Posaconazole versus voriconazole for primary treatment of invasive aspergillosis: a phase 3, randomised, controlled, non-inferiority trial.

BACKGROUND: Voriconazole has been recommended as primary treatment for patients with invasive aspergillosis. Intravenous and tablet formulations of posaconazole that have improved systemic absorption could be an effective alternative to voriconazole. We aimed to assess non-inferiority of posaconazole to voriconazole for the primary treatment of invasive aspergillosis. METHODS: We did a randomised, prospective, double-blind, double-dummy, controlled trial comparing posaconazole (intravenous or oral posaconazole 300 mg twice on day 1, followed by 300 mg once a day for days 2-84) with voriconazole (6 mg/kg intravenous or 300 mg oral twice on day 1 followed by 4 mg/kg intravenously or 200 mg orally twice a day for days 2-84) for 12 weeks or less in the primary treatment of invasive aspergillosis. Participants were from 91 study sites in 26 countries, were aged 13 years or older, weighed at least 40 kg, and met criteria for proven, probable, or possible fungal disease. Participants were randomly assigned (1:1) via a computer-generated randomisation schedule with stratification by risk status. The primary endpoint was cumulative all-cause mortality up until day 42 in the intention-to-treat (ITT) population (defined as randomly assigned participants who received ≥1 dose of study drug), with a 10% non-inferiority margin. The ITT population was also evaluated for safety. This study is registered with ClinicalTrials.gov, NCT01782131, and EudraCT, 2011-003938-14. FINDINGS: Between Oct 25, 2013, and Sept 10, 2019, of 653 individuals assessed for eligibility, 575 ITT participants were randomly assigned and received one or more doses of study drug (n=288 [50%] posaconazole, n=287 [50%] voriconazole). Mortality up until day 42 was 15% (44 of 288) in the posaconazole group and 21% (59 of 287) in the voriconazole group (treatment difference -5·3% [95% CI -11·6 to 1·0]; p<0·0001). Mortality up until day 42 in the full-analysis-set subpopulation (ITT participants with proven or probable invasive aspergillosis) supported this conclusion: 31 (19%) of 163 participants in the posaconazole group and 32 (19%) of 171 participants in the voriconazole group (treatment difference 0·3% [95% CI -8·2 to 8·8]). The most frequently reported treatment-related adverse events (incidence >3%) were increased aspartate aminotransferase (AST) or alanine aminotransferase (ALT), nausea, hypokalaemia, and vomiting in the posaconazole group and increased ALT, AST, or alkaline phosphatase, hallucination, increased γ-glutamyltransferase peptidase, nausea, and blurred vision in the voriconazole group. The overall incidence of treatment-related adverse event rates in the ITT population was 30% for posaconazole and 40% for voriconazole (treatment difference -10·2% [95% CI -17·9 to -2·4]). INTERPRETATION: Posaconazole was non-inferior to voriconazole for all-cause mortality up until day 42 in participants with invasive aspergillosis. Posaconazole was well tolerated, and participants had fewer treatment-related adverse events than in the voriconazole group. This study supports the use of posaconazole as a first-line treatment for the condition. FUNDING: Merck Sharp & Dohme, a subsidiary of Merck & Co, Inc.

Inhibition of cholesteryl ester transfer protein by anacetrapib does not impair the anti-inflammatory properties of high density lipoprotein.

Cholesteryl ester transfer protein (CETP) is a target of therapeutic intervention for coronary heart disease. Anacetrapib, a potent inhibitor of CETP, has been shown to reduce LDL-cholesterol by 40% and increase HDL-cholesterol by 140% in patients, and is currently being evaluated in a phase III cardiovascular outcomes trial. HDL is known to possess anti-inflammatory properties, however with such large increases in HDL-cholesterol, it is unclear whether CETP inhibition perturbs HDL functionality such as anti-inflammatory effects on endothelial cells. The purpose of the present study was to determine whether CETP inhibition by anacetrapib affects the anti-inflammatory properties of HDL. HDL was isolated from either hamsters treated with vehicle or anacetrapib for 2weeks, or from normal human subjects treated either placebo, 20mg, or 150mg anacetrapib daily for 2weeks. Anacetrapib treatment increased plasma HDL cholesterol levels by 65% and between 48 and 82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated hamsters suppressed TNFα induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Similar results were obtained with human HDL samples pre and post treatment with placebo or anacetrapib. Further, HDL inhibited TNFα-induced MCP-1 secretion, monocyte adhesion and NF-κB activation in endothelial cells, and the inhibition was similar between control and anacetrapib treated groups. These studies demonstrate that anacetrapib treatment does not impair the ability of HDL to suppress an inflammatory response in endothelial cells.

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice.

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.

Pharmacokinetic and Exposure Response Analysis of the Double-Blind Randomized Study of Posaconazole and Voriconazole for Treatment of Invasive Aspergillosis.

BACKGROUND AND OBJECTIVE: A double-blind phase 3 study was conducted to compare posaconazole 300 mg intravenously (IV)/300 mg orally once daily (twice daily day 1) with voriconazole 4 mg/kg IV twice daily/200 mg orally twice daily (6 mg/kg day 1) for treatment of invasive aspergillosis. This analysis was conducted to summarize the pharmacokinetics and exposure-response relationships of posaconazole and voriconazole using plasma trough concentration (Ctrough) as a surrogate for exposure from the double-blind phase 3 study. METHODS: The pharmacokinetic evaluable population included all intention-to-treat (ITT) participants with at least one plasma concentration during the treatment period. Treatment blinding was maintained without therapeutic drug monitoring. Ctrough sampling occurred throughout treatment; efficacy and safety were evaluated using quartiles determined by mean Ctrough concentrations. Exposure efficacy variables included day 42 all-cause mortality (primary study endpoint) and global clinical response. Exposure safety variables included all adverse events and treatment-related adverse events. RESULTS: The pharmacokinetic analysis population included 506 of 575 ITT participants (437 with Ctrough concentrations: 228 posaconazole, 209 voriconazole). No trend was seen across quartiles of posaconazole Ctrough for the key efficacy endpoint of all-cause mortality through day 42. Participants in the highest quartile of voriconazole Ctrough had higher all-cause mortality through day 42 than participants in the lower three quartiles of voriconazole Ctrough. Similar findings were observed for global clinical response and Ctrough. No clear exposure safety trend by quartile was seen for posaconazole or voriconazole. CONCLUSIONS: A strong exposure-response relationship was not observed across the range of exposure from the administered doses and formulations for posaconazole or voriconazole. TRIAL REGISTRATION: NCT01782131; registered January 30, 2013.

Macrophage insulin receptor deficiency increases ER stress-induced apoptosis and necrotic core formation in advanced atherosclerotic lesions.

Insulin resistance in diabetes and metabolic syndrome is thought to increase susceptibility to atherosclerotic cardiovascular disease, but the underlying mechanisms are poorly understood. To evaluate the possibility that decreased insulin signaling in macrophage foam cells might worsen atherosclerosis, Ldlr(-/-) mice were transplanted with insulin receptor Insr(+/+) or Insr(-/-) bone marrow. Western diet-fed Insr(-/-) recipients developed larger, more complex lesions with increased necrotic cores and increased numbers of apoptotic cells. Insr(-/-) macrophages showed diminished Akt phosphorylation and an augmented ER stress response, leading to induction of scavenger receptor A and increased apoptosis when challenged with cholesterol loading or nutrient deprivation. These studies suggest that defective insulin signaling and reduced Akt activity impair the ability of macrophages to deal with ER stress-induced apoptosis within atherosclerotic plaques.

Hepatic FoxO1 ablation exacerbates lipid abnormalities during hyperglycemia.

Patients with diabetes suffer disproportionately from impaired lipid metabolism and cardiovascular disease, but the relevant roles of insulin resistance and hyperglycemia in these processes are unclear. Transcription factor FoxO1 is regulated dually by insulin and nutrients. In this study, we addressed the hypothesis that, in addition to its established role to regulate hepatic glucose production, FoxO1 controls aspects of lipid metabolism in the diabetic liver. Mice with a liver-specific deletion of FoxO1 (L-FoxO1) and their control littermates were rendered hyperglycemic by streptozotocin administration. Subsequently, we monitored serum lipids, liver VLDL secretion, and hepatic expression of genes related to lipid metabolism. Hepatic FoxO1 ablation resulted in increased VLDL secretion, increased cholesterol, and increased plasma free fatty acids, three hallmarks of the diabetic state. l-FoxO1 mice expressed increased levels of SREBP-2 and FGF21 without affecting lipogenic genes. We propose that FoxO1 fine tunes lipolysis through its actions on FGF21 and that hepatic FoxO1 ablation increases availability of substrates for hepatic triglyceride and cholesterol synthesis and VLDL secretion. The implications of these findings are that FoxO1 protects against excessive hepatic lipid production during hyperglycemia and that its inhibition by intensive insulin treatment may exacerbate paradoxically the lipid abnormalities of diabetes.

Defective phagocytosis of apoptotic cells by macrophages in atherosclerotic lesions of ob/ob mice and reversal by a fish oil diet.

RATIONALE: The complications of atherosclerosis are a major cause of death and disability in type 2 diabetes. Defective clearance of apoptotic cells by macrophages (efferocytosis) is thought to lead to increased necrotic core formation and inflammation in atherosclerotic lesions. OBJECTIVE: To determine whether there is defective efferocytosis in a mouse model of obesity and atherosclerosis. METHODS AND RESULTS: We quantified efferocytosis in peritoneal macrophages and in atherosclerotic lesions of obese ob/ob or ob/ob;Ldlr(-/-) mice and littermate controls. Peritoneal macrophages from ob/ob and ob/ob;Ldlr(-/-) mice showed impaired efferocytosis, reflecting defective phosphatidylinositol 3-kinase activation during uptake of apoptotic cells. Membrane lipid composition of ob/ob and ob/ob;Ldlr(-/-) macrophages showed an increased content of saturated fatty acids (FAs) and decreased omega-3 FAs (eicosapentaenoic acid and docosahexaenoic acid) compared to controls. A similar defect in efferocytosis was induced by treating control macrophages with saturated free FA/BSA complexes, whereas the defect in ob/ob macrophages was reversed by treatment with eicosapentaenoic acid/BSA or by feeding ob/ob mice a fish oil diet rich in omega-3 FAs. There was also defective macrophage efferocytosis in atherosclerotic lesions of ob/ob;Ldlr(-/-) mice and this was reversed by a fish oil-rich diet. CONCLUSIONS: The findings suggest that in obesity and type 2 diabetes elevated levels of saturated FAs and/or decreased levels of omega-3 FAs contribute to decreased macrophage efferocytosis. Beneficial effects of fish oil diets in atherosclerotic cardiovascular disease may involve improvements in macrophage function related to reversal of defective efferocytosis and could be particularly important in type 2 diabetes and obesity.

Increased atherosclerosis in mice with vascular ATP-binding cassette transporter G1 deficiency--brief report.

OBJECTIVE: The objective of this study was to investigate the role of vascular ATP-binding cassette transporter G1 (ABCG1) in atherogenesis without a confounding difference in macrophage ABCG1 expression. ABCG1 is highly expressed in macrophages and endothelial cells. ABCG1 preserves endothelial function by maintaining endothelial NO synthase activity and by reducing adhesion molecule expression and monocyte adhesion. METHODS AND RESULTS: To investigate the role of vascular ABCG1 in atherosclerosis in vivo Abcg1(-/-)/Ldlr(-/-) and Ldlr(-/-) mice were transplanted with wild-type bone marrow and fed a Western-type diet for 12 or 23 weeks. The atherosclerotic lesion area was similar in both groups after 12 weeks but was increased in Abcg1(-/-)/Ldlr(-/-) recipients after 23 weeks, especially in the aortic arch (2.2-fold; P<0.01). Endothelial NO synthase-mediated vascular relaxation was impaired in male Abcg1(-/-)/Ldlr(-/-) recipients. CONCLUSIONS: Our data show an atheroprotective role of vascular ABCG1, especially in the aortic arch, likely related to its role in the preservation of endothelial NO synthase activity.

Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice.

HDLs protect against the development of atherosclerosis, but the underlying mechanisms are poorly understood. HDL and its apolipoproteins can promote cholesterol efflux from macrophage foam cells via the ATP-binding cassette transporters ABCA1 and ABCG1. Experiments addressing the individual roles of ABCA1 and ABCG1 in the development of atherosclerosis have produced mixed results, perhaps because of compensatory upregulation in the individual KO models. To clarify the role of transporter-mediated sterol efflux in this disease process, we transplanted BM from Abca1(-/-)Abcg1(-/-) mice into LDL receptor-deficient mice and administered a high-cholesterol diet. Compared with control and single-KO BM recipients, Abca1(-/-)Abcg1(-/-) BM recipients showed accelerated atherosclerosis and extensive infiltration of the myocardium and spleen with macrophage foam cells. In experiments with isolated macrophages, combined ABCA1 and ABCG1 deficiency resulted in impaired cholesterol efflux to HDL or apoA-1, profoundly decreased apoE secretion, and increased secretion of inflammatory cytokines and chemokines. In addition, these cells showed increased apoptosis when challenged with free cholesterol or oxidized LDL loading. These results suggest that the combined effects of ABCA1 and ABCG1 in mediating macrophage sterol efflux are central to the antiatherogenic properties of HDL.

Molecular Profiling Reveals a Common Metabolic Signature of Tissue Fibrosis.

Fibrosis, or the accumulation of extracellular matrix, is a common feature of many chronic diseases. To interrogate core molecular pathways underlying fibrosis, we cross-examine human primary cells from various tissues treated with TGF-ß, as well as kidney and liver fibrosis models. Transcriptome analyses reveal that genes involved in fatty acid oxidation are significantly perturbed. Furthermore, mitochondrial dysfunction and acylcarnitine accumulation are found in fibrotic tissues. Substantial downregulation of the PGC1α gene is evident in both in vitro and in vivo fibrosis models, suggesting a common node of metabolic signature for tissue fibrosis. In order to identify suppressors of fibrosis, we carry out a compound library phenotypic screen and identify AMPK and PPAR as highly enriched targets. We further show that pharmacological treatment of MK-8722 (AMPK activator) and MK-4074 (ACC inhibitor) reduce fibrosis in vivo. Altogether, our work demonstrate that metabolic defect is integral to TGF-ß signaling and fibrosis.

Increased inflammatory gene expression in ABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling via toll-like receptors, and neutrophil infiltration of atherosclerotic lesions.

BACKGROUND: Two macrophage ABC transporters, ABCA1 and ABCG1, have a major role in promoting cholesterol efflux from macrophages. Peritoneal macrophages deficient in ABCA1, ABCG1, or both show enhanced expression of inflammatory and chemokine genes. This study was undertaken to elucidate the mechanisms and consequences of enhanced inflammatory gene expression in ABC transporter-deficient macrophages. METHODS AND RESULTS: Basal and lipopolysaccharide-stimulated thioglycollate-elicited peritoneal macrophages showed increased inflammatory gene expression in the order Abca1(-/-)Abcg1(-/-)>Abcg1(-/-)>Abca1(-/-)>wild-type. The increased inflammatory gene expression was abolished in macrophages deficient in Toll-like receptor 4 (TLR4) or MyD88/TRIF. TLR4 cell surface concentration was increased in Abca1(-/-)Abcg1(-/-)>Abcg1(-/-)> Abca1(-/-)> wild-type macrophages. Treatment of transporter-deficient cells with cyclodextrin reduced and cholesterol-cyclodextrin loading increased inflammatory gene expression. Abca1(-/-)Abcg1(-) bone marrow-derived macrophages showed enhanced inflammatory gene responses to TLR2, TLR3, and TLR4 ligands. To assess in vivo relevance, we injected intraperitoneally thioglycollate in Abcg1(-/-) bone marrow-transplanted, Western diet-fed, Ldlr-deficient mice. This resulted in a profound inflammatory infiltrate in the adventitia and necrotic core region of atherosclerotic lesions, consisting primarily of neutrophils. CONCLUSIONS: The results suggest that high-density lipoprotein and apolipoprotein A-1 exert anti-inflammatory effects by promoting cholesterol efflux via ABCG1 and ABCA1 with consequent attenuation of signaling via Toll-like receptors. In response to a peripheral inflammatory stimulus, atherosclerotic lesions containing Abcg1(-/-) macrophages experience an inflammatory "echo," suggesting a possible mechanism of plaque destabilization in subjects with low high-density lipoprotein levels.

Dual role of transcription factor FoxO1 in controlling hepatic insulin sensitivity and lipid metabolism.

Hepatic insulin resistance affects both carbohydrate and lipid metabolism. It has been proposed that insulin controls these 2 metabolic branches through distinct signaling pathways. FoxO transcription factors are considered effectors of the pathway regulating hepatic glucose production. Here we show that adenoviral delivery of constitutively nuclear forkhead box O1 (FoxO1) to mouse liver results in steatosis arising from increased triglyceride accumulation and decreased fatty acid oxidation. FoxO1 gain of function paradoxically increased insulin sensitivity by promoting Akt phosphorylation, while FoxO1 inhibition via siRNA decreased it. We show that FoxO1 regulation of Akt phosphorylation does not require DNA binding and is associated with repression of the pseudokinase tribble 3 (Trb3), a modulator of Akt activity. This unexpected dual role of FoxO1 in promoting insulin sensitivity and lipid synthesis in addition to glucose production has the potential to explain the peculiar admixture of insulin resistance and sensitivity that is commonly observed in the metabolic syndrome.

The macrophage at the crossroads of insulin resistance and atherosclerosis.

The macrophage has emerged as an important player in the pathogenesis of both atherosclerosis and insulin resistance. Cross-talk between inflammatory macrophages and adipocytes may be involved in insulin resistance in peripheral tissues. Defective insulin signaling in cells of the arterial wall including macrophages may promote the development of atherosclerosis. Insulin resistant macrophages are more susceptible to endoplasmic reticulum stress and apoptosis in response to various stimuli such as nutrient deprivation, free cholesterol loading, and oxidized LDL. Increased apoptosis of insulin resistant macrophages and impaired phagocytic clearance of apoptotic cells by insulin resistant macrophages in atherosclerotic lesions may lead to enhanced postapoptotic necrosis, larger lipid-rich cores, increased inflammation, and more complex vulnerable plaques.

DGAT2 Inhibition Alters Aspects of Triglyceride Metabolism in Rodents but Not in Non-human Primates.

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters. In contrast, experiments in primary human, rhesus, and cynomolgus hepatocytes demonstrated that selective inhibition of DGAT2 has only a modest effect. Acute and chronic inhibition of DGAT2 in rhesus primates recapitulated the in vitro data yielding no significant effects on production of plasma TG or VLDL apolipoprotein B. These results call into question whether selective inhibition of DGAT2 is sufficient for remediation of dyslipidemia.

Increased CD36 protein as a response to defective insulin signaling in macrophages.

Accelerated atherosclerosis is a major cause of morbidity and death in insulin-resistant states such as obesity and the metabolic syndrome, but the underlying mechanisms are poorly understood. We show that macrophages from obese (ob/ob) mice have increased binding and uptake of oxidized LDL, in part due to a post-transcriptional increase in CD36 protein. Macrophages from ob/ob mice are also insulin resistant, as shown by reduced expression and signaling of insulin receptors. Three lines of evidence indicate that the increase in CD36 is caused by defective insulin signaling: (a) Treatment of wild-type macrophages with LY294002, an inhibitor of insulin signaling via PI3K, results in an increase in CD36; (b) insulin receptor knockout macrophages show a post-transcriptional increase in CD36 protein; and (c) administration of thiazolidinediones to intact ob/ob mice and ob/ob, LDL receptor-deficient mice results in a reversal of macrophage insulin receptor defects and decreases CD36 protein. The last finding contrasts with the increase in CD36 that results from treatment of macrophages with these drugs ex vivo. The results suggest that defective macrophage insulin signaling predisposes to foam cell formation and atherosclerosis in insulin-resistant states and that this is reversed in vivo by treatment with PPAR-gamma activators.

The impact of insulin resistance on macrophage death pathways in advanced atherosclerosis.

Macrophage death in advanced atherosclerosis causes plaque necrosis, which promotes plaque rupture and acute atherothrombotic vascular events. Of interest, plaque necrosis and atherothrombotic disease are markedly increased in diabetes and metabolic syndrome. We discovered a novel 'multi-hit' macrophage apoptosis pathway that appears to be highly relevant to advanced atherosclerosis. The elements of the pathway include: (a) activation of the unfolded protein response (UPR) by cholesterol overloading of the endoplasmic reticulum or by other UPR activators known to exist in atheromata; and (b) pro-apoptotic signalling involving the type A scavenger receptor (SRA). The downstream apoptosis effectors include CHOP (GADD153) for the UPR and JNK for SRA signalling. Remarkably, components of this pathway are enhanced in macrophages with defective insulin signalling, including UPR activation and SRA expression. As a result, insulin-resistant macrophages show increased susceptibility to apoptosis when exposed to UPR activators and SRA ligands. Moreover, the advanced lesions of atherosclerosis-prone mice reconstituted with insulin-resistant macrophages show increased macrophage apoptosis and plaque necrosis. Based on these findings, we propose that one mechanism of increased plaque necrosis and atherothrombotic vascular disease in insulin resistant syndromes is up-regulation of a two-hit signal transduction pathway involved in advanced lesional macrophage death.

Decreased atherosclerosis in low-density lipoprotein receptor knockout mice transplanted with Abcg1-/- bone marrow.

OBJECTIVE: Recent studies indicate that the ATP-binding cassette transporter ABCG1 can promote cholesterol efflux from macrophages to high-density lipoprotein. This study was designed to assess the in vivo role of macrophage ABCG1 in atherosclerosis. METHODS AND RESULTS: Bone marrow from Abcg1-/- mice was transplanted into irradiated Ldlr-/- recipients, and atherosclerosis was evaluated by aortic root assay after 7 or 11 weeks of feeding on a Western diet. After 7 weeks, there was no difference in lesion area in mice receiving either wild-type or Abcg1-/- bone marrow, whereas after 11 weeks, lesion area was moderately but significantly reduced in Abcg1-/- recipients. ABCG1-deficient peritoneal macrophages showed induction of several liver X receptor target genes, such as Abca1 and Srebp1c, and a dramatic increase in apolipoprotein E (apoE) protein both in cell media and lysates, without parallel change in apoE mRNA. Abca1 knockdown prevented the increase in apoE secretion but had minimal effects on apoE accumulation in cell lysates of Abcg1-/- macrophages. Plasma apoE levels were markedly increased in recipients of Abcg1-/- bone marrow. CONCLUSIONS: These studies reveal an inverse relationship between Abcg1 expression and apoE accumulation and secretion in macrophages. The reduced atherosclerosis in recipients of Abcg1-deficient bone marrow may be explained by induction of Abca1 and an associated increase in macrophage apoE secretion.

Discovery and Pharmacology of a Novel Class of Diacylglycerol Acyltransferase 2 Inhibitors.

DGAT2 plays a critical role in hepatic triglyceride production, and data suggests that inhibition of DGAT2 could prove to be beneficial in treating a number of disease states. This article documents the discovery and optimization of a selective small molecule inhibitor of DGAT2 as well as pharmacological proof of biology in a mouse model of triglyceride production.

Impaired MEK signaling and SERCA expression promote ER stress and apoptosis in insulin-resistant macrophages and are reversed by exenatide treatment.

Accumulation of toxic lipids evokes the unfolded protein response (UPR) and apoptotic death of macrophages and vascular cells in atherosclerotic plaques. Primary macrophages from insulin-resistant ob/ob and insulin receptor (Insr)(-/-) mice display increased apoptosis in response to loading with free cholesterol or oxysterol, but underlying mechanisms have not been elucidated. We show increased activation of all three major branches of the UPR in response to free cholesterol or oxysterol loading in insulin-resistant macrophages. Inhibition and rescue experiments revealed that defective MEK/extracellular signal\x{2013}related kinase (ERK)/cAMP-responsive element-binding protein (CREBP) signaling in insulin-resistant macrophages leads to decreased expression of sarcoplasmic endoplasmic reticulum (ER) Ca(2+)-ATPase, depletion of ER calcium stores, PKR-like ER kinase activation, and ER stress-associated apoptosis. Activation of macrophage glucagon-like peptide 1 (GLP-1) receptor via the antidiabetic drug exenatide led to improvements in both ERK and AKT signaling and reversed the increase in UPR and apoptosis of insulin-resistant macrophages in atherosclerotic lesions of ob/ob.Ldlr(-/-) and Insr(-/-).Ldlr(-/-) mice. Increased signaling via GLP-1 receptor or the CREBP activator protein kinase A thus offers a way to rescue insulin-resistant macrophages from excessive ER stress responses and apoptosis in insulin resistance and type 2 diabetes.

Regulation of hepatic LDL receptors by mTORC1 and PCSK9 in mice.

Individuals with type 2 diabetes have an increased risk of atherosclerosis. One factor underlying this is dyslipidemia, which in hyperinsulinemic subjects with early type 2 diabetes is typically characterized by increased VLDL secretion but normal LDL cholesterol levels, possibly reflecting enhanced catabolism of LDL via hepatic LDLRs. Recent studies have also suggested that hepatic insulin signaling sustains LDLR levels. We therefore sought to elucidate the mechanisms linking hepatic insulin signaling to regulation of LDLR levels. In WT mice, insulin receptor knockdown by shRNA resulted in decreased hepatic mTORC1 signaling and LDLR protein levels. It also led to increased expression of PCSK9, a known post-transcriptional regulator of LDLR expression. Administration of the mTORC1 inhibitor rapamycin caused increased expression of PCSK9, decreased levels of hepatic LDLR protein, and increased levels of VLDL/LDL cholesterol in WT but not Pcsk9-/- mice. Conversely, mice with increased hepatic mTORC1 activity exhibited decreased expression of PCSK9 and increased levels of hepatic LDLR protein levels. Pcsk9 is regulated by the transcription factor HNF1α, and our further detailed analyses suggest that increased mTORC1 activity leads to activation of PKCδ, reduced activity of HNF4α and HNF1α, decreased PCSK9 expression, and ultimately increased hepatic LDLR protein levels, which result in decreased circulating LDL levels. We therefore suggest that PCSK9 inhibition could be an effective way to reduce the adverse side effect of increased LDL levels that is observed in transplant patients taking rapamycin as immunosuppressive therapy.