Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids

Macrophages accumulate triglycerides under certain pathological conditions such as atherosclerosis. Triglycerides are carried in the bloodstream as part of very low-density lipoproteins (VLDL) and chylomicrons. How macrophages take up and process VLDL-lipids is not very well known. Here, using VLDL-sized triglyceride-rich emulsion particles, we aimed to study the mechanism by which VLDL-triglycerides are taken up, processed, and stored in macrophages. Our results show that macrophage uptake of emulsion particles mimicking VLDL (VLDLm) is dependent on lipoproteins lipase (LPL) and requires the lipoprotein-binding C-terminal domain of LPL but not the catalytic N-terminal domain. Subsequent internalization of VLDLm-triglycerides by macrophages is carried out by caveolae-mediated endocytosis, followed by triglyceride hydrolysis catalyzed by lysosomal acid lipase. Transfer of lysosomal fatty acids to the ER for subsequent storage as triglycerides is mediated by Stard3, whereas NPC1 was found to promote the extracellular efflux of fatty acids from lysosomes. Our data provide novel insights into how macrophages process VLDL-derived triglycerides and suggest that macrophages have the remarkable capacity to excrete part of the internalized triglycerides as fatty acids. SummaryTriglyceride-rich lipoproteins and their remnants contribute to atherosclerosis, possibly by carrying remnant cholesterol and/or by exerting a pro-inflammatory effect on macrophage. Nevertheless, little is known about how macrophages process triglyceride-rich lipoproteins. We show that uptake by macrophages of VLDL-like particles is dependent on the enzyme lipoproteins lipase via its C-terminal domain. Subsequent internalization of VLDL-triglycerides by macrophages is carried out by caveolae-mediated endocytosis, followed by hydrolysis by lysosomal acid lipase. Transfer of lysosomal fatty acids to the ER for lipid storage is mediated by Stard3, while NPC1 promotes the extracellular efflux of fatty acids. Our data provide novel insights into how macrophages process VLDL-derived triglycerides and suggest that macrophages have the remarkable capacity to excrete internalized triglycerides as fatty acids. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Choline is an essential nutrient that is critical component of the membrane phospholipid phosphatidylcholine (PC), the neurotransmitter acetylcholine and the methylation pathway. In the liver specifically, PC is the major membrane constituent and can be synthesized by the CDP-choline or the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. With the continuing global rise in the rates of obesity and non-alcoholic fatty liver disease, we sought to explore how excess fatty acids (FA), typical of an obesity and hepatic steatosis, affect choline uptake and metabolism in primary hepatocytes. Our results demonstrate that hepatocytes chronically treated with palmitate, but not oleate or a mixture, had decreased choline uptake, which was associated with lower choline incorporation into PC and lower expression of choline transport proteins. Interestingly, a reduction in the rate of degradation spared PC levels in response to palmitate when compared to control. PE synthesis was slightly diminished; however, no compensatory changes in the PEMT pathway were observed. We next hypothesized that ER stress may be a potential mechanism by which palmitate treatment diminished choline. However, when we exposed primary hepatocytes to the common ER stress inducing compound tunicamycin, choline uptake, contrary to our expectation was augmented, concomitant with the transcript expression of choline transporters. Moreover, tunicamycin-induced ER stress divorced the observed increase in choline uptake from CDP-choline pathway flux since ER stress significantly diminished the incorporation and total PC content, similar to PE. Conclusion: Therefore, our results suggest that the altered FA milieu seen in obesity and fatty liver disease progression may adversely affect choline metabolism, but that compensatory mechanisms work to maintain phospholipid homeostasis.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC=\"FIGDIR/small/746750v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (42K):\norg.highwire.dtl.DTLVardef@1090f2aorg.highwire.dtl.DTLVardef@1c28eedorg.highwire.dtl.DTLVardef@35a80eorg.highwire.dtl.DTLVardef@ac5a53_HPS_FORMAT_FIGEXP M_FIG C_FIG

Structured AbstractO_ST_ABSObjectivesC_ST_ABSTriglyceride (TG) association with apolipoprotein B100 (apoB100) serves to form very low density lipoproteins (VLDL) in the liver. The repertoire of factors that facilitate this association is incompletely defined. FITM2, an integral endoplasmic reticulum (ER) protein, was originally discovered as a factor participating in cytoplasmic lipid droplets (LDs) in tissues that do not form VLDL. We hypothesized that in the liver, in addition to promoting cytosolic LD formation, FITM2 would also transfer TG from its site of synthesis in the ER membrane to nascent VLDL particles within the ER lumen. MethodsExperiments were conducted using a rat hepatic cell line (McArdle-RH7777, or McA cells), an established model of mammalian lipoprotein metabolism, and mice. FITM2 expression was reduced using siRNA in cells and by liver specific cre-recombinase mediated deletion of the Fitm2 gene in mice. Effects of FITM2 deficiency on VLDL assembly and secretion in vitro and in vivo were measured by multiple methods, including density gradient ultracentrifugation, chromatography, mass spectrometry, simulated Raman spectroscopy (SRS) microscopy, sub-cellular fractionation, immunoprecipitation, immunofluorescence, and electron microscopy. Main findings1) FITM2-deficient hepatic cells in vitro and in vivo secrete TG-depleted VLDL particles, but the number of particles is unchanged compared to controls; 2) FITM2 deficiency in mice on a high fat diet (HFD) results in decreased plasma TG levels. The number of apoB100-containing lipoproteins remains similar, but shift from VLDL to LDL density; 3) Both in vitro and in vivo, when TG synthesis is stimulated and FITM2 is deficient, TG accumulates in the ER, and despite its availability this pool is unable to fully lipidate apoB100 particles; 4) FITM2 deficiency disrupts ER morphology and results in ER stress. Principal conclusionsThe results suggest that FITM2 contributes to VLDL lipidation, especially when newly synthesized hepatic TG is in abundance. In addition to its fundamental importance in VLDL assembly, the results also suggest that under dysmetabolic conditions, FITM2 may be a limiting factor that ultimately contributes to non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH).

The two major products of intestinal triacylglycerol digestion and lipoprotein lipolysis are monoacylglycerols (MAG) and fatty acids. In the gut, these products are taken up by enterocytes and packaged into perilipin-coated cytosolic lipid droplets and then secreted as chylomicrons. We observed that fat feeding or intragastric administration of triacylglycerol oil caused the enterocyte Golgi to fragment into submicron puncta dispersed throughout the cytosol. Further, this apparent Golgi dispersion was also observed in cultured fibroblasts after treatment with fat (cream) and pancreatic lipase, but not when treated with deactivated lipase. We therefore hypothesized that a hydrolytic fat product, specifically monoacylglycerols, fatty acids or a combination of these molecules can trigger Golgi fragmentation. Disruption of coatomer function is known to cause Golgi to fuse with the ER, and blocks perilipin 2 delivery to lipid droplets. Thus, we assessed the effects of MAG on coatomer distribution, Golgi structure and perilipin 2 localization. We found that MAG, but not fatty acids, dispersed coatomer from the Golgi, fragmented the Golgi and caused perilipin 2 to accumulate on cellular membranes. Thus, our findings suggest that monoacylglycerol production during digestion disperses the Golgi, possibly by altering coatomer function, which may regulate metabolite transport between the ER and Golgi.

Dehydrogenase/reductase (SDR family) member1, DHRS1, a member of the conserved short chain dehydrogenase/reductase (SDR) superfamily, has been identified in lipid droplets proteome of different cells and tissues. However, until now, little is known about the potential role of DHRS1 on the lipid droplet (LD). Here, we report that DHRS1 localized to the lipid droplet in Huh7 and HeLa cells and ectopic expression of DHRS1 in HeLa cell induced a significant change in the lipid droplet morphology resulting in nearly 2 fold increase both in lipid droplet size and total triacylglycerol level independent of oleic acid treatment. DHRS1 interacted with ERLIN1, a non-caveolae lipid raft-like domain marker, in HeLa cell and ERLIN1 deficient HeLa cells displayed no detectable change in LD morphology. Although ectopic expression of DHRS1-GFP fusion protein in ERLIN1 deficient HeLa cells resulted in fewer GFP-labeled ring structures relative to WT HeLa cell; thus suggesting that ERLIN1 may be involved in regulating DHRS1 protein turnover. Taken together, these data showed that DHRS1 localized to the LD and induced a significant change in LD morphology which may be regulated by ERLIN1.

Lipoprotein lipase (LPL) is critical for clearance of circulating triglycerides and for tissue fatty acid supply. LPL is primarily synthesized and secreted by adipocytes into the interstitium and must traffic from there to the abluminal/basolateral side of capillary endothelial cells. There, LPL binds glycosylphosphatidylinositol-anchored protein 1, GPIHBP1, which stabilizes the protein and facilitates its movement across the endothelial cells to the luminal side where it functions in hydrolysis of lipoprotein triglycerides. Importance of LPL traffic is supported by findings that rare mutations in GPIHBP1 cause hypertriglyceridemia. However our understanding of how LPL is secreted by adipocytes and traffics to endothelial cells is incomplete. Here we examined the possibility that secretion and traffic of adipocyte LPL might involve generation of small extracellular vesicles (sEVs/exosomes) which often mediate cell-cell communication. Proteomic analysis of sEVs secreted by adipocytes showed them enriched in LPL. To study LPL secretion and transfer we generated human derived pre-adipocytes (HPA) that stably express tagged LPL (FLAG and His epitopes). LPL pulldown and sEV isolation from HPA conditioned media documented that greater than 70% of secreted LPL is present in sEVs. The mechanism for LPL secretion in sEVs was found to involve the ESCRT-independent neutral sphingomyelinase 2 (nSMase2) pathway, as treatment with the nSMase2 inhibitor GW4869 reduced secretion by 80%. The above observations were reproduced using highly sensitive nanoparticle flow cytometry. The sEV associated LPL has lipolytic activity and it is released by heparin addition indicating it is on the sEV surface. In addition, using human derived microvascular endothelial cells with stable lentiviral expression of GPIHBP1 we show that LPL positive sEVs transfer LPL to these cells, but not to control cells without GPIHBP1. Our findings suggest that sEV formation by nSMase2 controls adipocyte LPL secretion and traffic, that sEVs protect LPL activity and facilitate LPL transfer to GPIHBP1 on endothelial cells.

Increased free fatty acids (FFA) are considered a key factor in the development of insulin resistance in muscle and liver; however, their role in regulating glucose uptake in adipocytes remains controversial. Here, the effects of palmitate (PA) and oleate (OA) were studied in 3T3-L1 adipocytes with a focus on lipid accumulation and glucose uptake. We found that glucose rather than FFA availability promotes adipocyte maturation and fat accumulation associated with increased basal glucose uptake and altered expression of fatty acid oxidation markers (CPT-1 isoforms and UCP-1) toward lipid storage phenotype. Neither PA, nor OA altered insulin-stimulated glucose uptake by immature or mature adipocytes. Adipogenic differentiation of preadipocytes in the presence of rosiglitazone led to appearance of double effect of PA on glucose uptake by differentiated adipocytes, i.e. (1) PA dose-dependently increased basal glucose uptake, and (2) at high concentration PA suppressed insulin-stimulated glucose uptake. The effect of PA on basal glucose uptake was independent of mTORC1-mediated feedback in insulin signaling and persisted even when insulin signaling was inhibited by high-dose PA. Nonetheless, it was associated with GLUT4 exposure at the plasma membrane as reported by PA-induced, insulin-independent translocation of cMyc-GLUT4-mCherry chimera expressed in 3T3-L1 adipocytes. Thus, we conclude that only excessive FFA may context-dependently trigger classic insulin resistance in adipocytes, but otherwise FFA increase glucose uptake in adipocytes via GLUT4 mobilization.

Polyunsaturated fatty acids (PUFAs) of the n-6 and n-3 series cannot be synthesized in mammals and therefore are called essential fatty acids (EFAs). Mead acid (20:3n-9) is an unusual n-9 PUFA, endogenously synthesized from oleic acid (18:1n-9) in an EFA-deficient state. Although Elovl5, a fatty acid elongase, has long been known to selectively elongate C18 and C20 PUFAs, it can use 18:1n-9 as a substrate for the synthesis of Mead acid under C20 PUFA-deficient, but not-sufficient, conditions. We found, by an in vitro enzyme assay, that the microsomal fraction obtained from PUFA-deficient, but not -sufficient, cells showed significant Elovl5 activity toward 18:1n-9, with no effect on its constitutive activity toward 18:3n-6, implying that Elovl5 acquires the activity toward 18:1n-9 under the PUFA-deficient conditions at the enzyme level. Further biochemical analysis revealed that Elovl5 was phosphorylated in the C20 PUFA-supplemented cells, and that treatment with an inhibitor of glycogen synthase kinase 3 (GSK3) completely abolished the phosphorylation of Elovl5 and retained the Elovl5 activity toward 18:1n-9, even in the presence of C20 PUFA. Finally, mutation of putative phosphorylation sites (T281A/S283A/S285A) on Elovl5 did not decrease the activity of Elovl5 toward 18:1n-9 by supplementation with C20 PUFA, suggesting that the phosphorylation of Elovl5 contributed to a change in substrate preference. Thus, by changing its substrate specificity in an EFA-deficient state, Elovl5 is able to regulate the synthesis of Mead acid to maintain levels of long-chain PUFAs.

Tribbles related homolog 1 (TRIB1) contributes to lipid and glucose homeostasis by facilitating the degradation of cognate cargos by the proteasome. We previously reported that TRIB1 was unstable in non-hepatic cellular models. Moreover, inclusion of proteasome inhibitors failed to prevent TRIB1 loss, consistent with the involvement of proteasome independent degradative processes. In view of the key role of TRIB1 in liver function, we continue our exploration of TRIB1 regulation pathways in two commonly used human hepatocyte models, HuH-7 and HepG2 cells. Proteasome inhibitors potently upregulated both endogenous and recombinant TRIB1 mRNA and protein levels. Increased transcript abundance was independent of MAPK activation while ER stress was a relatively mild inducer. Despite increasing TRIB1 protein abundance and stabilizing bulk ubiquitination, proteasome inhibition failed to stabilize TRIB1, pointing to the predominance of proteasome independent protein degradation processes controlling TRIB1 protein abundance in hepatomas. Proteasome inhibition via downregulation of its PSMB3 regulatory subunit, in contrast to its chemical inhibition, had minimal impact on TRIB1 levels. Moreover, immunoprecipitation experiments showed no evidence of TRIB1 ubiquitination. Cytoplasmic retained TRIB1 was unstable, indicating that TRIB1 lability is regulated prior to its nuclear import. Substitution of the TRIB1 PEST-like region with a GST helical region or N-terminal deletions failed to fully stabilize TRIB1. Finally, inclusion of protease or autophagy inhibitors in vivo did not rescue TRIB1 stability. This work excludes proteasome-mediated degradation as a significant contributor to TRIB1 instability and identifies transcriptional regulation as a prominent mechanism regulating TRIB1 abundance in liver models in response to proteasome inhibition.

Insulin resistance is a critical mediator of the development of non-alcoholic fatty liver disease (NAFLD). An excess influx of fatty acids to the liver is thought to be a pathogenic cause of insulin resistance and the development of non-alcoholic fatty liver disease (NAFLD). Although elevated levels of free fatty acids (FFA) in plasma contribute to inducing insulin resistance and NAFLD, the molecular mechanism is not completely understood. This study aimed to determine whether inositol polyphosphate multikinase (IPMK), a regulator of insulin signaling, plays any role in FFA-induced insulin resistance in primary hepatocytes. Here, we show that excess FFA decreased IPMK expression, and blockade of IPMK decrease attenuated the FFA-induced suppression of Akt phosphorylation in primary mouse hepatocytes (PMH). Moreover, overexpression of IPMK prevented the FFA-induced suppression of Akt phosphorylation by insulin, while knockout of IPMK exacerbated insulin resistance in PMH. In addition, treatment with MG132, a proteasomal inhibitor, inhibits FFA-induced decrease in IPMK expression and Akt phosphorylation in PMH. Furthermore, treatment with the antioxidant N-Acetyl Cysteine (NAC) significantly attenuated the FFA-induced reduction of IPMK and restored FFA-induced insulin resistance in PMH. In conclusion, our findings suggest that excess FFA reduces IPMK expression and contributes to the FFA-induced decrease in Akt phosphorylation in PMH, leading to insulin resistance. Our study highlights IPMK as a potential therapeutic target for preventing insulin resistance and NAFLD.

Glycerophospholipid (GPL) homeostasis in eukaryotic cells is thought to be maintained via biosynthesis, degradation and acyl chain remodeling. Here we provide evidence for an additional process termed "head-group remodeling" where other GPLs, when in excess, are rapidly converted to phosphatidylcholine and triacylglycerol. Mass spectrometric studies showed the formation of diacylglycerol, but not phosphatidic acid, from the exogenous GPL thus indicating that the first step is catalyzed by a phospholipase C-type enzyme. Consistently, triacylglycerol formation was significantly inhibited by the knock-down of several PLCs, but not phospholipase Ds. Second, we found that each exogenous GPL strongly inhibited the synthesis of the corresponding endogenous GPL class. Based on these and previous data we hypothesize how mammalian cells could coordinate the multiple processes contributing to GPL homeostasis in mammalian cells. In conclusion, this study provides the first evidence that head group remodeling plays an important role in GPL homeostasis in mammalian cells.

Distinct plasma microRNA profiles associate with different disease features. Elevated plasma microRNA hsa-miR-193b-3p has been reported in patients with pre-diabetes where early liver dysmetabolism plays a crucial role. MicroRNA target databases revealed that hsa-miR-193b-3p could potentially target PPARGC1A, a master switch transcriptional co-activator that orchestrates the expression of genes involved in multiple metabolic pathways. In this study we evaluated the effects of hsa-miR-193b-3p in the human HepG2 hepatocyte cell line. We show that hsa-miR-193b-3p targets PPARGC1A/PGC1 mRNA 3UTR and reduces its expression. Overexpression of hsa-miR-193b-3p under hyperglycemia resulted in increased accumulation of intracellular lipid droplets; and quantitative real-time RT-PCR of a panel of 24 genes revealed significant disruption in the expression of genes coordinating glucose uptake, glycolysis, pyruvate metabolism, fatty acid synthesis, fatty acid oxidation, triglyceride synthesis, VLDL secretion, insulin signalling, and mitochondrial biogenesis, dynamics and function. These results provide in vitro evidence that microRNA hsa-miR-193b-3p downregulates PPARGC1A/PGC1, disrupts downstream metabolic pathways and leads to the accumulation of intracellular lipids in hepatocytes. We propose that microRNA hsa-miR-193b-3p may have potential as a clinically relevant plasma biomarker for metabolic associated fatty liver disease in a pre-diabetic dysglycemic context.

Sphingolipids are crucial components of cell membranes. Sphingolipid {Delta}8-unsaturation is more specific to plants and is involved in the regulation of stress responses. The structure and functions of sphingolipids in microalgae are still poorly understood. Ostreococus tauri is a minimal microalga at the base of the green lineage, and is therefore a key organism for understanding lipid evolution. The present work reports the characterisation as well as the temperature regulation of sphingolipids and {Delta}8-sphingolipid desaturase from O. tauri. Complex sphingolipids are glycosylceramides with unique glycosyl moieties encompassing hexuronic acid residues, reminiscent of bacterial glucuronosylceramides, with up to three additional hexose residues. In contrast, the ceramide backbones show limited variety, with dihydroxylated C18/C18:1E{Delta}8 sphingoid bases and C16:0 fatty-acyl chain being the main compounds. The sphingolipid {Delta}8-desaturase from O. tauri, although phylogenetically related to plant homologues has a substrate preference similar to the diatom homologue. Both sphingolipid {Delta}8-desaturase transcripts and sphingolipid {Delta}8-unsaturation are regulated in a temperature- dependent manner being higher at 14{degrees}C than 24{degrees}C. Overexpressing the sphingolipid {Delta}8- desaturase in O. tauri at 24{degrees}C results in higher sphingolipid unsaturation and impairs the increase in cell size, structure and chlorophyll. In particular, the cell-size defect is not detected in cells acclimated to 14{degrees}C and is furthermore suppressed upon transfer from 24{degrees}C to 14{degrees}C. Our work provides the first functional evidence for the involvement of sphingolipid {Delta}8-unsaturation for temperature acclimation in microalgae, suggesting that this function is an ancestral feature in the green lineage.

The storage and release of triacylglycerol (TAG) in lipid droplets (LDs) is regulated by dynamic protein interactions. /{beta} hydrolase domain-containing protein 5 (ABHD5; also known as CGI-58) is a membrane/LD bound protein that functions as a co-activator of Patatin Like Phospholipase Domain Containing 2 (PNPLA2; also known as Adipose triglyceride lipase, ATGL) the rate-limiting enzyme for TAG hydrolysis. The dysregulation of TAG hydrolysis is involved in various metabolic diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD). We previously demonstrated that ABHD5 interacted with PNPLA3, a closely related family member to PNPLA2. Importantly, a common missense variant in PNPLA3 (I148M) is the greatest genetic risk factor for MASLD. PNPLA3 148M functions to sequester ABHD5 and prevent co-activation of PNPLA2, which has implications for initiating MASLD; however, the exact mechanisms involved are not understood. Here we demonstrate that LD targeting of both ABHD5 and PNPLA3 I148M is required for the interaction. Molecular modeling demonstrates important resides in the C-terminus of PNPLA3 for LD binding and fluorescence cross-correlation spectroscopy demonstrates that PNPLA3 I148M greater associates with ABHD5 than WT PNPLA3. Moreover, the C-terminus of PNPLA3 is sufficient for functional targeting of PNPLAs to LD and the interaction with ABHD5. In addition, ABHD5 is a general binding partner of LD-bound PNPLAs. Finally, PNPLA3 I148M targeting to LD is required to promote steatosis in vitro and in the liver. Overall results suggest that PNPLA3 I148M is a gain of function mutation and that the interaction with ABHD5 on LD is required to promote liver steatosis.

Ceramides (Cer) have been shown as lipotoxic inducers, which disturb numerous cell signalling pathways especially insulin signalling pathway leading to metabolic disorders such as type 2 diabetes. In this study, we aimed to determine the role of de novo hepatic Cer synthesis on energy and liver homeostasis in mice. We generated mice lacking serine palmitoyltransferase 2 (Sptlc2), the rate limiting enzyme of Cer de novo synthesis, in hepatocytes. Despite lower expression of hepatic Sptlc2, we observed an increased concentration of hepatic Cer, especially C16:0-Cer and C18:0-Cer associated with an increased neutral sphingomyelinase 2 expression, and a decreased sphingomyelin content in the liver. Sptlc2{Delta}Hep mice were protected against obesity induced by high fat diet. Bile acid (BA) hydrophobicity was drastically decreased in KO mice, and was associated with a defect in lipid absorption. In addition, an important increase of tauro-muricholic acid in BA pool composition was associated with a downregulation of the nuclear BA receptor FXR target genes. Sptlc2 deficiency also enhanced glucose tolerance and attenuated hepatic glucose production. Finally, Sptlc2 disruption promoted apoptosis, inflammation and progressive development of hepatic fibrosis worsening with age. Our data suggest a compensatory mechanism to regulate hepatic Cer content from sphingomyelin hydrolysis, with deleterious impact on liver homeostasis. In addition, our results show the implication of hepatic sphingolipid modulation on BA metabolism and hepatic glucose production in an insulinin-dependent manner, which demonstrates the role of Cer in many metabolic functions still under-researched.

Hepatocyte nuclear factor 4 (HNF4) and glucocorticoid receptor (GR), master regulators of liver metabolism, are down-regulated in fatty liver diseases. The present study was aimed to elucidate the role of down-regulation of HNF4 and GR in fatty liver and hyperlipidemia. Adult mice with liver-specific heterozygote and knockout (knockout) of HNF4 were fed a low-fat diet (LFD) or a high-fat-high-sugar diet (HFHS) for 15 days. Compared to LFD-fed mice, HFHS-fed wildtype mice had hepatic induction of lipid catabolic genes and down-regulation of lipogenic genes. Compared to HFHS-fed wildtype mice, HNF4 heterozygote mice had down-regulation of lipid catabolic genes, induction of lipogenic genes, and increased hepatic and blood levels of lipids, whereas HNF4 knockout mice had mild hypolipidemia, down-regulation of lipid-efflux genes, but induction of genes for uptake/storage of lipids. Sterol-regulatory-element-binding protein-1c (SREBP-1C), a master lipogenic regulator, was induced in HFHS-fed HNF4 heterozygote mice. In reporter assays, HNF4 potently inhibited the transactivation of mouse and human SREBP-1C promoter by liver X receptor. Surprisingly, nuclear GR proteins were gene-dosage-dependently decreased in HNF4 heterozygote and knockout mice. HFHS-fed mice with liver-specific knockout of GR had increased hepatic lipids and induction of SREBP-1C and PPAR{gamma}. In reporter assays, GR and HNF4 synergistically/additively induced lipid catabolic genes. Phosphorylation of AMP-activated protein kinase (AMPK), a key GR modulator, was dramatically decreased in HNF4 knockout mice. Thus, cooperative induction of lipid catabolic genes and suppression of lipogenic genes by HNF4 and GR, modulated by AMPK, may mediate the early resistance to HFHS-induced fatty liver and hyperlipidemia.

BackgroundNon-Alcoholic Fatty Liver Disease (NAFLD), now referred to as Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), is a widespread and complex health issue characterized by the accumulation of lipids in liver cells, not linked to alcohol consumption or other known liver problems. Despite its strong association with insulin resistance and obesity, a notable proportion of lean individuals develop NAFLD, suggesting the involvement of additional factors. Mitochondrial dysfunction has emerged as a potential contributor, given its role in lipid homeostasis and the generation of reactive oxidative species (ROS). MethodsIn this study, we took advantage of the mtDNA mutator mouse model, characterized by oxidative phosphorylation deficiency due to accelerated accumulation of mitochondrial DNA (mtDNA) mutations, to investigate how mitochondrial dysfunction affects liver homeostasis triggering fat accumulation. ResultsWe found that mitochondrial dysfunction induces liver steatosis and inflammation in this mouse model at a young age, independent of obesity. Using quantitative mass spectrometry, we reveal that mitochondrial dysfunction alters the levels of multiple proteins involved in lipid metabolism, cholesterol homeostasis, and inflammation. In vitro data show a shift in cellular metabolism toward the glycolytic pathway and confirm the upregulation of inflammatory genes. These changes are associated with oxidative stress and occur independently of mtDNA molecule release into the cytoplasm. ConclusionOur findings demonstrate that liver steatosis might develop as result of mitochondrial dysfunction without obesity and insulin resistance, underscoring the importance of mitochondrial dysfunction in NAFLD development in lean individuals and providing valuable insights into the molecular mechanisms underlying this complex metabolic disorder. This mouse model offers a unique platform for further investigations aimed at unraveling the intricacies of NAFLD pathogenesis and potential therapeutic targets beyond conventional risk factors.

Zebrafish are an ideal model organism to study lipid metabolism and to elucidate the molecular underpinnings of human lipid-associated disorders. In this study, we provide an improved protocol to assay the impact of a high-cholesterol diet (HCD) on zebrafish lipid deposition and lipoprotein regulation. Fish fed HCD developed hypercholesterolemia as indicated by significantly elevated ApoB-containing lipoproteins (ApoB-LP) and increased plasma levels of cholesterol and cholesterol esters. Feeding of the HCD to larvae (8 days followed by a 1 day fast) and adult female fish (2 weeks, followed by 3 days of fasting) was also associated with a fatty liver phenotype that presented as severe hepatic steatosis. The HCD feeding paradigm doubled the levels of liver triacylglycerol (TG), which was striking because our HCD was only supplemented with cholesterol. The accumulated liver TG was unlikely due to increased de novo lipogenesis or inhibited {beta}-oxidation since no differentially expressed genes in these pathways were found between the livers of fish fed the HCD versus control diets. However, fasted HCD fish had significantly increased lipogenesis gene fasn in adipose tissue and higher free fatty acids (FFA) in plasma. This suggested that elevated dietary cholesterol resulted in lipid accumulation in adipocytes, which supplied more FFA during fasting, promoting hepatic steatosis. In conclusion, our HCD zebrafish protocol represents an effective and reliable approach for studying the temporal characteristics of the physiological and biochemical responses to high levels of dietary cholesterol and provides insights into the mechanisms that may underlie fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) and Metabolic syndrome (MS) have become a global health concern as incidence of these metabolic disorders is growing rapidly in developing countries particularly in the Middle East, South America and Africa. Studies have shown that protein restriction is associated with increased risk of metabolic diseases, possibly through effects on fatty acid (FA) metabolism. In the present study, we investigated whether a low protein diet modulates FA metabolism and whether methyl donor supplementation can ameliorate these effects and improve metabolic health. Male C57BL/6 mice were fed either a low protein diet (LPD, 90 g/kg protein, n=8), a LPD supplemented with methyl donors (MD-LPD; choline chloride, betaine, methionine, folic acid, vitamin B12, n=8) or normal protein diet (NPD, 180 g/kg protein, n=8) for 7 weeks prior to analysis of serum fatty acid profiles by GC FID and MS and liver fatty acid synthesis and uptake gene expression by RT-qPCR. We observed significant depletion of serum C15:0 and C17:0 in LPD-fed males compared to NPD. Serum long chain saturated FAs C18:0 and C24:0 were increased in LPD male mice compared to NPD. Gene expression analysis revealed an upregulation of hepatic cluster of differentiation 36 (CD36) expression in LPD mice compared to NPD suggesting increased fat uptake in the liver. However, when LPD diet was supplemented with methyl donors, we observed either no change in serum C15: 0 and an increased serum C17:0 compared to LPD with no methyl donor supplementation. Again, methyl donor supplementation upregulated fatty acid desaturase 1 (FADS1), thioredoxin-1 (TRX1) and catalase (CAT) expression in the liver of MD-LPD fed mice compared to LPD mice. Altogether, our study revealed that odd chain fatty acids (OCFA)s are key early markers observed in a suboptimal diet-induced metabolic changes and may be potential targets to improve metabolic health outcomes.

Obesity caused by overnutrition is a major risk factor for non-alcoholic fatty liver disease (NAFLD). Several lipid intermediates such as fatty acids, glycerophospholipids and sphingolipids are implicated in NAFLD, but detailed characterization of lipids and their functional links to proteome and phosphoproteome remain to be elucidated. To characterize this complex molecular relationship, we used multi-omics approach by conducting comparative proteomic, phoshoproteomic and lipidomic analyses of high fat (HFD) and low fat (LFD) diet fed mice livers. We quantified 2447 proteins and 1339 phosphoproteins containing 1650 class I phosphosites (with localization probability > 0.75), of which 669 phosphosites were significantly different between HFD and LFD mice livers. We detected alterations of proteins associated with cellular metabolic processes such as small molecule catabolic process, monocarboxylic acid, long- and medium-chain fatty acid, and ketone body metabolic processes, and peroxisome organization. We observed significant downregulation of protein phosphorylation in HFD fed mice liver in general. Untargeted lipidomics identified upregulation of triacylglycerols, glycerolipids and ether glycerophosphocholines and downregulation of glycerophospholipids such as lysoglycerophospholipids, as well as ceramides and acylcarnitines. Analysis of differentially regulated phosphosites revealed phosphorylation dependent deregulation of insulin signaling as well as lipogenic and lipolytic pathways during HFD induced obesity. Thus, this study reveals a molecular connection between decreased protein phosphorylation and lipolysis, as well as lipid-mediated signaling in diet-induced obesity.