Probing the regulation of cellular lipid metabolism by trans fatty acids and the lipid-sensitive ANGPTL4 protein

A coordinated regulation of lipid metabolism is important in maintaining cardiometabolic health, since a dysregulation in lipid metabolism is implicated in hyperlipidaemia, insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), atherosclerosis and coronary artery disease. This thesis has investigated the pre-clinical effects of dietary trans fatty acids, lipid-sensitive angiopoietin-like 4 (ANGPTL4) and muscle inflammation on cardiometabolic health. The aim is to further understand the metabolic and molecular mechanisms by which trans fatty acids, ANGPTL4 and muscle-specific inflammation impact cardiometabolic health in order to devise therapeutic strategies. Trans fatty acids are unsaturated fatty acids that contain at least one double bond in the trans configuration. In humans, the consumption of certain trans fatty acids is implicated in the onset and progression of cardiovascular diseases due in part to the ability of such trans fatty acids to raise the plasma levels of total and LDL-cholesterol, and decrease the levels of HDL-cholesterol. However, the underlying mechanisms for the deleterious effects of trans fatty acids are not fully understood. We show in C57Bl/6 mice that a diet enriched in industrial trans fatty acids from partially hydrogenated soybean oil promotes non-alcoholic fatty liver disease (NAFLD) compared to cis-unsaturated and saturated fatty acid diets. At the cellular level, we demonstrate in murine hepatocyte and adipocyte cell lines that the industrial fatty acid elaidate induces cholesterogenesis through enhanced expression and activity of sterol regulatory element binding protein 2 (SREBP2). ANGPTL4 is an inhibitor of lipoprotein lipase (LPL). LPL hydrolyses circulating triglycerides to generate fatty acids for uptake into tissues. Therefore, as an inhibitor of LPL, ANGPTL4 indirectly raises the levels of circulating triglycerides leading to an associated increased risk for cardiovascular diseases. However, the potential of inhibiting ANGPTL4 to lower the risk of cardiovascular diseases is hampered by lethal side-effects in ANGPTL4-deficient mice fed diets rich in saturated fatty acids. In this thesis, we demonstrate that ANGPTL4-deficient mice respond favourably to a trans fat diet than a saturated fat diet. Furthermore, we show that saturated fatty acids but not trans fatty acids induce inflammation and endoplasmic reticulum (ER) stress in murine macrophages. This thesis also characterised a novel ANGPTL4-hypomorphic mice model with only a partial expression of the N-terminal domain of ANGPTL4. Following a chronic high saturated fat diet challenge, the hypomorphs showed reduced levels of plasma triglycerides as the complete ANGPTL4-deficient mice. Interestingly, the hypomorphs showed attenuated and milder forms of the side-effects that occur in the complete ANGPTL4-deficient mice. Finally, this thesis has also examined the role of muscle specific inflammation on systemic glucose homeostasis. We found that skeletal muscle-specific inflammation via over-expression of monocyte chemoattractant protein-1 (MCP-1) does not affect systemic glucose and insulin sensitivity in mice. Taken together, this thesis has provided insight into the molecular mechanisms of trans fatty acids and highlighted the prospect of harnessing the therapeutic potential of ANGPTL4 inactivation for correcting cardiometabolic abnormalities.</p

The effects of a novel oral nutritional supplement as compared to standard care on body composition, physical function and skeletal muscle mRNA expression in Dutch older adults with (or at risk of) undernutrition

In a randomized controlled trial, 82 older adults (>65y) with (or at risk of) undernutrition (n=82) were randomly allocated to 12 weeks of supplementation with a novel supplement (586 kcal, 22 g protein of which 50% whey and 50% casein, 206 mg ursolic acid, 7 g free BCAAs, 11 µg vitamin D) or standard care (600 kcal, 24g protein of which 100% casein, 4 µg vitamin D). Body weight increased significantly in the 12 weeks, both in the intervention group (+1.6 ± 0.2 kg, p<.0001) and in the standard care group (+1.8 ± 0.2 kg, p<.0001). Gait speed during 4m and 400m tests improved over time in the intervention group, whereas the standard care showed no improvements (time*treatment effects 400m: p=0.038 and 4m: p=0.048). Gene sets related to mitochondrial functioning were strongly upregulated in the participants receiving the intervention product. We showed that a novel oral nutritional supplement improves gait speed in older adults via improvements in mitochondrial functioning

The effects of a novel oral nutritional supplement as compared to standard care on body composition, physical function and skeletal muscle mRNA expression in Dutch older adults with (or at risk of) undernutrition

In a randomized controlled trial, 82 older adults (>65y) with (or at risk of) undernutrition (n=82) were randomly allocated to 12 weeks of supplementation with a novel supplement (586 kcal, 22 g protein of which 50% whey and 50% casein, 206 mg ursolic acid, 7 g free BCAAs, 11 µg vitamin D) or standard care (600 kcal, 24g protein of which 100% casein, 4 µg vitamin D). Body weight increased significantly in the 12 weeks, both in the intervention group (+1.6 ± 0.2 kg, p<.0001) and in the standard care group (+1.8 ± 0.2 kg, p<.0001). Gait speed during 4m and 400m tests improved over time in the intervention group, whereas the standard care showed no improvements (time*treatment effects 400m: p=0.038 and 4m: p=0.048). Gene sets related to mitochondrial functioning were strongly upregulated in the participants receiving the intervention product. We showed that a novel oral nutritional supplement improves gait speed in older adults via improvements in mitochondrial functioning

A novel oral nutritional supplement improves gait speed and mitochondrial functioning compared to standard care in older adults with (or at risk of) undernutrition: results from a randomized controlled trial

Undernutrition in older adults is mainly addressed by oral nutritional supplements, which do not affect physical functioning. In this study, we tested a novel oral nutritional supplement that included whey and casein protein, ursolic acid, free branch-chained amino acids and vitamin D against a standard supplement. We included older adults (>65y) with (or at risk of) undernutrition (n=82) and randomized them to 12 weeks of novel or standard supplement. Both groups showed significant increases in body mass. No within or between-group differences in lean body mass were observed. Fat mass increased significantly more in the standard than the novel supplement group (time*treatment effect P=0.045). The novel supplement group showed a larger improvement in walking performance on distances of 4m (treatment x time interaction P=0.048) and 400m (treatment x time interaction P=0.038) than the standard treatment group. Gene sets related to mitochondrial functioning and oxidative phosphorylation were upregulated in the novel supplement group and downregulated in the standard supplement group. We conclude that a 12-week intervention with the novel supplement improved walking performance both during short and long distance as compared to a standard supplement, which can largely be explained by increased mitochondrial functioning in the group receiving the novel supplement

Industrial Trans Fatty Acids Stimulate SREBP2-Mediated Cholesterogenesis and Promote Non-Alcoholic Fatty Liver Disease

Scope: The mechanisms underlying the deleterious effects of trans fatty acids on plasma cholesterol and non-alcoholic fatty liver disease (NAFLD) are unclear. Here, the aim is to investigate the molecular mechanisms of action of industrial trans fatty acids. Methods and results: Hepa1-6 hepatoma cells were incubated with elaidate, oleate, or palmitate. C57Bl/6 mice were fed diets rich in trans-unsaturated, cis-unsaturated, or saturated fatty acids. Transcriptomics analysis of Hepa1-6 cells shows that elaidate but not oleate or palmitate induces expression of genes involved in cholesterol biosynthesis. Induction of cholesterogenesis by elaidate is mediated by increased sterol regulatory element-binding protein 2 (SREBP2) activity and is dependent on SREBP cleavage–activating protein (SCAP), yet independent of liver-X receptor and ubiquitin regulatory X domain-containing protein 8. Elaidate decreases intracellular free cholesterol levels and represses the anticholesterogenic effect of exogenous cholesterol. In mice, the trans-unsaturated diet increases the ratio of liver to gonadal fat mass, steatosis, hepatic cholesterol levels, alanine aminotransferase activity, and fibrosis markers, suggesting enhanced NAFLD, compared to the cis-unsaturated and saturated diets. Conclusion: Elaidate induces cholesterogenesis in vitro by activating the SCAP–SREBP2 axis, likely by lowering intracellular free cholesterol and attenuating cholesterol-dependent repression of SCAP. This pathway potentially underlies the increase in liver cholesterol and NAFLD by industrial trans fatty acids

Industrial Trans Fatty Acids Stimulate SREBP2-Mediated Cholesterogenesis and Promote Non-Alcoholic Fatty Liver Disease

Scope: The mechanisms underlying the deleterious effects of trans fatty acids on plasma cholesterol and non-alcoholic fatty liver disease (NAFLD) are unclear. Here, the aim is to investigate the molecular mechanisms of action of industrial trans fatty acids. Methods and results: Hepa1-6 hepatoma cells were incubated with elaidate, oleate, or palmitate. C57Bl/6 mice were fed diets rich in trans-unsaturated, cis-unsaturated, or saturated fatty acids. Transcriptomics analysis of Hepa1-6 cells shows that elaidate but not oleate or palmitate induces expression of genes involved in cholesterol biosynthesis. Induction of cholesterogenesis by elaidate is mediated by increased sterol regulatory element-binding protein 2 (SREBP2) activity and is dependent on SREBP cleavage–activating protein (SCAP), yet independent of liver-X receptor and ubiquitin regulatory X domain-containing protein 8. Elaidate decreases intracellular free cholesterol levels and represses the anticholesterogenic effect of exogenous cholesterol. In mice, the trans-unsaturated diet increases the ratio of liver to gonadal fat mass, steatosis, hepatic cholesterol levels, alanine aminotransferase activity, and fibrosis markers, suggesting enhanced NAFLD, compared to the cis-unsaturated and saturated diets. Conclusion: Elaidate induces cholesterogenesis in vitro by activating the SCAP–SREBP2 axis, likely by lowering intracellular free cholesterol and attenuating cholesterol-dependent repression of SCAP. This pathway potentially underlies the increase in liver cholesterol and NAFLD by industrial trans fatty acids.</p

Feeding Angptl4(-/-) mice trans fat promotes foam cell formation in mesenteric lymph nodes without leading to ascites

Angiopoietin-like 4 (ANGPTL4) regulates plasma triglyceride levels by inhibiting LPL. Inactivation of ANG-PTL4 decreases plasma triglycerides and reduces the risk of coronary artery disease. Unfortunately, targeting ANGPTL4 for the therapeutic management of dyslipidemia and atherosclerosis is hampered by the observation that mice and monkeys in which ANGPTL4 is inactivated exhibit lipid accumulation in the mesenteric lymph nodes (MLNs). In mice these pathological events exclusively unfold upon feeding a high saturated FA diet and are followed by an ultimately lethal pro-inflammatory response and chylous ascites. Here, we show that Angptl4(-/-) mice fed a diet rich in trans FAs develop numerous lipid-filled giant cells in their MLNs, yet do not have elevated serum amyloid and haptoglobin, do not exhibit ascites, and survive, unlike Angptl4(-/-) mice fed a saturated FA-rich diet. In RAW264.7 macrophages, the saturated FA, palmitate, markedly increased markers of inflammation and the unfolded protein response, whereas the trans-unsaturated elaidate and the cis-unsaturated oleate had the opposite effect. In conclusion, trans and saturated FAs have very distinct biological effects in macrophages. Furthermore, lipid accumulation in MLNs is uncoupled from activation of an acute-phase response and chylous ascites, suggesting that ANGPTL4 should not be fully dismissed as target for dyslipidemia

Characterization of ANGPTL4 function in macrophages and adipocytes using Angptl4-knockout and Angptl4-hypomorphic mice

ANGPTL4 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 non-esterified fatty acid levels, 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

Muscle-specific inflammation induced by MCP-1 overexpression does not affect whole-body insulin sensitivity in mice

Aims/hypothesis: Obesity is associated with a state of chronic low-grade inflammation that is believed to contribute to the development of skeletal muscle insulin resistance. However, the extent to which local and systemic elevation of cytokines, such as monocyte chemoattractant protein 1 (MCP-1), interferes with the action of insulin and promotes insulin resistance and glucose intolerance in muscle remains unclear. Here, we aim to investigate the effect of muscle-specific overexpression of MCP-1 on insulin sensitivity and glucose tolerance in lean and obese mice. Methods: We used Mck–Mcp-1 transgenic (Tg) mice characterised by muscle-specific overexpression of Mcp-1 (also known as Ccl2) and elevated plasma MCP-1 levels. Mice were fed either chow or high-fat diet for 10 weeks. Numerous metabolic variables were measured, including glucose and insulin tolerance tests, muscle insulin signalling and plasma NEFA, triacylglycerol, cholesterol, glucose and insulin. Results: Despite clearly promoting skeletal muscle inflammation, muscle-specific overexpression of Mcp-1 did not influence glucose tolerance or insulin sensitivity in either lean chow-fed or diet-induced obese mice. In addition, plasma NEFA, triacylglycerol, cholesterol, glucose and insulin were not affected by MCP-1 overexpression. Finally, in vivo insulin-induced Akt phosphorylation in skeletal muscle did not differ between Mcp-1-Tg and wild-type mice. Conclusions/interpretation: We show that increased MCP-1 production in skeletal muscle and concomitant elevated MCP-1 levels in plasma promote inflammation in skeletal muscle but do not influence insulin signalling and have no effect on insulin resistance and glucose tolerance in lean and obese mice. Overall, our data argue against MCP-1 promoting insulin resistance in skeletal muscle and raise questions about the impact of inflammation on insulin sensitivity in muscle

Pain Burden in the CASiRe International Cohort of Sickle Cell Patients: United States and Ghana

Objectives: Sickle Cell Disease (SCD) is a genetic blood disorder affecting over 1 million people globally. The aim of this analysis is to explore the pain burden of patients with SCD in two countries: the United States and Ghana. Methods: The Consortium for the Advancement of Sickle Cell Research (CASiRe) was created to better understand the clinical severity of patients with SCD worldwide. Data regarding gender, SCD genotype, prior medical diagnoses, and validated pain burden measures were analyzed from the CASiRe database. The Sickle Cell Pain Burden Interview (SCPBI) was used to assess pain burden, the impact of pain on physical, emotional, and social function. Results: Most subjects identified as Black/African American (n = 298, 97.0%). Patient ages ranged from 6 to 73 years. 35.9% resided in the United States, 64.1% resided in Ghana, 40.9% were men, and 58.7% were women. The mean SCPBI score for US SCD patients was 6.53(±5.89) vs 4.04(±5.10) for Ghanaian patients, P <0.001. Pain burden was higher in US men vs Ghanaian men (6.74(±5.68) vs 3.54(±4.46), P = .003) and in US women vs Ghanaian women (6.37 ± 6.06 vs 4.44(±5.54), P = .032). Pain burden was higher in US patients than Ghanaian patients for both the Hb SC/SBeta+ genotype (5.40(±5.29) vs 2.82(±4.86), P = .054) and Hb SS/SBeta0 genotype (6.79(±6.01) vs 4.49(±5.13), P = .003). Pain burden was significantly higher in SCD patients with comorbid conditions independent of geographic origin including stroke, cholecystectomy, gallstones, depression, and headache. Discussion: US patients with SCD have a higher pain burden than Ghanaian patients. Further studies should investigate underlying contributors to pain burden in these populations and further explore the etiology of geographic differences in pain