type 1 collagenase (Worthington Biochemical, Lakewood, NJ), 1mg/ml dispase 2 (Sigma) and 0.5 mg/ml DNase 1 (Sigma) using a GentleMacs tissue dissociator (Miltenyi Biotec). The single cell suspension was filtered through a 70 uM filter. The washed cells were incubated with biotinylated anti-F4/80 antibody (clone BM8; BioLegend, San Diego, CA) followed by magnetically coupled streptavidin microbeads (Miltenyi Biotec). F4/80⁺ cells were positively selected by magnetic separation (Miltenyi BIotec) to > 97% purity. The flow through fraction, containing the non-macrophages was saved for analysis.

Quantitative PCR

Total RNA was isolated using High Pure RNA Isolation kits (Roche Diagnostics). RNA was reverse transcribed using the High Capacity cDNA Archive kit (Applied Biosystems). Murine and human Irg1 and IL-6 expression was examined using validated gene expression assays (Applied Biosystems). Briefly, 10 ng cDNA was put in a final volume of 20 µl containing 10 µl Taqman Universal PCR mix and 1 µl primer/probe gene expression assay. All samples were run on an ABI 7300 real-time PCR system and analyzed using the ΔΔCT method ⁴⁵. Gene expression was normalized to the level of the housekeeping gene HPRT or GAPDH.

Metabolomics

For detection of itaconate and TCA intermediates, cell pellets (~ 1x10^6 cells) were washed and resuspended in 80% methanol. Phase separation was achieved by centrifugation at 4°C. The methanol-water phase containing polar metabolites was dried using a vacuum concentrator. Targeted measurements on the resuspended metabolite samples were performed through electrospray ionization mass spectrometry (ESI-LC–MS/MS) analysis as described ⁴⁶.

For broad metabolomics analysis, cells were washed with cold 150 mM ammonium acetate and resuspended in cold 80% methanol/0.1M formic acid. Phase separation and lyophilization of samples was performed as described above. Lyophilized samples were reconstituted in 50% acetonitrile and analyzed in an Agilent 1290 Infinity II ultra-high performance liquid chromatography (UHPLC) system coupled with an Agilent 6546 quadrupole time-of-flight (Q-TOF) mass spectrometer. Acquired data was analyzed using Agilent MassHunter Profinder 8.0, which performed data processing of > 500 target metabolites derived from an in-house Accurate Mass Retention Time (AMRT) metabolite standard library ⁴⁷.

Lipidomics

Cell pellets (~ 1x10^6 cells) were resuspended in methanol, extracted with chloroform and centrifuged for 5 min at 4C. Dried lipid extracts were reconstituted with 50 µL methanol/chloroform (9:1, v/v). Analysis was carried out an Agilent 1290 Infinity II ultra-high performance liquid chromatography (UHPLC) system coupled with an Agilent 6546 quadrupole time-of-flight (Q-TOF) mass spectrometer. Final curation of annotated lipids was performed with Agilent MassHunter Profinder (Version 10.0.2) as described ⁴⁸. Agilent MassHunter Mass Profiler (Professional Version 15.1) was used for statistical analysis.

Acyl-CoA Analysis

Acyl-CoAs were extracted by resuspension of the cell pellets in 10% trichloroacetic acid, sonication, centrifugation at 17,000 x g 10min 4C, and extraction of the supernatant by 80% MetOH (final concentration) containing 25mM ammonium acetate. Supernatant from further centrifugation at 17,000 x g 10 min 4C was dried using a vacuum concentrator and then resuspended in 50 µL of water. Liquid chromatography-high resolution mass spectrometry analysis was conducted as previously described ⁴⁹ on a Q Exactive Plus operating in positive ion mode coupled to a Vanquish Duo LC. Analysts were blinded to sample identity.

Biochemical analyses on Serum and Tissue Samples

Blood was collected at the indicated times and placed into serum separator tubes which were spun 12,000 x g for 5 min and stored frozen until use. Serum insulin levels were determined using a commercial ELISA (Cayman Chemical). Free fatty acids were determined using an enzyme-based assay kit (Abcam). A triglyceride colorimetric assay kit (Cayman Chemical) was used to determine triglycerides in serum samples or homogenized tissues.

For glucose tolerance testing, mice were fasted 16 hr. Blood glucose levels were tested using a commercially available blood glucose meter and test strips (CVS Health). Baseline glucose levels were read and then mice were challenged i.p. with 2 g/kG sterile glucose in 100 ul saline. For insulin tolerance testing, 5 hr fasted mice were injected i.p. with a single dose of insulin (0.5U/kG). Blood glucose was read every 15 min for 2 hr.

Preparation of hepatocytes

Hepatocytes were isolated from freshly isolated mouse livers (Wildtype mice) as described ⁴². Unless indicated otherwise, cultured hepatocytes were treated overnight in complete media (DMEM supplemented with 5% FBS; 1x insulin-transferrin-selenium (Thermo Fisher Scientific) with 10 mM sodium itaconate in the presence or absence of a 1:10 dilution of sterile, chemically defined lipid mixture 1 (2 ug/ml arachidonic acid; 10 ug/ml each linoleic, myristic, oleic, palmitic and stearic acids; 0.22 mg/ml cholesterol; Sigma-Aldrich, St. Louis, MO).

Extracellular flux analysis

Isolated hepatocytes were seeded at 1.7×10⁴ cells per 96 well in complete media and treated as described overnight. Plated cells were washed gently with PBS and incubated with Seahorse assay media supplemented with 2mM glutamine and 25mM Glucose for 1h at 37°C with no CO₂. Extracellular flux analysis was performed at 37°C with no CO₂ using the XF-96 analyzer (Seahorse Bioscience) per the manufacturer’s instructions. Port additions and times were used as indicated in the figures.

Flow cytometric analysis

Cells (1 x 10⁶) were incubated in cell staining buffer (0.1% BSA, 0.1% sodium azide) containing 250 µg/ml 2.4G2 ascites for 15 min. Cells were stained with either fluorescently conjugated antibodies to F4/80 (clone BM8) or Ly6 (clone 1A8), or the fluorescent probe BODIPY 493/503 (Thermo Fisher Scientific) for 20 min. After washing, the labeled cells were analyzed on an LSR II flow cytometer using FACS-DIVA software (Becton Dickinson).

Bulk RNA Sequencing

Total RNA was isolated from treated hepatocytes using a PureLink RNA Mini kit with on-column DNase digestion (Invitrogen). All samples met quality control of RIN > 8.0 using a Bioanalyzer. 500 ng total RNA was used via the stranded mRNA ligation kit (Illumina) as the input to an mRNA capture, cDNA synthesis and standard Illumina library prep prior to sequencing on the NextSeq 2000 instrument (Illumina, Inc). HiSeq Real Time Analysis software (RTA 3.9) was used for processing raw data files. Illumina bcl2fastq2.20 was used to demultiplex and convert binary base calls and qualities to fastq format. Trimmed sequencing reads were mapped to mouse reference genome mm10 and annotated using GENCODE STAR aligner (version 2.7.0f) with two-pass alignment. RSEM (version 1.3.1) was used for gene and transcript quantification based on GENCODE annotation files.

For RNAseq data processing, raw counts were uploaded to the Partek Flow platform and all analysis was carried out using default parameters. Counts were normalized and filtered to remove low expressed features (< 1.0). Batch removal was carried out using a general linear model. Data was visualized using principal component analysis and differential gene expression analysis was carried out using analysis of variance (ANOVA) testing comparing different treatment conditions. Gene set enrichment analysis was done as described ⁵⁰. Briefly, raw counts were normalized with the median ratio method from DESeq2. Batch effects were removed and visualized using PCA. Normalized and batch corrected counts were imported to the GSEA software version 4.2.3 (Broad Institute). Gene sets were obtained from MSigDB (Broad Institute) or generated de novo in Gene Matrix Transposed (GMT) file format. Analysis was run using default parameters, with the exception of “Min size,” which was reduced to 2.

Statistical analysis

Statistical differences between groups were analyzed using GraphPad Prism software. Significance is indicated by *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 values.

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Itaconic acid underpins hepatocyte lipid metabolism in non-alcoholic fatty liver disease

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