VLDL-TG Production Research Articles

Lactation alters the relationship between liver lipid synthesis and hepatic fat stores in the postpartum period

In mothers who are nursing their infants, increased clearance of plasma metabolites into the mammary gland may reduce ectopic lipid in the liver. No study to date has investigated the role of lactation on liver lipid synthesis in humans, and we hypothesized that lactation would modify fatty acid and glucose handling to support liver metabolism in a manner synchronized with the demands of milk production. Lactating (n = 18) and formula-feeding women (n = 10) underwent metabolic testing at 6-week postpartum to determine whether lactation modified intrahepatic triacylglycerols (IHTGs), measured by proton magnetic resonance spectroscopy. Subjects ingested oral deuterated water to measure fractional de novo lipogenesis (DNL) in VLDL-TG during fasting and during an isotope-labeled clamp at an insulin infusion rate of 10 mU/m2/min. Compared with formula-feeding women, we found that lactating women exhibited lower plasma VLDL-TG concentrations, similar IHTG content and similar contribution of DNL to total VLDL-TG production. These findings suggest that lactation lowers plasma VLDL-TG concentrations for reasons that are unrelated to IHTG and DNL. Surprisingly, we determined that the rate of appearance of nonesterified fatty acids was not related to IHTG in either group, and the expected positive association between DNL and IHTG was only significant in formula-feeding women. Further, in lactating women only, the higher the prolactin concentration, the lower the IHTG, while greater DNL strongly associated with elevations in VLDL-TG. In conclusion, we suggest that future studies should investigate the role of lactation and prolactin in liver lipid secretion and metabolism.

Physiological interindividual variability in endogenous estradiol concentration does not influence adipose tissue and hepatic lipid kinetics in women.

Increased triglyceride (TG) and apolipoprotein B-100 (apoB-100) concentrations in plasma are important risk factors for cardiovascular disease in women. Administration of some estrogen preparations raises plasma TG and apoB-100 concentrations by increasing hepatic very low-density lipoprotein (VLDL) TG and apoB-100 secretion rates. However, the influence of physiological variation in endogenous estradiol on VLDL-TG and VLDL-apoB-100 metabolism and on free fatty acid (FFA) release into plasma (the major source of fatty acids for VLDL-TG production) is not known. We measured basal VLDL-TG, VLDL-apoB-100, and plasma FFA kinetics by using stable isotopically labeled tracers in 36 eumenorrheic, premenopausal women (age: 33 ± 2 years, BMI: 31 ± 1 kg/m2; mean ± s.e.m.) during the follicular phase of the menstrual cycle; participants were divided into two groups based on low (n = 18) or high (n = 18) plasma estradiol concentrations (defined as below or above the median value of 140 pmol/L in the whole group). Mean plasma estradiol concentration was >3-fold higher in the high-estradiol than in the low-estradiol group (299 ± 37 and 96 ± 7 pmol/L, P < 0.001); there was no difference in plasma progesterone concentrations between the two groups (P = 0.976). There were no significant differences in plasma FFA concentration, FFA rate of appearance in plasma, VLDL-TG and VLDL-apoB-100 concentrations, hepatic VLDL-TG and VLDL-apoB-100 secretion rates, VLDL-TG and VLDL-apoB-100 plasma clearance rates, and mean residence times (all P ≥ 0.45). No significant associations were found between plasma estradiol concentration and FFA, VLDL-TG, and VLDL-apoB-100 concentrations and kinetics (all P > 0.19). Plasma estradiol concentration is not an important correlate of basal plasma FFA, VLDL-TG, and VLDL-apoB-100 kinetics in premenopausal women.

Cannabinoid type 1 receptor inverse agonism attenuates dyslipidemia and atherosclerosis in APOE∗3-Leiden.CETP mice

Pharmacological blockade of the cannabinoid type 1 receptor, a G protein-coupled receptor expressed in the central nervous system and various peripheral tissues, reverses diet-induced obesity and dyslipidemia through the reduction of food intake and altered nutrient partitioning. This strategy is being explored for a number of therapeutic applications; however, its potency for the treatment of atherosclerotic cardiovascular disease via improvements in lipid metabolism remains unclear. Therefore, here, we aimed to investigate whether inhibition of the endocannabinoid system can attenuate atherosclerosis development through improvement of dyslipidemia. Lean, dyslipidemic female APOE∗3-Leiden.CETP transgenic mice were fed a Western-type diet supplemented with or without the cannabinoid type 1 receptor inverse agonist rimonabant (20 mg·kg body weight−1 day−1) for up to 20 weeks. Plasma lipids and bile acids were determined, and atherosclerotic lesions were scored in the aortic valve region. Rimonabant lowered plasma levels of triglyceride (TG) (−56%) and non-HDL-C (−19%) and increased HDL-C (+57%). These effects were explained by decreased VLDL-TG production (−52%) and accelerated VLDL-TG turnover accompanied by pronounced browning of white adipose tissue. In addition, rimonabant attenuated reverse cholesterol transport (−30%), increased plasma bile acid levels (+160%), and increased hepatic cholesterol accumulation (+88%). Importantly, rimonabant markedly lowered atherosclerotic lesion size (−64%), which coincided with decreased lesion severity (28% vs. 56% severe lesions) and which strongly correlated with non-HDL-C exposure (R2 = 0.60). Taken together, inhibition of the endocannabinoid system potently reverses dyslipidemia and prevents atherogenesis, even in the absence of obesity.

312-OR: Gender-Specific Effects on Hepatic Fat Metabolism in Type 2 Diabetes before and after Remission

Men are more susceptible to type 2 diabetes and NAFLD than women, possibly secondary to differences in hepatic lipoprotein metabolism. The Tyneside cohort of Diabetes Remission Clinical Trial (34M/30F, 52.3±8.0 years, BMI 35.1±4.5kg/m2) were studied at baseline and to 24 months after weight loss. 3-point Dixon MRI was used for intra-organ fat. Hepatic VLDL-TG production was measured using a competitive blocking method. In the nondiabetic group, both liver and pancreas fat differed markedly in men and women (5.4± 1.1 vs. 3.4±0.1%, p=0.005, and 7.6±0.5 vs. 4.7±0.4%, p=0.0006, respectively). In diabetes, both liver and pancreas fat were similar in men and women at baseline (15.4±1.9 vs. 16.9±1.9%, p=0.57; and 8.5±0.4 vs. 8.4±0.5%, p=0.66, respectively). They decreased similarly after weight loss in both men and women (liver fat: 2.6±0.6% and 3.6±0.7%; pancreas fat: 7.6 ±0.4% and 7.5±0.5%, p&amp;lt;0.0001 vs. baseline for both). VLDL-TG production was also similar at baseline (554±39 vs. 559±33mg/kg/day, p=0.96) and after weight loss (418±28 vs. 448±37mg/kg/day, p=0.29). Women had a lower VLDL-TG pool size (p=0.002), lower plasma VLDL-TG (p&amp;lt;0.0001), lower fasting glucagon (p&amp;lt;0.05), higher adiponectin (p&amp;lt;0.001), and lower visceral adipose tissue (p&amp;lt;0.0001) volumes than men regardless of diabetes status. However, diabetic women had higher: subcutaneous fat (p&amp;lt;0.0001), leptin (p&amp;lt;0.0001), fasting NEFA (p=0.004), GDF-15 (p=0.044), and FGF-21 (p=0.008). After 24 months of intervention, men and women remained significantly different in VLDL-TG pool, NEFA, SAT, VAT, leptin, and adiponectin (p&amp;lt;0.05 for all). Women are likely to process lipoproteins more rapidly and safely store VLDL-TG. This minimizes delivery of toxic lipid metabolites from VLDL-TG to pancreatic islets. These data demonstrate important gender differences in hepatic fat handling, and underscore the importance of considering this when designing metabolic studies. Disclosure A. Al-Mrabeh: None. S. Melhem: None. S.V. Zhyzhneuskaya: None. C. Peters: Speaker’s Bureau; Self; Sanofi-Aventis. A.C. Barnes: None. K.G. Hollingsworth: None. N. Sattar: Advisory Panel; Self; Amgen, AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk A/S, Pfizer Inc., Sanofi. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. M.E. Lean: None. R. Taylor: Speaker’s Bureau; Self; Novo Nordisk Foundation. Funding Diabetes UK

Niacin increases diet-induced hepatic steatosis in B6129 mice

Nonalcoholic fatty liver disease (NAFLD) is a very common disorder affecting between 20 and 30% of adults in the United States. However, there is no effective pharmacotherapy for treating NAFLD. Niacin, a water-soluble vitamin (B3), at pharmacological doses, decreases hepatic triglyceride (TG) content in NAFLD through inhibition of diacylglycerol acyltransferase 2, a key enzyme that catalyzes the final step in TG synthesis. Alternatively, some studies indicate that niacin induces fatty liver in high-fat diet (HFD)-fed rats. Therefore, in this study we investigated whether niacin is beneficial in treating NAFLD in two strains of mice, C57BL/6J (B6) and B6129SF2/J (B6129) mice, with 20 weeks of HFD feeding. Niacin treatment was started from week 5 until the end of the study. Niacin treatment increased normalized liver weight, hepatic TG content and NAFLD score in HFD-fed B6129 mice but had no impact on B6 mice. Metabolomics analysis revealed that in B6129 mice, 4-hydroxyphenylpyruvic acid (4-HPP), which is associated with fatty acid oxidation, did not change with HFD feeding but significantly decreased with niacin treatment. Lipidomics analysis discovered that the abundance of phosphocholine (PC), which is critical for very low-density lipoprotein (VLDL)–TG production and secretion, was decreased in HFD-fed B6129 with niacin treatment. In conclusion, niacin had no impact on diet-induced NAFLD development in B6 mice but potentiated hepatic steatosis in HFD-fed B6129 mice due to impaired fatty acid oxidation and decreased VLDL-TG production and secretion.

1844-P: Long-Term Remission of Type 2 Diabetes after Weight Loss Requires Effective Clearance of Hepatic VLDL-TG and Maintenance of Low Intrapancreatic Fat

The Diabetes Remission Clinical Trial (DiRECT) demonstrated initial remission of type 2 diabetes in about half of a large primary care population, and this was associated with decrease in intrapancreatic fat content and return of beta cell function. We have carried out detailed metabolic studies on a subgroup at 2 years, comparing those who remained in remission (HbA1c &amp;lt;6.5%; n=20, 7F/13M, 54.6±7.3 years, BMI 34.5±4.9kg/m2), and those who failed to maintain the initial remission (n=13, 6F/7M, 52.6±8.3 years, BMI 34.9±3.7kg/m2). Pancreas and liver fat were quantified by 3-point Dixon MRI. Hepatic VLDL-triglyceride (VLDL1-TG) production was measured in vivo using a non-isotopic competitive blocking method. Remission of diabetes was associated with major decreases in liver fat, pancreas fat, and hepatic VLDL-TG production during the weight loss phase between baseline to 5 months in both groups. However, those who failed to maintain remission were characterized by more weight regain (11. 3±1.9 vs. 6.6±1.0 kg, p=0.036), and greater increases in both liver (6.2±1.1 vs. 3.6±0.9%, p=0.05), and pancreas fat (+1.0±0.39 vs. -0.48±0.25%, p=0.005) between 5 months and 24 months. Hepatic VLDL-TG pool level remained stable under remission conditions (1046.4± 204.6 to 1638.6± 372.11 mg, p=0.35), whereas there was a significant increase when remission was lost (1328.0± 271.8 to 2865.6±692.4mg, p=0.04). In those who failed to maintain remission, fasting VLDL-TG increased (0.46±0.10 to 0.84±0.17 mmol/l, p&amp;lt;0.05), and there was a positive correlation between the rise in liver fat and intrapancreatic fat (r=0.64, p=0.018). These data emphasize the link between hepatic VLDL-TG-metabolism and accumulation of fat within the liver and pancreas in the pathogenesis of type 2 diabetes, provide further evidence for the role of fat in its pathophysiology, and show that longer term remission of type 2 diabetes is dependent upon avoidance of weight regain. Disclosure A. Al-Mrabeh: None. S.V. Zhyzhneuskaya: None. A.C. Barnes: None. K.G. Hollingsworth: None. N. Sattar: Advisory Panel; Self; Amgen Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Novo Nordisk A/S. Consultant; Self; NAPP Pharmaceuticals Limited. Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. Speaker's Bureau; Self; Amgen Inc., Eli Lilly and Company, Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Roche Diagnostics France, Sanofi. M.E. Lean: Consultant; Self; Counterweight Ltd., Novo Nordisk A/S. Research Support; Self; Novo Nordisk A/S. R. Taylor: Advisory Panel; Self; Wilmington Healthcare. Speaker's Bureau; Self; Lilly Diabetes, Novartis AG. Funding Diabetes UK

Cholesteryl ester transfer protein alters liver and plasma triglyceride metabolism through two liver networks in female mice

Elevated plasma TGs increase risk of cardiovascular disease in women. Estrogen treatment raises plasma TGs in women, but molecular mechanisms remain poorly understood. Here we explore the role of cholesteryl ester transfer protein (CETP) in the regulation of TG metabolism in female mice, which naturally lack CETP. In transgenic CETP females, acute estrogen treatment raised plasma TGs 50%, increased TG production, and increased expression of genes involved in VLDL synthesis, but not in nontransgenic littermate females. In CETP females, estrogen enhanced expression of small heterodimer partner (SHP), a nuclear receptor regulating VLDL production. Deletion of liver SHP prevented increases in TG production and expression of genes involved in VLDL synthesis in CETP mice with estrogen treatment. We also examined whether CETP expression had effects on TG metabolism independent of estrogen treatment. CETP increased liver β-oxidation and reduced liver TG content by 60%. Liver estrogen receptor α (ERα) was required for CETP expression to enhance β-oxidation and reduce liver TG content. Thus, CETP alters at least two networks governing TG metabolism, one involving SHP to increase VLDL-TG production in response to estrogen, and another involving ERα to enhance β-oxidation and lower liver TG content. These findings demonstrate a novel role for CETP in estrogen-mediated increases in TG production and a broader role for CETP in TG metabolism.

Abstract 397: Transgenic Expression of Cholesteryl Ester Transfer Protein in Female Mice Disrupts Liver and Plasma Triglyceride Metabolism With Estrogen Treatment in a Liver Network Involving Estrogen Receptor Alpha and Small Heterodimer Partner

Background: Triglycerides are an important risk factor for cardiovascular disease, especially in women. Hormone replacement therapy raises triglycerides (TG) in postmenopausal women, but mechanisms responsible remain poorly understood. Cholesteryl Ester Transfer Protein (CETP) is a serum protein that shuttles TG and cholesteryl ester between lipoproteins in humans and plays an important role in lipoprotein metabolism. The role of CETP in regulating whole body triglyceride homeostasis remains unclear. Mice (which naturally lack CETP) expressing a transgenic copy of CETP or their wild-type (WT) littermates were used to determine the role of CETP in regulating plasma and liver TG metabolism in response to estrogen. Methods and Results: Female CETP and WT mice were ovariectomized and given vehicle or estrogen treatment. Estrogen raised plasma TG in CETP females, but not in WT females. CETP expression impaired TG clearance independent of estrogen. Estrogen treatment raised very-low density lipoprotein (VLDL) TG production in CETP females, but not in WT females. Estrogen treatment augmented mRNA expression and protein activity of VLDL assembly and secretion targets (Apob, Mttp, PDI) in CETP females, but not in WT females. Additionally, CETP expression lowered liver TG content and increased mRNA expression of TG oxidation targets (Ppara, Acox1, Acadm, Cpt2). Deletion of liver estrogen receptor alpha prevented CETP-mediated decreases in liver TG content and increases in mRNA expression of TG oxidation targets. Liver-specific deletion of estrogen receptor alpha did not attenuate estrogen-mediated increases in plasma TG and VLDL-TG production in female mice expressing CETP. Deletion of liver small heterodimer partner attenuated CETP-mediated increases in plasma TG and VLDL-TG production with estrogen treatment. Deletion of small heterodimer partner also attenuated CETP-mediated increases in mRNA expression and protein activity of VLDL synthesis and assembly targets with estrogen treatment. Conclusion: These studies indicate several novel functions of CETP in regulating plasma and liver TG homeostasis. We also discovered that CETP expression in female mice also augments several aspects of estrogen action in liver that impact liver TG biology.

Sex-Specific Differences in Hepatic Fat Oxidation and Synthesis May Explain the Higher Propensity for NAFLD in Men.

In most populations a greater proportion of men have hepatic steatosis than women. Sex-specific differences in hepatic dietary fatty acid (FA) metabolism have not been well characterized. We compared fasting and postprandial hepatic FA synthesis (de novo lipogenesis [DNL]) and oxidation in men and women. Fasting and postprandial hepatic FA metabolism was studied in 22 healthy men (n = 11) and women with similar age, body mass index, and liver fat content using metabolic substrates labeled with stable-isotope tracers ((2)H2O and [U(13)C]palmitate). Dietary FA oxidation was assessed by appearance of (13)C into plasma 3-hydroxybutyrate and breath CO2 as markers of liver and whole-body FA oxidation, respectively. Despite similar liver fat content, fasting and postprandial plasma triacylglycerol (TG) concentrations were significantly (P < .05) higher in men compared with women. The appearance of (13)C from dietary FA into plasma 3-hydroxybutyrate and breath CO2 was greater (P < .05) in women compared with men. Although the contribution of DNL into very low-density lipoprotein (VLDL)-TG was similar (∼ 10%) in the fasting state, there was a divergence in pattern over the course of the study, with men maintaining a higher contribution of DNL to VLDL-TG than women (P = .006 time x sex interaction). The combination of lower dietary FA oxidation and a prolonged increase in DNL observed in men may represent partitioning of FA into esterification and storage pathways within the liver, leading to greater VLDL-TG production, and predispose to the sex difference in hepatic steatosis.

Inactivation of ANGPTL3 reduces hepatic VLDL-triglyceride secretion

Humans and mice lacking angiopoietin-like protein 3 (ANGPTL3) have pan-hypolipidemia. ANGPTL3 inhibits two intravascular lipases, LPL and endothelial lipase, and the low plasma TG and HDL-cholesterol levels in ANGPTL3 deficiency reflect increased activity of these enzymes. The mechanism responsible for the low LDL-cholesterol levels associated with ANGPTL3 deficiency is not known. Here we used an anti-ANGPTL3 monoclonal antibody (REGN1500) to inactivate ANGPTL3 in mice with genetic deficiencies in key proteins involved in clearance of ApoB-containing lipoproteins. REGN1500 treatment consistently reduced plasma cholesterol levels in mice in which Apoe, Ldlr, Lrp1, and Sdc1 were inactivated singly or in combination, but did not alter clearance of rabbit (125)I-βVLDL or mouse (125)I-LDL. Despite a 61% reduction in VLDL-TG production, VLDL-ApoB-100 production was unchanged in REGN1500-treated animals. Hepatic TG content, fatty acid synthesis, and fatty acid oxidation were similar in REGN1500 and control antibody-treated animals. Taken together, our findings indicate that inactivation of ANGPTL3 does not affect the number of ApoB-containing lipoproteins secreted by the liver but alters the particles that are made such that they are cleared more rapidly from the circulation via a noncanonical pathway(s). The increased clearance of lipolytic remnants results in decreased production of LDL in ANGPTL3-deficient animals.

Effect of GLP-1 based therapies on diabetic dyslipidemia.

Glucagon-like peptide-1 (GLP-1), is a hormone secreted by small intestine. Consumption of food or glucose stimulates synthesis and secretion of GLP-1 in the bloodstream, which in turn stimulates insulin secretion from pancreas and delays gastric emptying. Owing to the favorable spectrum of effects on reduction of hyperglycemia and body weight, GLP-1 mimetics are intensely pursued as therapies for the treatment of type 2 diabetes (T2DM). Even after intensive control of hyperglycemia, the propensity for cardiovascular disease cannot be totally negated in diabetic patients. A major reason for the cardiovascular disease risk in diabetic patients is underlying dyslipidemia, also termed as diabetic dyslipidemia. It is characterized by high concentrations of triglycerides and LDL cholesterol, and lowered HDL cholesterol in plasma, which are associated with hyperglycemia. Increased insulin resistance gives rise to increased free fatty acids in bloodstream, which is the main reason for the lipid changes appearing in diabetic dyslipidemia. The secondary complications like atherosclerosis and other cardiovascular diseases may be predicted with the blood concentrations of triglycerides and cholesterol, due to the correlation proven in clinic. Hence, new drugs that target diabetic dyslipidemia will always be useful in therapy. Apart from its actions on body weight and glucose, GLP-1 can also regulate cholesterol and triglycerides by numerous ways. Acute and long term treatment with either GLP-1 or its stable analogs reduced fasting as well as postprandial lipids in healthy as well as T2DM patients. GLP-1R signaling reduces VLDL-TG production rate from liver, reduces hepatic TG content by modulating key enzymes of lipid metabolism in liver, and impairs hepatocyte de novo lipogenesis and β-oxidation. GLP-1 can also modulate reverse cholesterol transport. Apart from these direct effects on lipid metabolism, GLP-1 also reduces atherosclerotic events by inhibiting expression of atherogenic inflammatory mediators, suppressing smooth muscle cell proliferation and stimulating NO production. This review mainly deliberates the association of GLP-1 in lipid regulation via lipid absorption, hepatic cholesterol metabolism, reverse cholesterol transport and progression of atherosclerosis.

Abstract 202: Inhibition of Central Melanocortin 4 Receptor Signaling Severely Impairs Brown Adipose Tissue Activity and VLDL Metabolism

Objectives: SNPs near the melanocortin 4 receptor (MC4R) gene are associated with cardiovascular diseases (CVD) and deficiency of MC4R is the most common monogenic cause of obesity, a major risk factor for the development of CVD. MC4R-deficient individuals not only show increased food intake but also lower basal energy expenditure. Furthermore, pharmacological inhibition of the central MC4R signaling by SHU9119 in mice increases weight gain independent of food intake. Since brown adipose tissue (BAT) importantly contributes to energy expenditure by combusting high amounts of triglycerides (TG) into heat, the aim of the study was to evaluate the effect of inhibiting MCR4 on VLDL-metabolism and BAT activity in mice. Methods and results: Female APOE*3-Leiden.CETP transgenic mice were fed a cholesterol-enriched Western-type diet for 4 weeks, matched on body weight and plasma lipids, and infused with either SHU9119 or vehicle for 2 weeks via an osmotic mini-pump into the lateral ventricle while being fed the same diet. SHU9119 increased body fat (+50%) and decreased fat oxidation (-42%). Shu9119 increased plasma VLDL-TG levels (+30%). Whereas hepatic VLDL-TG production rate was not affected, Shu9119 severely impaired clearance of VLDL-TG, explained by reduced uptake of [ 3 H]TG by BAT (-25%). In BAT, SHU9119 decreased UCP-1 protein (-60%) and induced large intracellular lipid droplets, indicative of severely disturbed BAT activity. Since SHU9119 increased food intake (+30%), SHU9119 was also administered in mice that were pair-fed with the vehicle-treated group. Under these conditions, SHU9119 still exhibited these effects, indicating MC4R inhibition impairs BAT activity independent of food intake. Conclusion: Inhibition of central MC4R signaling by SHU9119 increases VLDL-TG levels and decreases energy expenditure specifically by impairing BAT function. We anticipate that MC4R regulates adiposity and VLDL metabolism not only by regulating food intake, but also by modulating BAT activity, and that activation of MC4R is a promising strategy to combat obesity and CVD by increasing BAT activity.

Abstract 621: Mice Lacking Prostaglandin E Receptor Subtype 4 Resist Diet-Induced Obesity Despite Hypertriglyceridemia

Objectives: Activation of prostaglandin E receptor subtype 4 (EP4) may suppress inflammation by regulating nuclear factor kappa B (NFκB). Indeed, chronic inflammation usually is accompanied by dyslipidemia. Thus, the present study tested the hypothesis that deletion of EP4 can accelerate the occurrence of metabolic syndrome Methods: EP4 wild-type and knockout mice were put on a high-fat diet (HFD) for thirty weeks. Body weight and food consumption were monitored. In addition, energy expenditure and fat composition were determined by indirect calorimetry and Minispec body composition analyzer, respectively. The impact of EP4 deletion on the ability to synthesize hepatic very low density lipoprotein (VLDL)-triglyceride (TG) and intestinal chylomicron (CM)-TG, as well as the ability to clear TG during HFD was examined. Circulating lipoprotein lipase (LPL) activity and tissue LPL activity were determined using a fluorometric assay kit and mRNA expression of lipid metabolism-related genes in adipose tissue was measured by quantitative polymerase chain reaction (Q-PCR). Results: After consuming a high-fat diet for thirty weeks, EP4 knockout mice exhibited reduced body weight gain and body fat mass, not attributable to changes in food intake, fat malabsorption and energy expenditure. However, EP4 knockout mice had a higher plasma TG level than wild-type mice. The deletion of EP4 did not influence hepatic VLDL-TG production or intestinal CM-TG synthesis but impaired TG clearance rate. EP4 knockout mice had a decreased circulating LPL activity, reduced LPL activity in epididymal fat and skeletal muscle, suggesting impaired hydrolysis and uptake of triglycerides in these tissues. Moreover, EP4 knockout mice had a reduced expression of CD36 in brown adipose tissue, which may indicate that the uptake of fatty acids is impaired. Conclusions: High-fat feeding of EP4 knockout mice resulted in a lean body phenotype, but caused hypertriglyceridemia resulting from impaired TG clearance, attributed to reduced LPL activity and expression of CD36. The results indicate that EP4 plays a critical role in systemic lipid homeostasis.

Abstract 400: Cannabinoid 1 Receptor Blockade Diminishes Obesity and Dyslipidemia via Peripheral Activation of Brown Adipose Tissue

Objectives: The endocannabinoid system is an important player in energy metabolism by regulating appetite, lipolysis and energy expenditure. Chronic blockade of the cannabinoid 1 receptor (CB1R) leads to long-term maintained weight loss and reduction of dyslipidemia in experimental and human obesity. Brown adipose tissue (BAT) that burns lipids towards heat using UCP1, recently emerged as a major player in lipoprotein metabolism and is present and active in human adults. The aim of the present study was to elucidate the mechanism by which CB1R blockade reverses dyslipidemia and obesity, with special focus on BAT. Methods and results: Diet-induced obese APOE*3-Leiden.CETP transgenic mice, a well-established model for human-like lipoprotein metabolism, were treated with the systemic CB1R blocker rimonabant (10 mg/kg/day) for 4 weeks. Rimonabant persistently decreased body weight (-25%, p&lt;0.001), fat mass (-32%, p&lt;0.001) and plasma triglyceride (TG) levels (-60%, p&lt;0.05), despite a modest and transient reduction in food intake. Interestingly, rimonabant reduced plasma TG levels, not by affecting VLDL-TG production by the liver, but rather by selectively increasing VLDL-TG clearance by BAT (+40%, p&lt;0.05). This was accompanied by increased energy expenditure (+20%, p&lt;0.05), decreased lipid droplet size and increased UCP1 content in BAT (+28%, p&lt;0.05), all pointing to increased BAT activity. Next, we demonstrated that the CB1R is highly expressed in BAT and that in vitro blockade of the CB1R in cultured brown adipocytes resulted in 2.5-fold upregulation of UCP1. Importantly, the in vivo results could be fully recapitulated using the strictly peripheral CB1R antagonist AM6545 (10 mg/kg/day) that does not induce hypophagia. Conclusion: CB1R blockade reduces dyslipidemia and obesity by peripheral activation of BAT. Selective targeting of peripheral CB1R in BAT has thus great therapeutic potential in decreasing dyslipidemia and obesity and ultimately cardiovascular diseases.

Metformin Lowers Plasma Triglycerides by Promoting VLDL-Triglyceride Clearance by Brown Adipose Tissue in Mice

Metformin is the first-line drug for the treatment of type 2 diabetes. Besides its well-characterized antihyperglycemic properties, metformin also lowers plasma VLDL triglyceride (TG). In this study, we investigated the underlying mechanisms in APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism. We found that metformin markedly lowered plasma total cholesterol and TG levels, an effect mostly due to a decrease in VLDL-TG, whereas HDL was slightly increased. Strikingly, metformin did not affect hepatic VLDL-TG production, VLDL particle composition, and hepatic lipid composition but selectively enhanced clearance of glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles into brown adipose tissue (BAT). BAT mass and lipid droplet content were reduced in metformin-treated mice, pointing to increased BAT activation. In addition, both AMP-activated protein kinase α1 (AMPKα1) expression and activity and HSL and mitochondrial content were increased in BAT. Furthermore, therapeutic concentrations of metformin increased AMPK and HSL activities and promoted lipolysis in T37i differentiated brown adipocytes. Collectively, our results identify BAT as an important player in the TG-lowering effect of metformin by enhancing VLDL-TG uptake, intracellular TG lipolysis, and subsequent mitochondrial fatty acid oxidation. Targeting BAT might therefore be considered as a future therapeutic strategy for the treatment of dyslipidemia.

Sympathetic nervous system control of triglyceride metabolism: novel concepts derived from recent studies

Important players in triglyceride (TG) metabolism include the liver (production), white adipose tissue (WAT) (storage), heart and skeletal muscle (combustion to generate ATP), and brown adipose tissue (BAT) (combustion toward heat), the collective action of which determine plasma TG levels. Interestingly, recent evidence points to a prominent role of the hypothalamus in TG metabolism through innervating the liver, WAT, and BAT mainly via sympathetic branches of the autonomic nervous system. Here, we review the recent findings in the area of sympathetic control of TG metabolism. Various neuronal populations, such as neuropeptide Y (NPY)-expressing neurons and melanocortin-expressing neurons, as well as peripherally produced hormones (i.e., GLP-1, leptin, and insulin), modulate sympathetic outflow from the hypothalamus toward target organs and thereby influence peripheral TG metabolism. We conclude that sympathetic stimulation in general increases lipolysis in WAT, enhances VLDL-TG production by the liver, and increases the activity of BAT with respect to lipolysis of TG, followed by combustion of fatty acids toward heat. Moreover, the increased knowledge about the involvement of the neuroendocrine system in TG metabolism presented in this review offers new therapeutic options to fight hypertriglyceridemia by specifically modulating sympathetic nervous system outflow toward liver, BAT, or WAT.

FoxO6 Integrates Insulin Signaling With MTP for Regulating VLDL Production in the Liver

Excessive production of triglyceride-rich very low-density lipoproteins (VLDL-TG) contributes to hypertriglyceridemia in obesity and type 2 diabetes. To understand the underlying mechanism, we studied hepatic regulation of VLDL-TG production by (forkhead box O6) FoxO6, a forkhead transcription factor that integrates insulin signaling to hepatic metabolism. We showed that transgenic mice expressing a constitutively active FoxO6 allele developed hypertriglyceridemia, culminating in elevated VLDL-TG levels and impaired postprandial TG clearance. This effect resulted in part from increased hepatic VLDL-TG production. We recapitulated these findings in cultured HepG2 cells and human primary hepatocytes, demonstrating that FoxO6 promoted hepatic VLDL-TG secretion. This action correlated with the ability of FoxO6 to stimulate hepatic production of microsomal triglyceride transfer protein (MTP), a molecular chaperone that catalyzes the rate-limiting step in VLDL-TG assembly and secretion. FoxO6 was shown to bind to the MTP promoter and stimulate MTP promoter activity in HepG2 cells. This effect was inhibited by insulin, consistent with the ability of insulin to promote FoxO6 phosphorylation and disable FoxO6 DNA-binding activity. Mutations of the FoxO6 target site within the MTP promoter abrogated FoxO6-mediated induction of MTP promoter activity. Hepatic FoxO6 expression became deregulated in insulin-resistant mice with obesity and type 2 diabetes. FoxO6 inhibition in insulin-resistant liver suppressed hepatic MTP expression and curbed VLDL-TG overproduction, contributing to the amelioration of hypertriglyceridemia in obese and diabetic db/db mice. These results characterize FoxO6 as an important signaling molecule upstream of MTP for regulating hepatic VLDL-TG production.

Atorvastatin plus omega‐3 fatty acid ethyl ester decreases very‐low‐density lipoprotein triglyceride production in insulin resistant obese men

To test the effect of atorvastatin (ATV) and ATV plus ω-3 FAEEs on VLDL-TG metabolism in obese, insulin resistant men. We carried out a 6-week randomized, placebo-controlled study to examine the effect of ATV (40 mg/day) and ATV plus ω-3 FAEEs (4 g/day) on VLDL-TG metabolism in 36 insulin resistant obese men. VLDL-TG kinetics were determined using d5 -glycerol, gas chromatography-mass spectrometry and compartmental modelling. Compared with the placebo, ATV significantly decreased VLDL-TG concentration (-40%, p < 0.001) by increasing VLDL-TG fractional catabolic rate (FCR) (+47%, p < 0.01). ATV plus ω-3 FAEEs lowered VLDL-TG concentration to a greater degree compared with placebo (-46%, p < 0.001) or ATV monotherapy (-13%, p = 0.04). This was achieved by a reduction in VLDL-TG production rate (PR) compared with placebo (-32%, p = 0.008) or ATV (-20%, p = 0.03) as well as a reciprocal increase in VLDL-TG FCR (+42%, p < 0.05) compared with placebo. In insulin resistant, dyslipidaemic, obese men, ATV improves VLDL-TG metabolism by increasing VLDL-TG FCR. The addition of 4 g/day ω-3 FAEE to statin therapy provides further TG-lowering by lowering VLDL-TG PR.

Independent Effects of Testosterone on Lipid Oxidation and VLDL-TG Production

Low testosterone (T) levels in men have been shown to predict development of the metabolic syndrome, but the effects of T on lipid metabolism are incompletely understood. In a randomized, double-blind, placebo-controlled, crossover study, 12 healthy, young males received gonadotropin-releasing hormone agonist treatment 1 month prior to 3 of 4 trial days to induce castrate levels of T. On trial days, T gel was applied to the body containing either high or low physiological T dose or placebo. On the 4th trial day, participants constituted their own eugonadal controls. Each study comprised a 5-h basal period and a 3-h hyperinsulinemic-euglycemic clamp. Short-term hypogonadism did not affect VLDL triglyceride (TG) secretion, nor did it affect VLDL-TG concentrations. It was, however, characterized by lower total lipid oxidation. In addition, acute rescue with high physiological T increased VLDL-TG secretion during both basal and clamp conditions. These data show that T can act through fast nongenomic pathways in the liver. In addition, the early hypogonadal state is characterized by decreased total lipid oxidation, but whether these changes represent early hypogonadal metabolic dysfunction warrants further investigations. T is not a major determinant of resting VLDL-TG kinetics in men.

The autonomic nervous system regulates postprandial hepatic lipid metabolism

The liver is a key organ in controlling glucose and lipid metabolism during feeding and fasting. In addition to hormones and nutrients, inputs from the autonomic nervous system are also involved in fine-tuning hepatic metabolic regulation. Previously, we have shown in rats that during fasting an intact sympathetic innervation of the liver is essential to maintain the secretion of triglycerides by the liver. In the current study, we hypothesized that in the postprandial condition the parasympathetic input to the liver inhibits hepatic VLDL-TG secretion. To test our hypothesis, we determined the effect of selective surgical hepatic denervations on triglyceride metabolism after a meal in male Wistar rats. We report that postprandial plasma triglyceride concentrations were significantly elevated in parasympathetically denervated rats compared with control rats (P = 0.008), and VLDL-TG production tended to be increased (P = 0.066). Sympathetically denervated rats also showed a small rise in postprandial triglyceride concentrations (P = 0.045). On the other hand, in rats fed on a six-meals-a-day schedule for several weeks, a parasympathetic denervation resulted in >70% higher plasma triglycerides during the day (P = 0.001), whereas a sympathetic denervation had no effect. Our results show that abolishing the parasympathetic input to the liver results in increased plasma triglyceride levels during postprandial conditions.