Dipeptidyl Peptidase 4/Midline-1 Axis Promotes T Lymphocyte Motility in Atherosclerosis.

T cells play a crucial role in atherosclerosis, with its infiltration preceding the formation of atheroma. However, how T-cell infiltration is regulated in atherosclerosis remains largely unknown. Here, this work demonstrates that dipeptidyl peptidase-4 (DPP4) is a novel regulator of T-cell motility in atherosclerosis. Single-cell ribonucleic acid (RNA) sequencing and flow cytometry show that CD4+ T cells in atherosclerotic patients display a marked increase of DPP4. Lack of DPP4 in hematopoietic cells or T cells reduces T-cell infiltration and atherosclerotic plaque volume in atherosclerosis mouse models. Mechanistically, DPP4 deficiency reduces T-cell motility by suppressing the expression of microtubule associated protein midline-1 (Mid1) in T cells. Deletion of either DPP4 or Mid1 inhibits chemokine-induced shape change and motility, while restitution of Mid1 in Dpp4-/- T cell largely restores its migratory ability. Thus, DPP4/Mid1, as a novel regulator of T-cell motility, may be a potential inflammatory target in atherosclerosis.

When MINMOD Artifactually Interprets Strong Insulin Secretion as Weak Insulin Action.

We address a problem with the Bergman-Cobelli Minimal Model, which has been used for 40 years to estimate S I during an intravenous glucose tolerance test (IVGTT). During the IVGTT blood glucose and insulin concentrations are measured in response to an acute intravenous glucose load. Insulin secretion is often assessed by the area under the insulin curve during the first few minutes (Acute Insulin Response, AIR). The issue addressed here is that we have found in simulated IVGTTs, representing certain contexts, Minimal Model estimates of S I are inversely related to AIR, resulting in artifactually lower S I . This may apply to Minimal Model studies reporting lower S I in Blacks than in Whites, a putative explanation for increased risk of T2D in Blacks. The hyperinsulinemic euglycemic clamp (HIEC), the reference method for assessing insulin sensitivity, by contrast generally does not show differences in insulin sensitivity between these groups. The reason for this difficulty is that glucose rises rapidly at the start of the IVGTT and reaches levels independent of S I , whereas insulin during this time is determined by AIR. The minimal model in effect interprets this combination as low insulin sensitivity even when actual insulin sensitivity is unchanged. This happens in particular when high AIR results from increased number of readily releasable insulin granules, which may occur in Blacks. We conclude that caution should be taken when comparing estimates of S I between Blacks and Whites.

Endothelial dysfunction due to selective insulin resistance in vascular endothelium: insights from mechanistic modeling.

Previously, we have used mathematical modeling to gain mechanistic insights into insulin-stimulated glucose uptake. Phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling required for metabolic actions of insulin also regulates endothelium-dependent production of the vasodilator nitric oxide (NO). Vasodilation increases blood flow that augments direct metabolic actions of insulin in skeletal muscle. This is counterbalanced by mitogen-activated protein kinase (MAPK)-dependent insulin signaling in endothelium that promotes secretion of the vasoconstrictor endothelin-1 (ET-1). In the present study, we extended our model of metabolic insulin signaling into a dynamic model of insulin signaling in vascular endothelium that explicitly represents opposing PI3K/NO and MAPK/ET-1 pathways. Novel NO and ET-1 subsystems were developed using published and new experimental data to generate model structures/parameters. The signal-response relationships of our model with respect to insulin-stimulated NO production, ET-1 secretion, and resultant vascular tone, agree with published experimental data, independent of those used for model development. Simulations of pathological stimuli directly impairing only insulin-stimulated PI3K/Akt activity predict altered dynamics of NO and ET-1 consistent with endothelial dysfunction in insulin-resistant states. Indeed, modeling pathway-selective impairment of PI3K/Akt pathways consistent with insulin resistance caused by glucotoxicity, lipotoxicity, or inflammation predict diminished NO production and increased ET-1 secretion characteristic of diabetes and endothelial dysfunction. We conclude that our mathematical model of insulin signaling in vascular endothelium supports the hypothesis that pathway-selective insulin resistance accounts, in part, for relationships between insulin resistance and endothelial dysfunction. This may be relevant for developing novel approaches for the treatment of diabetes and its cardiovascular complications.

Race affects the association of obesity measures with insulin sensitivity.

BACKGROUND: Race differences in body composition and fat distribution may in part explain the differences in insulin sensitivity and the disproportionate burden of type 2 diabetes in African Americans. OBJECTIVE: To determine if differences in body composition and fat distribution explain race differences in insulin sensitivity and identify obesity measures that were independently associated with insulin sensitivity. METHODS: Participants were 113 lean, overweight, and obese African-American and Caucasian-American adults without diabetes. Skeletal muscle insulin sensitivity was determined using a hyperinsulinemic-euglycemic clamp (SIClamp, insulin rate:120 mU/m2/min). Subcutaneous abdominal adipose tissue (SAAT), intra-abdominal adipose tissue (IAAT), and liver fat were measured by MRI; leg fat, total fat, and lean mass were measured by DXA. RESULTS: Race-by-adiposity interactions were significant in cross-sectional analyses utilizing multiple linear regression models for SIClamp (P < 0.05); higher BMI, fat mass, SAAT, leg fat, and liver fat were associated with lower SIClamp in Caucasian Americans but not African Americans. Race-by-IAAT interaction was not significant (P = 0.65). A central fat distribution (SAAT adjusted for leg fat) was associated with lower SIClamp in African Americans (ß = -0.45, SE = 0.11, P < 0.001) but not Caucasian Americans (ß = -0.42, SE = 0.30, P = 0.17). A peripheral fat distribution (leg fat adjusted for IAAT/SAAT) was associated with a higher SIClamp in African Americans (ß = 0.11, SE = 0.05, P = 0.02) but lower SIClamp in Caucasian Americans (ß = -0.28, SE = 0.14, P = 0.049). Lean mass was inversely associated with SIClamp in African Americans (ß = -0.05, SE = 0.03, P = 0.04) but not Caucasian Americans (ß = 0.08, SE = 0.05, P = 0.10) in the model for leg fat. CONCLUSIONS: Measures of overall adiposity were more strongly associated with SIClamp in Caucasian Americans, whereas body fat distribution and lean mass showed stronger correlations with SIClamp in African Americans. Insulin sensitivity may have a genetic basis in African Americans that is reflected in the pattern of body fat distribution. These findings suggest a race-specific pathophysiology of insulin resistance, which has implications for the prevention of diabetes and related cardiometabolic diseases.

Anticoagulant Use and Risk of Ischemic Stroke and Bleeding in Patients With Secondary Atrial Fibrillation Associated With Acute Coronary Syndromes, Acute Pulmonary Disease, or Sepsis.

OBJECTIVES: The purpose of this study was to determine if anticoagulation of patients with new onset secondary atrial fibrillation (AF) occurring with acute coronary syndromes (ACS), acute pulmonary disease, or sepsis is associated with a reduction in ischemic stroke or an increase in bleeding risk. BACKGROUND: Studies evaluating the benefits and risks of anticoagulation in secondary AF are infrequent, and the optimal management of these patients is not well understood. METHODS: A retrospective study cohort was identified of 2,304 patients age 65 years or older, hospitalized with a primary diagnosis of ACS, acute pulmonary disease (chronic obstructive pulmonary disease, pneumonia/influenza, pulmonary embolism, or pleural effusion) or sepsis, and a complication of new-onset AF during admission from 1999 to 2015. RESULTS: Over a follow-up of ∼3 years, we did not identify any association between anticoagulation and a lower incidence of ischemic stroke in patients with new-onset AF occurring with ACS, acute pulmonary disease, or sepsis (odds ratio [OR]: 1.22 [95% confidence interval (CI): 0.65 to 2.27], OR: 0.97 [95% CI: 0.53 to 1.77], and OR: 1.98 [95% CI: 0.29 to 13.47]), after adjusting for confounders. However, anticoagulation was associated with a higher risk of bleeding in patients with AF associated with acute pulmonary disease (OR: 1.72 [95% CI: 1.23 to 2.39]), but not in ACS or sepsis (OR: 1.42 [95% CI: 0.94 to 2.14], OR: 0.96 [95% CI: 0.29 to 3.21]). CONCLUSIONS: Our study demonstrates that the benefit of anticoagulation in secondary AF is not strong and can be associated with a higher risk of bleeding. Careful individual assessment regarding decisions on anticoagulation is warranted in these patients.

Simvastatin Treatment Protects Myocardium in Noncoronary Artery Cardiac Surgery by Inhibiting Apoptosis Through miR-15a-5p Targeting.

Simvastatin treatment is cardioprotective in patients undergoing noncoronary artery cardiac surgery. However, the mechanisms by which simvastatin treatment protects the myocardium under these conditions are not fully understood. Seventy patients undergoing noncoronary cardiac surgery, 35 from a simvastatin treatment group and 35 from a control treatment group, were enrolled in our clinical study. Simvastatin (20 mg/d) was administered preoperatively for 5-7 days. Myocardial tissue biopsies were taken before and after surgery. Apoptosis was detected by TUNEL staining. The expressions of Bcl-2 and Bak in myocardial tissue were detected by immunoblotting. The expressions of miRNA and Bcl-2 mRNA were detected by quantitative real-time polymerase chain reaction assays. Cardiomyocytes were isolated from rat and cultured cells. MiR-15a-5p mimic was transfected into cardiomyocytes, and the Bcl-2 was detected by immunoblotting. TUNEL staining showed significantly less myocardial apoptosis in the simvastatin treatment group when compared with the control treatment group. Protein expression of Bcl-2 was increased in the simvastatin treatment group before surgery, and Bak expression was increased in the control treatment group after surgery. Further comparisons showed that Bcl-2/Bak ratios were reduced in the control treatment group but were not significantly changed in the simvastatin treatment group after surgery. Furthermore, microarray assays revealed that miR-15a-5p was significantly decreased by simvastatin treatment. This was validated by quantitative real-time polymerase chain reaction analysis. MiR-15a-5p was predicted to target Bcl-2 mRNA at nucleotide positions 2529-2536. This was validated by luciferase binding assays. Coincident with the change in miR-15a-5p, the mRNA expression of Bcl-2 was increased in the simvastatin treatment group. MiR-15a-5p mimic significantly inhibited Bcl-2 expression in cardiomyocytes. Our findings strongly suggest that simvastatin treatment preoperatively protected the myocardium in patients undergoing noncoronary artery cardiac surgery, at least in part, by inhibiting apoptosis via suppressing miR-15a-5p expression, leading to increasing expression of Bcl-2 and decreasing expression of Bak.

Transgenic mice with ectopic expression of constitutively active TLR4 in adipose tissues do not show impaired insulin sensitivity.

INTRODUCTION: Chronic low-grade inflammation is associated with obesity and diabetes. However, what causes and mediates chronic inflammation in metabolic disorders is not well understood. Toll-like receptor 4 (TLR4) mediates both infection-induced and sterile inflammation by recognizing pathogen-associated molecular patterns and endogenous molecules, respectively. Saturated fatty acids can activate TLR4, and TLR4-deficient mice were protected from high fat diet (HFD)-induced obesity and insulin resistance, suggesting that TLR4-mediated inflammation may cause metabolic dysfunction, such as obesity and insulin resistance. METHODS: We generated two transgenic (TG) mouse lines expressing a constitutively active TLR4 in adipose tissue and determined whether these TG mice would show increased insulin resistance. RESULTS: TG mice fed a high fat or a normal chow diet did not exhibit increased insulin resistance compared to their wild-type controls despite increased localized inflammation in white adipose tissue. Furthermore, females of one TG line fed a normal chow diet had improved insulin sensitivity with reduction in both adiposity and body weight when compared with wild-type littermates. There were significant differences between female and male mice in metabolic biomarkers and mRNA expression in proinflammatory genes and negative regulators of TLR4 signaling, regardless of genotype and diet. CONCLUSIONS: Together, these results suggest that constitutively active TLR4-induced inflammation in white adipose tissue is not sufficient to induce systemic insulin resistance, and that high fat diet-induced insulin resistance may require other signals in addition to TLR4-mediated inflammation.

Combining Potent Statin Therapy with Other Drugs to Optimize Simultaneous Cardiovascular and Metabolic Benefits while Minimizing Adverse Events.

Hypercholesterolemia and hypertension are among the most important risk factors for cardiovascular (CV) disease. They are also important contributors to metabolic diseases including diabetes that further increase CV risk. Updated guidelines emphasize targeted reduction of overall CV risks but do not explicitly incorporate potential adverse metabolic outcomes that also influence CV health. Hypercholesterolemia and hypertension have synergistic deleterious effects on interrelated insulin resistance and endothelial dysfunction. Dysregulation of the renin-angiotensin system is an important pathophysiological mechanism linking insulin resistance and endothelial dysfunction to atherogenesis. Statins are the reference standard treatment to prevent CV disease in patients with hypercholesterolemia. Statins work best for secondary CV prevention. Unfortunately, most statin therapies dose-dependently cause insulin resistance, increase new onset diabetes risk and exacerbate existing type 2 diabetes mellitus. Pravastatin is often too weak to achieve target low-density lipoprotein cholesterol levels despite having beneficial metabolic actions. Renin-angiotensin system inhibitors improve both endothelial dysfunction and insulin resistance in addition to controlling blood pressure. In this regard, combined statin-based and renin-angiotensin system (RAS) inhibitor therapies demonstrate additive/synergistic beneficial effects on endothelial dysfunction, insulin resistance, and other metabolic parameters in addition to lowering both cholesterol levels and blood pressure. This combined therapy simultaneously reduces CV events when compared to either drug type used as monotherapy. This is mediated by both separate and interrelated mechanisms. Therefore, statin-based therapy combined with RAS inhibitors is important for developing optimal management strategies in patients with hypertension, hypercholesterolemia, diabetes, metabolic syndrome, or obesity. This combined therapy can help prevent or treat CV disease while minimizing adverse metabolic consequences.

Deletion of interleukin 1 receptor-associated kinase 1 (Irak1) improves glucose tolerance primarily by increasing insulin sensitivity in skeletal muscle.

Chronic inflammation may contribute to insulin resistance via molecular cross-talk between pathways for pro-inflammatory and insulin signaling. Interleukin 1 receptor-associated kinase 1 (IRAK-1) mediates pro-inflammatory signaling via IL-1 receptor/Toll-like receptors, which may contribute to insulin resistance, but this hypothesis is untested. Here, we used male Irak1 null (k/o) mice to investigate the metabolic role of IRAK-1. C57BL/6 wild-type (WT) and k/o mice had comparable body weights on low-fat and high-fat diets (LFD and HFD, respectively). After 12 weeks on LFD (but not HFD), k/o mice (versus WT) had substantially improved glucose tolerance (assessed by the intraperitoneal glucose tolerance test (IPGTT)). As assessed with the hyperinsulinemic euglycemic glucose clamp technique, insulin sensitivity was 30% higher in the Irak1 k/o mice on chow diet, but the Irak1 deletion did not affect IPGTT outcomes in mice on HFD, suggesting that the deletion did not overcome the impact of obesity on glucose tolerance. Moreover, insulin-stimulated glucose-disposal rates were higher in the k/o mice, but we detected no significant difference in hepatic glucose production rates (± insulin infusion). Positron emission/computed tomography scans indicated higher insulin-stimulated glucose uptake in muscle, but not liver, in Irak1 k/o mice in vivo Moreover, insulin-stimulated phosphorylation of Akt was higher in muscle, but not in liver, from Irak1 k/o mice ex vivo In conclusion, Irak1 deletion improved muscle insulin sensitivity, with the effect being most apparent in LFD mice.

Acute vascular and metabolic actions of the green tea polyphenol epigallocatechin 3-gallate in rat skeletal muscle.

Epidemiological studies show a dose-dependent relationship between green tea consumption and reduced risk for type 2 diabetes and cardiovascular disease. Bioactive compounds in green tea including the polyphenol epigallocatechin 3-gallate (EGCG) have insulin-mimetic actions on glucose metabolism and vascular function in isolated cell culture studies. The aim of this study is to explore acute vascular and metabolic actions of EGCG in skeletal muscle of Sprague-Dawley rats. Direct vascular and metabolic actions of EGCG were investigated using surgically isolated constant-flow perfused rat hindlimbs. EGCG infused at 0.1, 1, 10 and 100 µM in 15 min step-wise increments caused dose-dependent vasodilation in 5-hydroxytryptamine pre-constricted hindlimbs. This response was not impaired by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin or the AMP-kinase inhibitor Compound C. The nitric oxide synthase (NOS) inhibitor NG-Nitro-l-Arginine Methyl Ester (L-NAME) completely blocked EGCG-mediated vasodilation at 0.1-10 µM, but not at 100 µM. EGCG at 10 µM did not alter muscle glucose uptake nor did it augment insulin-stimulated muscle glucose uptake. The acute metabolic and vascular actions of 10 µM EGCG in vivo were investigated in anaesthetised rats during a hyperinsulinemic-euglycemic clamp (10 mU min-1 kg-1 insulin). EGCG and insulin both stimulated comparable increases in muscle microvascular blood flow without an additive effect. EGCG-mediated microvascular action occurred without altering whole body or muscle glucose uptake. We concluded that EGCG has direct NOS-dependent vasodilator actions in skeletal muscle that do not acutely alter muscle glucose uptake or enhance the vascular and metabolic actions of insulin in healthy rats.

Infliximab therapy restores adiponectin expression in perivascular adipose tissue and improves endothelial nitric oxide-mediated vasodilation in mice with type 1 diabetes.

Increased TNFα-mediated JNK signaling in the perivascular adipose tissue (PVAT) may contribute to the pathogenesis of vascular complications in T1DM by reducing adiponectin (Ad) synthesis and therefore impairing Ad-mediated activity in the contiguous blood vessel system. We evaluated whether in vivo treatment with the TNFα blocking antibody infliximab normalized expression of Ad and Ad receptors in various fat depots, and whether this effect correlated with improved endothelial activity and vasodilator function in streptozotocin (STZ)-induced diabetic mice. STZ mice were studied at 1 and 2weeks after diabetes onset, and compared to age-matched infliximab-treated diabetic (I-STZ) and control animals (CTRL) (n=10 each group). In STZ mice, activation of pro-inflammatory JNK signaling was faster in PVAT (P<0.01) than in visceral (VAT), epididymal (EAT) and subcutaneous (SAT) adipose depots, and associated with decreased Ad synthesis and dysregulated AdipoR1/R2 levels. In parallel, activation of JNK in aortic endothelial cells and mesenteric arteries was associated with decreased expression/phosphorylation of eNOS and impaired ACh-mediated vasodilation (P<0.05 vs. CTRL). Treatment with infliximab abrogated JNK activation, ameliorated Ad protein expression, and normalized expression of both AdipoR1 and AdipoR2 in PVAT, concomitantly improving eNOS expression and vessel relaxation in mesenteric arteries from I-STZ mice (P<0.01 vs. STZ). These observations underline the early susceptibility of PVAT to activation of pro-inflammatory JNK signaling, and highlight its potential importance in early vascular changes of T1DM. Further elucidation of the role of PVAT in cardiovascular complications may allow for the design of novel therapeutic strategies directly addressing PVAT pathophysiology.

Estrogen deprivation in primate pregnancy leads to insulin resistance in offspring.

This study tested the hypothesis that estrogen programs mechanisms within the primate fetus that promote insulin sensitivity and glucose homeostasis in offspring. Glucose tolerance tests were performed longitudinally in prepubertal offspring of baboons untreated or treated on days 100 to 165/175 of gestation (term is 184 days) with the aromatase inhibitor letrozole, which decreased fetal estradiol levels by 95%. Basal plasma insulin levels were over two-fold greater in offspring delivered to letrozole-treated than untreated animals. Moreover, the peak 1min, average of the 1, 3, and 5min, and area under the curve blood glucose and plasma insulin levels after an i.v. bolus of glucose were greater (P<0.05 and P<0.01, respectively) in offspring deprived of estrogen in utero than in untreated animals and partially or completely restored in letrozole plus estradiol-treated baboons. The value for the homeostasis model assessment of insulin resistance was 2.5-fold greater (P<0.02) and quantitative insulin sensitivity check index lower (P<0.01) in offspring of letrozole-treated versus untreated animals and returned to almost normal in letrozole plus estradiol-treated animals. The exaggerated rise in glucose and insulin levels after glucose challenge in baboon offspring deprived of estrogen in utero indicates that pancreatic beta cells had the capacity to secrete insulin, but that peripheral glucose uptake and/or metabolism were impaired, indicative of insulin resistance and glucose intolerance. We propose that estrogen normally programs mechanisms in utero within the developing primate fetus that lead to insulin sensitivity, normal glucose tolerance, and the capacity to metabolize glucose after birth.

Direct Evidence that Myocardial Insulin Resistance following Myocardial Ischemia Contributes to Post-Ischemic Heart Failure.

A close link between heart failure (HF) and systemic insulin resistance has been well documented, whereas myocardial insulin resistance and its association with HF are inadequately investigated. This study aims to determine the role of myocardial insulin resistance in ischemic HF and its underlying mechanisms. Male Sprague-Dawley rats subjected to myocardial infarction (MI) developed progressive left ventricular dilation with dysfunction and HF at 4 wk post-MI. Of note, myocardial insulin sensitivity was decreased as early as 1 wk after MI, which was accompanied by increased production of myocardial TNF-α. Overexpression of TNF-α in heart mimicked impaired insulin signaling and cardiac dysfunction leading to HF observed after MI. Treatment of rats with a specific TNF-α inhibitor improved myocardial insulin signaling post-MI. Insulin treatment given immediately following MI suppressed myocardial TNF-α production and improved cardiac insulin sensitivity and opposed cardiac dysfunction/remodeling. Moreover, tamoxifen-induced cardiomyocyte-specific insulin receptor knockout mice exhibited aggravated post-ischemic ventricular remodeling and dysfunction compared with controls. In conclusion, MI induces myocardial insulin resistance (without systemic insulin resistance) mediated partly by ischemia-induced myocardial TNF-α overproduction and promotes the development of HF. Our findings underscore the direct and essential role of myocardial insulin signaling in protection against post-ischemic HF.

Dominant negative FADD dissipates the proapoptotic signalosome of the unfolded protein response in diabetic embryopathy.

Endoplasmic reticulum (ER) stress and caspase 8-dependent apoptosis are two interlinked causal events in maternal diabetes-induced neural tube defects (NTDs). The inositol-requiring enzyme 1α (IRE1α) signalosome mediates the proapoptotic effect of ER stress. Diabetes increases tumor necrosis factor receptor type 1R-associated death domain (TRADD) expression. Here, we revealed two new unfolded protein response (UPR) regulators, TRADD and Fas-associated protein with death domain (FADD). TRADD interacted with both the IRE1α-TRAF2-ASK1 complex and FADD. In vivo overexpression of a FADD dominant negative (FADD-DN) mutant lacking the death effector domain disrupted diabetes-induced IRE1α signalosome and suppressed ER stress and caspase 8-dependent apoptosis, leading to NTD prevention. FADD-DN abrogated ER stress markers and blocked the JNK1/2-ASK1 pathway. Diabetes-induced mitochondrial translocation of proapoptotic Bcl-2 members mitochondrial dysfunction and caspase cleavage were also alleviated by FADD-DN. In vitro TRADD overexpression triggered UPR and ER stress before manifestation of caspase 3 and caspase 8 cleavage and apoptosis. FADD-DN overexpression repressed high glucose- or TRADD overexpression-induced IRE1α phosphorylation, its downstream proapoptotic kinase activation and endonuclease activities, and apoptosis. FADD-DN also attenuated tunicamycin-induced UPR and ER stress. These findings suggest that TRADD participates in the IRE1α signalosome and induces UPR and ER stress and that the association between TRADD and FADD is essential for diabetes- or high glucose-induced UPR and ER stress.

ASK1 mediates the teratogenicity of diabetes in the developing heart by inducing ER stress and inhibiting critical factors essential for cardiac development.

Maternal diabetes in mice induces heart defects similar to those observed in human diabetic pregnancies. Diabetes enhances apoptosis and suppresses cell proliferation in the developing heart, yet the underlying mechanism remains elusive. Apoptosis signal-regulating kinase 1 (ASK1) activates the proapoptotic c-Jun NH2-terminal kinase 1/2 (JNK1/2) leading to apoptosis, suggesting a possible role of ASK1 in diabetes-induced heart defects. We aimed to investigate whether ASK1 is activated in the heart and whether deleting the Ask1 gene blocks diabetes-induced adverse events and heart defect formation. The ASK1-JNK1/2 pathway was activated by diabetes. Deleting Ask1 gene significantly reduced the rate of heart defects, including ventricular septal defects (VSDs) and persistent truncus arteriosus (PTA). Additionally, Ask1 deletion diminished diabetes-induced JNK1/2 phosphorylation and its downstream transcription factors and endoplasmic reticulum (ER) stress markers. Consistent with this, caspase activation and apoptosis were blunted. Ask1 deletion blocked the increase in cell cycle inhibitors (p21 and p27) and the decrease in cyclin D1 and D3 and reversed diabetes-repressed cell proliferation. Ask1 deletion also restored the expression of BMP4, NKX2.5, and GATA5, Smad1/5/8 phosphorylation, whose mutations or deletion result in reduced cell proliferation, VSD, and PTA formation. We conclude that ASK1 may mediate the teratogenicity of diabetes through activating the JNK1/2-ER stress pathway and inhibiting cell cycle progression, thereby impeding the cardiogenesis pathways essential for ventricular septation and outflow tract development.

Cellular Stress, Excessive Apoptosis, and the Effect of Metformin in a Mouse Model of Type 2 Diabetic Embryopathy.

Increasing prevalence of type 2 diabetes in women of childbearing age has led to a higher incidence of diabetes-associated birth defects. We established a model of type 2 diabetic embryopathy by feeding 4-week-old female mice a high-fat diet (HFD) (60% fat). After 15 weeks on HFD, the mice showed characteristics of type 2 diabetes mellitus (DM) and were mated with lean male mice. During pregnancy, control dams fed a normal diet (10% fat) were maintained on either normal diet or HFD, serving as a control group with elevated circulating free fatty acids. DM dams produced offspring at a rate of 11.3% for neural tube defect (NTD) formation, whereas no embryos in the control groups developed NTDs. Elevated markers of oxidative stress, endoplasmic reticulum stress, caspase activation, and neuroepithelial cell apoptosis (causal events in type 1 diabetic embryopathy) were observed in embryos of DM dams. DM dams treated with 200 mg/kg metformin in drinking water ameliorated fasting hyperglycemia, glucose intolerance, and insulin resistance with consequent reduction of cellular stress, apoptosis, and NTDs in their embryos. We conclude that cellular stress and apoptosis occur and that metformin effectively reduces type 2 diabetic embryopathy in a useful rodent model.

Evidence for several independent genetic variants affecting lipoprotein (a) cholesterol levels.

Lipoprotein (a) [Lp(a)] is an independent risk factor for atherosclerosis-related events that is under strong genetic control (heritability = 0.68-0.98). However, causal mutations and functional validation of biological pathways modulating Lp(a) metabolism are lacking. We performed a genome-wide association scan to identify genetic variants associated with Lp(a)-cholesterol levels in the Old Order Amish. We confirmed a previously known locus on chromosome 6q25-26 and found Lp(a) levels also to be significantly associated with a SNP near the APOA5-APOA4-APOC3-APOA1 gene cluster on chromosome 11q23 linked in the Amish to the APOC3 R19X null mutation. On 6q locus, we detected associations of Lp(a)-cholesterol with 118 common variants (P = 5 × 10(-8) to 3.91 × 10(-19)) spanning a ∼5.3 Mb region that included the LPA gene. To further elucidate variation within LPA, we sequenced LPA and identified two variants most strongly associated with Lp(a)-cholesterol, rs3798220 (P = 1.07 × 10(-14)) and rs10455872 (P = 1.85 × 10(-12)). We also measured copy numbers of kringle IV-2 (KIV-2) in LPA using qPCR. KIV-2 numbers were significantly associated with Lp(a)-cholesterol (P = 2.28 × 10(-9)). Conditional analyses revealed that rs3798220 and rs10455872 were associated with Lp(a)-cholesterol levels independent of each other and KIV-2 copy number. Furthermore, we determined for the first time that levels of LPA mRNA were higher in the carriers than non-carriers of rs10455872 (P = 0.0001) and were not different between carriers and non-carriers of rs3798220. Protein levels of apo(a) were higher in the carriers than non-carriers of both rs10455872 and rs3798220. In summary, we identified multiple independent genetic determinants for Lp(a)-cholesterol. These findings provide new insights into Lp(a) regulation.