Plasma Triglyceride Clearance Research Articles

Effects of age and diet on triglyceride metabolism in mice

Both age and diet can contribute to alterations in triglyceride metabolism and subsequent metabolic disease. In humans, plasma triglyceride levels increase with age. Diets high in saturated fats can increase triglyceride levels while diets high in omega-3 fatty acids decrease triglyceride levels. Here we asked how age and long-term diet altered triglyceride metabolism in mice. We fed male and female C57Bl/6 mice a low-fat diet, a western diet (WD), or a diet high in polyunsaturated and omega-3 fatty acids (n3D) for up to 2 years. We measured survival, body composition, plasma triglyceride levels, chylomicron clearance, and oral fat, glucose, and insulin tolerance. Triglyceride levels in mice did not increase with age, regardless of diet. Oral fat tolerance increased with age, while chylomicron clearance remained unchanged. Decreased survival was observed in WD-fed mice. Interestingly, n3D-fed mice diet gained more lean mass and had lower insulin levels than WD-fed or LFD-fed mice. Moreover, triglyceride uptake into the hearts of n3D-fed mice was strikingly higher than in other groups. Our data indicate that in C57Bl/6 mice, age-induced changes in triglyceride metabolism differ from those observed in humans. Mice, like humans, appeared to have decreased fat absorption with age, but in mice plasma triglyceride clearance did not decrease with age, resulting in lower plasma triglyceride levels and improved fat tolerance with age. Although a chronic diet high in omega-3 fatty acids increased insulin sensitivity and triglyceride uptake specifically into the heart, how these observations are connected is unclear.

Development of a hamster model of spontaneous hypertriglyceridemia in diabetes.

Hypertriglyceridemia (HTG) often accompanies diabetes and is considered a risk factor for diabetic vascular complications. However, inducing diabetic HTG typically requires high-fat diets in certain animal models. Leveraging our newly developed LDL receptor knockout hamster model, which exhibits features akin to human lipid metabolism, we sought to determine whether these animals would develop HTG without dietary manipulations in diabetes. Diabetes was induced via intraperitoneal injection of STZ in wild type and heterozygous LDL receptor deficient hamsters. Blood glucose, triglyceride, and cholesterol were measured over 60 days. Plasma TG clearance was determined via olive oil gavage. The effect of insulin on diabetic HTG was assessed on Day 60 post-diabetes induction. Blood glucose increased over threefold, while plasma insulin decreased to 30% of controls after STZ injection in both wild type and heterozygous hamsters by Day 7, remaining stable for 60 days. Plasma TG in wild-type hamsters remained unchanged at Day 7 post-STZ injection but increased slightly thereafter. Conversely, heterozygous hamsters exhibited severe HTG by Day 7 until the end of the study. Olive oil gavage revealed much slower plasma triglyceride clearance in heterozygous hamsters compared to WT animals, despite significantly reduced lipoprotein lipase activity in post-heparin plasma in both animals. Hyperglycemia and HTG in heterozygous hamsters were reversed to pre-diabetic levels following intraperitoneal insulin administration. In conclusion, severe HTG in diabetic heterozygous LDL receptor deficient hamsters developed spontaneously and was insulin-dependent. Thus, this hamster model holds promise for effectively studying the complications associated with human diabetic HTG.

Effects of Age and Diet on Triglyceride Metabolism in Mice.

Both age and diet can contribute to alterations in triglyceride metabolism and subsequent metabolic disease. In humans, plasma triglyceride levels increase with age. Diets high in saturated fats can increase triglyceride levels while diets high in omega-3 fatty acids decrease triglyceride levels. Here we asked how age and long-term diet effected triglyceride metabolism in mice. We fed male and female mice a low-fat diet, a western diet, or a diet high in polyunsaturated and omega-3 (n-3) fatty acids for up to 2 years. We measured survival, body composition, plasma triglyceride levels, chylomicron clearance, and oral fat, glucose, and insulin tolerance. Triglyceride levels in mice did not increase with age, regardless of diet. Oral fat tolerance increased with age, while chylomicron clearance remained unchanged. Mice fed western diet had decreased survival. Interestingly, mice fed the n-3 diet gained more lean mass, and had lower insulin levels than mice fed either low-fat or western diet. Moreover, triglyceride uptake into the hearts of mice fed the n-3 diet was strikingly higher than in other groups. In mice, age-induced changes in triglyceride metabolism did not match those in humans. Our data suggested that mice, like humans, had decreased fat absorption with age, but plasma triglyceride clearance did not decrease with age in mice, resulting in lower plasma triglyceride levels and improved oral fat tolerance with age. A chronic diet high in n-3 fatty acids increased insulin sensitivity and uptake of triglycerides specifically into the heart but how these observations are connected is unclear.

Postprandial Triglyceride, Glucose and Insulin Levels 10 Years After Bariatric Surgery in Women With Severe Obesity - A Pilot Study: Part 2 - Biliopancreatic Diversion.

Obesity significantly increases the risk of developing metabolic and cardiovascular diseases. The most effective management tool for both obesity and type 2 diabetes (T2D) is bariatric/metabolic surgery. Delayed postprandial plasma triglyceride clearance contributes to the development of atherosclerosis in patients with T2D. Biliopancreatic diversion (BPD) was shown to be the most effective procedure in long-term T2D remission. However, the effect of BPD on postprandial metabolic profile has not been studied so far. In this pilot study, we therefore examined the changes in postprandial glucose, insulin, and triglyceride in women with severe obesity and T2D before surgery and then two and ten years after BPD. The studied cohort included 7 women (mean age at baseline=49.3±8.2 years) with severe obesity (mean BMI= 45.7±2.9 kg/m?) and T2D. A standardized liquid mixed-meal test was carried out in all subjects and the mean postprandial levels of plasma glucose, insulin, and triglyceride were analyzed by standard laboratory procedures. For statistical evaluation, ANOVA with Bonferroni multiple comparisons was used. Ten years after BPD not only a significant reduction of an average BMI (F=32.9, p<0.001) but also significant declines in mean postprandial plasma levels of glucose (F=155.3, p<0.001), insulin (F=69.8, p<0.001), and triglyceride (F=139.9, p<0.001) were demonstrated. The observed changes in postprandial metabolic profile may contribute to improved cardiometabolic health after bariatric surgery.

ANGPTL3 negatively regulates IL-1β-induced NF-κB activation by inhibiting the IL1R1-associated signaling complex assembly.

Interleukin-1β (IL-1β)-induced signaling is one of the most important pathways in regulating inflammation and immunity. The assembly of the receptor complex, consisting of the ligand IL-1β, the IL-1 receptor (IL-1R) type 1 (IL1R1), and the IL-1R accessory protein (IL1RAP), initiates this signaling. However, how the IL1R1-associated complex is regulated remains elusive. Angiopoietin like 3 (ANGPTL3), a key inhibitor of plasma triglyceride clearance, is mainly expressed in the liver and exists in both intracellular and extracellular secreted forms. Currently, ANGPTL3 has emerged as a highly promising drug target for hypertriglyceridemia and associated cardiovascular diseases. However, most studies have focused on the secreted form of ANGPTL3, while its intracellular role is still largely unknown. Here, we report that intracellular ANGPTL3 acts as a negative regulator of IL-1β-triggered signaling. Overexpression of ANGPTL3 inhibited IL-1β-induced NF-κB activation and the transcription of inflammatory genes in HepG2, THP1, and HEK293T cells, while knockdown or knockout of ANGPTL3 resulted in opposite effects. Mechanistically, ANGPTL3 interacted with IL1R1 and IL1RAP through its intracellular C-terminal fibrinogen-like domain and disrupted the assembly of the IL1R1-associated complex. Taken together, our study reveals a novel role for ANGPTL3 in inflammation, whereby it inhibits the physiological interaction between IL1R1 and IL1RAP to maintain immune tolerance and homeostasis in the liver.

A hepatokine derived from the ER protein CREBH promotes triglyceride metabolism by stimulating lipoprotein lipase activity.

The endoplasmic reticulum (ER)-tethered, liver-enriched stress sensor CREBH is processed in response to increased energy demands or hepatic stress to release an amino-terminal fragment that functions as a transcription factor for hepatic genes encoding lipid and glucose metabolic factors. Here, we discovered that the carboxyl-terminal fragment of CREBH (CREBH-C) derived from membrane-bound, full-length CREBH was secreted as a hepatokine in response to fasting or hepatic stress. Phosphorylation of CREBH-C mediated by the kinase CaMKII was required for efficient secretion of CREBH-C through exocytosis. Lipoprotein lipase (LPL) mediates the lipolysis of circulating triglycerides for tissue uptake and is inhibited by a complex consisting of angiopoietin-like (ANGPTL) 3 and ANGPTL8. Secreted CREBH-C blocked the formation of ANGPTL3-ANGPTL8 complexes, leading to increased LPL activity in plasma and metabolic tissues in mice. CREBH-C administration promoted plasma triglyceride clearance and partitioning into peripheral tissues and mitigated hypertriglyceridemia and hepatic steatosis in mice fed a high-fat diet. Individuals with obesity had higher circulating amounts of CREBH-C than control individuals, and human CREBH loss-of-function variants were associated with dysregulated plasma triglycerides. These results identify a stress-induced, secreted protein fragment derived from CREBH that functions as a hepatokine to stimulate LPL activity and triglyceride homeostasis.

The zinc finger and BTB domain containing protein ZBTB20 regulates plasma triglyceride metabolism by repressing lipoprotein lipase gene transcription in hepatocytes.

Lipoprotein lipase (LPL) is responsible for the lipolytic processing of triglyceride-rich lipoproteins, the deficiency of which causes severe hypertriglyceridemia. Liver LPL expression is high in suckling rodents but relatively low at adulthood. However, the regulatory mechanism and functional significance of liver LPL expression are incompletely understood. We have established the zinc finger protein ZBTB20 as a critical factor for hepatic lipogenesis. Here, we evaluated the role of ZBTB20 in regulating liver Lpl gene transcription and plasma triglyceride metabolism. Hepatocyte-specific inactivation of ZBTB20 in mice led to a remarkable increase in LPL expression at the mRNA and protein levels in adult liver, in which LPL protein was mainly localized onto sinusoidal epithelial cells and Kupffer cells. As a result, the LPL activity in postheparin plasma was substantially increased, and postprandial plasma triglyceride clearance was significantly enhanced, whereas plasma triglyceride levels were decreased. The dysregulated liver LPL expression and low plasma triglyceride levels in ZBTB20-deficient mice were normalized by inactivating hepatic LPL expression. ZBTB20 deficiency protected the mice against high-fat diet-induced hyperlipidemia without causing excessive triglyceride accumulation in the liver. Chromatin immunoprecipitation and gel-shift assay studies revealed that ZBTB20 binds to the LPL promoter in the liver. A luciferase reporter assay revealed that ZBTB20 inhibits the transcriptional activity of LPL promoter. The regulation of LPL expression by ZBTB20 is liver-specific under physiological conditions. Liver ZBTB20 serves as a key regulator of LPL expression and plasma triglyceride metabolism and could be a therapeutic target for hypertriglyceridemia.

Regulation of plasma triglyceride partitioning by adipose-derived ANGPTL4 in mice

Elevated plasma triglyceride levels are associated with metabolic disease. Angiopoietin-like protein 4 (ANGPTL4) regulates plasma triglyceride levels by inhibiting lipoprotein lipase (LPL). Our aim was to investigate the role of adipocyte-specific deficiency of ANGPTL4 in mice during high fat diet feeding. Adipocyte-specific ANGPTL4 deficient mice were fed a high fat diet (60% kCal from fat) for either 12 weeks or 6 months. We performed plasma metabolic measurements, triglyceride clearance and uptake assays, LPL activity assays, and assessed glucose homeostasis. Mice lacking adipocyte ANGPTL4 recapitulated the triglyceride phenotypes of whole-body ANGPTL4 deficiency, including increased adipose LPL activity, lower plasma triglyceride levels, and increased uptake of triglycerides into adipose tissue. When fed a high fat diet (HFD), these mice continued to display enhanced adipose LPL activity and initially had improved glucose and insulin sensitivity. However, after 6 months on HFD, the improvements in glucose homeostasis were largely lost. Moreover, despite higher adipose LPL activity levels, mice lacking adipocyte ANGPTL4 no longer had increased triglyceride uptake into adipose compared to littermate controls after chronic high-fat feeding. These observations suggest that after chronic high-fat feeding LPL is no longer rate-limiting for triglyceride delivery to adipocytes. We conclude that while adipocyte-derived ANGPTL4 is an important regulator of plasma triglyceride levels and triglyceride partitioning under normal diet conditions, its role is diminished after chronic high-fat feeding.

Liver-selective γ-secretase inhibition ameliorates diet-induced hepatic steatosis, dyslipidemia and atherosclerosis

Hepatic γ-secretase regulates low-density lipoprotein receptor (LDLR) cleavage and degradation, affecting clearance of plasma triglyceride (TG)-rich lipoproteins (TRLs). In this study, we investigated whether γ-secretase inhibition modulates risk of Western (high-fat/sucrose and high-cholesterol)-type diet (WTD)-induced hepatic steatosis, dyslipidemia and atherosclerosis. We evaluated liver and plasma lipids in WTD-fed mice with hepatocyte-specific ablation of the non-redundant γ-secretase-targeting subunit Nicastrin (L-Ncst). In parallel, we investigated the effect of liver-selective Ncst antisense oligonucleotides (ASO) on lipid metabolism and atherosclerosis in wildtype (WT) and ApoE knockout (ApoE−/-) mice fed normal chow or WTD. WTD-fed L-Ncst and Ncst ASO-treated WT mice showed reduced total cholesterol and LDL-cholesterol (LDL-C), as well as reduced hepatic lipid content as compared to Cre- and control ASO-treated WT mice. Treatment of WTD-fed ApoE−/- mice with Ncst ASO markedly lowered total and LDL cholesterol, hepatic TG and attenuated atherosclerotic lesions in the aorta, as compared to control ASO-treated mice. L-Ncst and Ncst ASO similarly showed reduced plasma glucose as compared to control mice. In conclusion, inhibition of hepatic γ-secretase reduces plasma glucose, and attenuates WTD-induced dyslipidemia, hepatic fat accumulation and atherosclerosis, suggesting potential pleiotropic application for diet-induced metabolic dysfunction.

The Effect of Acute Continuous Hypoxia on Triglyceride Levels in Constantly Fed Healthy Men.

IntroductionElevated plasma triglyceride (TG) concentrations are an important contributor to deleterious metabolic alterations. Evidence in animals suggest that acute exposure to an environment with reduced oxygen inhibits plasma TG clearance and causes important rise in plasma TG, especially in the postprandial state. The objective of this study was to characterize the effects of an acute exposure to normobaric hypoxia on prandial TG levels in 2 distinct lipoprotein subtypes in healthy humans: chylomicrons which are secreted by the intestine and carry dietary lipids, and denser TG carriers (mainly VLDL), which are secreted by the liver and carry endogenous lipids. Plasma lipolytic activity was also assessed. It was hypothesized that hypoxia would reduce prandial plasma lipolytic activity and raise prandial TG levels in both lipoprotein subtypes.MethodsUsing a randomized crossover design, 9 healthy young men were studied for 6 h in a constantly fed state while being exposed to either normobaric hypoxia (FiO2 = 0.12) and normoxic conditions on two different days. Prandial glucose, TG, non-esterified fatty acid (NEFA), and post-heparin plasma lipolytic activity were measured during each session.ResultsSix hours of exposure to hypoxia marginally increase prandial glycemia (+5%, p = 0.06) while increasing insulinemia by 40% (p = 0.04). Hypoxia induced a 30% rise in prandial NEFA levels and tended to slightly increased total prandial TG levels by 15% (p = 0.11). No difference was observed in TG concentrations and metabolism of chylomicrons between conditions. However, TG in the VLDL containing fraction decreased significantly overtime under normoxia but not under hypoxia (time × condition interaction, p = 0.02). No difference was observed in post-heparin plasmatic lipolytic activity between conditions.ConclusionAcute hypoxia in healthy men tends to increase prandial VLDL-TG levels. These results lend support to the increased blood lipid levels reported in animals exposed acutely to lower partial pressures of oxygen.

Novel GPIHBP1-independent pathway for clearance of plasma TGs in Angptl4−/−Gpihbp1−/− mice

Mice lacking glycosylphosphatidylinositol-anchored HDL-binding protein 1 (GPIHBP1) are unable to traffic LPL to the vascular lumen. Thus, triglyceride (TG) clearance is severely blunted, and mice are extremely hypertriglyceridemic. Paradoxically, mice lacking both GPIHBP1 and the LPL regulator, angiopoietin-like 4 (ANGPTL4), are far less hypertriglyceridemic. We sought to determine the mechanism by which Angptl4-/-Gpihbp1-/- double-knockout mice clear plasma TGs. We confirmed that, on a normal chow diet, plasma TG levels were lower in Angptl4-/-Gpihbp1-/- mice than in Gpihbp1-/- mice; however, the difference disappeared with administration of a high-fat diet. Although LPL remained mislocalized in double-knockout mice, plasma TG clearance in brown adipose tissue (BAT) increased compared with Gpihbp1-/- mice. Whole lipoprotein uptake was observed in the BAT of both Gpihbp1-/- and Angptl4-/-Gpihbp1-/- mice, but BAT lipase activity was significantly higher in the double-knockout mice. We conclude that Angptl4-/-Gpihbp1-/- mice clear plasma TGs primarily through a slow and noncanonical pathway that includes the uptake of whole lipoprotein particles.

Chylomicron-Derived Fatty Acid Spillover in Adipose Tissue: A Signature of Metabolic Health?

Context and Objectives:Spillover of fatty acids (FAs) into the plasma nonesterified fatty acid (NEFA) pool, because of an inability of adipose tissue (AT) to accommodate sufficient fat uptake, has been suggested to contribute to obesity-related insulin resistance. Using specific labeling techniques, we compared the proportion of spillover-derived NEFA across a range of adiposity.Participants and Methods:Seventy-one healthy men and women were fed a mixed meal (40 g fat) containing [U13C]palmitate to assess the contribution of chylomicron-derived spillover FAs. To investigate subcutaneous abdominal-specific spillover, arteriovenous difference and stable-isotope methodologies were used in substudy (six men, six women).Results:Chylomicron-derived FA spillover was higher in individuals with a BMI <25 kg/m2 (n = 18) compared with those with a BMI ≥25 kg/m2 (n = 53) (22.2 ± 1.6% vs 18.6 ± 0.7%, P = 0.02). Women had higher chylomicron-derived FA spillover than age- and BMI-matched men (21.9 ± 1.1% vs 15.0 ± 1.6%, P = 0.001). Assessing spillover across subcutaneous abdominal AT showed higher proportions in women than in men (28.5 ± 6.1% vs 9.9 ± 1.3%, P = 0.01).Conclusion:There is a considerable degree of spillover FA into the systemic NEFA pool in the postprandial state; this process is greater and more dynamic in lean individuals and women. Contrary to general perception, spillover of chylomicron-derived FA into systemic circulation is a physiologically normal feature most easily observed in people with a higher capacity for clearance of plasma triglycerides, but does not appear to be a pathway providing excess NEFA in obesity.

ANGPTL8 promotes the ability of ANGPTL3 to bind and inhibit lipoprotein lipase

ObjectiveSeveral members of the angiopoietin-like (ANGPTL) family of proteins, including ANGPTL3 and ANGPTL8, regulate lipoprotein lipase (LPL) activity. Deficiency in either ANGPTL3 or ANGPTL8 reduces plasma triglyceride levels and increases LPL activity, whereas overexpression of either protein does the opposite. Recent studies suggest that ANGPTL8 may functionally interact with ANGPTL3 to alter clearance of plasma triglycerides; however, the nature of this interaction has remained elusive. We tested the hypothesis that ANGPTL8 forms a complex with ANGPTL3 and that this complex is necessary for the inhibition of vascular LPL by ANGPTL3. MethodsWe analyzed the interactions of ANGPTL3 and ANGPTL8 with each other and with LPL using co-immunoprecipitation, western blotting, lipase activity assays, and the NanoBiT split-luciferase system. We also used adenovirus injection to overexpress ANGPTL3 in mice that lacked ANGPTL8. ResultsWe found that ANGPTL3 or ANGPTL8 alone could only inhibit LPL at concentrations that far exceeded physiological levels, especially when LPL was bound to its endothelial cell receptor/transporter GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1). Physical interaction was observed between ANGPTL3 and ANGPTL8 when the proteins were co-expressed, and co-expression with ANGPTL3 greatly enhanced the secretion of ANGPTL8. Importantly, ANGPTL3–ANGPTL8 complexes had a dramatically increased ability to inhibit LPL compared to either protein alone. Adenovirus experiments showed that 2-fold overexpression of ANGPTL3 significantly increased plasma triglycerides only in the presence of ANGPTL8. Protein interaction assays showed that ANGPTL8 greatly increased the ability of ANGPTL3 to bind LPL. ConclusionsTogether, these data indicate that ANGPTL8 binds to ANGPTL3 and that this complex is necessary for ANGPTL3 to efficiently bind and inhibit LPL.

ANGPTL8 requires ANGPTL3 to inhibit lipoprotein lipase and plasma triglyceride clearance

Angiopoietin-like (ANGPTL)3 and ANGPTL8 are secreted proteins and inhibitors of LPL-mediated plasma triglyceride (TG) clearance. It is unclear how these two ANGPTL proteins interact to regulate LPL activity. ANGPTL3 inhibits LPL activity and increases serum TG independent of ANGPTL8. These effects are reversed with an ANGPTL3 blocking antibody. Here, we show that ANGPTL8, although it possesses a functional inhibitory motif, is inactive by itself and requires ANGPTL3 expression to inhibit LPL and increase plasma TG. Using a mutated form of ANGPTL3 that lacks LPL inhibitory activity, we demonstrate that ANGPTL3 activity is not required for its ability to activate ANGPTL8. Moreover, coexpression of ANGPTL3 and ANGPTL8 leads to a far more efficacious increase in TG in mice than ANGPTL3 alone, suggesting the major inhibitory activity of this complex derives from ANGPTL8. An antibody to the C terminus of ANGPTL8 reversed LPL inhibition by ANGPTL8 in the presence of ANGPTL3. The antibody did not disrupt the ANGPTL8:ANGPTL3 complex, but came in close proximity to the LPL inhibitory motif in the N terminus of ANGPTL8. Collectively, these data show that ANGPTL8 has a functional LPL inhibitory motif, but only inhibits LPL and increases plasma TG levels in mice in the presence of ANGPTL3.

ANGPTL8 Promotes the Ability of ANGPTL3 to Inhibit Lipoprotein Lipase

Lipoprotein lipase (LPL) is critically important for the delivery of plasma lipoprotein–derived triglycerides to peripheral tissues. Activity of LPL is carefully regulated and misregulation of LPL can contribute to metabolic diseases including obesity, metabolic syndrome, and cardiovascular disease. Several members of the angiopoietin‐like (ANGPTL) family of proteins, including ANGPTL3 and ANGPTL8, regulate LPL activity. Deficiency in either ANGPTL3 or ANGPTL8 reduces plasma triglyceride levels and increases LPL activity, whereas overexpression of either protein does the opposite. Recent studies showed that ANGPTL8 overexpression only raises plasma triglycerides when ANGPTL3 is present suggesting that ANGPTL3 and ANGPTL8 work together to alter clearance of plasma triglycerides, however the nature of this interaction has remained elusive. We tested the hypothesis that ANGPTL8 forms a complex with ANGPTL3 and that this complex is necessary for efficient inhibition of LPL. We found that ANGPTL3 or ANGPTL8 alone could inhibit LPL only at concentrations that far exceeded physiological levels, especially when LPL was bound to its endothelial cell receptor/transporter GPIHBP1 (glycosylphosphatidylinositol‐anchored high‐density lipoprotein binding protein 1). Physical interaction was observed between ANGPTL3 and ANGPTL8 when the proteins were co‐expressed, and co‐expression with ANGPTL3 greatly enhanced the secretion of ANGPTL8. Importantly, the ability of ANGPTL3‐ANGPTL8 complexes to inhibit LPL was dramatically increased compared to either protein alone. Part of this improvement could be ascribed to an improved ability to bind LPL as protein interaction assays showed that ANGPTL8 increased the ability of ANGPTL3 to bind LPL. Together, these data suggest that ANGPTL8 binds to ANGPTL3, forming a complex that has an increased ability to bind and inhibit LPL.Support or Funding InformationThis work was supported by grants from the NHLBI (HL130146) and the Carver Medical Research Initiative

ANGPTL8 Blockade With a Monoclonal Antibody Promotes Triglyceride Clearance, Energy Expenditure, and Weight Loss in Mice

Angiopoietin-like protein (ANGPTL)8 is a negative regulator of lipoprotein lipase-mediated plasma triglyceride (TG) clearance. In this study, we describe a fully human monoclonal antibody (REGN3776) that binds monkey and human ANGPTL8 with high affinity. Inhibition of ANGPTL8 with REGN3776 in humanized ANGPTL8 mice decreased plasma TGs and increased lipoprotein lipase activity. Additionally, REGN3776 reduced body weight and fat content. The reduction in body weight was secondary to increased energy expenditure. Finally, single administration of REGN3776 normalized plasma TGs in dyslipidemic cynomolgus monkeys. In conclusion, we show that blockade of ANGPTL8 with monoclonal antibody strongly reduced plasma TGs in mice and monkeys. These data suggest that inhibition of ANGPTL8 may provide a new therapeutic avenue for the treatment of dyslipidemia with beneficial effects on body weight.

Angptl4 does not control hyperglucagonemia or α-cell hyperplasia following glucagon receptor inhibition

Genetic disruption or pharmacologic inhibition of glucagon signaling effectively lowers blood glucose but results in compensatory glucagon hypersecretion involving expansion of pancreatic α-cell mass. Ben-Zvi et al. recently reported that angiopoietin-like protein 4 (Angptl4) links glucagon receptor inhibition to hyperglucagonemia and α-cell proliferation [Ben-Zvi et al. (2015) Proc Natl Acad Sci USA 112:15498-15503]. Angptl4 is a secreted protein and inhibitor of lipoprotein lipase-mediated plasma triglyceride clearance. We report that Angptl4-/- mice treated with an anti-glucagon receptor monoclonal antibody undergo elevation of plasma glucagon levels and α-cell expansion similar to wild-type mice. Overexpression of Angptl4 in liver of mice caused a 8.6-fold elevation in plasma triglyceride levels, but did not alter plasma glucagon levels or α-cell mass. Furthermore, administration of glucagon receptor-blocking antibody to healthy individuals increased plasma glucagon and amino acid levels, but did not change circulating Angptl4 concentration. These data show that Angptl4 does not link glucagon receptor inhibition to compensatory hyperglucagonemia or expansion of α-cell mass, and that it cannot be given to induce such secretion and growth. The reduction of plasma triglyceride levels in Angptl4-/- mice and increase following Angptl4 overexpression suggest that changes in plasma triglyceride metabolism do not regulate α-cells in the pancreas. Our findings corroborate recent data showing that increased plasma amino acids and their transport into α-cells link glucagon receptor blockage to α-cell hyperplasia.

Loss of Transcription Factor CREBH Accelerates Diet-Induced Atherosclerosis in Ldlr-/- Mice.

Liver-enriched transcription factor cAMP-responsive element-binding protein H (CREBH) regulates plasma triglyceride clearance by inducing lipoprotein lipase cofactors, such as apolipoprotein A-IV (apoA-IV), apoA-V, and apoC-II. CREBH also regulates apoA-I transcription. This study aims to determine whether CREBH has a role in lipoprotein metabolism and development of atherosclerosis. CREBH-deficient Creb3l3(-/-) mice were bred with Ldlr(-/-) mice creating Ldlr(-/-) Creb3l3(-/-) double knockout mice. Mice were fed on a high-fat and high-sucrose Western diet for 20 weeks. We showed that CREBH deletion in Ldlr(-/-) mice increased very low-density lipoprotein-associated triglyceride and cholesterol levels, consistent with the impairment of lipoprotein lipase-mediated triglyceride clearance in these mice. In contrast, high-density lipoprotein cholesterol levels were decreased in CREBH-deficient mice, which was associated with decreased production of apoA-I from the liver. The results indicate that CREBH directly activated Apoa1 gene transcription. Accompanied by the worsened atherogenic lipid profile, Ldlr(-/-) Creb3l3(-/-) mice developed significantly more atherosclerotic lesions in the aortas than Ldlr(-/-) mice. We identified CREBH as an important regulator of lipoprotein metabolism and suggest that increasing hepatic CREBH activity may be a novel strategy for prevention and treatment of atherosclerosis.

Regulation of lipid metabolism by angiopoietin-like proteins.

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. 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. 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.

Abstract 199: The Regulation of Intestinal apoC-III by Dietary Fat: Key Differences From Hepatic apoC-III Regulation and Implications for Lipoprotein Synthesis and Secretion

Apolipoprotein C-III (apoC-III) influences both plasma triglycerides and inflammation, and as such is a particularly important target for mediating cardiovascular disease (CVD). ApoC-III is an exchangeable apolipoprotein expressed in both liver and intestine; in humans, apoC-III levels in plasma are an independent predictor of CVD risk. ApoC-III is a potent regulator of plasma triglycerides through effects on intestinal lipid absorption, plasma triglyceride clearance, hepatic lipoprotein uptake, and VLDL secretion. ApoC-III also acts as a signaling molecule modulating vascular function. Hepatic apoC-III is regulated by insulin via the transcription factor, forkhead box protein O1 (FOXO1), and by glucose via carbohydrate-responsive element-binding protein (ChREBP). Though much is known about hepatic apoC-III, little is known about the regulation of intestinal apoC-III (including the primary stimulus for its expression). We have found that apoC-III is secreted from the intestine in response to dietary carbohydrate, and that a fish oil-enriched diet lowers intestinal apoC-III mRNA expression. Our findings suggest that ChREBP is the primary modulator of apoC-III expression in the intestine. While dietary fish oil lowers both hepatic and intestinal apoC-III mRNA levels, it appears to do so through distinctive mechanisms. This is a potentially important difference since apoC-III is a key regulator of post-prandial plasma lipid levels and inflammation.