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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.

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.

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.

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.

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

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.

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

High-protein diets are known to reduce adiposity in the context of high carbohydrate and Western diets. However, few studies have investigated the specific high-protein effect on lipogenesis induced by a high-sucrose (HS) diet or fat deposition induced by high-fat feeding. We aimed to determine the effects of high protein intake on the development of fat deposition and partitioning in response to high-fat and/or HS feeding. A total of thirty adult male Wistar rats were assigned to one of the six dietary regimens with low and high protein, sucrose and fat contents for 5 weeks. Body weight (BW) and food intake were measured weekly. Oral glucose tolerance tests and meal tolerance tests were performed after 4th and 5th weeks of the regimen, respectively. At the end of the study, the rats were killed 2 h after ingestion of a calibrated meal. Blood, tissues and organs were collected for analysis of circulating metabolites and hormones, body composition and mRNA expression in the liver and adipose tissues. No changes were observed in cumulative energy intake and BW gain after 5 weeks of dietary treatment. However, high-protein diets reduced by 20 % the adiposity gain induced by HS and high-sucrose high-fat (HS-HF) diets. Gene expression and transcriptomic analysis suggested that high protein intake reduced liver capacity for lipogenesis by reducing mRNA expressions of fatty acid synthase (fasn), acetyl-CoA carboxylase a and b (Acaca and Acacb) and sterol regulatory element binding transcription factor 1c (Srebf-1c). Moreover, ketogenesis, as indicated by plasma β-hydroxybutyrate levels, was higher in HS-HF-fed mice that were also fed high protein levels. Taken together, these results suggest that high-protein diets may reduce adiposity by inhibiting lipogenesis and stimulating ketogenesis in the liver.

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.