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35 Pág. Centro de Biotecnología y Genómica de Plantas, New types of protein sources will enter our diet in a near future, reinforcing the need for a straightforward in vitro (cell-based) screening model to test and predict the safety of these novel proteins, in particular their potential risk for de novo allergic sensitization. The Adverse Outcome Pathway (AOP) for allergen sensitization describes the current knowledge of key events underlying the complex cellular interactions that proceed allergic food sensitization. Currently, there is no consensus on the in vitro model to study the intestinal translocation of proteins as well as the epithelial activation, which comprise the first molecular initiation events (ME1-3) and the first key event of the AOP, respectively. As members of INFOGEST, we have highlighted several critical features that should be considered for any proposed in vitro model to study epithelial protein transport in the context of allergic sensitization. In addition, we defined which intestinal cell types are indispensable in a consensus model of the first steps of the AOP, and which cell types are optional or desired when there is the possibility to create a more complex cell model. A model of these first key aspects of the AOP can be used to study the gut epithelial translocation behavior of known hypo- and hyperallergens, juxtaposed to the transport behavior of novel proteins as a first screen for risk management of dietary proteins. Indeed, this disquisition forms a basis for the development of a future consensus model of the allergic sensitization cascade, comprising also the other key events (KE2-5).

Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene,Using next-generation sequencing, we identified a novel dominant rare variant in theFamily members carrying theWe identified and characterized a novel rare variant in the

Diabetic dyslipidaemia, characterized by quantitative, qualitative and kinetic changes in all major circulating lipids, contributes to the increased cardiovascular risk in patients with type 2 diabetes mellitus (T2DM). A promising therapeutic avenue is the inhibition of the proprotein convertase subtilisin kexin 9 (PCSK9) with human monoclonal antibodies (mAbs) that potently reduce plasma low-density lipoprotein cholesterol (LDL-C) levels on top of statin treatment. The aim of this review is to evaluate the efficacy of PCSK9 inhibitors to lower the residual cardiovascular risk of T2DM patients and to discuss the safety of PCSK9 inhibition in these patients. PCSK9 inhibitors potently lower plasma LDL-C levels in T2DM patients and reduce risk for the development of cardiovascular disease. Anti-PCSK9 mAbs are generally not more or less effective in T2DM patients compared to a general high-risk population. Nevertheless, due to their higher cardiovascular risk, the absolute risk reduction of major cardiovascular events is more significant in T2DM patients. This suggests that treatment of T2DM patients with anti-PCSK9 mAbs could be attractive from a cost-effectiveness perspective. Treatment with anti-PCSK9 mAbs did not result in significant treatment-emergent adverse effects. While genetic studies suggest a potential link between PCSK9 inhibition and glucose homeostasis, anti-PCSK9 mAbs did not worsen glycaemic control in T2DM patients, but their safety should be verified after a longer-term follow-up.

Resume La dyslipidemie diabetique est caracterisee par des alterations quantitatives, qualitatives, et cinetiques des lipoproteines, contribuant a l’augmentation du risque cardiovasculaire des patients diabetiques de type 2 (DT2). En cas d’echec du traitement par statines, l’inhibition de PCSK9 (pour proprotein convertase subtilisin/kexin 9) par des anticorps humains monoclonaux (Ac), qui reduit fortement les concentrations de LDL-cholesterol (LDL-C), represente une option therapeutique interessante. L’objectif de cette revue est de discuter l’interet des Ac anti-PCSK9 pour reduire le risque cardiovasculaire chez les patients DT2. De maniere globale, les Ac anti-PCSK9 sont aussi efficaces sur la reduction du LDL-C chez les personnes diabetiques que dans la population generale. En revanche, les Ac anti-PCSK9 n’ont que peu d’effet sur l’hypertriglyceridemie des patients DT2. Les analyses en sous-groupes des etudes de morbi-mortalite cardiovasculaire ont confirme le benefice cardiovasculaire des Ac anti-PCSK9, avec une reduction plus importante du risque absolu. La tolerance des Ac anti-PCSK9 est excellente, sans survenue d’evenements indesirables graves. Bien que des donnees genetiques suggerent un lien potentiel entre l’inhibition de PCSK9 et le risque de diabete, les Ac anti-PCSK9 ne s’accompagnent pas d’une degradation de l’homeostasie du glucose, ni d’une augmentation des nouveaux cas de diabete. Neanmoins, des donnees de suivi a plus long-terme seront necessaires pour confirmer ces premieres donnees encourageantes.

International audience; Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes lysosomal degradation of the LDL receptor and is a key regulator of cholesterol metabolism. After the liver, the small intestine is the second organ that highly expresses PCSK9. However, the small intestine's ability to secrete PCSK9 remains a matter of debate. While liver-specific PCSK9-deficient mice present no PCSK9 in systemic blood, human intestinal Caco-2 cells can actively secrete PCSK9. This raises the possibility for active intestinal secretion via the portal blood. Here, we aimed to determine whether enterocytes can secrete PCSK9 using in vitro, ex vivo, and in vivo approaches. We first observed that PCSK9 secretion from Caco-2 cells was biphasic and dependent on Caco-2 maturation status. Transcriptional analysis suggested that this transient reduction in PCSK9 secretion might be due to loss of SREBP2-mediated transcription of PCSK9. Consistently, PCSK9 secretion was not detected ex vivo in human or mouse intestinal biopsies mounted in Ussing chambers. Finally, direct comparison of systemic versus portal blood PCSK9 concentrations in WT or liver-specific PCSK9-deficient mice confirmed the inability of the small intestine to secrete PCSK9 into the portal compartment. Altogether, our data demonstrate that mature enterocytes do not secrete PCSK9 and reinforce the central role of the liver in the regulation of the concentration of circulating PCSK9 and consequently of cellular LDL receptors.

International audience; Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) with anti-PCSK9 monoclonal antibodies (alirocumab and evolocumab) or with small-interfering RNAs (inclisiran) lowers plasma low-density lipoprotein cholesterol (LDL-C) levels, a major risk factor for the development of atherosclerotic cardiovascular disease (ASCVD). 1,2 So far, the pharmacologic inhibition of PCSK9 presents with a favorable safety profile, but longer-term safety remains to be proven. Studies in preclinical models and in humans have suggested a potential link between PCSK9 deficiency and the risk of nonalcoholic fatty liver disease (NAFLD), 3,4 a spectrum of progressive liver diseases ranging from simple steatosis to fibrosis that can lead to cirrhosis and hepatocellular carcinoma. Notably, it has been shown that PCSK9 knockout mice are more prone to develop severe hepatic steatosis and fibrosis when receiving a high-fat diet. 3 This study aims to study the associations of the lifelong genetic inhibition of PCSK9 with plasma liver enzymes and NAFLD by using the loss-of-function variant PCSK9p.Arg46Leu as a genetic instrument. For comparison, we used 2 variants in PNPLA3 and TM6SF2, known to be involved in the pathogenesis of NAFLD. 5 Methods | Approval. Both the UK Biobank and the Electronic Medical Record and Genomics (eMERGE) cohort have obtained regulatory approvals, and all participants provided written informed consent. Because the data were publicly available, we did not obtain specific approval for this study. UK-Biobank Data Sets. Genetic associations with biologic traits (plasma circulating liver enzymes) in the UK-Biobank cohort were extracted from the second-round results (released August 1, 2018) by the Nealelab (see description in http://www. nealelab.is/uk-biobank/). These data were obtained from 361 194 participants (194 174 women and 167 020 men) of the UK-Biobank cohort (https://www.ukbiobank.ac.uk).

Objective: Increased postprandial lipemia (PPL) is an independent risk factor for atherosclerotic cardiovascular diseases. PCSK9 (Proprotein convertase subtilisin kexin type 9) is an endogenous inhibitor of the LDLR (low-density lipoprotein receptor) pathway. We previously showed that PCSK9 inhibition in mice reduces PPL. However, the relative contribution of intracellular intestinal PCSK9 or liver-derived circulating PCSK9 to this effect is still unclear. Approach and Results: To address this issue, we generated the first intestine-specific Pcsk9 -deficient (i- Pcsk9 −/− ) mouse model. PPL was measured in i- Pcsk9 −/− as well as in wild-type and streptozotocin-induced diabetic mice following treatment with a PCSK9 monoclonal antibody (alirocumab). Blocking the circulating form of PCSK9 with alirocumab significantly reduced PPL, while overexpressing human PCSK9 in the liver of full Pcsk9 −/− mice had the opposite effect. Alirocumab regulated PPL in a LDLR-dependent manner as this effect was abolished in Ldlr −/− mice. In contrast, i- Pcsk9 −/− mice did not exhibit alterations in plasma lipid parameters nor in PPL. Finally, PPL was highly exacerbated by streptozotocin-induced diabetes mellitus in Pcsk9 +/+ but not in Pcsk9 −/− mice, an effect that was mimicked by the use of alirocumab in streptozotocin-treated Pcsk9 +/+ mice. Conclusions: Taken together, our data demonstrate that PPL is significantly altered by full but not intestinal PCSK9 deficiency. Treatment with a PCSK9 monoclonal antibody mimics the effect of PCSK9 deficiency on PPL suggesting that circulating PCSK9 rather than intestinal PCSK9 is a critical regulator of PPL. These data validate the clinical relevance of PCSK9 inhibitors to reduce PPL, especially in patients with type 2 diabetes mellitus.

Background Elevated plasma triglycerides are increasingly viewed as a causal risk factor for coronary artery disease. One protein that raises plasma triglyceride levels and that has emerged as a modulator of coronary artery disease risk is angiopoietin-like 4 (ANGPTL4). ANGPTL4 raises plasma triglyceride levels by inhibiting lipoprotein lipase (LPL), the enzyme that catalyzes the hydrolysis of circulating triglycerides on the capillary endothelium. Objective The objective of the present study was to assess the association between ANGPTL4 and LPL in human adipose tissue, and to examine the influence of nutritional status on ANGPTL4 expression. Methods We determined ANGPTL4 and LPL mRNA and protein levels in different adipose tissue depots in a large number of severely obese patients who underwent bariatric surgery. Furthermore, in 72 abdominally obese subjects, we measured ANGPTL4 and LPL mRNA levels in subcutaneous adipose tissue in the fasted and postprandial state. Results ANGPTL4 mRNA levels were highest in subcutaneous adipose tissue, whereas LPL mRNA levels were highest in mesenteric adipose tissue. ANGPTL4 and LPL mRNA levels were strongly positively correlated in the omental and subcutaneous adipose tissue depots. In contrast, ANGPTL4 and LPL protein levels were negatively correlated in subcutaneous adipose tissue, suggesting a suppressive effect of ANGPTL4 on LPL protein abundance in subcutaneous adipose tissue. ANGPTL4 mRNA levels were 38% higher in the fasted compared to the postprandial state. Conclusion Our data provide valuable insights into the relationship between ANGPTL4 and LPL in human adipose tissue, as well as the physiological function and regulation of ANGPTL4 in humans.

Angiopoietin-like 4 (ANGPTL4) raises plasma triglyceride levels by inhibiting lipoprotein lipase. A set of compounds that are able to reduce plasma triglyceride levels are bile acids (BA). Because BA have been shown to decrease ANGPTL4 secretion by intestinal cells, we hypothesized that BA lower plasma triglycerides (partly) via ANGPTL4. To test that hypothesis, wild-type and Angptl4(-/-) mice were fed chow supplemented with taurocholic acid (TCA) for seven days. TCA supplementation effectively lowered plasma triglycerides in wild-type and Angptl4(-/-) mice, indicating that ANGPTL4 is not required for plasma triglyceride-lowering by BA. Intriguingly, however, plasma and hepatic BA concentrations were significantly lower in TCA-supplemented Angptl4(-/-) mice than in TCA-supplemented wild-type mice. These changes in the Angptl4(-/-) mice were accompanied by lower BA levels in ileal scrapings and decreased expression of FXR-target genes in the ileum, including the BA transporter Slc10a2. By contrast, faecal excretion of specifically primary BA was higher in the Angptl4(-/-) mice, suggesting that loss of ANGPTL4 impairs intestinal BA absorption. Since the gut microbiota converts primary BA into secondary BA, elevated excretion of primary BA in Angptl4(-/-) mice may reflect differences in gut microbial composition and/or functionality. Indeed, colonic microbial composition was markedly different between Angptl4(-/-) and wild-type mice. Suppression of the gut bacteria using antibiotics abolished differences in plasma, hepatic, and faecal BA levels between TCA-supplemented Angptl4(-/-) and wild-type mice. In conclusion, 1) ANGPTL4 is not involved in the triglyceride-lowering effect of BA; 2) ANGPTL4 promotes BA absorption during TCA supplementation via a mechanism dependent on the gut microbiota.

Obesity promotes accumulation of lipid-laden macrophages in adipose tissue. Here, we determined the role of macrophage lipid accumulation in the development of obesity-induced adipose tissue inflammation, using mice with myeloid-specific deficiency of the lipid-inducible HILPDA protein. HILPDA deficiency in bone marrow-derived macrophages markedly reduced intracellular lipid levels and accumulation of fluorescently-labeled fatty acids in lipid droplets. Decreased lipid storage in HILPDA-deficient macrophages could be almost completely rescued by inhibition of adipose triglyceride lipase (ATGL) and was associated with increased oxidative metabolism. In diet-induced obese mice, HILPDA deficiency did not alter inflammatory or metabolic parameters, despite markedly reducing lipid storage in adipose tissue macrophages. Our data indicate that HILPDA is a lipid-induced physiological inhibitor of ATGL-mediated lipolysis that uncouples lipid storage in adipose tissue macrophages from inflammation and metabolic dysregulation. Overall, our data question the importance of lipid storage in adipose tissue macrophages in obesity-induced inflammation and metabolic dysregulation.

Contains fulltext : 208777.pdf (Publisher’s version ) (Open Access) Angiopoietin-like protein (ANGPTL)4 regulates plasma lipids, making it an attractive target for correcting dyslipidemia. However, ANGPTL4 inactivation in mice fed a high fat diet causes chylous ascites, an acute-phase response, and mesenteric lymphadenopathy. Here, we studied the role of ANGPTL4 in lipid uptake in macrophages and in the above-mentioned pathologies using Angptl4-hypomorphic and Angptl4 (-/-) mice. Angptl4 expression in peritoneal and bone marrow-derived macrophages was highly induced by lipids. Recombinant ANGPTL4 decreased lipid uptake in macrophages, whereas deficiency of ANGPTL4 increased lipid uptake, upregulated lipid-induced genes, and increased respiration. ANGPTL4 deficiency did not alter LPL protein levels in macrophages. Angptl4-hypomorphic mice with partial expression of a truncated N-terminal ANGPTL4 exhibited reduced fasting plasma triglyceride, cholesterol, and NEFAs, strongly resembling Angptl4 (-/-) mice. However, during high fat feeding, Angptl4-hypomorphic mice showed markedly delayed and attenuated elevation in plasma serum amyloid A and much milder chylous ascites than Angptl4 (-/-) mice, despite similar abundance of lipid-laden giant cells in mesenteric lymph nodes. In conclusion, ANGPTL4 deficiency increases lipid uptake and respiration in macrophages without affecting LPL protein levels. Compared with the absence of ANGPTL4, low levels of N-terminal ANGPTL4 mitigate the development of chylous ascites and an acute-phase response in mice.

Summary: Obesity leads to a state of chronic, low-grade inflammation that features the accumulation of lipid-laden macrophages in adipose tissue. Here, we determined the role of macrophage lipid-droplet accumulation in the development of obesity-induced adipose-tissue inflammation, using mice with myeloid-specific deficiency of the lipid-inducible HILPDA protein. HILPDA deficiency markedly reduced intracellular lipid levels and accumulation of fluorescently labeled fatty acids. Decreased lipid storage in HILPDA-deficient macrophages can be rescued by inhibition of adipose triglyceride lipase (ATGL) and is associated with increased oxidative metabolism. In diet-induced obese mice, HILPDA deficiency does not alter inflammatory and metabolic parameters, despite markedly reducing lipid accumulation in macrophages. Overall, we find that HILPDA is a lipid-inducible, physiological inhibitor of ATGL-mediated lipolysis in macrophages and uncouples lipid storage in adipose tissue macrophages from inflammation and metabolic dysregulation. Our data question the contribution of lipid droplet accumulation in adipose tissue macrophages in obesity-induced inflammation and metabolic dysregulation. : Lipid-laden macrophages are believed to play a key role in obesity-induced adipose tissue inflammation. Van Dierendonck et al. show that the lipid-droplet-associated protein HILPDA regulates intracellular triglyceride breakdown by directly inhibiting ATGL and that reduced lipid accumulation in adipose tissue macrophages surprisingly does not affect adipose tissue inflammation. Keywords: Hilpda, ATGL, fatty acid metabolism, lipid droplets, macrophages, inflammation, obesity

PURPOSE OF REVIEW: The angiopoietin-like proteins (ANGPTLs) 3, 4 and 8 have emerged as key regulators of plasma lipid metabolism by serving as potent inhibitors of the enzyme lipoprotein lipase (LPL). In this review, we provide an integrated picture of the role of ANGPTL3, ANGPTL4 and ANGPTL8 in lipid metabolism by focusing on their impact on LPL activity and plasma triglyceride clearance during physiological conditions such as fasting, refeeding, exercise and cold exposure. RECENT FINDINGS: Upon refeeding, circulating ANGPTL3 and ANGPTL8 promote the replenishment of white adipose tissue depots by specifically inhibiting LPL activity in oxidative tissues. During exercise and cold exposure, ANGPTL4 represses local LPL activity to assure that plasma triglycerides are specifically shuttled to exercising muscle and brown adipose tissue, respectively. Overall, ANGPTL4 is the central component of a fatty acid-driven feedback mechanism that regulates plasma triglyceride hydrolysis and subsequent tissue fatty acid uptake in response to changes in lipid availability and cellular fuel demand. SUMMARY: ANGPTL3, ANGPTL4 and ANGPTL8 together ensure that triglycerides from triglyceride-rich lipoproteins are adequately distributed during different physiological conditions. The impact of the ANGPTLs on plasma lipid levels has led to scrutiny of ANGPTLs as therapeutic targets for dyslipidemia.

LPL hydrolyzes triglycerides in triglyceride-rich lipoproteins along the capillaries of heart, skeletal muscle, and adipose tissue. The activity of LPL is repressed by angiopoietin-like 4 (ANGPTL4) but the underlying mechanisms have not been fully elucidated. Our objective was to study the cellular location and mechanism for LPL inhibition by ANGPTL4. We performed studies in transfected cells, ex vivo studies, and in vivo studies with Angptl4(-/-) mice. Cotransfection of CHO pgsA-745 cells with ANGPTL4 and LPL reduced intracellular LPL protein levels, suggesting that ANGPTL4 promotes LPL degradation. This conclusion was supported by studies of primary adipocytes and adipose tissue explants from wild-type and Angptl4(-/-) mice. Absence of ANGPTL4 resulted in accumulation of the mature-glycosylated form of LPL and increased secretion of LPL. Blocking endoplasmic reticulum (ER)-Golgi transport abolished differences in LPL abundance between wild-type and Angptl4(-/-) adipocytes, suggesting that ANGPTL4 acts upon LPL after LPL processing in the ER. Finally, physiological changes in adipose tissue ANGPTL4 expression during fasting and cold resulted in inverse changes in the amount of mature-glycosylated LPL in wild-type mice, but not Angptl4(-/-) mice. We conclude that ANGPTL4 promotes loss of intracellular LPL by stimulating LPL degradation after LPL processing in the ER.

Objective Chronic cold exposure causes white adipose tissue (WAT) to adopt features of brown adipose tissue (BAT), a process known as browning. Previous studies have hinted at a possible role for the transcription factor Peroxisome Proliferator-Activated Receptor alpha (PPARα) in cold-induced browning. Here we aimed to investigate the importance of PPARα in driving transcriptional changes during cold-induced browning in mice. Methods Male wildtype and PPARα−/− mice were housed at thermoneutrality (28 °C) or cold (5 °C) for 10 days. Whole genome expression analysis was performed on inguinal WAT. In addition, other analyses were carried out. Whole genome expression data of livers of wildtype and PPARα−/− mice fasted for 24 h served as positive control for PPARα-dependent gene regulation. Results Cold exposure increased food intake and decreased weight of BAT and WAT to a similar extent in wildtype and PPARα−/− mice. Except for plasma non-esterified fatty acids, none of the cold-induced changes in plasma metabolites were dependent on PPARα genotype. Histological analysis of inguinal WAT showed clear browning upon cold exposure but did not reveal any morphological differences between wildtype and PPARα−/− mice. Transcriptomics analysis of inguinal WAT showed a marked effect of cold on overall gene expression, as revealed by principle component analysis and hierarchical clustering. However, wildtype and PPARα−/− mice clustered together, even after cold exposure, indicating a similar overall gene expression profile in the two genotypes. Pathway analysis revealed that cold upregulated pathways involved in energy usage, oxidative phosphorylation, and fatty acid β-oxidation to a similar extent in wildtype and PPARα−/− mice. Furthermore, cold-mediated induction of genes related to thermogenesis such as Ucp1, Elovl3, Cox7a1, Cox8, and Cidea, as well as many PPAR target genes, was similar in wildtype and PPARα−/− mice. Finally, pharmacological PPARα activation had a minimal effect on expression of cold-induced genes in murine WAT. Conclusion Cold-induced changes in gene expression in inguinal WAT are unaltered in mice lacking PPARα, indicating that PPARα is dispensable for cold-induced browning., Highlights • Chronic cold markedly induces PPARα expression in inguinal fat. • Cold exposure causes transient hypothermia in PPARα−/− mice. • Chronic cold-induced changes in gene expression in inguinal fat are unaltered in PPARα−/− mice. • Chronic cold does not lead to PPARα activation in liver.

International audience; Elevated plasma triglyceride (TG) levels are an independent risk factor for cardiovascular disease (CVD). Proprotein convertase subtilisin-kexin 9 (PCSK9) - a protein therapeutically targeted to lower plasma cholesterol levels - might regulate plasma TG-rich lipoprotein (TRL) levels. We provide a timely and critical review of the current evidence for a role of PCSK9 in TRL metabolism by assessing the impact of PCSK9 gene variants, by reviewing recent clinical data with PCSK9 inhibitors, and by describing the potential mechanisms by which PCSK9 might regulate TRL metabolism. We conclude that the impact of PCSK9 on TRL metabolism is relatively modest, especially compared to its impact on cholesterol metabolism.

Contains fulltext : 197192.pdf (Publisher’s version ) (Open Access) Lipoprotein lipase (LPL) catalyzes the breakdown of circulating triglycerides in muscle and fat. LPL is inhibited by several proteins, including angiopoietin-like 4 (ANGPTL4), and may be cleaved by members of the proprotein convertase subtilisin/kexin (PCSK) family. Here, we aimed to investigate the cleavage of LPL in adipocytes by PCSKs and study the potential involvement of ANGPTL4. A substantial portion of LPL in mouse and human adipose tissue was cleaved into N- and C-terminal fragments. Treatment of different adipocytes with the PCSK inhibitor decanoyl-RVKR-chloromethyl ketone markedly decreased LPL cleavage, indicating that LPL is cleaved by PCSKs. Silencing of Pcsk3/furin significantly decreased LPL cleavage in cell culture medium and lysates of 3T3-L1 adipocytes. Remarkably, PCSK-mediated cleavage of LPL in adipocytes was diminished by Angptl4 silencing and was decreased in adipocytes and adipose tissue of Angptl4(-/-) mice. Differences in LPL cleavage between Angptl4(-/-) and WT mice were abrogated by treatment with decanoyl-RVKR-chloromethyl ketone. Induction of ANGPTL4 in adipose tissue during fasting enhanced PCSK-mediated LPL cleavage, concurrent with decreased LPL activity, in WT but not Angptl4(-/-) mice. In adipocytes, after removal of cell surface LPL by heparin, levels of N-terminal LPL were still markedly higher in WT compared with Angptl4(-/-) adipocytes, suggesting that stimulation of PCSK-mediated LPL cleavage by ANGPTL4 occurs intracellularly. Finally, treating adipocytes with insulin increased full-length LPL and decreased N-terminal LPL in an ANGPTL4-dependent manner. In conclusion, ANGPTL4 promotes PCSK-mediated intracellular cleavage of LPL in adipocytes, likely contributing to regulation of LPL in adipose tissue. Our data provide further support for an intracellular action of ANGPTL4 in adipocytes.

Cold exposure of mice is a common method to stimulate brown adipose tissue (BAT) activity and induce browning of white adipose tissue (WAT) that has beneficial effects on whole-body lipid metabolism, including reduced plasma triglyceride (TG) concentrations. The liver is a key regulatory organ in lipid metabolism as it can take up as well as oxidize fatty acids. The liver can also synthesize, store and secrete TGs in VLDL particles. The effects of cold exposure on murine hepatic lipid metabolism have not been addressed. Here, we report the effects of 24-h exposure to 4°C on parameters of hepatic lipid metabolism of male C57BL/6J mice. Cold exposure increased hepatic TG concentrations by 2-fold (P Cyp7a1encoding the rate-limiting enzyme in the classical bile acid (BA) synthesis pathway was increased by 4.3-fold (P P P

Summary: Many favorable metabolic effects have been attributed to thermogenic activity of brown adipose tissue (BAT). Yet, time of day has rarely been considered in this field of research. Here, we show that a diurnal rhythm in BAT activity regulates plasma lipid metabolism. We observed a high-amplitude rhythm in fatty acid uptake by BAT that synchronized with the light/dark cycle. Highest uptake was found at the onset of the active period, which coincided with high lipoprotein lipase expression and low angiopoietin-like 4 expression by BAT. Diurnal rhythmicity in BAT activity determined the rate at which lipids were cleared from the circulation, thereby imposing the daily rhythm in plasma lipid concentrations. In mice as well as humans, postprandial lipid excursions were nearly absent at waking. We anticipate that diurnal BAT activity is an important factor to consider when studying the therapeutic potential of promoting BAT activity. : van den Berg et al. show a strong circadian rhythm in fatty acid uptake by brown adipose tissue that peaks at wakening regardless of the light exposure period. Consequently, postprandial lipid handling by brown adipose tissue is highest at wakening, resulting in the lowest postprandial plasma lipid excursions. Keywords: circadian rhythm, diurnal rhythm, brown adipose tissue, triglycerides, fatty acids, lipoprotein lipase, angiopoietin-like 4, postprandial lipid response, APOE∗3-Leiden.CETP mice

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, yet the pathogenesis of NAFLD is only partially understood. Here, we investigated the role of the gut bacteria in NAFLD by stimulating the gut bacteria via feeding mice the fermentable dietary fiber, guar gum (GG), and suppressing the gut bacteria via chronic oral administration of antibiotics. GG feeding profoundly altered the gut microbiota composition, in parallel with reduced diet-induced obesity and improved glucose tolerance. Strikingly, despite reducing adipose tissue mass and inflammation, GG enhanced hepatic inflammation and fibrosis, concurrent with markedly elevated plasma and hepatic bile acid levels. Consistent with a role of elevated bile acids in the liver phenotype, treatment of mice with taurocholic acid stimulated hepatic inflammation and fibrosis. In contrast to GG, chronic oral administration of antibiotics effectively suppressed the gut bacteria, decreased portal secondary bile acid levels, and attenuated hepatic inflammation and fibrosis. Neither GG nor antibiotics influenced plasma lipopolysaccharide levels. In conclusion, our data indicate a causal link between changes in gut microbiota and hepatic inflammation and fibrosis in a mouse model of NAFLD, possibly via alterations in bile acids.-Janssen, A. W. F., T. Houben, S. Katiraei, W. Dijk, L. Boutens, N. van der Bolt, Z. Wang, J. M. Brown, S. L. Hazen, S. Mandard, R. Shiri-Sverdlov, F. Kuipers, K. Willems van Dijk, J. Vervoort, R. Stienstra, G. J. E. J. Hooiveld, and S. Kersten. Modulation of the gut microbiota impacts nonalcoholic fatty liver disease: a potential role for bile acids.

Triglycerides are stored in specialized organelles called lipid droplets. Numerous proteins have been shown to be physically associated with lipid droplets and govern their function. Previously, the protein hypoxia-inducible lipid droplet-associated (HILPDA) was localized to lipid droplets and was suggested to inhibit triglyceride lipolysis in hepatocytes. We confirm the partial localization of HILPDA to lipid droplets and show that HILPDA is highly abundant in adipose tissue, where its expression is controlled by the peroxisome proliferator-activated receptor γ and by β-adrenergic stimulation. Levels of HILPDA markedly increased during 3T3-L1 adipocyte differentiation. Nevertheless, silencing of Hilpda using small interfering RNA or overexpression of Hilpda using adenovirus did not show a clear impact on 3T3-L1 adipogenesis. Following β-adrenergic stimulation, the silencing of Hilpda in adipocytes did not significantly alter the release of nonesterified fatty acids (NEFA) and glycerol. By contrast, adenoviral-mediated overexpression of Hilpda modestly attenuated the release of NEFA from adipocytes following β-adrenergic stimulation. In mice, adipocyte-specific inactivation of Hilpda had no effect on plasma levels of NEFA and glycerol after fasting, cold exposure, or pharmacological β-adrenergic stimulation. In addition, other relevant metabolic parameters were unchanged by adipocyte-specific inactivation of Hilpda. Taken together, we find that HILPDA is highly abundant in adipose tissue, where its levels are induced by peroxisome proliferator-activated receptor γ and β-adrenergic stimulation. In contrast to the reported inhibition of lipolysis by HILPDA in hepatocytes, our data do not support an important direct role of HILPDA in the regulation of lipolysis in adipocytes in vivo and in vitro.

The body efficiently stores energy in the form of triglyceride (fat) molecules. However, triglycerides cannot directly enter or exit our cells, but first need to be degraded to so-called fatty acids before moving in or out a cell. This degradation process, called lipolysis, is crucial for human physiology and is tightly regulated to prevent the accumulation of fats either within organs or within the bloodstream - hallmarks of diseases such as obesity and cardiovascular disease. To allow for uptake by underlying organs, triglycerides in the circulation are efficiently broken down by an enzyme called lipoprotein lipase (LPL) that sits in the bloodstream of multiple organs (extracellular lipolysis). In this thesis, we characterized a protein named angiopoietin-like 4 (ANGPTL4) that potently inhibits LPL and, thereby, inhibits the breakdown of triglycerides in the bloodstream. Our data show that by adjusting the tissue expression levels of ANGPTL4, different organs collaborate to ensure that triglycerides are distributed to organs in need of energy. Moreover, we uncovered that, in the fat tissue, ANGPTL4 starts to inhibit LPL before LPL arrives in the bloodstream. By preventing the arrival of LPL in the bloodstream, ANGPTL4 is capable of rapidly adjusting the rates of triglyceride degradation and the concomitant uptake of fatty acids from the circulation to the energy requirements of the underlying organ. To exit our cells, stored triglycerides, such as present in our fat tissue, need to be broken down to fatty acids. Subsequently, the released fatty acids can fuel other organs in need of energy. To further clarify the mechanisms underlying this process of intracellular lipolysis, we investigated the role of a promising new protein called HILPDA. Our data show, however, that loss of HILPDA did not impact the release of fatty acids from the fat tissue, while a high abundance of HILPDA only had a mild attenuating effect on the release of fatty acids. This suggests that HILPDA is not a major physiological regulator of intracellular lipolysis in fat cells. In conclusion, in this thesis, we have clarified the regulation of intracellular and extracellular lipolysis by studying the respective roles of the proteins ANGPTL4 and HILPDA. Such efforts are clinically relevant, as regulators of lipolysis are potential therapeutic targets to lower cardiovascular disease risk.

The pathophysiology of disease has dominated the biomedical world during the past few centuries. Lately, however, renewed interest has been observed in the phenomenon of 'health'. Even though this phenomenon has been defined in many different ways, an encompassing definition that can be aptly used by the scientific medical community to measure and quantify health has yet to be found. Processes known to underlie health are homeostasis, allostasis (homeodynamics), salutogenesis, adaptability, robustness and resilience. Together, they become especially important during times of stress and disbalance, allowing the body to adapt to a certain 'threat' and ensuring proper functioning of the body. Focus on this ability to adapt could aid in defining health. In addition, it would allow health, as such, measurable and quantifiable. Bodily adaptations can, for example, be measured via a variety of changes in biomarkers and parameters, which reflect the capacity of various organ systems to respond and adapt to stress conditions. Potentially, specific stressors could be applied in research, to which allostatic and homeostatic responses of tested individuals might be measured. The addition of this dynamic component to the current definition of health would, thereby, mean a step forward in health research and could stimulate the development of new therapeutic strategies, optimizing current and future (preventive) health care.

Brown adipose tissue (BAT) activation via cold exposure is increasingly scrutinized as a potential approach to ameliorate cardio-metabolic risk. Transition to cold temperatures requires changes in the partitioning of energy substrates, re-routing fatty acids to BAT to fuel non-shivering thermogenesis. However, the mechanisms behind the redistribution of energy substrates to BAT remain largely unknown. Angiopoietin-like 4 (ANGPTL4), a protein that inhibits lipoprotein lipase (LPL) activity, is highly expressed in BAT. Here, we demonstrate that ANGPTL4 is part of a shuttling mechanism that directs fatty acids derived from circulating triglyceride-rich lipoproteins to BAT during cold. Specifically, we show that cold markedly down-regulates ANGPTL4 in BAT, likely via activation of AMPK, enhancing LPL activity and uptake of plasma triglyceride-derived fatty acids. In contrast, cold up-regulates ANGPTL4 in WAT, abolishing a cold-induced increase in LPL activity. Together, our data indicate that ANGPTL4 is an important regulator of plasma lipid partitioning during sustained cold. DOI: http://dx.doi.org/10.7554/eLife.08428.001, eLife digest The body stores energy in the form of fat molecules. Most of these molecules are stored in white fat cells. Other fat cells, the so-called brown fat cells, consume fats and produce heat to maintain body temperature in cold conditions. The capacity of brown fat cells to consume fats has led researchers to investigate whether brown fat cells might be a key to combat obesity. When an organism is cold, fat is shuttled to the brown fat cells. An enzyme called lipoprotein lipase is involved in a process that allows these fat molecules to be taken up by brown fat cells. However, it was not clear exactly how this process works. A protein called Angiopoietin-like 4 (ANGPTL4) inhibits the activity of lipoprotein lipase in white fat cells and is also found at high levels in brown fat cells. Here, Dijk et al. used genetic and biochemical approaches to study the role of ANGPTL4 in the fat cells of mice. The experiments show that when mice are exposed to cold, the levels of ANGPTL4 decrease in the brown fat cells. This allows the activity of lipoprotein lipase to increase so that these cells are able to take up more fat molecules. However, the opposite happens in white fat cells during cold exposure. The levels of ANGPTL4 increase, which decreases the activity of lipoprotein lipase in white fat cells to allow fat molecules to be shuttled specifically to the brown fat cells. Further experiments suggest that the opposite regulation of ANGPTL4 in brown and white fat cells could be due to a protein called AMPK. This protein is found at higher levels in brown fat cells than in white fat cells and is produced by brown fat cells during cold exposure. Taken together, Dijk et al. show that organs and cells work together to ensure that fat molecules are appropriately distributed to cells in need of energy, such as to brown fat cells during cold. How these findings could be used to stimulate fat consumption by brown fat cells in humans remains open for further investigation. DOI: http://dx.doi.org/10.7554/eLife.08428.002

Brown adipose tissue (BAT) produces heat by burning TGs that are stored within intracellular lipid droplets and need to be replenished by the uptake of TG-derived FA from plasma. It is currently unclear whether BAT takes up FA via uptake of TG-rich lipoproteins (TRLs), after lipolysis-mediated liberation of FA, or via a combination of both. Therefore, we generated glycerol tri[(3)H]oleate and [(14)C]cholesteryl oleate double-labeled TRL-mimicking particles with an average diameter of 45, 80, and 150 nm (representing small VLDL to chylomicrons) and injected these intravenously into male C57Bl/6J mice. At room temperature (21°C), the uptake of (3)H-activity by BAT, expressed per gram of tissue, was much higher than the uptake of (14)C-activity, irrespective of particle size, indicating lipolysis-mediated uptake of TG-derived FA rather than whole particle uptake. Cold exposure (7°C) increased the uptake of FA derived from the differently sized particles by BAT, while retaining the selectivity for uptake of FA over cholesteryl ester (CE). At thermoneutrality (28°C), total FA uptake by BAT was attenuated, but the specificity of uptake of FA over CE was again largely retained. Altogether, we conclude that, in our model, BAT takes up plasma TG preferentially by means of lipolysis-mediated uptake of FA.

Item does not contain fulltext Obesity is accompanied by the presence of chronic low-grade inflammation manifested by infiltration of macrophages into adipose tissue. Mannose-binding lectin (MBL), a soluble mediator of innate immunity, promotes phagocytosis and alters macrophage function. To assess the function of MBL in the development of obesity, we studied wild-type and MBL(-/-) mice rendered obese using a high-fat diet (HFD). Whereas no gross morphological differences were observed in liver, an HFD provoked distinct changes in the adipose tissue morphology of MBL(-/-) mice. In parallel with increased adipocyte size, MBL(-/-) mice displayed an increased influx of macrophages into adipose tissue. Macrophages were polarized toward an alternatively activated phenotype known to modulate apoptotic cell clearance. MBL deficiency also significantly increased the number of apoptotic cells in adipose tissue. Consistent with these observations, recombinant MBL enhanced phagocytic capacity of the stromal vascular fraction isolated from adipose tissue and modulated uptake of apoptotic adipocytes by macrophages. Despite changes in macrophage abundance and polarity, the absence of MBL did not affect systemic insulin resistance. Finally, in humans, lower levels of circulating MBL were accompanied by enhanced macrophage influx in subcutaneous adipose tissue. We propose a novel role for MBL in the recognition and clearance of apoptotic adipocytes during obesity.

Gut hormones produced by entero-endocrine cells (EEC) located throughout the gastrointestinal tract play a major role in the regulation of glucose and energy homeostasis. Angiopoietin-like 4 (ANGPTL4, also referred to as fasting induced adipose factor) is a secreted factor involved in regulation of lipid homeostasis and has been proposed as circulating mediator between the gut microbiota and fat storage in adipose tissue, although discordant data exist. Currently, little is known about the site and regulation of ANGPTL4 production in the intestine. Here, we show using immunohistochemistry and immunofluorescence that cells positive for ANGPTL4 are scattered along the epithelial layer in the human small and large intestine. ANGPTL4-positive cells exhibit typical features of EEC characterized by large ANGPTL4-positive secretory granules directed towards the basolateral side. In support, extensive overlap was observed between ANGPTL4-positive cells and cells positive for the entero-endocrine marker chromogranin A. Higher resolution images revealed that ANGPTL4 and chromogranin A are partially present in distinct intracellular vesicles. Using entero-endocrine HuTu-80 cells, ANGPTL4 secretion was shown to be induced by short chain fatty acids and reduced by bile acids. Finally, levels of ANGPTL4 in human plasma were significantly decreased following meal consumption. In conclusion, ANGPTL4 is produced by EEC in human intestine and expression may be regulated by short chain fatty acids and bile acids.

Triglyceride (TG)-rich chylomicrons and very low density lipoproteins (VLDL) distribute fatty acids (FA) to various tissues by interacting with the enzyme lipoprotein lipase (LPL). The protein angiopoietin-like 4 (Angptl4) is under sensitive transcriptional control by FA and the FA-activated peroxisome proliferator activated receptors (PPARs), and its tissue expression largely overlaps with that of LPL. Growing evidence indicates that Angptl4 mediates the physiological fluctuations in LPL activity, including the decrease in adipose tissue LPL activity during fasting. This review focuses on the major ambiguities concerning the mechanism of LPL inhibition by Angptl4, as well as on the physiological role of Angptl4 in lipid metabolism, highlighting its function in a variety of tissues, and uses this information to make suggestions for further research.

The health benefits of consuming organically produced foods compared with conventional foods are unclear. Important obstacles to drawing clear conclusions in this field of research are (1) the lack of a clear operational definition of health and (2) the inability to distinguish between different levels of health using valid biomarkers. In this paper, some shortcomings of the current definition of health are outlined and the relevance of integrating a more dynamic and functional component is emphasised, which is reflected by the ability to adapt. The state of health could then be determined by challenging an individual with some form of stressor and by subsequent quantification and evaluation of the coherence in recovery of various physiological processes and parameters. A set of relevant parameters includes the activity of the immune system and the activity of the autonomous nervous system. A good recovery towards homeostasis is suggested to reflect a qualitatively good state of health. Furthermore, it would enable objective evaluation of health-optimising strategies, including the consumption of organically produced foods that aim to strengthen health. (C) 2012 Society of Chemical Industry

Angiopoietin-like factor 4 (Angptl4) is a secreted protein that inhibits lipoprotein lipase (LPL) activity in various tissues, such as white adipose tissue (WAT) and muscle. In mice, Angptl4 is most highly expressed in brown adipose tissue (BAT), the tissue responsible for non-shivering thermogenesis.

Obesity is known to be accompanied by the presence of a chronic low grade inflammation manifested by the infiltration of macrophages into white adipose tissue.