ANGPTL8/betatrophin does not control pancreatic beta cell expansion.

Recently, it was reported that angiopoietin-like protein 8 (ANGPTL8) was the long-sought "betatrophin" that could control pancreatic beta cell proliferation. However, studies of Angptl8(?/?) mice revealed profound reduction of triglyceride levels, but no abnormalities in glucose homeostasis. We now report that Angptl8(?/?) mice undergo entirely normal beta cell expansion in response to insulin resistance resulting from either a high-fat diet or from the administration of the insulin receptor antagonist S961. Furthermore, overexpression of ANGPTL8 in livers of mice doubles plasma triglyceride levels, but does not alter beta cell expansion nor glucose metabolism. These data indicate that ANGPTL8 does not play a role in controlling beta cell growth, nor can it be given to induce such expansion. The findings that plasma triglyceride levels are reduced by Angptl8 deletion and increased following ANGPTL8 overexpression support the possibility that inhibition of ANGPTL8 represents a therapeutic strategy for hypertriglyceridemia.

Discordance between GLP-1R gene and protein expression in mouse pancreatic islet cells.

The insulinotropic actions of glucagon-like peptide 1 receptor (GLP-1R) in ß-cells have made it a useful target to manage type 2 diabetes. Metabolic stress reduces ß-cell sensitivity to GLP-1, yet the underlying mechanisms are unknown. We hypothesized that Glp1r expression is heterogeneous among ß-cells and that metabolic stress decreases the number of GLP-1R-positive ß-cells. Here, analyses of publicly available single-cell RNA-Seq sequencing (scRNASeq) data from mouse and human ß-cells indicated that significant populations of ß-cells do not express the Glp1r gene, supporting heterogeneous GLP-1R expression. To check these results, we used complementary approaches employing FACS coupled with quantitative RT-PCR, a validated GLP-1R antibody, and flow cytometry to quantify GLP-1R promoter activity, gene expression, and protein expression in mouse α-, ß-, and δ-cells. Experiments with Glp1r reporter mice and a validated GLP-1R antibody indicated that >90% of the ß-cells are GLP-1R positive, contradicting the findings with the scRNASeq data. α-cells did not express Glp1r mRNA and δ-cells expressed Glp1r mRNA but not protein. We also examined the expression patterns of GLP-1R in mouse models of metabolic stress. Multiparous female mice had significantly decreased ß-cell Glp1r expression, but no reduction in GLP-1R protein levels or GLP-1R-mediated insulin secretion. These findings suggest caution in interpreting the results of scRNASeq for low-abundance transcripts such as the incretin receptors and indicate that GLP-1R is widely expressed in ß-cells, absent in α-cells, and expressed at the mRNA, but not protein, level in δ-cells.

A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease.

BACKGROUND: Elucidation of the genetic factors underlying chronic liver disease may reveal new therapeutic targets. METHODS: We used exome sequence data and electronic health records from 46,544 participants in the DiscovEHR human genetics study to identify genetic variants associated with serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Variants that were replicated in three additional cohorts (12,527 persons) were evaluated for association with clinical diagnoses of chronic liver disease in DiscovEHR study participants and two independent cohorts (total of 37,173 persons) and with histopathological severity of liver disease in 2391 human liver samples. RESULTS: A splice variant (rs72613567:TA) in HSD17B13, encoding the hepatic lipid droplet protein hydroxysteroid 17-beta dehydrogenase 13, was associated with reduced levels of ALT (P=4.2×10-12) and AST (P=6.2×10-10). Among DiscovEHR study participants, this variant was associated with a reduced risk of alcoholic liver disease (by 42% [95% confidence interval {CI}, 20 to 58] among heterozygotes and by 53% [95% CI, 3 to 77] among homozygotes), nonalcoholic liver disease (by 17% [95% CI, 8 to 25] among heterozygotes and by 30% [95% CI, 13 to 43] among homozygotes), alcoholic cirrhosis (by 42% [95% CI, 14 to 61] among heterozygotes and by 73% [95% CI, 15 to 91] among homozygotes), and nonalcoholic cirrhosis (by 26% [95% CI, 7 to 40] among heterozygotes and by 49% [95% CI, 15 to 69] among homozygotes). Associations were confirmed in two independent cohorts. The rs72613567:TA variant was associated with a reduced risk of nonalcoholic steatohepatitis, but not steatosis, in human liver samples. The rs72613567:TA variant mitigated liver injury associated with the risk-increasing PNPLA3 p.I148M allele and resulted in an unstable and truncated protein with reduced enzymatic activity. CONCLUSIONS: A loss-of-function variant in HSD17B13 was associated with a reduced risk of chronic liver disease and of progression from steatosis to steatohepatitis. (Funded by Regeneron Pharmaceuticals and others.).

Hepatic Glucagon Signaling Regulates PCSK9 and Low-Density Lipoprotein Cholesterol.

RATIONALE: Glucagon is a key hormone that regulates the adaptive metabolic responses to fasting. In addition to maintaining glucose homeostasis, glucagon participates in the regulation of cholesterol metabolism; however, the molecular pathways underlying this effect are incompletely understood. OBJECTIVE: We sought to determine the role of hepatic Gcgr (glucagon receptor) signaling in plasma cholesterol regulation and identify its underlying molecular mechanisms. METHODS AND RESULTS: We show that Gcgr signaling plays an essential role in LDL-C (low-density lipoprotein cholesterol) homeostasis through regulating the PCSK9 (proprotein convertase subtilisin/kexin type 9) levels. Silencing of hepatic Gcgr or inhibition of glucagon action increased hepatic and plasma PCSK9 and resulted in lower LDLR (LDL receptor) protein and increased plasma LDL-C. Conversely, treatment of wild-type (WT) mice with glucagon lowered LDL-C levels, whereas this response was abrogated in Pcsk9-/- and Ldlr-/- mice. Our gain- and loss-of-function studies identified Epac2 (exchange protein activated by cAMP-2) and Rap1 (Ras-related protein-1) as the downstream mediators of glucagon's action on PCSK9 homeostasis. Moreover, mechanistic studies revealed that glucagon affected the half-life of PCSK9 protein without changing the level of its mRNA, indicating that Gcgr signaling regulates PCSK9 degradation. CONCLUSIONS: These findings provide novel insights into the molecular interplay between hepatic glucagon signaling and lipid metabolism and describe a new posttranscriptional mechanism of PCSK9 regulation.

Increased thermogenesis by a noncanonical pathway in ANGPTL3/8-deficient mice.

Dietary triglyceride (TG) is the most efficient energy substrate. It is processed and stored at substantially lower metabolic cost than is protein or carbohydrate. In fed animals, circulating TGs are preferentially routed for storage to white adipose tissue (WAT) by angiopoietin-like proteins 3 (A3) and 8 (A8). Here, we show that mice lacking A3 and A8 (A3-/-A8-/- mice) have decreased fat mass and a striking increase in temperature (+1 °C) in the fed (but not fasted) state, without alterations in food intake or physical activity. Subcutaneous WAT (WAT-SQ) from these animals had morphologic and metabolic changes characteristic of beiging. O2 consumption rates (OCRs) and expression of genes involved in both fatty acid synthesis and fatty acid oxidation were increased in WAT-SQ of A3-/-A8-/- mice, but not in their epididymal or brown adipose tissue (BAT). The hyperthermic response to feeding was blocked by maintaining A3-/-A8-/- mice at thermoneutrality or by treating with a ß3-adrenergic receptor (AR) antagonist. To determine if sympathetic stimulation was sufficient to increase body temperature in A3-/-A8-/- mice, WT and A3-/-A8-/- animals were maintained at thermoneutrality and then treated with a ß3-AR agonist; treatment induced hyperthermia in A3-/-A8-/- , but not WT, mice. Antibody-mediated inactivation of both circulating A3 and A8 induced hyperthermia in WT mice. Together, these data indicate that A3 and A8 are essential for efficient storage of dietary TG and that disruption of these genes increases feeding-induced thermogenesis and energy utilization.

Glucagon contributes to liver zonation.

Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal function. Wnt/ß-catenin signaling controls metabolic zonation by activating genes in the perivenous hepatocytes, while suppressing genes in the periportal counterparts. We now demonstrate that glucagon opposes the actions of Wnt/ß-catenin signaling on gene expression and metabolic zonation pattern. The effects were more pronounced in the periportal hepatocytes where 28% of all genes were activated by glucagon and inhibited by Wnt/ß-catenin. The glucagon and Wnt/ß-catenin receptors and their signaling pathways are uniformly distributed in periportal and perivenous hepatocytes and the expression is not regulated by the opposing signal. Collectively, our results show that glucagon controls gene expression and metabolic zonation in the liver through a counterplay with the Wnt/ß-catenin signaling pathway.

Mice harboring the human SLC30A8 R138X loss-of-function mutation have increased insulin secretory capacity.

SLC30A8 encodes a zinc transporter that is primarily expressed in the pancreatic islets of Langerhans. In ß-cells it transports zinc into insulin-containing secretory granules. Loss-of-function (LOF) mutations in SLC30A8 protect against type 2 diabetes in humans. In this study, we generated a knockin mouse model carrying one of the most common human LOF mutations for SLC30A8, R138X. The R138X mice had normal body weight, glucose tolerance, and pancreatic ß-cell mass. Interestingly, in hyperglycemic conditions induced by the insulin receptor antagonist S961, the R138X mice showed a 50% increase in insulin secretion. This effect was not associated with enhanced ß-cell proliferation or mass. Our data suggest that the SLC30A8 R138X LOF mutation may exert beneficial effects on glucose metabolism by increasing the capacity of ß-cells to secrete insulin under hyperglycemic conditions.

Alirocumab, evinacumab, and atorvastatin triple therapy regresses plaque lesions and improves lesion composition in mice.

Atherosclerosis-related CVD causes nearly 20 million deaths annually. Most patients are treated after plaques develop, so therapies must regress existing lesions. Current therapies reduce plaque volume, but targeting all apoB-containing lipoproteins with intensive combinations that include alirocumab or evinacumab, monoclonal antibodies against cholesterol-regulating proprotein convertase subtilisin/kexin type 9 and angiopoietin-like protein 3, may provide more benefit. We investigated the effect of such lipid-lowering interventions on atherosclerosis in APOE*3-Leiden.CETP mice, a well-established model for hyperlipidemia. Mice were fed a Western-type diet for 13 weeks and thereafter matched into a baseline group (euthanized at 13 weeks) and five groups that received diet alone (control) or with treatment [atorvastatin; atorvastatin and alirocumab; atorvastatin and evinacumab; or atorvastatin, alirocumab, and evinacumab (triple therapy)] for 25 weeks. We measured effects on cholesterol levels, plaque composition and morphology, monocyte adherence, and macrophage proliferation. All interventions reduced plasma total cholesterol (37% with atorvastatin to 80% with triple treatment; all P < 0.001). Triple treatment decreased non-HDL-C to 1.0 mmol/l (91% difference from control; P < 0.001). Atorvastatin reduced atherosclerosis progression by 28% versus control (P < 0.001); double treatment completely blocked progression and diminished lesion severity. Triple treatment regressed lesion size versus baseline in the thoracic aorta by 50% and the aortic root by 36% (both P < 0.05 vs. baseline), decreased macrophage accumulation through reduced proliferation, and abated lesion severity. Thus, high-intensive cholesterol-lowering triple treatment targeting all apoB-containing lipoproteins regresses atherosclerotic lesion area and improves lesion composition in mice, making it a promising potential approach for treating atherosclerosis.

Angiopoietin-like protein 3 governs LDL-cholesterol levels through endothelial lipase-dependent VLDL clearance.

Angiopoietin-like protein (ANGPTL)3 regulates plasma lipids by inhibiting LPL and endothelial lipase (EL). ANGPTL3 inactivation lowers LDL-C independently of the classical LDLR-mediated pathway and represents a promising therapeutic approach for individuals with homozygous familial hypercholesterolemia due to LDLR mutations. Yet, how ANGPTL3 regulates LDL-C levels is unknown. Here, we demonstrate in hyperlipidemic humans and mice that ANGPTL3 controls VLDL catabolism upstream of LDL. Using kinetic, lipidomic, and biophysical studies, we show that ANGPTL3 inhibition reduces VLDL-lipid content and size, generating remnant particles that are efficiently removed from the circulation. This suggests that ANGPTL3 inhibition lowers LDL-C by limiting LDL particle production. Mechanistically, we discovered that EL is a key mediator of ANGPTL3's novel pathway. Our experiments revealed that, although dispensable in the presence of LDLR, EL-mediated processing of VLDL becomes critical for LDLR-independent particle clearance. In the absence of EL and LDLR, ANGPTL3 inhibition perturbed VLDL catabolism, promoted accumulation of atypical remnants, and failed to reduce LDL-C. Taken together, we uncover ANGPTL3 at the helm of a novel EL-dependent pathway that lowers LDL-C in the absence of LDLR.

Glucagon receptor inhibition normalizes blood glucose in severe insulin-resistant mice.

Inactivating mutations in the insulin receptor results in extreme insulin resistance. The resulting hyperglycemia is very difficult to treat, and patients are at risk for early morbidity and mortality from complications of diabetes. We used the insulin receptor antagonist S961 to induce severe insulin resistance, hyperglycemia, and ketonemia in mice. Using this model, we show that glucagon receptor (GCGR) inhibition with a monoclonal antibody normalized blood glucose and ß-hydroxybutyrate levels. Insulin receptor antagonism increased pancreatic ß-cell mass threefold. Normalization of blood glucose levels with GCGR-blocking antibody unexpectedly doubled ß-cell mass relative to that observed with S961 alone and 5.8-fold over control. GCGR antibody blockage expanded α-cell mass 5.7-fold, and S961 had no additional effects. Collectively, these data show that GCGR antibody inhibition represents a potential therapeutic option for treatment of patients with extreme insulin-resistance syndromes.

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.

Inactivating Variants in ANGPTL4 and Risk of Coronary Artery Disease.

BACKGROUND: Higher-than-normal levels of circulating triglycerides are a risk factor for ischemic cardiovascular disease. Activation of lipoprotein lipase, an enzyme that is inhibited by angiopoietin-like 4 (ANGPTL4), has been shown to reduce levels of circulating triglycerides. METHODS: We sequenced the exons of ANGPTL4 in samples obtain from 42,930 participants of predominantly European ancestry in the DiscovEHR human genetics study. We performed tests of association between lipid levels and the missense E40K variant (which has been associated with reduced plasma triglyceride levels) and other inactivating mutations. We then tested for associations between coronary artery disease and the E40K variant and other inactivating mutations in 10,552 participants with coronary artery disease and 29,223 controls. We also tested the effect of a human monoclonal antibody against ANGPTL4 on lipid levels in mice and monkeys. RESULTS: We identified 1661 heterozygotes and 17 homozygotes for the E40K variant and 75 participants who had 13 other monoallelic inactivating mutations in ANGPTL4. The levels of triglycerides were 13% lower and the levels of high-density lipoprotein (HDL) cholesterol were 7% higher among carriers of the E40K variant than among noncarriers. Carriers of the E40K variant were also significantly less likely than noncarriers to have coronary artery disease (odds ratio, 0.81; 95% confidence interval, 0.70 to 0.92; P=0.002). K40 homozygotes had markedly lower levels of triglycerides and higher levels of HDL cholesterol than did heterozygotes. Carriers of other inactivating mutations also had lower triglyceride levels and higher HDL cholesterol levels and were less likely to have coronary artery disease than were noncarriers. Monoclonal antibody inhibition of Angptl4 in mice and monkeys reduced triglyceride levels. CONCLUSIONS: Carriers of E40K and other inactivating mutations in ANGPTL4 had lower levels of triglycerides and a lower risk of coronary artery disease than did noncarriers. The inhibition of Angptl4 in mice and monkeys also resulted in corresponding reductions in these values. (Funded by Regeneron Pharmaceuticals.).

Preemptive Activation of the Integrated Stress Response Protects Mice From Diet-Induced Obesity and Insulin Resistance by Fibroblast Growth Factor 21 Induction.

Integrated stress response (ISR) is a signaling system in which phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by stress-specific kinases and subsequent activation of activation transcription factor (ATF) 4 help restore cellular homeostasis following exposure to environmental stresses. ISR activation has been observed in metabolic diseases, including hepatic steatosis (HS), steatohepatitis (SH), and insulin resistance (IR), but it remains unclear whether ISR contributes to disease pathogenesis or represents an innate defense mechanism against metabolic stresses. Constitutive repressor of eIF2α phosphorylation (CReP) is a critical regulatory subunit of the eIF2α phosphatase complex. Here, we show that CReP ablation causes constitutive eIF2α phosphorylation in the liver, which leads to activation of the ATF4 transcriptional program including increased fibroblast growth factor 21 (FGF21) production. Liver-specific CReP knockout (CRePLKO ) mice exhibited marked browning of white adipose tissue (WAT) and increased energy expenditure and insulin sensitivity in an FGF21-dependent manner. Furthermore, CRePLKO mice were protected from high-fat diet (HFD)-induced obesity, HS, and IR. Acute CReP ablation in liver of HFD-induced obese mice also reduced adiposity and improved glucose homeostasis. Conclusion: These data suggest that CReP abundance is a critical determinant for eIF2α phosphorylation and ensuing ISR activation in the liver. Constitutive ISR activation in the liver induces FGF21 and confers protection from HFD-induced adiposity, IR, and HS in mice. Augmenting hepatic ISR may represent a therapeutic approach to treat metabolic disorders.

Characterization of glucose-stimulated insulin release protocols in african green monkeys (Chlorocebus aethiops).

BACKGROUND: Management of diabetes remains a major health and economic challenge, demanding test systems in which to develop new therapies. These studies assessed different methodologies for determining glucose tolerance in green monkeys. METHODS: Twenty-eight African green monkeys between 4 and 24 years old underwent single or repeat intravenous glucose tolerance testing (IVGTT), oral glucose tolerance testing (OGTT), and/or graded glucose infusion testing. RESULTS: Geriatric monkeys exhibited glucose intolerance with impaired glucose-stimulated insulin secretion following IVGTT. Repeat IVGTT and OGTT assessments were inconsistent. Monkeys with low glucose-stimulated insulin secretion after graded glucose infusion exhibited elevated blood glucose levels. CONCLUSION: IVGTT and graded glucose infusion protocols revealed differences in glucose tolerance among green monkeys at single time points, including age-dependent differences suggestive of shifts in pancreatic beta-cell functional capacity, but care should be applied to study design and the interpretation of data in the setting of longitudinal studies.

Use of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells.

This study provides an assessment of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells. The system combines microfluidic technology and nanoliter-scale reactions. We sequenced 622 cells, allowing identification of 341 islet cells with high-quality gene expression profiles. The cells clustered into populations of α-cells (5%), ß-cells (92%), δ-cells (1%), and pancreatic polypeptide cells (2%). We identified cell-type-specific transcription factors and pathways primarily involved in nutrient sensing and oxidation and cell signaling. Unexpectedly, 281 cells had to be removed from the analysis due to low viability, low sequencing quality, or contamination resulting in the detection of more than one islet hormone. Collectively, we provide a resource for identification of high-quality gene expression datasets to help expand insights into genes and pathways characterizing islet cell types. We reveal limitations in the C1 Fluidigm cell capture process resulting in contaminated cells with altered gene expression patterns. This calls for caution when interpreting single-cell transcriptomics data using the C1 Fluidigm system.

A first-in-human pharmacodynamic and pharmacokinetic study of a fully human anti-glucagon receptor monoclonal antibody in normal healthy volunteers.

AIMS: Glucagon receptor (GCGR) blockers are being investigated as potential therapeutics for type 1 and type 2 diabetes. Here we report the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of REGN1193, a fully human glucagon receptor blocking monoclonal antibody from a first-in-human healthy volunteer randomized double-blinded trial. METHODS: Healthy men and women received single ascending doses of REGN1193 ranging from 0.05 to 0.6 mg/kg (n = 42) or placebo (n = 14) intravenously. Safety, tolerability and PK were assessed over 106 days. The glucose-lowering effect of REGN1193 was assessed after induction of hyperglycaemia by serial glucagon challenges. RESULTS: REGN1193 was generally well tolerated. There were small (<3× the upper limit of normal) and transient dose-dependent increases in hepatic aminotransferases. No increase in LDL-C was observed. Hypoglycaemia, assessed as laboratory blood glucose ≤70 mg/dL, occurred in 6/14 (43%) subjects on placebo and 27/42 (57%) on REGN1193 across all dose groups. All episodes of hypoglycaemia were asymptomatic, >50 mg/dL, and did not require treatment or medical assistance. Concentration-time profiles suggest a 2-compartment disposition and marked nonlinearity, consistent with target-mediated clearance. REGN1193 inhibited the glucagon-stimulated glucose increase in a dose-dependent manner. The 0.6 mg/kg dose inhibited the glucagon-induced glucose area under the curve for 0 to 90 minutes (AUC0-90 minutes ) by 80% to 90% on days 3 and 15, while blunting the increase in C-peptide. REGN1193 dose-dependently increased total GLP-1, GLP-2 and glucagon, with plasma levels returning to baseline by day 29 in all dose groups. CONCLUSION: REGN1193, a GCGR-blocking monoclonal antibody, produced a safety, tolerability and PK/PD profile suitable for further clinical development. The occurrence of transient elevations in serum hepatic aminotransferases observed here and reported with several small molecule glucagon receptor antagonists suggests an on-target effect of glucagon receptor blockade. The underlying mechanism is unknown.

GPR17 gene disruption does not alter food intake or glucose homeostasis in mice.

G protein-coupled receptor 17 (GPR17) was recently reported to be a Foxo1 target in agouti-related peptide (AGRP) neurons. Intracerebroventricular injection of GPR17 agonists induced food intake, whereas administration of an antagonist to the receptor reduced feeding. These data lead to the conclusion that pharmacological modulation of GPR17 has therapeutic potential to treat obesity. Here we report that mice deficient in Gpr17 (Gpr17(-/-)) have similar food intake and body weight compared with their wild-type littermates. Gpr17(-/-) mice have normal hypothalamic Agrp mRNA expression, AGRP plasma levels, and metabolic rate. GPR17 deficiency in mice did not affect glucose homeostasis or prevent fat-induced insulin resistance. These data do not support a role for GPR17 in the control of food intake, body weight, or glycemic control.

Hepatic ANGPTL3 regulates adipose tissue energy homeostasis.

Angiopoietin-like protein 3 (ANGPTL3) is a circulating inhibitor of lipoprotein and endothelial lipase whose physiological function has remained obscure. Here we show that ANGPTL3 plays a major role in promoting uptake of circulating very low density lipoprotein-triglycerides (VLDL-TGs) into white adipose tissue (WAT) rather than oxidative tissues (skeletal muscle, heart brown adipose tissue) in the fed state. This conclusion emerged from studies of Angptl3(-/-) mice. Whereas feeding increased VLDL-TG uptake into WAT eightfold in wild-type mice, no increase occurred in fed Angptl3(-/-) animals. Despite the reduction in delivery to and retention of TG in WAT, fat mass was largely preserved by a compensatory increase in de novo lipogenesis in Angptl3(-/-) mice. Glucose uptake into WAT was increased 10-fold in KO mice, and tracer studies revealed increased conversion of glucose to fatty acids in WAT but not liver. It is likely that the increased uptake of glucose into WAT explains the increased insulin sensitivity associated with inactivation of ANGPTL3. The beneficial effects of ANGPTL3 deficiency on both glucose and lipoprotein metabolism make it an attractive therapeutic target.

Inhibition of PCSK9 does not improve lipopolysaccharide-induced mortality in mice.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that targets LDL receptors (LDLRs) for degradation in liver. Blocking the interaction of PCSK9 with the LDLR potently reduces plasma LDL cholesterol levels and cardiovascular events. Recently, it has been suggested that inhibition of PCSK9 might also improve outcomes in mice and humans with sepsis, possibly by increasing LDLR-mediated clearance of endotoxins. Sepsis is a complication of a severe microbial infection that has shared pathways with lipid metabolism. Here, we tested whether anti-PCSK9 antibodies prevent death from lipopolysaccharide (LPS)-induced endotoxemia. Mice were administered PCSK9 antibodies prior to, or shortly after, injecting LPS. In both scenarios, the administration of PCSK9 antibodies did not alter endotoxemia-induced mortality. Afterward, we determined whether the complete absence of PCSK9 improved endotoxemia-induced mortality in mice with the germ-line deletion of Pcsk9 Similarly, PCSK9 knockout mice were not protected from LPS-induced death. To determine whether low LDLR expression increased LPS-induced mortality, Ldlr-/- mice and PCSK9 transgenic mice were studied after injection of LPS. Endotoxemia-induced mortality was not altered in either mouse model. In a human cohort, we observed no correlation between plasma inflammation markers with total cholesterol levels, LDL cholesterol, and PCSK9. Combined, our data demonstrate that PCSK9 inhibition provides no protection from LPS-induced mortality in mice.

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.