Lipid Overload Research Articles

A diet-independent zebrafish model for NAFLD recapitulates patient lipid profiles and offers a system for small molecule screening

Non-alcoholic Fatty Liver Disease (NAFLD) or pathological hepatic lipid overload, is considered to affect obese individuals. However, NAFLD in lean individuals is prevalent, especially in South Asian population. The pathophysiology of lean NAFLD is not well understood and most animal models of NAFLD use the high-fat diet paradigm. To bridge this gap, we have developed a diet-independent model of NAFLD in zebrafish. We have previously shown that chronic systemic inflammation causes metabolic changes in the liver leading to hepatic fat accumulation in an IL6 overexpressing (IL6-OE) zebrafish model. In the present study, we compared the hepatic lipid composition of adult IL6-OE zebrafish to the controls and found an accumulation of saturated triacylglycerols and a reduction in the unsaturated triacylglycerol species reminiscent of NAFLD patients. Zebrafish is an ideal system for chemical genetic screens. We tested whether the hepatic lipid accumulation in the IL6-OE is responsive to chemical treatment. We found that PPAR-gamma agonist Rosiglitazone, known to reduce lipid overload in the high-fat diet models of NAFLD, could ameliorate the fatty liver phenotype of the IL6-OE fish. Rosiglitazone treatment reduced the accumulation of saturated lipids and showed a concomitant increase in unsaturated TAG species in our inflammation-induced NAFLD model. Our observations suggest that the IL6-OE model can be effective for small molecule screening to identify compounds that can reverse hepatic lipid accumulation, especially relevant to lean NAFLD.

The role of endothelial adipose triglyceride lipase (ecATGL) in heart failure with preserved ejection fraction (HFpEF)

Abstract Background and objectives Obesity is one of the major drivers of heart failure with preserved ejection fraction (HFpEF). Endothelial cells (EC) have an important barrier function including the control of lipid transport from the coronary circulation to the myocardium. This EC lipid transport involves, in addition to fast transcellular transport, processes of EC-lipogenesis and -lipolysis, and is likely involved in the development of cardiac lipid accumulation in obese phenotypes of HFpEF. In order to determine the role of EC lipid metabolism/transport in HFpEF, we generated mice with an EC-specific deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme of lipolysis, and subjected these mice to a hypertensive-obese two-hit HFpEF model. Methods and results Inducible endothelial-specific ATGL knockout (ecATGL-KO) mice were generated by crossbreeding Atgl fl/fl mice with Cdh5(PAC)-CreERT2 mice. For the induction of HFpEF, mice received a high fat diet (HFD, 60% kcal from fat) and L-NAME for 15 weeks, while control animals (Ctrl) received low fat diet (LFD, 10% kcal from fat) and water, as previously described. To analyse cardiac function, standard and speckle-tracking echocardiography were performed. After 15 weeks, Cre− littermates (wild type, wt) with HFpEF developed obesity and hypertension (body weight: wt-Ctrl: 33.8±2.5g vs. wt-HFpEF: 45.1±4.4g, p<0.0001; systolic blood pressure: wt-Ctrl: 99.9±3.7mmHg vs. wt-HFpEF: 135.5±5.6mmHg, p<0.0001), which did not significantly differ in ecATGL-KO mice. In parallel, wt-HFpEF mice developed established echocardiographic signs of HFpEF including diastolic dysfunction: E/é: (wt-Ctrl: −22.3±1.3 vs. wt-HFpEF: −45.2±3.8; p<0.0001), IVRT: (wt-Ctrl: 17.8±0.7ms vs. wt-HFpEF: 19.3±0.4ms; p= p<0.0001), and an impaired global longitudinal strain (GLS) (wt-Ctrl: −20.7±1.1% vs. wt-HFpEF 12.9±0.7%; p<0.0001). HFpEF parameters were significantly improved in ecATGL-KO mice after 15 weeks of HFD/L-NAME: (E/é: −28.9±2.8; p<0.0001 vs. wt-HFpEF; IVRT: 18.3±0.6; p<0.05 vs. wt-HFpEF; GLS: −19.1±1.7; p<0.0001 vs. wt-HFpEF). No changes in systolic function (EF) were observed. Electron microscopical investigations of cardiac (LV) tissue sections showed enhanced lipid droplet accumulation in coronary ECs of ecATGL-KO mice when compared to wt mice. Complementary in vitro experiments in HUVECs using an ATGL inhibitor or genetic deletion of Atgl using siRNA resulted in significant lipid droplet accumulation which did not affect EC survival and function. Conclusion This study demonstrates that the lack of ATGL in ECs attenuates the development of HFpEF. Enhanced lipid droplet accumulation in coronary ECs in the absence of ATGL may protect the myocardium from lipid overload, as seen in obese phenotypes of HFpEF. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Einstein Foundation Berlin;DZHK (German Centre for Cardiovascular Research), partner site Berlin;DFG (Deutsche Forschungsgemeinschaft)

Chronic insulin resistance deteriorates cardiac mitochondrial quality control in a mouse model of SEC-NAFLD-IR

Abstract Background Insulin resistance and nonalcoholic fatty liver disease (NAFLD) both relate to cardiovascular mortality. Using a mouse model of chronic lipid overload and secondary-NAFLD-induced insulin resistance (SEC-NAFLD-IR), we recently deciphered that SEC-NAFLD-IR already at young age provoked myocardial lipotoxicity with reduced mitochondrial efficiency and increased vulnerability to cardiac ischemia. However, long-term consequences of SEC-NAFLD-IR remain elusive. Purpose Here we aimed to elucidate the impact of long-term SEC-NAFLD-IR on multiple mitochondrial quality control (mQC) mechanisms in the heart and its consequences for cardiac function. Methods We studied 36 SEC-NAFLD-IR mice (72-week-old). For mechanistic experiments, we applied palmitate-induced insulin resistant murine HL-1 cells. Cardiac mitochondrial dynamics were measured via quantification of mitochondrial morphology and expression of mitochondrial fusion and fission factors (Opa1, Drp1, Fis1, Mfn 1 & 2). Mitophagy level was evaluated via immunofluorescence and protein expression of key mitophagy-related genes (Parkin, NIX, LC3). Mitochondrial biogenesis and mass were examined via quantitation of PGC-1α expression, mtDNA and citrate synthase activity. Results 72-week-old SEC-NAFLD-IR mice exhibited 21% (p=0.001) and 32% (p<0.001) higher body weight and heart weight compared with controls. Along with elevated oxidative stress, hepatic lipid accumulation and inflammation, 6h-fasted SEC-NAFLD-IR mice were characterized by increased plasma glucose, insulin and cholesterol. SEC-NAFLD-IR mice displayed a cardiac phenotype with 21% higher left ventricular mass (normalized to body weight, p<0.001) and 6% lower ejection fraction compared to controls (73.5% SEM 0.90 vs 69.4% SEM 1.65, p=0.04). We found several advantageous mQC mechanisms suppressed in aged SEC-NAFLD-IR mice including long form OPA1-mediated mitochondrial fusion, Parkin- and NIX-mediated mitophagy. Likewise, mitochondrial biogenesis was suppressed in the aged insulin-resistant heart, which was connected to a 65% downregulation of PGC-1α1 expression (p=0.01). Interestingly, downregulation of cardiac PGC-1α1 in aged SEC-NAFLD-IR mice coincided with upregulation of PARIS, indicating the crucial participation of the Parkin/PARIS pathway in mQC of the insulin-resistant heart. In addition, induction of insulin resistance in murine HL-1 cardiomyocytes also led to increased mitochondrial fragmentation and decreased PGC-1α1 expression. Conclusion This study demonstrated that regulation of mitochondrial network and turnover is hampered by SEC-NAFLD-IR in the hearts of aged mice, which may contribute to hypertrophy and cardiac dysfunction in insulin resistance. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)

Immune mechanisms linking metabolic injury to inflammation and fibrosis in fatty liver disease - novel insights into cellular communication circuits.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease and is emerging as the leading cause of cirrhosis, liver transplantation and hepatocellular carcinoma (HCC). NAFLD is a metabolic disease that is considered the hepatic manifestation of the metabolic syndrome; however, during the evolution of NAFLD from steatosis to non-alcoholic steatohepatitis (NASH), to more advanced stages of NASH with liver fibrosis, the immune system plays an integral role. Triggers for inflammation are rooted in hepatic (lipid overload, lipotoxicity, oxidative stress) and extrahepatic (gut-liver axis, adipose tissue, skeletal muscle) systems, resulting in unique immune-mediated pathomechanisms in NAFLD. In recent years, the implementation of single-cell RNA-sequencing and high dimensional multi-omics (proteogenomics, lipidomics) and spatial transcriptomics have tremendously advanced our understanding of the complex heterogeneity of various liver immune cell subsets in health and disease. In NAFLD, several emerging inflammatory mechanisms have been uncovered, including profound macrophage heterogeneity, auto-aggressive T cells, the role of unconventional T cells and platelet-immune cell interactions, potentially yielding novel therapeutics. In this review, we will highlight the recent discoveries related to inflammation in NAFLD, discuss the role of immune cell subsets during the different stages of the disease (including disease regression) and integrate the multiple systems driving inflammation. We propose a refined concept by which the immune system contributes to all stages of NAFLD and discuss open scientific questions arising from this paradigm shift that need to be unravelled in the coming years. Finally, we discuss novel therapeutic approaches to target the multiple triggers of inflammation, including combination therapy via nuclear receptors (FXR agonists, PPAR agonists).

The Role of Forkhead Box O in Pathogenesis and Therapy of Diabetes Mellitus.

Type 2 diabetes is a disease that causes numerous complications disrupting the functioning of the entire body. Therefore, new treatments for the disease are being sought. Studies in recent years have shown that forkhead box O (FOXO) proteins may be a promising target for diabetes therapy. FOXO proteins are transcription factors involved in numerous physiological processes and in various pathological conditions, including cardiovascular diseases and diabetes. Their roles include regulating the cell cycle, DNA repair, influencing apoptosis, glucose metabolism, autophagy processes and ageing. FOXO1 is an important regulator of pancreatic beta-cell function affecting pancreatic beta cells under conditions of insulin resistance. FOXO1 also protects beta cells from damage resulting from oxidative stress associated with glucose and lipid overload. FOXO has been shown to affect a number of processes involved in the development of diabetes and its complications. FOXO regulates pancreatic β-cell function during metabolic stress and also plays an important role in regulating wound healing. Therefore, the pharmacological regulation of FOXO proteins is a promising approach to developing treatments for many diseases, including diabetes mellitus. In this review, we describe the role of FOXO proteins in the pathogenesis of diabetes and the role of the modulation of FOXO function in the therapy of this disease.

Glycerolipid Synthesis and Lipid Droplet Formation in the Endoplasmic Reticulum.

More than 60 years ago, Eugene Kennedy and coworkers elucidated the endoplasmic reticulum (ER)-based pathways of glycerolipid synthesis, including the synthesis of phospholipids and triacylglycerols (TGs). The reactions of the Kennedy pathway were identified by studying the conversion of lipid intermediates and the isolation of biochemical enzymatic activities, but the molecular basis for most of these reactions was unknown. With recent progress in the cell biology, biochemistry, and structural biology in this area, we have a much more mechanistic understanding of this pathway and its reactions. In this review, we provide an overview of molecular aspects of glycerolipid synthesis, focusing on recent insights into the synthesis of TGs. Further, we go beyond the Kennedy pathway to describe the mechanisms for storage of TG in cytosolic lipid droplets and discuss how overwhelming these pathways leads to ER stress and cellular toxicity, as seen in diseases linked to lipid overload and obesity.

Exercise inhibits JNK pathway activation and lipotoxicity via macrophage migration inhibitory factor in nonalcoholic fatty liver disease.

The macrophage migration inhibitory factor (MIF) expressed in hepatocytes can limit steatosis during obesity. Lipotoxicity in nonalcoholic fatty liver disease is mediated in part by the activation of the stress kinase JNK, but whether MIF modulates JNK in lipotoxicity is unknown. In this study, we investigated the role of MIF in regulating JNK activation and high-fat fostered liver lipotoxicity during simultaneous exercise treatment. Fifteen mice were equally divided into three groups: normal diet, high-fat diet, and high-fat and exercise groups. High-fat feeding for extended periods elicited evident hyperlipemia, liver steatosis, and cell apoptosis in mice, with inhibited MIF and activated downstream MAPK kinase 4 phosphorylation and JNK. These effects were then reversed following prescribed swimming exercise, indicating that the advent of exercise could prevent liver lipotoxicity induced by lipid overload and might correlate to the action of modulating MIF and its downstream JNK pathway. Similar detrimental effects of lipotoxicity were observed in in vitro HepG2 cells palmitic acid treatment. Suppressed JNK reduced the hepatocyte lipotoxicity by regulating the BCL family, and the excess JNK activation could also be attenuated through MIF supplementation or exacerbated by MIF siRNA administration. The results found suggest that exercise reduces lipotoxicity and inhibits JNK activation by modulating endogenous hepatic MIF in NAFLD. These findings have clinical implications for the prevention and intervention of patients with immoderate diet evoked NAFLD.

Mechanisms of Myocardial Damage Due to Hyperlipidemia: A Review of Recent Studies.

Myocardial injury and necrosis caused by hyperlipidemia have been investigated by several researchers. Their pathogenesis and molecular basis are different from those of the more common clinical ischemic myocardial injury. Hyperlipidemia leads to peroxide accumulation in the cardiomyocytes, causes lipid overload, decreases the antioxidant capacity of the body, and promotes the inflammatory response. Furthermore, hyperlipidemia causes changes in the structure and function of mitochondria in the cardiomyocytes, which results in their injury and necrosis. Many previous studies have shown that metabolic diseases (eg, obesity and diabetes) and chemical poisoning can lead to hyperlipidemic myocardial injury and necrosis. Moreover, it has been observed that this pathological process can be inhibited by many small molecular substances. In the clinic, myocardial damage can be prevented or reduced by lowering the levels of triglyceride and cholesterol. Myocardial damage can also be regulated via the molecular pathway of myocardial injury caused by hyperlipidemia so that the disease can be treated. The present article reviewed the recent findings reported on the mechanisms of myocardial damage due to hyperlipidemia.

Abstract IA03: Polyploidy, DNA damage response driver or gatekeeper of chronic liver diseases

Abstract Polyploidy, also known as whole-genome amplification, is a condition in which the organism has more than the two basic sets of chromosomes. Polyploidy frequently arises during tissue development and repair, and in age-associated diseases, such as cancer. Its consequences are diverse and clearly different between systems. The liver is a particularly fascinating organ in that it can adapt its ploidy to the physiological and pathological context. In human liver parenchyma, polyploidy is mainly cellular (binuclear hepatocytes) with no specific zonation across the liver lobule. During liver tumorigenesis, cellular ploidy is drastically reduced; binuclear polyploid hepatocytes are barely present in HCC tumors. Remarkably, nuclear ploidy is specifically amplified in HCC tumors. In fact, nuclear ploidy is amplified in HCCs harboring a low degree of differentiation and TP53 mutations. Highly polyploid tumors are associated with a poor prognosis. Hepatocytes increase their ploidy also during the proliferative stress that underlies various liver disease conditions. Recently, we investigated mechanisms underlying activation of the DNA damage response during Non-alcoholic Steatotic Liver Disease. We demonstrated that proliferating mouse NAFLD hepatocytes harbor replication stress with an alteration of the replication fork’s speed and activation of ATR pathway which is sufficient to cause DNA breaks. Nucleotide pool imbalance occurring during NAFLD is the key driver of replication stress. Remarkably, DNA lesions drive cGAS/STING pathway activation, a major component of cells’ intrinsic immune response. The translational significance of this study was reiterated by showing that lipid overload in proliferating HepaRG was sufficient to induce replication stress and nucleotide pool imbalance. Moreover, livers from NAFLD human patients displayed nucleotide pathways deregulation and cGAS/STING genes alteration. Altogether, our findings shed new light on the mechanisms by which damaged NAFLD hepatocytes might promote disease progression. Citation Format: Chantal Desdouets. Polyploidy, DNA damage response driver or gatekeeper of chronic liver diseases [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr IA03.

CREBH alleviates mitochondrial oxidative stress through SIRT3 mediating deacetylation of MnSOD and suppression of Nlrp3 inflammasome in NASH.

Lipotoxicity and unresolved oxidative stress are key drivers of metabolic inflammation in nonalcoholic steatohepatitis (NASH). cAMP-response element binding protein H(CREBH) is a liver-specific transcription factor and regulates the glucose and lipid metabolism of NASH. However, its role in mitochondrial oxidative stress and its association with sirtuin 3 (SIRT3), a master regulator of deacetylation for mitochondrial proteins, remains elusive. In this study, AML-12cells were treated with palmitic acid to imitate the pathological changes of NASH in vitro and 8-week-old male C57BL/6J mice were fed with a high-fat (HF) diet or a methionine-choline-deficient (MCD) diet to build the widely accepted in vivo model of NASH. We found that lipid overload induced mitochondrial oxidative stress and stimulated the expression of CREBH and SIRT3. CREBH overexpression alleviated the mitochondrial oxidative stress. Moreover, CREBH promoted SIRT3 expression, which regulated the deacetylation of manganese superoxide dismutase (MnSOD) and inhibited NOD-Like Receptor Pyrin Domain Containing 3 (Nlrp3) inflammasome activation whereas suppression of SIRT3 damaged the protecting ability of CREBH in mitochondrial oxidative stress. CREBH knockout mice were highly susceptible to HF and MCD diet-induced NASH with more severe oxidative stress. Collectively, our results firstly provided the support that CREBH could serve as a protective factor in the progression of NASH by regulating the acetylation of MnSOD and the activation of Nlrp3 inflammasome through SIRT3. These results suggest that CREBH might be a valuable therapeutic candidate for NASH.

Silibinin Suppresses the Hyperlipidemic Effects of the ALK-Tyrosine Kinase Inhibitor Lorlatinib in Hepatic Cells.

The third-generation anaplastic lymphoma tyrosine kinase inhibitor (ALK-TKI) lorlatinib has a unique side effect profile that includes hypercholesteremia and hypertriglyceridemia in >80% of lung cancer patients. Here, we tested the hypothesis that lorlatinib might directly promote the accumulation of cholesterol and/or triglycerides in human hepatic cells. We investigated the capacity of the hepatoprotectant silibinin to modify the lipid-modifying activity of lorlatinib. To predict clinically relevant drug–drug interactions if silibinin were used to clinically manage lorlatinib-induced hyperlipidemic effects in hepatic cells, we also explored the capacity of silibinin to interact with and block CYP3A4 activity using in silico computational descriptions and in vitro biochemical assays. A semi-targeted ultrahigh pressure liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry with electrospray ionization (UHPLC-ESI-QTOF-MS/MS)-based lipidomic approach revealed that short-term treatment of hepatic cells with lorlatinib promotes the accumulation of numerous molecular species of cholesteryl esters and triglycerides. Silibinin treatment significantly protected the steady-state lipidome of hepatocytes against the hyperlipidemic actions of lorlatinib. Lipid staining confirmed the ability of lorlatinib to promote neutral lipid overload in hepatocytes upon long-term exposure, which was prevented by co-treatment with silibinin. Computational analyses and cell-free biochemical assays predicted a weak to moderate inhibitory activity of clinically relevant concentrations of silibinin against CYP3A4 when compared with recommended (rosuvastatin) and non-recommended (simvastatin) statins for lorlatinib-associated dyslipidemia. The elevated plasma cholesterol and triglyceride levels in lorlatinib-treated lung cancer patients might involve primary alterations in the hepatic accumulation of lipid intermediates. Silibinin could be clinically explored to reduce the undesirable hyperlipidemic activity of lorlatinib in lung cancer patients.

Paraoxonase 1 Ameliorates Renal Lipotoxicity by Activating Lipophagy and Inhibiting Pyroptosis

Several studies in recent years have shown that lipid overload causes lipotoxic damage to the kidney, and oxidative stress, inflammation, and autophagic arrest are all important mechanisms of renal lipotoxicity. However, effective measures with therapeutic effects on renal lipotoxicity are limited. The present study indicated the protective effect of the paraoxonase 1 (PON1) against renal lipotoxicity in high-fat diet-fed scavenger receptor class B type I-deficient (SR-BI-/-) mice. The results showed that SR-BI-/- mice exhibited significant renal pathologic characteristics, such as oxidative stress, inflammation, and fibrosis, under a normal chow diet, and were accompanied by dyslipidemia and reduced plasma PON1 activity and renal PON1 levels. PON1 overexpression significantly attenuated the above pathologic changes in the kidneys of SR-BI-/- mice fed with a high-fat diet. Mechanistically, PON1 may ameliorate renal oxidative stress by reducing reactive oxygen species production, reduce renal lipid accumulation by inhibiting AKT/mechanistic target of rapamycin kinase pathway to activate lipophagy, and reduce the occurrence of inflammation and cell death by inhibiting Nod-like receptor family protein 3 inflammasome-mediated pyroptosis. The present study is the first to show that PON1 overexpression can effectively alleviate renal lipotoxicity injury, and PON1 may be a promising therapeutic strategy for the treatment of renal lipotoxicity-related diseases.

High-fat diet affects autophagy and mitochondrial compartment in rat Harderiangland.

The Harderian gland (HG) of Rattus norvegicus is an orbital gland secreting lipids that accumulate in excess under condition of increased lipid metabolism. To study the response elicitated by lipid overload in rat HG, we housed the animals in thermoneutral conditions (28-30°C) in association to high fat diet (HFD). In HFD rats alterated blood lipid levels result in lipid accumulation in HG as demonstrated by the increased gland weight and histochemical/ultrastructural analyses. The HFD-caused oxidative stress forces the gland to trigger antioxidant defense mechanisms and autophagic process, such as lipophagy and mitophagy. Induction of mitochondrial DNA(mtDNA) damage and repair was stronger in HFD-rat HGs. An increase in marker expression levels of mitochondrial biogenesis, fission, and fusion occurred to counteract mtDNA copy number reduction and mitophagy. Therefore, the results demonstrate that rat HG activates autophagy as survival strategy under conditions of increased lipid metabolism and suggest a key role for mitophagy and membrane dynamics in the mitochondrial adaptive response to HFD.

Exogenous ANP Treatment Ameliorates Myocardial Insulin Resistance and Protects against Ischemia-Reperfusion Injury in Diet-Induced Obesity.

Increasing evidence suggests natriuretic peptides (NPs) coordinate interorgan metabolic crosstalk. We recently reported exogenous ANP treatment ameliorated systemic insulin resistance by inducing adipose tissue browning and attenuating hepatic steatosis in diet-induced obesity (DIO). We herein investigated whether ANP treatment also ameliorates myocardial insulin resistance, leading to cardioprotection during ischemia–reperfusion injury (IRI) in DIO. Mice fed a high-fat diet (HFD) or normal-fat diet for 13 weeks were treated with or without ANP infusion subcutaneously for another 3 weeks. Left ventricular BNP expression was substantially reduced in HFD hearts. Intraperitoneal-insulin-administration-induced Akt phosphorylation was impaired in HFD hearts, which was restored by ANP treatment, suggesting that ANP treatment ameliorated myocardial insulin resistance. After ischemia–reperfusion using the Langendorff model, HFD impaired cardiac functional recovery with a corresponding increased infarct size. However, ANP treatment improved functional recovery and reduced injury while restoring impaired IRI-induced Akt phosphorylation in HFD hearts. Myocardial ultrastructural analyses showed increased peri-mitochondrial lipid droplets with concomitantly decreased ATGL and HSL phosphorylation levels in ANP-treated HFD, suggesting that ANP protects mitochondria from lipid overload by trapping lipids. Accordingly, ANP treatment attenuated mitochondria cristae disruption after IRI in HFD hearts. In summary, exogenous ANP treatment ameliorates myocardial insulin resistance and protects against IRI associated with mitochondrial ultrastructure modifications in DIO. Replenishing biologically active NPs substantially affects HFD hearts in which endogenous NP production is impaired.

The Role and Mechanism of CREBH Regulating SIRT3 in Metabolic Associated Fatty Liver Disease

AimsTo investigate the effect of cAMP response element-binding protein H (CREBH) on metabolic associated fatty liver disease by regulating sirtuin 3 (SIRT3). Main methodsTwo mouse models of fatty liver induced by a methionine-choline deficient (MCD) diet and a high-fat (HF) diet and an in vitro model of palmitic acid (PA) induced lipid-overloaded hepatocytes were constructed to detect the expression of CREBH, SIRT3, total acetylation, and downstream protein interactions and lipid metabolism phenotype, which were further validated in CREBH−/− mice and lentivirus-overexpressing CREBH hepatocytes. Key findingsIn fatty liver and lipid overload models, the expressions of CREBH and SIRT3 were down-regulated and their expression was positively correlated, accompanied by an increase in the level of total protein acetylation. Overexpression of CREBH alleviated excess lipid accumulation, impaired viability, and the ability to metabolize energy through the fatty acid oxidation pathway in hepatocytes in vitro. Furthermore, overexpression of CREBH restored the interaction of the deacetylase SIRT3 with the molecules carnitine palmitoyl-transferase 2 (CPT2) and long-chain acyl CoA dehydrogenase (ACADL) involved in the fatty acid oxidation pathway and their deacetylation status. However, CREBH−/− aggravated the damage of lipid metabolism in the liver tissue of mice. SignificanceCREBH increased the enzymatic activity of downstream factors by positively regulating the expression of SIRT3, which promoted the oxidative decomposition of fatty acids in hepatocytes and played an important role in fatty acid oxidation in MAFLD.

Replication stress triggered by nucleotide pool imbalance drives DNA damage and cGAS-STING pathway activation in NAFLD.

Non-alcoholic steatotic liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. NAFLD has a major effect on the intrinsic proliferative properties of hepatocytes. Here, we investigated the mechanisms underlying the activation of DNA damage response during NAFLD. Proliferating mouse NAFLD hepatocytes harbor replication stress (RS) with an alteration of the replication fork's speed and activation of ATR pathway, which is sufficient to cause DNA breaks. Nucleotide pool imbalance occurring during NAFLD is the key driver of RS. Remarkably, DNA lesions drive cGAS/STING pathway activation, a major component of cells' intrinsic immune response. The translational significance of this study was reiterated by showing that lipid overload in proliferating HepaRG was sufficient to induce RS and nucleotide pool imbalance. Moreover, livers from NAFLD patients displayed nucleotide pathway deregulation and cGAS/STING gene alteration. Altogether, our findings shed light on the mechanisms by which damaged NAFLD hepatocytes might promote disease progression.

Chrysophanol exerts protective effect against atherosclerosis via NFκB-mediated signaling in LDLR-/- mice model

Purpose: To study the therapeutic effect of chrysophanol (CHR) on diet-induced atherogenesis in LDLR-/- mice.Methods: Mice were fed atherogenic diet for 12 weeks after which some lipid profile markers such as total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c) and triglyceride (TG) were measured. The mRNA expression levels of lipid synthesis genes and lipid overload-related inflammatory indicator molecules were assayed with quantitative real time polymerase chain reaction (qRT-PCR), while the corresponding protein expressions were determined with western blotting assay. The therapeutic effect of CHR on atherogenesis was confirmed using H & E and Oil red O stainings of mice aortic sections.Results: CHR administration significantly reduced levels of TC, LDL-c, HDL-c and TG (p ≤ 0.05), and restored the mRNA and protein expressions of genes involved in lipid and glucose homeostasis, namely, AdipoR1, PPAR-Ƴ and HMco-A (p < 0.05). Moreover, CHR potentially alleviated diet-induced inflammation, as is evident in reduced levels of molecular inflammatory signaling factors NF-κB and TLR-4, and significant down-regulations of the proinflammatory cytokines, TNF-α, IL-6 and IL-1β (p < 0.05). Furthermore, aorta histology revealed that CHR significantly reduced lipid storage in the arteries of mice fed atherogenic diet (p < 0.05).Conclusion: These results indicate that CHR reduces diet-induced lipid storage in LDLR-/- mice and also controlled inflammation-associated lipid overload. These findings may provide a molecular basis for potential application of chrysophanol in the treatment of atherosclerosis.

Increased plasma fatty acid clearance, not fatty acid concentration, is associated with muscle insulin resistance in people with obesity

BackgroundAlthough it is well-accepted that increased plasma free fatty acid (FFA) concentration causes lipid overload and muscle insulin resistance in people with obesity, plasma FFA concentration poorly predicts insulin-resistant glucose metabolism. It has been proposed that hyperinsulinemia in people with obesity sufficiently inhibits adipose tissue triglyceride lipolysis to prevent FFA-induced insulin resistance. However, we hypothesized enhanced FFA clearance in people with obesity, compared with lean people, prevents a marked increase in plasma FFA even when FFA appearance is high. MethodsWe assessed FFA kinetics during basal conditions and during a hyperinsulinemic-euglycemic clamp procedure in 14 lean people and 46 people with obesity by using [13C]palmitate tracer infusion. Insulin-stimulated muscle glucose uptake rate was evaluated by dynamic PET-imaging of skeletal muscles after [18F]fluorodeoxyglucose injection. ResultsPlasma FFA clearance was accelerated in participants with obesity and correlated negatively with muscle insulin sensitivity without a difference between lean and obese participants. Furthermore, insulin infusion increased FFA clearance and the increase was greater in obese than lean participants. ConclusionsOur findings suggest plasma FFA extraction efficiency, not just plasma FFA concentration, is an important determinant of the cellular fatty acid load and the stimulatory effect of insulin on FFA clearance counteracts some of its antilipolytic effect.

Insulin Signaling Induces AKR1C3 to Promote Lipid Overload through FASN in SGBS Cells, as a Model of PCOS Adipocytes

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age, affecting 8‐13% of women. The characteristic androgen excess (AE) of PCOS is believed to promote comorbidities, such as obesity and type 2 diabetes. AE correlates with insulin resistance (IR) in these women and may be the driving factor for lipid overload. Aldo‐keto reductase family 1 member C3 (AKR1C3) is induced by insulin in PCOS adipocytes and may be central in driving the AE of PCOS. AKR1C3 predominantly catalyzes formation of androgens such as testosterone and 5α‐dihydrotestosterone in peripheral tissues, which are potent agonists for the androgen receptor (AR) and upregulate de novo lipogenesis. Fatty acid synthase (FASN) is induced by androgens and may be responsible for an altered lipid profile in mature adipocytes. Nuclear factor erythroid 2‐related factor 2 (NRF2) is a known transcription factor for AKR1C3 and is regulated by insulin. AKR1C3 overexpression in adipocytes by insulin activation of NRF2 could increase androgen receptor signaling and promote de novo lipogenesis. The resulting lipid overflow could exacerbate insulin resistance through a feedforward mechanism and potentiate the cardio‐metabolic risk seen in PCOS. We hypothesize that insulin signaling induces AKR1C3 to promote lipid overload through FASN in PCOS adipocytes. Using differentiated human Simpson‐Golabi‐Behmel Syndrome (SGBS) adipocytes as a model for PCOS adipocytes, we have confirmed that AKR1C3 is induced >600 fold by insulin. Using pharmacological inhibition, we found that the insulin induction of AKR1C3 and FASN is dependent on PI3K/AKT/mTOR measured by RT‐qPCR and immunoblot. Using NRF2 siRNA and ML‐385, a NRF2 inhibitor, we have determined that AKR1C3 and FASN induction are NRF2 dependent, indicating NRF2 may be one transcription factor that binds to the AKR1C3 promotor region in adipocytes. We also determined that FASN induction is both AKR1C3 and AR dependent using pharmacological inhibition/antagonism and siRNA, where its effect was measured by RT‐qPCR and immunoblot. Pharmacological inhibition with NRF2 or AKR1C3 inhibitors additionally downregulated de novo lipogenesis measured by Oil Red O staining, which is a phenotypic readout for FASN activity. These studies elucidate the signaling mechanism by which AKR1C3 and FASN are induced by insulin in adipocytes. We conclude that upregulation of AKR1C3 may promote lipid overload in PCOS adipocytes by induction of FASN, and that AKR1C3 is a possible therapeutic target to prevent the risk of cardio‐metabolic disease in PCOS women.

Subcellular Recycling of CD36 as Target to Rescue Lipid Overload‐induced Myocardial Contractile Dysfunction

BackgroundDisturbances in cardiac metabolism are increasingly being recognized as crucial drivers of the development and progression of heart disease and ageing. In particular, lipid overload due to Western life style or obesity eventually elicits cardiac insulin resistance and contractile dysfunction (referred to as diabetic cardiomyopathy). As a result, there is an urgent unmet clinical need for treatments that can directly target metabolic defects in heart failure. The transmembrane protein CD36 both facilitates myocardial fatty acid uptake and regulates this process by its subcellular recycling between endosomes and the sarcolemma. In turn, CD36 recycling was recently found to be regulated by instantaneous changes in activity of vacuolar‐type H+‐ATPase (v‐ATPase) in the endosomes, as controlled by rapid assembly and disassembly of V0 and V1 subunits of v‐ATPase.HypothesisRegulation of subcellular recycling of CD36 by v‐ATPase is an effective target for metabolic modulation therapy in diabetic cardiomyopathy.ObjectiveTo investigate whether manipulating v‐ATPase activity, and thereby CD36 recycling, rescues insulin resistance and contractile dysfunction of the diabetic heart.MethodsIsolated rat cardiomyocytes and human (iPSC‐derived and differentiated) cardiomyocytes were treated with bafilomycin (v‐ATPase inhibitor) or energy substrates, i.e., fatty acids, glucose, amino acids (AAs) or b‐hydroxy butyrate (HB). In addition, rats in vivo were treated with AAs. v‐ATPase activity (assembly status), substrate transporter localization (CD36 and glucose transporter GLUT4) and fatty acid and glucose uptake rates (in absence or presence of insulin) were measured.ResultsIn rat and human cardiomyocytes both bafilomycin, excess palmitate and HB inhibited v‐ATPase activity, increased sarcolemmal CD36 content, increased fatty acid uptake and lipid accumulation, and elicited contractile dysfunction. However, the latter could be rescued by excess glucose or AAs, concomitant with in each case a similar increase of v‐ATPase activity and CD36 reinternalization, while glucose uptake was increased. Hence, v‐ATPase activity/assembly is required for retention of CD36 in endosomes. In case of AAs, v‐ATPase (re)assembly was dependent on mTORC1 activation. In high fat diet‐fed rats, AAs had a similar beneficial action at the myocellular level.ConclusionsThese novel data suggest a pivotal role for endosomal v‐ATPase activity and subcellular CD36 recycling in determining myocardial substrate preference, in particular fatty acids versus glucose. The molecular mechanism involves assembly/disassembly of the V0 and V1 subunits of v‐ATPase, allowing a rapid change in activity. Lipid accumulation and contractile dysfunction in diabetic cardiomyopathy is aggravated by HB but resolved by AAs.PerspectiveSpecific amino acids acting through v‐ATPase reassembly may be an effective nutraceutical therapy in diabetic cardiomyopathy, but ketone bodies should not be used as these may aggravate lipid accumulation and contractile dysfunction.