Toll-like receptors TLR2 and TLR4 block the replication of pancreatic ß cells in diet-induced obesity.

Consumption of a high-energy Western diet triggers mild adaptive ß cell proliferation to compensate for peripheral insulin resistance; however, the underlying molecular mechanism remains unclear. In the present study we show that the toll-like receptors TLR2 and TLR4 inhibited the diet-induced replication of ß cells in mice and humans. The combined, but not the individual, loss of TLR2 and TLR4 increased the replication of ß cells, but not that of α cells, leading to enlarged ß cell area and hyperinsulinemia in diet-induced obesity. Loss of TLR2 and TLR4 increased the nuclear abundance of the cell cycle regulators cyclin D2 and Cdk4 in a manner dependent on the signaling mediator Erk. These data reveal a regulatory mechanism controlling the proliferation of ß cells in diet-induced obesity and suggest that selective targeting of the TLR2/TLR4 pathways may reverse ß cell failure in patients with diabetes.

Macrophages take up VLDL-sized emulsion particles through caveolae-mediated endocytosis and excrete part of the internalized triglycerides as fatty acids.

Triglycerides are carried in the bloodstream as part of very low-density lipoproteins (VLDLs) and chylomicrons, which represent the triglyceride-rich lipoproteins. Triglyceride-rich lipoproteins and their remnants contribute to atherosclerosis, possibly by carrying remnant cholesterol and/or by exerting a proinflammatory effect on macrophages. Nevertheless, little is known about how macrophages process triglyceride-rich lipoproteins. 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 VLDL-sized emulsion particles is dependent on lipoprotein lipase (LPL) and requires the lipoprotein-binding C-terminal domain but not the catalytic N-terminal domain of LPL. Subsequent internalization of VLDL-sized emulsion particles by macrophages is carried out by caveolae-mediated endocytosis, followed by triglyceride hydrolysis catalyzed by lysosomal acid lipase. It is shown that STARD3 is required for the transfer of lysosomal fatty acids to the ER for subsequent storage as triglycerides, while NPC1 likely is involved in promoting the extracellular efflux of fatty acids from lysosomes. Our data provide novel insights into how macrophages process VLDL triglycerides and suggest that macrophages have the remarkable capacity to excrete part of the internalized triglycerides as fatty acids.

Triglyceride breakdown from lipid droplets regulates the inflammatory response in macrophages.

In response to inflammatory activation by pathogens, macrophages accumulate triglycerides in intracellular lipid droplets. The mechanisms underlying triglyceride accumulation and its exact role in the inflammatory response of macrophages are not fully understood. Here, we aim to further elucidate the mechanism and function of triglyceride accumulation in the inflammatory response of activated macrophages. Lipopolysaccharide (LPS)-mediated activation markedly increased triglyceride accumulation in macrophages. This increase could be attributed to up-regulation of the hypoxia-inducible lipid droplet­associated (HILPDA) protein, which down-regulated adipose triglyceride lipase (ATGL) protein levels, in turn leading to decreased ATGL-mediated triglyceride hydrolysis. The reduction in ATGL-mediated lipolysis attenuated the inflammatory response in macrophages after ex vivo and in vitro activation, and was accompanied by decreased production of prostaglandin-E2 (PGE2) and interleukin-6 (IL-6). Overall, we provide evidence that LPS-mediated activation of macrophages suppresses lipolysis via induction of HILPDA, thereby reducing the availability of proinflammatory lipid precursors and suppressing the production of PGE2 and IL-6.

Characterization of sexual dimorphism in ANGPTL4 levels and function.

ANGPTL4 is an attractive pharmacological target for lowering plasma triglycerides and cardiovascular risk. Since most preclinical studies on ANGPTL4 were performed in male mice, little is known about sexual dimorphism in ANGPTL4 regulation and function. Here, we aimed to study potential sexual dimorphism in ANGPTL4 mRNA and protein levels and ANGPTL4 function. Additionally, we performed exploratory studies on the function of ANGPTL4 in the liver during fasting using Angptl4-transgenic and Angptl4-/- mice. Compared to female mice, male mice showed higher hepatic and adipose ANGPTL4 mRNA and protein levels, as well as a more pronounced effect of genetic ANGPTL4 modulation on plasma lipids. By contrast, very limited sexual dimorphism in ANGPTL4 levels was observed in human liver and adipose tissue. In human and mouse adipose tissue, ANGPTL8 mRNA and/or protein levels were significantly higher in females than males. Adipose LPL protein levels were higher in female than male Angptl4-/- mice, which was abolished by ANGPTL4 (over) expression. At the human genetic level, the ANGPTL4 E40K loss-of-function variant was associated with similar plasma triglyceride reductions in women and men. Finally, ANGPTL4 ablation in fasted mice was associated with changes in hepatic gene expression consistent with PPARα activation. In conclusion, the levels of ANGPTL4 and the magnitude of the effect of ANGPTL4 on plasma lipids exhibit sexual dimorphism. Nonetheless, inactivation of ANGPTL4 should confer a similar metabolic benefit in women and men. Expression levels of ANGPTL8 in human and mouse adipose tissue are highly sexually dimorphic, showing higher levels in females than males.

Metabolic effects of nuclear receptor activation in vivo after 28-day oral exposure to three endocrine-disrupting chemicals.

Environmental exposure to endocrine-disrupting chemicals (EDCs) can lead to metabolic disruption, resulting in metabolic complications including adiposity, dyslipidemia, hepatic lipid accumulation, and glucose intolerance. Hepatic nuclear receptor activation is one of the mechanisms mediating metabolic effects of EDCs. Here, we investigated the potential to use a repeated dose 28-day oral toxicity test for identification of EDCs with metabolic endpoints. Bisphenol A (BPA), pregnenolone-16α-carbonitrile (PCN), and perfluorooctanoic acid (PFOA) were used as reference compounds. Male and female wild-type C57BL/6 mice were orally exposed to 5, 50, and 500 µg/kg of BPA, 1000, 10 000, and 100 000 µg/kg of PCN and 50 and 300 µg/kg of PFOA for 28 days next to normal chow diet. Primary endpoints were glucose tolerance, hepatic lipid accumulation, and plasma lipids. After 28-day exposure, no changes in body weight and glucose tolerance were observed in BPA-, PCN-, or PFOA-treated males or females. PCN and PFOA at the highest dose in both sexes and BPA at the middle and high dose in males increased relative liver weight. PFOA reduced plasma triglycerides in males and females, and increased hepatic triglyceride content in males. PCN and PFOA induced hepatic expression of typical pregnane X receptor (PXR) and peroxisome proliferator-activated receptor (PPAR)α target genes, respectively. Exposure to BPA resulted in limited gene expression changes. In conclusion, the observed changes on metabolic health parameters were modest, suggesting that a standard repeated dose 28-day oral toxicity test is not a sensitive method for the detection of the metabolic effect of EDCs.

Genetic Mimicry Analysis Reveals the Specific Lipases Targeted by the ANGPTL3-ANGPTL8 Complex and ANGPTL4.

Angiopoietin-like proteins, ANGPTL3, ANGPTL4, and ANGPTL8, are involved in regulating plasma lipids. In vitro and animal-based studies point to LPL and endothelial lipase (EL, LIPG) as key targets of ANGPTLs. To examine the ANGPTL mechanisms for plasma lipid modulation in humans, we pursued a genetic mimicry analysis of enhancing or suppressing variants in the LPL, LIPG, lipase C hepatic type (LIPC), ANGPTL3, ANGPTL4, and ANGPTL8 genes using data on 248 metabolic parameters derived from over 110,000 nonfasted individuals in the UK Biobank and validated in over 13,000 overnight fasted individuals from 11 other European populations. ANGPTL4 suppression was highly concordant with LPL enhancement but not HL or EL, suggesting ANGPTL4 impacts plasma metabolic parameters exclusively via LPL. The LPL-independent effects of ANGPTL3 suppression on plasma metabolic parameters showed a striking inverse resemblance with EL suppression, suggesting ANGPTL3 not only targets LPL but also targets EL. Investigation of the impact of the ANGPTL3-ANGPTL8 complex on plasma metabolite traits via the ANGPTL8 R59W substitution as an instrumental variable showed a much higher concordance between R59W and EL activity than between R59W and LPL activity, suggesting the R59W substitution more strongly affects EL inhibition than LPL inhibition. Meanwhile, when using a rare and deleterious protein-truncating ANGPTL8 variant as an instrumental variable, the ANGPTL3-ANGPTL8 complex was very LPL specific. In conclusion, our analysis provides strong human genetic evidence that the ANGPTL3-ANGPTL8 complex regulates plasma metabolic parameters, which is achieved by impacting LPL and EL. By contrast, ANGPTL4 influences plasma metabolic parameters exclusively via LPL.

ANGPTL4 silencing via antisense oligonucleotides reduces plasma triglycerides and glucose in mice without causing lymphadenopathy.

Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice. In separate experiments, injection of two different doses of ANGPTL4 ASO over 20 weeks of high-fat feeding reduced hepatic and adipose ANGPTL4 levels but did not trigger mesenteric lymphadenopathy, an acute phase response, chylous ascites, or any other pathological phenotypes. Compared with mice injected with negative control ASO, mice injected with ANGPTL4 ASO showed reduced food intake, reduced weight gain, and improved glucose tolerance. In addition, they exhibited lower plasma TGs, total cholesterol, LDL-C, glucose, serum amyloid A, and liver TG levels. By contrast, no significant difference in plasma alanine aminotransferase activity was observed. Overall, these data suggest that ASOs targeting ANGPTL4 effectively reduce plasma TG levels in mice without raising major safety concerns.

Inducible hepatic expression of CREBH mitigates diet-induced obesity, insulin resistance, and hepatic steatosis in mice.

Cyclic AMP-responsive element-binding protein H (CREBH encoded by Creb3l3) is a transcription factor that regulates the expression of genes that control lipid and glucose metabolism as well as inflammation. CREBH is upregulated in the liver under conditions of overnutrition, and mice globally lacking the gene (CREBH-/-) are highly susceptible to diet-induced obesity, insulin resistance, and hepatic steatosis. The net protective effects of CREBH have been attributed in large part to the activities of fibroblast growth factor (Fgf)-21 (Fgf21), a target gene that promotes weight loss, improves glucose homeostasis, and reduces hepatic lipid accumulation. To explore the possibility that activation of the CREBH-Fgf21 axis could ameliorate established effects of high-fat feeding, we generated an inducible transgenic hepatocyte-specific CREBH overexpression mouse model (Tg-rtTA). Acute overexpression of CREBH in livers of Tg-rtTA mice effectively reversed diet-induced obesity, insulin resistance, and hepatic steatosis. These changes were associated with increased activities of thermogenic brown and beige adipose tissues in Tg-rtTA mice, leading to reductions in fat mass, along with enhanced insulin sensitivity and glucose tolerance. Genetically silencing Fgf21 in Tg-rtTA mice abrogated the CREBH-mediated reductions in body weight loss, but only partially reversed the observed improvements in glucose metabolism. These findings reveal that the protective effects of CREBH activation may be leveraged to mitigate diet-induced obesity and associated metabolic abnormalities in both Fgf21-dependent and Fgf21-independent pathways.

Hepatic Sel1L-Hrd1 ER-associated degradation (ERAD) manages FGF21 levels and systemic metabolism via CREBH.

Fibroblast growth factor 21 (Fgf21) is a liver-derived, fasting-induced hormone with broad effects on growth, nutrient metabolism, and insulin sensitivity. Here, we report the discovery of a novel mechanism regulating Fgf21 expression under growth and fasting-feeding. The Sel1L-Hrd1 complex is the most conserved branch of mammalian endoplasmic reticulum (ER)-associated degradation (ERAD) machinery. Mice with liver-specific deletion of Sel1L exhibit growth retardation with markedly elevated circulating Fgf21, reaching levels close to those in Fgf21 transgenic mice or pharmacological models. Mechanistically, we show that the Sel1L-Hrd1 ERAD complex controls Fgf21 transcription by regulating the ubiquitination and turnover (and thus nuclear abundance) of ER-resident transcription factor Crebh, while having no effect on the other well-known Fgf21 transcription factor Pparα. Our data reveal a physiologically regulated, inverse correlation between Sel1L-Hrd1 ERAD and Crebh-Fgf21 levels under fasting-feeding and growth. This study not only establishes the importance of Sel1L-Hrd1 ERAD in the liver in the regulation of systemic energy metabolism, but also reveals a novel hepatic "ERAD-Crebh-Fgf21" axis directly linking ER protein turnover to gene transcription and systemic metabolic regulation.

Triglyceride-rich lipoproteins and their remnants: metabolic insights, role in atherosclerotic cardiovascular disease, and emerging therapeutic strategies-a consensus statement from the European Atherosclerosis Society.

Recent advances in human genetics, together with a large body of epidemiologic, preclinical, and clinical trial results, provide strong support for a causal association between triglycerides (TG), TG-rich lipoproteins (TRL), and TRL remnants, and increased risk of myocardial infarction, ischaemic stroke, and aortic valve stenosis. These data also indicate that TRL and their remnants may contribute significantly to residual cardiovascular risk in patients on optimized low-density lipoprotein (LDL)-lowering therapy. This statement critically appraises current understanding of the structure, function, and metabolism of TRL, and their pathophysiological role in atherosclerotic cardiovascular disease (ASCVD). Key points are (i) a working definition of normo- and hypertriglyceridaemic states and their relation to risk of ASCVD, (ii) a conceptual framework for the generation of remnants due to dysregulation of TRL production, lipolysis, and remodelling, as well as clearance of remnant lipoproteins from the circulation, (iii) the pleiotropic proatherogenic actions of TRL and remnants at the arterial wall, (iv) challenges in defining, quantitating, and assessing the atherogenic properties of remnant particles, and (v) exploration of the relative atherogenicity of TRL and remnants compared to LDL. Assessment of these issues provides a foundation for evaluating approaches to effectively reduce levels of TRL and remnants by targeting either production, lipolysis, or hepatic clearance, or a combination of these mechanisms. This consensus statement updates current understanding in an integrated manner, thereby providing a platform for new therapeutic paradigms targeting TRL and their remnants, with the aim of reducing the risk of ASCVD.

ANGPTL3 as therapeutic target.

PURPOSE OF REVIEW: Elevated LDL-C and triglycerides are important risk factors for the development of atherosclerotic cardiovascular disease. Although effective therapies for lipid lowering exist, many people do not reach their treatment targets. In the last two decades, ANGPTL3 has emerged as a novel therapeutic target for lowering plasma LDL-C and triglycerides. Here, an overview of the recent literature on ANGPTL3 is provided, focusing on the therapeutic benefits of inactivation of ANGPTL3 via monoclonal antibodies, antisense oligonucleotides, and other more nascent approaches. In addition, the potential mechanisms by which ANGPTL3 inactivation lowers plasma LDL-C are discussed. RECENT FINDINGS: ANGPTL3 is a factor secreted by the liver that inhibits lipoprotein lipase and other lipases via the formation of a complex with the related protein ANGPTL8. Large-scale genetic studies in humans have shown that carriers of loss-of-function variants in ANGPTL3 have lower plasma LDL-C and triglyceride levels, and are at reduced risk of atherosclerotic cardiovascular disease. Clinical studies in patients with different forms of dyslipidemia have demonstrated that inactivation of ANGPTL3 using monoclonal antibodies or antisense oligonucleotides markedly lowers plasma LDL-C and triglyceride levels. SUMMARY: Anti-ANGPTL3 therapies hold considerable promise for reducing plasma LDL-C and triglycerides in selected patient groups.

Hepatic ADTRP overexpression does not influence lipid and glucose metabolism.

The peroxisome proliferator-activated receptors (PPARs) are a group of transcription factors belonging to the nuclear receptor superfamily. Since most target genes of PPARs are implicated in lipid and glucose metabolism, regulation by PPARs could be used as a screening tool to identify novel genes involved in lipid or glucose metabolism. Here, we identify Adtrp, a serine hydrolase enzyme that was reported to catalyze the hydrolysis of fatty acid esters of hydroxy fatty acids (FAHFAs), as a novel PPAR-regulated gene. Adtrp was significantly upregulated by PPARα activation in mouse primary hepatocytes, liver slices, and whole liver. In addition, Adtrp was upregulated by PPARγ activation in 3L3-L1 adipocytes and in white adipose tissue. ChIP-SEQ identified a strong PPAR-binding site in the immediate upstream promoter of the Adtrp gene. Adenoviral-mediated hepatic overexpression of Adtrp in diet-induced obese mice caused a modest increase in plasma nonesterified fatty acids but did not influence diet-induced obesity, liver triglyceride levels, liver lipidomic profiles, liver transcriptomic profiles, plasma cholesterol, triglyceride, glycerol, and glucose levels. Moreover, hepatic Adtrp overexpression did not lead to significant changes in FAHFA levels in plasma or liver and did not influence glucose and insulin tolerance. Finally, hepatic overexpression of Adtrp did not influence liver triglycerides and levels of plasma metabolites after a 24-h fast. Taken together, our data suggest that despite being a PPAR-regulated gene, hepatic Adtrp does not seem to play a major role in lipid and glucose metabolism and does not regulate FAHFA levels.

Role and mechanism of the action of angiopoietin-like protein ANGPTL4 in plasma lipid metabolism.

Triglycerides are carried in the bloodstream as the components of very low-density lipoproteins and chylomicrons. These circulating triglycerides are primarily hydrolyzed in muscle and adipose tissue by the enzyme lipoprotein lipase (LPL). The activity of LPL is regulated by numerous mechanisms, including by three members of the angiopoietin-like protein family: ANGPTL3, ANGPTL4, and ANGPTL8. In this review, we discuss the recent literature concerning the role and mechanism of action of ANGPTL4 in lipid metabolism. ANGPTL4 is a fasting- and lipid-induced factor secreted by numerous cells, including adipocytes, hepatocytes, (cardio)myocytes, and macrophages. In adipocytes, ANGPTL4 mediates the fasting-induced repression of LPL activity by promoting the unfolding of LPL, leading to the cleavage and subsequent degradation of LPL. The inhibition of LPL by ANGPTL4 is opposed by ANGPTL8, which keeps the LPL active after feeding. In macrophages and (cardio)myocytes, ANGPTL4 functions as a lipid-inducible feedback regulator of LPL-mediated lipid uptake. In comparison, in hepatocytes, ANGPTL4 functions as a local inhibitor of hepatic lipase and possibly as an endocrine inhibitor of LPL in extra-hepatic tissues. At the genetic level, loss-of-function mutations in ANGPTL4 are associated with lower plasma triglycerides and higher plasma HDL-C levels, and a reduced risk of coronary artery disease, suggesting that ANGPTL4 is a viable pharmacological target for reducing cardiovascular risk. Whole-body targeting of ANGPTL4 is contraindicated because of severe pathological complications, whereas liver-specific inactivation of ANGPTL4, either as monotherapy or coupled to anti-ANGPTL3 therapies might be a suitable strategy for lowering plasma triglycerides in selected patient groups. In conclusion, the tissue-specific targeting of ANGPTL4 appears to be a viable pharmacological approach to reduce circulating triglycerides.

A lipase fusion feasts on fat.

The enzyme lipoprotein lipase (LPL) is responsible for breaking down triglycerides in the blood. Mutations in LPL cause a rare but debilitating disorder characterized by excessive plasma triglyceride levels for which treatment options are limited. Nimonkar et al. now present a fusion protein between LPL and its physiological transporter GBIHBP1 that is highly active and largely resistant to physiological inhibitors of LPL. Injecting this fusion protein effectively lowers plasma triglycerides in mice and represents a promising new approach for lowering triglycerides in patients with familial chylomicronemia syndrome.

Systemic PFOS and PFOA exposure and disturbed lipid homeostasis in humans: what do we know and what not?

Associations between per- and polyfluoroalkyl substances (PFASs) and increased blood lipids have been repeatedly observed in humans, but a causal relation has been debated. Rodent studies show reverse effects, i.e. decreased blood cholesterol and triglycerides, occurring however at PFAS serum levels at least 100-fold higher than those in humans. This paper aims to present the main issues regarding the modulation of lipid homeostasis by the two most common PFASs, PFOS and PFOA, with emphasis on the underlying mechanisms relevant for humans. Overall, the apparent contrast between human and animal data may be an artifact of dose, with different molecular pathways coming into play upon exposure to PFASs at very low versus high levels. Altogether, the interpretation of existing rodent data on PFOS/PFOA-induced lipid perturbations with respect to the human situation is complex. From a mechanistic perspective, research on human liver cells shows that PFOS/PFOA activate the PPARα pathway, whereas studies on the involvement of other nuclear receptors, like PXR, are less conclusive. Other data indicate that suppression of the nuclear receptor HNF4α signaling pathway, as well as perturbations of bile acid metabolism and transport might be important cellular events that require further investigation. Future studies with human-relevant test systems would help to obtain more insight into the mechanistic pathways pertinent for humans. These studies shall be designed with a careful consideration of appropriate dosing and toxicokinetics, so as to enable biologically plausible quantitative extrapolations. Such research will increase the understanding of possible perturbed lipid homeostasis related to PFOS/ PFOA exposure and the potential implications for human health.

Circadian misalignment induces fatty acid metabolism gene profiles and compromises insulin sensitivity in human skeletal muscle.

Circadian misalignment, such as in shift work, has been associated with obesity and type 2 diabetes. However, direct effects of circadian misalignment on skeletal muscle insulin sensitivity and the muscle molecular circadian clock have never been studied in humans. Here, we investigated insulin sensitivity and muscle metabolism in 14 healthy young lean men [age 22.4 ± 2.8 years; body mass index (BMI) 22.3 ± 2.1 kg/m2 (mean ± SD)] after a 3-d control protocol and a 3.5-d misalignment protocol induced by a 12-h rapid shift of the behavioral cycle. We show that short-term circadian misalignment results in a significant decrease in muscle insulin sensitivity due to a reduced skeletal muscle nonoxidative glucose disposal (rate of disappearance: 23.7 ± 2.4 vs. 18.4 ± 1.4 mg/kg per minute; control vs. misalignment; P = 0.024). Fasting glucose and free fatty acid levels as well as sleeping metabolic rate were higher during circadian misalignment. Molecular analysis of skeletal muscle biopsies revealed that the molecular circadian clock was not aligned to the inverted behavioral cycle, and transcriptome analysis revealed the human PPAR pathway as a key player in the disturbed energy metabolism upon circadian misalignment. Our findings may provide a mechanism underlying the increased risk of type 2 diabetes among shift workers.

Probing metabolic memory in the hepatic response to fasting.

Tissues may respond differently to a particular stimulus if they have been previously exposed to that same stimulus. Here, we tested the hypothesis that a strong metabolic stimulus such as fasting may influence the hepatic response to a subsequent fast and thus elicit a memory effect. Overnight fasting in mice significantly increased plasma free fatty acids, glycerol, ß-hydroxybutyrate, and liver triglycerides, and decreased plasma glucose, plasma triglycerides, and liver glycogen levels. In addition, fasting dramatically changed the liver transcriptome, upregulating genes involved in gluconeogenesis and in uptake, oxidation, storage, and mobilization of fatty acids, and downregulating genes involved in fatty acid synthesis, fatty acid elongation/desaturation, and cholesterol synthesis. Fasting also markedly impacted the liver metabolome, causing a decrease in the levels of numerous amino acids, glycolytic-intermediates, TCA cycle intermediates, and nucleotides. However, these fasting-induced changes were unaffected by two previous overnight fasts. Also, no significant effect was observed of prior fasting on glucose tolerance. Finally, analysis of the effect of fasting on the transcriptome in hepatocyte humanized mouse livers indicated modest similarity in gene regulation in mouse and human liver cells. In general, genes involved in metabolic pathways were upregulated or downregulated to a lesser extent in human liver cells than in mouse liver cells. In conclusion, we found that previous exposure to fasting in mice did not influence the hepatic response to a subsequent fast, arguing against the concept of metabolic memory in the liver. Our data provide a useful resource for the study of liver metabolism during fasting.

Transcriptomic signature of fasting in human adipose tissue.

Little is known about gene regulation by fasting in human adipose tissue. Accordingly, the objective of this study was to investigate the effects of fasting on adipose tissue gene expression in humans. To that end, subcutaneous adipose tissue biopsies were collected from 11 volunteers 2 and 26 h after consumption of a standardized meal. For comparison, epididymal adipose tissue was collected from C57Bl/6J mice in the ab libitum-fed state and after a 16 h fast. The timing of sampling adipose tissue roughly corresponds with the near depletion of liver glycogen. Transcriptome analysis was carried out using Affymetrix microarrays. We found that, 1) fasting downregulated numerous metabolic pathways in human adipose tissue, including triglyceride and fatty acid synthesis, glycolysis and glycogen synthesis, TCA cycle, oxidative phosphorylation, mitochondrial translation, and insulin signaling; 2) fasting downregulated genes involved in proteasomal degradation in human adipose tissue; 3) fasting had much less pronounced effects on the adipose tissue transcriptome in humans than mice; 4) although major overlap in fasting-induced gene regulation was observed between human and mouse adipose tissue, many genes were differentially regulated in the two species, including genes involved in insulin signaling (PRKAG2, PFKFB3), PPAR signaling (PPARG, ACSL1, HMGCS2, SLC22A5, ACOT1), glycogen metabolism (PCK1, PYGB), and lipid droplets (PLIN1, PNPLA2, CIDEA, CIDEC). In conclusion, although numerous genes and pathways are regulated similarly by fasting in human and mouse adipose tissue, many genes show very distinct responses to fasting in humans and mice. Our data provide a useful resource to study adipose tissue function during fasting.

MicroRNA-204-5p modulates mitochondrial biogenesis in C2C12 myotubes and associates with oxidative capacity in humans.

Using an unbiased high-throughput microRNA (miRNA)-silencing screen combined with functional readouts for mitochondrial oxidative capacity in C2C12 myocytes, we previously identified 19 miRNAs as putative regulators of skeletal muscle mitochondrial metabolism. In the current study, we highlight miRNA-204-5p, identified from this screen, and further studied its role in the regulation of skeletal muscle mitochondrial function. Following silencing of miRNA-204-5p in C2C12 myotubes, gene and protein expression were assessed using quantitative polymerase chain reaction, microarray analysis, and western blot analysis, while morphological changes were studied by confocal microscopy. In addition, miRNA-204-5p expression was quantified in human skeletal muscle biopsies and associated with in vivo mitochondrial oxidative capacity. Transcript levels of PGC-1α (3.71-fold; p < .01), predicted as an miR-204-5p target, as well as mitochondrial DNA copy number (p < .05) and citrate synthase activity (p = .06) were increased upon miRNA-204-5p silencing in C2C12 myotubes. Silencing of miRNA-204-5p further resulted in morphological changes, induced gene expression of autophagy marker light chain 3 protein b (LC3B; q = .05), and reduced expression of the mitophagy marker FUNDC1 (q = .01). Confocal imaging revealed colocalization between the autophagosome marker LC3B and the mitochondrial marker OxPhos upon miRNA-204-5p silencing. Finally, miRNA-204-5p was differentially expressed in human subjects displaying large variation in oxidative capacity and its expression levels associated with in vivo measures of skeletal muscle mitochondrial function. In summary, silencing of miRNA-204-5p in C2C12 myotubes stimulated mitochondrial biogenesis, impacted on cellular morphology, and altered expression of markers related to autophagy and mitophagy. The association between miRNA-204-5p and in vivo mitochondrial function in human skeletal muscle further identifies miRNA-204-5p as an interesting modulator of skeletal muscle mitochondrial metabolism.

Perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), and perfluorononanoic acid (PFNA) increase triglyceride levels and decrease cholesterogenic gene expression in human HepaRG liver cells.

Per- and polyfluoroalkyl substances (PFASs) are omnipresent in the environment, food chain, and humans. Epidemiological studies have shown a positive association between serum levels of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), and increased serum cholesterol and, in some cases, also triglyceride levels. However, causality has been questioned, as animal studies, as well as a human trial, showed a decrease in serum cholesterol and no effects or a decrease in plasma triglycerides. To obtain more insight into the effects of PFASs on these processes, the present study investigated the effects of PFOA, PFOS, and perfluorononanoic acid (PFNA) on intracellular triglyceride and cholesterol levels in human HepaRG liver cells. DNA microarray analyses were performed to provide insight into underlying mechanisms. All PFASs induced an increase in cellular triglyceride levels, but had no effect on cholesterol levels. Gene set enrichment analysis (GSEA) of the microarray data indicated that gene sets related to cholesterol biosynthesis were repressed by PFOA, PFOS, and PFNA. Other gene sets commonly affected by all PFAS were related to PERK/ATF4 signaling (induced), tRNA amino-acylation (induced), amino acid transport (induced), and glycolysis/gluconeogenesis (repressed). Moreover, numerous target genes of peroxisome proliferator-activated receptor α (PPARα) were found to be upregulated. Altogether, the present study shows that PFOA, PFOS, and PFNA increase triglyceride levels and inhibit cholesterogenic gene expression in HepaRG cells. In addition, the present study indicates that PFASs induce endoplasmic reticulum stress, which may be an important mechanism underlying some of the toxic effects of these chemicals.