Hepatic lipid droplet-associated proteome changes distinguish dietary-induced fatty liver from glucose tolerance in male mice.

Fatty liver is characterized by the expansion of lipid droplets (LDs) and is associated with the development of many metabolic diseases. We assessed the morphology of hepatic LDs and performed quantitative proteomics in lean, glucose-tolerant mice compared to high-fat diet (HFD) fed mice that displayed hepatic steatosis and glucose intolerance as well as high-starch diet (HStD) fed mice who exhibited similar levels of hepatic steatosis but remained glucose tolerant. Both HFD and HStD-fed mice had more and larger LDs than Chow-fed animals. We observed striking differences in liver LD proteomes of HFD and HStD-fed mice compared to Chow-fed mice, with fewer differences between HFD and HStD. Taking advantage of our diet strategy, we identified a fatty liver LD proteome consisting of proteins common in HFD- and HStD-fed mice, as well as a proteome associated with glucose tolerance that included proteins shared in Chow and HStD but not HFD-fed mice. Notably, glucose intolerance was associated with changes in the ratio of adipose triglyceride lipase to perilipin 5 in the LD proteome, suggesting dysregulation of neutral lipid homeostasis in glucose-intolerant fatty liver. We conclude that our novel dietary approach uncouples ectopic lipid burden from insulin resistance-associated changes in the hepatic lipid droplet proteome.

Hepatocyte mARC1 promotes fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) has a prevalence of ∼25% worldwide, with significant public health consequences yet few effective treatments. Human genetics can help elucidate novel biology and identify targets for new therapeutics. Genetic variants in mitochondrial amidoxime-reducing component 1 (MTARC1) have been associated with NAFLD and liver-related mortality; however, its pathophysiological role and the cell type(s) mediating these effects remain unclear. We aimed to investigate how MTARC1 exerts its effects on NAFLD by integrating human genetics with in vitro and in vivo studies of mARC1 knockdown. Methods: Analyses including multi-trait colocalisation and Mendelian randomisation were used to assess the genetic associations of MTARC1. In addition, we established an in vitro long-term primary human hepatocyte model with metabolic readouts and used the Gubra Amylin NASH (GAN)-diet non-alcoholic steatohepatitis mouse model treated with hepatocyte-specific N-acetylgalactosamine (GalNAc)-siRNA to understand the in vivo impacts of MTARC1. Results: We showed that genetic variants within the MTARC1 locus are associated with liver enzymes, liver fat, plasma lipids, and body composition, and these associations are attributable to the same causal variant (p.A165T, rs2642438 G>A), suggesting a shared mechanism. We demonstrated that increased MTARC1 mRNA had an adverse effect on these traits using Mendelian randomisation, implying therapeutic inhibition of mARC1 could be beneficial. In vitro mARC1 knockdown decreased lipid accumulation and increased triglyceride secretion, and in vivo GalNAc-siRNA-mediated knockdown of mARC1 lowered hepatic but increased plasma triglycerides. We found alterations in pathways regulating lipid metabolism and decreased secretion of 3-hydroxybutyrate upon mARC1 knockdown in vitro and in vivo. Conclusions: Collectively, our findings from human genetics, and in vitro and in vivo hepatocyte-specific mARC1 knockdown support the potential efficacy of hepatocyte-specific targeting of mARC1 for treatment of NAFLD. Impact and implications: We report that genetically predicted increases in MTARC1 mRNA associate with poor liver health. Furthermore, knockdown of mARC1 reduces hepatic steatosis in primary human hepatocytes and a murine NASH model. Together, these findings further underscore the therapeutic potential of targeting hepatocyte MTARC1 for NAFLD.

MLX plays a key role in lipid and glucose metabolism in humans: Evidence from in vitro and in vivo studies.

BACKGROUND AND AIM: Enhanced hepatic de novo lipogenesis (DNL) has been proposed as an underlying mechanism for the development of NAFLD and insulin resistance. Max-like protein factor X (MLX) acts as a heterodimer binding partner for glucose sensing transcription factors and inhibition of MLX or downstream targets has been shown to alleviate intrahepatic triglyceride (IHTG) accumulation in mice. However, its effect on insulin sensitivity remains unclear. As human data is lacking, the aim of the present work was to investigate the role of MLX in regulating lipid and glucose metabolism in primary human hepatocytes (PHH) and in healthy participants with and without MLX polymorphisms. METHODS: PHH were transfected with non-targeting or MLX siRNA to assess the effect of MLX knockdown on lipid and glucose metabolism, insulin signalling and the hepatocellular transcriptome. A targeted association analysis on imputed genotype data for MLX on healthy individuals was undertaken to assess associations between specific MLX SNPs (rs665268, rs632758 and rs1474040), plasma biochemistry, IHTG content, DNL and gluconeogenesis. RESULTS: MLX knockdown in PHH altered lipid metabolism (decreased DNL (p < 0.05), increased fatty acid oxidation and ketogenesis (p < 0.05), and reduced lipid accumulation (p < 0.001)). Additionally, MLX knockdown increased glycolysis, lactate secretion and glucose production (p < 0.001) and insulin-stimulated pAKT levels (p < 0.01) as assessed by transcriptomic, steady-state and dynamic measurements. Consistent with the in vitro data, individuals with the rs1474040-A and rs632758-C variants had lower fasting plasma insulin (p < 0.05 and p < 0.01, respectively) and TG (p < 0.05 and p < 0.01, respectively). Although there was no difference in IHTG or gluconeogenesis, individuals with rs632758 SNP had notably lower hepatic DNL (p < 0.01). CONCLUSION: We have demonstrated using human in vitro and in vivo models that MLX inhibition favored lipid catabolism over anabolism and increased glucose production, despite increased glycolysis and phosphorylation of Akt, suggesting a metabolic mechanism that involves futile cycling.

Determining the temporal, dose, and composition effects of nutritional substrates in an in vitro model of intrahepatocellular triglyceride accumulation.

Pathological accumulation of intrahepatic triglyceride underpins the early stages of nonalcoholic fatty liver disease (NAFLD) and can progress to fibrosis, cirrhosis, and cancer of the liver. Studies in humans suggest that consumption of a diet enriched in saturated compared to unsaturated fatty acids (FAs), is more detrimental to liver fat accumulation and metabolism. However, the reasons for the divergence remain unclear and physiologically-relevant cellular models are required. Therefore, the aims of this study were to investigate the effect of modifying media composition, concentration, and treatment frequency of sugars, FAs and insulin on intrahepatocellular triglyceride content and intracellular glucose, FA and circadian function. Huh7 cells were treated with 2% human serum and a combination of sugars and FAs (low fat low sugar [LFLS], high fat low sugar [HFLS], or high fat high sugar [HFHS]) enriched in either unsaturated (OPLA) or saturated (POLA) FAs for 2, 4, or 7 days with a daily or alternating treatment regime. Stable isotope tracers were utilized to investigate basal and/or insulin-responsive changes in hepatocyte metabolism in response to different treatment regimes. Cell viability, media biochemistry, intracellular metabolism, and circadian biology were quantified. The FA composition of the media (OPLA vs. POLA) did not influence cell viability or intracellular triglyceride content in hepatocytes. In contrast, POLA-treated cells had lower FA oxidation and media acetate, and with higher FA concentrations, displayed lower intracellular glycogen content and diminished insulin stimulation of glycogenesis, compared to OPLA-treated cells. The addition of HFHS also had profound effects on circadian oscillation and gene expression. Cells treated daily with HFHS for at least 4 days resulted in a cellular model displaying characteristics of early stage NAFLD seen in humans. Repeated treatment for longer durations (≥7 days) may provide opportunities to investigate lipid and glucose metabolism in more severe stages of NAFLD.

Prostate cancer cell proliferation is influenced by LDL-cholesterol availability and cholesteryl ester turnover.

BACKGROUND: Prostate cancer growth is driven by androgen receptor signaling, and advanced disease is initially treatable by depleting circulating androgens. However, prostate cancer cells inevitably adapt, resulting in disease relapse with incurable castrate-resistant prostate cancer. Androgen deprivation therapy has many side effects, including hypercholesterolemia, and more aggressive and castrate-resistant prostate cancers typically feature cellular accumulation of cholesterol stored in the form of cholesteryl esters. As cholesterol is a key substrate for de novo steroidogenesis in prostate cells, this study hypothesized that castrate-resistant/advanced prostate cancer cell growth is influenced by the availability of extracellular, low-density lipoprotein (LDL)-derived, cholesterol, which is coupled to intracellular cholesteryl ester homeostasis. METHODS: C4-2B and PC3 prostate cancer cells were cultured in media supplemented with fetal calf serum (FCS), charcoal-stripped FCS (CS-FCS), lipoprotein-deficient FCS (LPDS), or charcoal-stripped LPDS (CS-LPDS) and analyzed by a variety of biochemical techniques. Cell viability and proliferation were measured by MTT assay and Incucyte, respectively. RESULTS: Reducing lipoprotein availability led to a reduction in cholesteryl ester levels and cell growth in C4-2B and PC3 cells, with concomitant reductions in PI3K/mTOR and p38MAPK signaling. This reduced growth in LPDS-containing media was fully recovered by supplementation of exogenous low-density lipoprotein (LDL), but LDL only partially rescued growth of cells cultured with CS-LPDS. This growth pattern was not associated with changes in androgen receptor signaling but rather increased p38MAPK and MEK1/ERK/MSK1 activation. The ability of LDL supplementation to rescue cell growth required cholesterol esterification as well as cholesteryl ester hydrolysis activity. Further, growth of cells cultured in low androgen levels (CS-FCS) was suppressed when cholesteryl ester hydrolysis was inhibited. CONCLUSIONS: Overall, these studies demonstrate that androgen-independent prostate cancer cell growth can be influenced by extracellular lipid levels and LDL-cholesterol availability and that uptake of extracellular cholesterol, through endocytosis of LDL-derived cholesterol and subsequent delivery and storage in the lipid droplet as cholesteryl esters, is required to support prostate cancer cell growth. This provides new insights into the relationship between extracellular cholesterol, intracellular cholesterol metabolism, and prostate cancer cell growth and the potential mechanisms linking hypercholesterolemia and more aggressive prostate cancer.

Tumour fatty acid metabolism in the context of therapy resistance and obesity.

Fatty acid metabolism is known to support tumorigenesis and disease progression as well as treatment resistance through enhanced lipid synthesis, storage and catabolism. More recently, the role of membrane fatty acid composition, for example, ratios of saturated, monounsaturated and polyunsaturated fatty acids, in promoting cell survival while limiting lipotoxicity and ferroptosis has been increasingly appreciated. Alongside these insights, it has become clear that tumour cells exhibit plasticity with respect to fatty acid metabolism, responding to extratumoural and systemic metabolic signals, such as obesity and cancer therapeutics, to promote the development of aggressive, treatment-resistant disease. Here, we describe cellular fatty acid metabolic changes that are connected to therapy resistance and contextualize obesity-associated changes in host fatty acid metabolism that likely influence the local tumour microenvironment to further modify cancer cell behaviour while simultaneously creating potential new vulnerabilities.

The diversity and breadth of cancer cell fatty acid metabolism.

Tumor cellular metabolism exhibits distinguishing features that collectively enhance biomass synthesis while maintaining redox balance and cellular homeostasis. These attributes reflect the complex interactions between cell-intrinsic factors such as genomic-transcriptomic regulation and cell-extrinsic influences, including growth factor and nutrient availability. Alongside glucose and amino acid metabolism, fatty acid metabolism supports tumorigenesis and disease progression through a range of processes including membrane biosynthesis, energy storage and production, and generation of signaling intermediates. Here, we highlight the complexity of cellular fatty acid metabolism in cancer, the various inputs and outputs of the intracellular free fatty acid pool, and the numerous ways that these pathways influence disease behavior.

Modifying nutritional substrates induces macrovesicular lipid droplet accumulation and metabolic alterations in a cellular model of hepatic steatosis.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) begins with steatosis, where a mixed macrovesicular pattern of large and small lipid droplets (LDs) develops. Since in vitro models recapitulating this are limited, the aims of this study were to develop mixed macrovesicular steatosis in immortalized hepatocytes and investigate effects on intracellular metabolism by altering nutritional substrates. METHODS: Huh7 cells were cultured in 11 mM glucose and 2% human serum (HS) for 7 days before additional sugars and fatty acids (FAs), either with 200 µM FAs (low fat low sugar; LFLS), 5.5 mM fructose + 200 µM FAs (low fat high sugar; LFHS), or 5.5 mM fructose + 800 µM FAs (high fat high sugar; HFHS), were added for 7 days. FA metabolism, lipid droplet characteristics, and transcriptomic signatures were investigated. RESULTS: Between the LFLS and LFHS conditions, there were few notable differences. In the HFHS condition, intracellular triacylglycerol (TAG) was increased and the LD pattern and distribution was similar to that found in primary steatotic hepatocytes. HFHS-treated cells had lower levels of de novo-derived FAs and secreted larger, TAG-rich lipoprotein particles. RNA sequencing and gene set enrichment analysis showed changes in several pathways including those involved in metabolism and cell cycle. CONCLUSIONS: Repeated doses of HFHS treatment resulted in a cellular model of NAFLD with a mixed macrovesicular LD pattern and metabolic dysfunction. Since these nutrients have been implicated in the development of NAFLD in humans, the model provides a good physiological basis for studying NAFLD development or regression in vitro.

Fatty Acid Oxidation Is an Adaptive Survival Pathway Induced in Prostate Tumors by HSP90 Inhibition.

HSP90 is a molecular chaperone required for stabilization and activation of hundreds of client proteins, including many known oncoproteins. AUY922 (luminespib), a new-generation HSP90 inhibitor, exhibits potent preclinical efficacy against several cancer types including prostate cancer. However, clinical use of HSP90 inhibitors for prostate cancer has been limited by toxicity and treatment resistance. Here, we aimed to design an effective combinatorial therapeutic regimen that utilizes subtoxic doses of AUY922, by identifying potential survival pathways induced by AUY922 in clinical prostate tumors. We conducted a proteomic analysis of 30 patient-derived explants (PDE) cultured in the absence and presence of AUY922, using quantitative mass spectrometry. AUY922 significantly increased the abundance of proteins involved in oxidative phosphorylation and fatty acid metabolism in the PDEs. Consistent with these findings, AUY922-treated prostate cancer cell lines exhibited increased mitochondrial mass and activated fatty acid metabolism processes. We hypothesized that activation of fatty acid oxidation is a potential adaptive response to AUY922 treatment and that cotargeting this process will sensitize prostate cancer cells to HSP90 inhibition. Combination treatment of AUY922 with a clinical inhibitor of fatty acid oxidation, perhexiline, synergistically decreased viability of several prostate cancer cell lines, and had significant efficacy in PDEs. The novel drug combination treatment induced cell-cycle arrest and apoptosis, and attenuated the heat shock response, a known mediator of HSP90 treatment resistance. This combination warrants further preclinical and clinical investigation as a novel strategy to overcome resistance to HSP90 inhibition. IMPLICATIONS: Metabolic pathways induced in tumor cells by therapeutic agents may be critical, but targetable, mediators of treatment resistance.

Insulin signaling requires glucose to promote lipid anabolism in adipocytes.

Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.

Extracellular Fatty Acids Are the Major Contributor to Lipid Synthesis in Prostate Cancer.

Prostate cancer cells exhibit altered cellular metabolism but, notably, not the hallmarks of Warburg metabolism. Prostate cancer cells exhibit increased de novo synthesis of fatty acids (FA); however, little is known about how extracellular FAs, such as those in the circulation, may support prostate cancer progression. Here, we show that increasing FA availability increased intracellular triacylglycerol content in cultured patient-derived tumor explants, LNCaP and C4-2B spheroids, a range of prostate cancer cells (LNCaP, C4-2B, 22Rv1, PC-3), and prostate epithelial cells (PNT1). Extracellular FAs are the major source (∼83%) of carbons to the total lipid pool in all cell lines, compared with glucose (∼13%) and glutamine (∼4%), and FA oxidation rates are greater in prostate cancer cells compared with PNT1 cells, which preferentially partitioned extracellular FAs into triacylglycerols. Because of the higher rates of FA oxidation in C4-2B cells, cells remained viable when challenged by the addition of palmitate to culture media and inhibition of mitochondrial FA oxidation sensitized C4-2B cells to palmitate-induced apoptosis. Whereas in PC-3 cells, palmitate induced apoptosis, which was prevented by pretreatment of PC-3 cells with FAs, and this protective effect required DGAT-1-mediated triacylglycerol synthesis. These outcomes highlight for the first-time heterogeneity of lipid metabolism in prostate cancer cells and the potential influence that obesity-associated dyslipidemia or host circulating has on prostate cancer progression. IMPLICATIONS: Extracellular-derived FAs are primary building blocks for complex lipids and heterogeneity in FA metabolism exists in prostate cancer that can influence tumor cell behavior.

Lipid and glucose metabolism in hepatocyte cell lines and primary mouse hepatocytes: a comprehensive resource for in vitro studies of hepatic metabolism.

The liver is a critical tissue for maintaining glucose, fatty acid, and cholesterol homeostasis. Primary hepatocytes represent the gold standard for studying the mechanisms controlling hepatic glucose, lipid, and cholesterol metabolism in vitro. However, access to primary hepatocytes can be limiting, and therefore, other immortalized hepatocyte models are commonly used. Here, we describe substrate metabolism of cultured AML12, IHH, and PH5CH8 cells, hepatocellular carcinoma-derived HepG2s, and primary mouse hepatocytes (PMH) to identify which of these cell lines most accurately phenocopy PMH basal and insulin-stimulated metabolism. Insulin-stimulated glucose metabolism in PH5CH8 cells, and to a lesser extent AML12 cells, responded most similarly to PMH. Notably, glucose incorporation in HepG2 cells were 14-fold greater than PMH. The differences in glucose metabolic activity were not explained by differential protein expression of key regulators of these pathways, for example glycogen synthase and glycogen content. In contrast, fatty acid metabolism in IHH cells was the closest to PMHs, yet insulin-responsive fatty acid metabolism in AML12 and HepG2 cells was most similar to PMH. Finally, incorporation of acetate into intracellular-free cholesterol was comparable for all cells to PMH; however, insulin-stimulated glucose conversion into lipids and the incorporation of acetate into intracellular cholesterol esters were strikingly different between PMHs and all tested cell lines. In general, AML12 cells most closely phenocopied PMH in vitro energy metabolism. However, the cell line most representative of PMHs differed depending on the mode of metabolism being investigated, and so careful consideration is needed in model selection.

Reporting of ethics in peer-reviewed verbal autopsy studies: a systematic review.

Introduction: Verbal autopsy (VA) is a method that determines the cause of death by interviewing a relative of the deceased about the events occurring before the death, in regions where medical certification of cause of death is incomplete. This paper aims to review the ethical standards reported in peer-reviewed VA studies. Methods: A systematic review of Medline and Ovid was conducted by two independent researchers. Data were extracted and analysed for articles based on three key areas: Institutional Review Board (IRB) clearance and consenting process; data collection and management procedures, including: time between death and interview; training and education of interviewer, confidentiality of data and data security; and declarations of funding and conflict of interest. Results: The review identified 802 articles, of which 288 were included. The review found that 48% all the studies reported having IRB clearance or obtaining consent of participants. The interviewer training and education levels were reported in 62% and 21% of the articles, respectively. Confidentiality of data was reported for 14% of all studies, 18% did not report the type of respondent interviewed and 51% reported time between death and the interview for the VA. Data security was reported in 8% of all studies. Funding was declared in 63% of all studies and conflict of interest in 42%. Reporting of all these variables increased over time. Conclusions: The results of this systematic review show that although there has been an increase in ethical reporting for VA studies, there still remains a large gap in reporting.

Correction: Insulin and diet-induced changes in the ubiquitin-modified proteome of rat liver.

[This corrects the article DOI: 10.1371/journal.pone.0174431.].

Role of hepatic Annexin A6 in fatty acid-induced lipid droplet formation.

Annexin A6 (AnxA6) has been implicated in the regulation of endo-/exocytic pathways, cholesterol transport, and the formation of multifactorial signaling complexes in many different cell types. More recently, AnxA6 has also been linked to triglyceride storage in adipocytes. Here we investigated the potential role of AnxA6 in fatty acid (FA) - induced lipid droplet (LD) formation in hepatocytes. AnxA6 was associated with LD from rat liver and HuH7 hepatocytes. In oleic acid (OA) -loaded HuH7 cells, substantial amounts of AnxA6 bound to LD in a Ca2+-independent manner. Remarkably, stable or transient AnxA6 overexpression in HuH7 cells led to elevated LD numbers/size and neutral lipid staining under control conditions as well as after OA loading compared to controls. In contrast, overexpression of AnxA1, AnxA2 and AnxA8 did not impact on OA-induced lipid accumulation. On the other hand, incubation of AnxA6-depleted HuH7 cells or primary hepatocytes from AnxA6 KO-mice with OA led to reduced FA accumulation and LD numbers. Furthermore, morphological analysis of liver sections from A6-KO mice revealed significantly lower LD numbers compared to wildtype animals. Interestingly, pharmacological inhibition of cytoplasmic phospholipase A2α (cPLA2α)-dependent LD formation was ineffective in AnxA6-depleted HuH7 cells. We conclude that cPLA2α-dependent pathways contribute to the novel regulatory role of hepatic AnxA6 in LD formation.

Insulin and diet-induced changes in the ubiquitin-modified proteome of rat liver.

Ubiquitin is a crucial post-translational modification regulating numerous cellular processes, but its role in metabolic disease is not well characterized. In this study, we identified the in vivo ubiquitin-modified proteome in rat liver and determined changes in this ubiquitome under acute insulin stimulation and high-fat and sucrose diet-induced insulin resistance. We identified 1267 ubiquitinated proteins in rat liver across diet and insulin-stimulated conditions, with 882 proteins common to all conditions. KEGG pathway analysis of these proteins identified enrichment of metabolic pathways, TCA cycle, glycolysis/gluconeogenesis, fatty acid metabolism, and carbon metabolism, with similar pathways altered by diet and insulin resistance. Thus, the rat liver ubiquitome is sensitive to diet and insulin stimulation and this is perturbed in insulin resistance.