ß-Cell Insulin Resistance Plays a Causal Role in Fat-Induced ß-Cell Dysfunction In Vitro and In Vivo.

In the classical insulin target tissues of liver, muscle, and adipose tissue, chronically elevated levels of free fatty acids (FFA) impair insulin signaling. Insulin signaling molecules are also present in ß-cells where they play a role in ß-cell function. Therefore, inhibition of the insulin/insulin-like growth factor 1 pathway may be involved in fat-induced ß-cell dysfunction. To address the role of ß-cell insulin resistance in FFA-induced ß-cell dysfunction we co-infused bisperoxovanadate (BPV) with oleate or olive oil for 48 hours in rats. BPV, a tyrosine phosphatase inhibitor, acts as an insulin mimetic and is devoid of any antioxidant effect that could prevent ß-cell dysfunction, unlike most insulin sensitizers. Following fat infusion, rats either underwent hyperglycemic clamps for assessment of ß-cell function in vivo or islets were isolated for ex vivo assessment of glucose-stimulated insulin secretion (GSIS). We also incubated islets with oleate or palmitate and BPV for in vitro assessment of GSIS and Akt (protein kinase B) phosphorylation. Next, mice with ß-cell specific deletion of PTEN (phosphatase and tensin homolog; negative regulator of insulin signaling) and littermate controls were infused with oleate for 48 hours, followed by hyperglycemic clamps or ex vivo evaluation of GSIS. In rat experiments, BPV protected against fat-induced impairment of ß-cell function in vivo, ex vivo, and in vitro. In mice, ß-cell specific deletion of PTEN protected against oleate-induced ß-cell dysfunction in vivo and ex vivo. These data support the hypothesis that ß-cell insulin resistance plays a causal role in FFA-induced ß-cell dysfunction.

Luseogliflozin inhibits high glucose-induced TGF-ß2 expression in mouse cardiomyocytes by suppressing NHE-1 activity.

OBJECTIVE: Sodium-glucose cotransporter-2 (SGLT2) inhibitors exhibit cardioprotective properties in patients with diabetes. However, SGLT2 is not expressed in the heart, and the underlying molecular mechanisms are not fully understood. We investigated whether the SGLT2 inhibitor luseogliflozin exerts beneficial effects on high glucose-exposed cardiomyocytes via the suppression of sodium-hydrogen exchanger-1 (NHE-1) activity. METHODS: Mouse cardiomyocytes were incubated under normal or high glucose conditions with vehicle, luseogliflozin, or the NHE-1 inhibitor cariporide. NHE-1 activity and gene expression were evaluated by the SNARF assay and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis, respectively. Six-week-old male db/db mice were treated with vehicle or luseogliflozin for 6 weeks, and the hearts were collected for histological, RT-PCR, and western blot analyses. RESULTS: High glucose increased NHE-1 activity and transforming growth factor (Tgf)-ß2 mRNA levels in cardiomyocytes, both of which were inhibited by luseogliflozin or cariporide, whereas their combination showed no additive suppression of Tgf-ß2 mRNA levels. Luseogliflozin attenuated cardiac hypertrophy and fibrosis in db/db mice in association with decreased mRNA and protein levels of TGF-ß2. CONCLUSIONS: Luseogliflozin may suppress cardiac hypertrophy in diabetes by reducing Tgf-ß2 expression in cardiomyocytes via the suppression of NHE-1 activity.

Nicotinamide Mononucleotide Prevents Free Fatty Acid-Induced Reduction in Glucose Tolerance by Decreasing Insulin Clearance.

The NAD-dependent deacetylase SIRT1 improves ß cell function. Accordingly, nicotinamide mononucleotide (NMN), the product of the rate-limiting step in NAD synthesis, prevents ß cell dysfunction and glucose intolerance in mice fed a high-fat diet. The current study was performed to assess the effects of NMN on ß cell dysfunction and glucose intolerance that are caused specifically by increased circulating free fatty acids (FFAs). NMN was intravenously infused, with or without oleate, in C57BL/6J mice over a 48-h-period to elevate intracellular NAD levels and consequently increase SIRT1 activity. Administration of NMN in the context of elevated plasma FFA levels considerably improved glucose tolerance. This was due not only to partial protection from FFA-induced ß cell dysfunction but also, unexpectedly, to a significant decrease in insulin clearance. However, in conditions of normal FFA levels, NMN impaired glucose tolerance due to decreased ß cell function. The presence of this dual action of NMN suggests caution in its proposed therapeutic use in humans.

Glucose-Dependent Insulinotropic Polypeptide Suppresses Foam Cell Formation of Macrophages through Inhibition of the Cyclin-Dependent Kinase 5-CD36 Pathway.

Glucose-dependent insulinotropic polypeptide (GIP) has been reported to have an atheroprotective property in animal models. However, the effect of GIP on macrophage foam cell formation, a crucial step of atherosclerosis, remains largely unknown. We investigated the effects of GIP on foam cell formation of, and CD36 expression in, macrophages extracted from GIP receptor-deficient (Gipr-/-) and Gipr+/+ mice and cultured human U937 macrophages by using an agonist for GIP receptor, [D-Ala2]GIP(1-42). Foam cell formation evaluated by esterification of free cholesterol to cholesteryl ester and CD36 gene expression in macrophages isolated from Gipr+/+ mice infused subcutaneously with [D-Ala2]GIP(1-42) were significantly suppressed compared with vehicle-treated mice, while these beneficial effects were not observed in macrophages isolated from Gipr-/- mice infused with [D-Ala2]GIP(1-42). When macrophages were isolated from Gipr+/+ and Gipr-/- mice, and then exposed to [D-Ala2]GIP(1-42), similar results were obtained. [D-Ala2]GIP(1-42) attenuated ox-LDL uptake of, and CD36 gene expression in, human U937 macrophages as well. Gene expression level of cyclin-dependent kinase 5 (Cdk5) was also suppressed by [D-Ala2]GIP(1-42) in U937 cells, which was corelated with that of CD36. A selective inhibitor of Cdk5, (R)-DRF053 mimicked the effects of [D-Ala2]GIP(1-42) in U937 cells. The present study suggests that GIP could inhibit foam cell formation of macrophages by suppressing the Cdk5-CD36 pathway via GIP receptor.

Glucose-dependent insulinotropic polypeptide inhibits cardiac hypertrophy and fibrosis in diabetic mice via suppression of TGF-ß2.

Diabetic cardiomyopathy is associated with an increased risk for heart failure and death in patients with diabetes. We investigated here whether and how GIP attenuated cardiac hypertrophy and fibrosis in diabetic mice with obesity. Diabetic db/db mice at 7 weeks old were infused with vehicle or GIP (50 nmol/kg/day) for 6 weeks, and hearts were collected for histological and RT-PCR analyzes. Cardiomyocytes isolated from neonatal mice were incubated with or without 300 nM [D-Ala2]-GIP, 30 mM glucose, or 100 µg/mL advanced glycation end products (AGEs) for RT-PCR and lucigenin assays. Compared with non-diabetic mice, diabetic mice exhibited larger left ventricle wall thickness and cardiomyocyte sizes and more fibrotic areas in association with up-regulation of myosin heavy chain ß (ß-Mhc) and transforming growth factor-beta2 (Tgf-ß2) mRNA levels, all of which were inhibited by GIP infusion. High glucose increased NADPH oxidase-driven superoxide generation and up-regulated ß-Mhc, Tgf-ß2, and receptor for AGEs mRNA levels in cardiomyocytes, and augmented the AGE-induced ß-Mhc gene expression. [D-Ala2]-GIP attenuated all of the deleterious effects of high glucose and/or AGEs on cardiomyocytes. Our present findings suggest that GIP could inhibit cardiac hypertrophy and fibrosis in diabetic mice via suppression of TGF-ß2.

AGE-RAGE Axis Stimulates Oxidized LDL Uptake into Macrophages through Cyclin-Dependent Kinase 5-CD36 Pathway via Oxidative Stress Generation.

Advanced glycation end products (AGEs) are localized in macrophage-derived foam cells within atherosclerotic lesions, which could be associated with the increased risk of atherosclerotic cardiovascular disease under diabetic conditions. Although foam cell formation of macrophages has been shown to be enhanced by AGEs, the underlying molecular mechanism remains unclear. Since cyclin-dependent kinase 5 (Cdk5) is reported to modulate inflammatory responses in macrophages, we investigated whether Cdk5 could be involved in AGE-induced CD36 gene expression and foam cell formation of macrophages. AGEs significantly increased Dil-oxidized low-density lipoprotein (ox-LDL) uptake, and Cdk5 and CD36 gene expression in U937 human macrophages, all of which were inhibited by DNA aptamer raised against RAGE (RAGE-aptamer). Cdk5 and CD36 gene expression levels were correlated with each other. An antioxidant, N-acetyl-l-cysteine, mimicked the effects of RAGE-aptamer on AGE-exposed U937 cells. A selective inhibitor of Cdk5, (R)-DRF053, attenuated the AGE-induced Dil-ox-LDL uptake and CD36 gene expression, whereas anti-CD36 antibody inhibited the Dil-ox-LDL uptake but not Cdk5 gene expression. The present study suggests that AGEs may stimulate ox-LDL uptake into macrophages through the Cdk5-CD36 pathway via RAGE-mediated oxidative stress.

GIP as a Potential Therapeutic Target for Atherosclerotic Cardiovascular Disease-A Systematic Review.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut hormones that are secreted from enteroendocrine L cells and K cells in response to digested nutrients, respectively. They are also referred to incretin for their ability to stimulate insulin secretion from pancreatic beta cells in a glucose-dependent manner. Furthermore, GLP-1 exerts anorexic effects via its actions in the central nervous system. Since native incretin is rapidly inactivated by dipeptidyl peptidase-4 (DPP-4), DPP-resistant GLP-1 receptor agonists (GLP-1RAs), and DPP-4 inhibitors are currently used for the treatment of type 2 diabetes as incretin-based therapy. These new-class agents have superiority to classical oral hypoglycemic agents such as sulfonylureas because of their low risks for hypoglycemia and body weight gain. In addition, a number of preclinical studies have shown the cardioprotective properties of incretin-based therapy, whose findings are further supported by several randomized clinical trials. Indeed, GLP-1RA has been significantly shown to reduce the risk of cardiovascular and renal events in patients with type 2 diabetes. However, the role of GIP in cardiovascular disease remains to be elucidated. Recently, pharmacological doses of GIP receptor agonists (GIPRAs) have been found to exert anti-obesity effects in animal models. These observations suggest that combination therapy of GLP-1R and GIPR may induce superior metabolic and anti-diabetic effects compared with each agonist individually. Clinical trials with GLP-1R/GIPR dual agonists are ongoing in diabetic patients. Therefore, in this review, we summarize the cardiovascular effects of GIP and GIPRAs in cell culture systems, animal models, and humans.

Lipocalin-2 exerts pro-atherosclerotic effects as evidenced by in vitro and in vivo experiments.

Lipocalin-2 (LCN2), a multiple bioactive hormone particularly expressed in adipose tissue, neutrophils, and macrophages, is known to exhibit anti-microbial effect, increase inflammatory cytokine levels, and maintain glucose homeostasis. Serum LCN2 level is positively correlated with the severity of coronary artery disease. However, it still remains unknown whether LCN2 affects atherogenesis. We assessed the effects of LCN2 on the inflammatory response and monocyte adhesion in human umbilical vein endothelial cells (HUVECs), inflammatory phenotype and foam cell formation in THP1 monocyte-derived macrophages, and migration and proliferation of human aortic smooth muscle cells (HASMCs) in vitro and aortic lesions in Apoe-/- mice in vivo. LCN2 and its receptor, low-density lipoprotein (LDL)-related protein-2, were expressed in THP1 monocytes, macrophages, HASMCs, and HUVECs. LCN2 significantly enhanced THP1 monocyte adhesion to HUVECs accompanied with upregulation of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin associated with nuclear factor-κB (NF-κB) upregulation in HUVECs. LCN2 significantly increased HUVEC proliferation and oxidized LDL-induced foam cell formation in THP1 monocyte-derived macrophages. LCN2 significantly increased the inflammatory M1 phenotype associated with NF-κB upregulation during differentiation of THP1 monocytes into macrophages. In HASMCs, LCN2 significantly promoted the migration and collagen-1 expression without inducing proliferation, which are associated with increased protein expression of phosphoinositide 3-kinase and phosphorylation of Akt, extracellular signal-regulated kinase, c-jun-N-terminal kinase, and NF-κB. Chronic LCN2 infusion into Apoe-/- mice significantly accelerated the development of aortic atherosclerotic lesions, with increased intraplaque monocyte/macrophage infiltration and pentraxin-3 and collagen-1 expressions. Our results suggested that LCN2 accelerates the development of atherosclerosis. Thus, LCN2 could serve as a novel therapeutic target for atherosclerotic diseases.

Luseogliflozin attenuates neointimal hyperplasia after wire injury in high-fat diet-fed mice via inhibition of perivascular adipose tissue remodeling.

BACKGROUND: Excess fat deposition could induce phenotypic changes of perivascular adipose tissue (PVAT remodeling), which may promote the progression of atherosclerosis via modulation of adipocytokine secretion. However, it remains unclear whether and how suppression of PVAT remodeling could attenuate vascular injury. In this study, we examined the effect of sodium-glucose cotransporter 2 (SGLT2) inhibitor, luseogliflozin on PVAT remodeling and neointima formation after wire injury in mice. METHODS: Wilt-type mice fed with low-fat diet (LFD) or high-fat diet (HFD) received oral administration of luseogliflozin (18 mg/kg/day) or vehicle. Mice underwent bilateral femoral artery wire injury followed by unilateral removal of surrounding PVAT. After 25 days, injured femoral arteries and surrounding PVAT were analyzed. RESULTS: In LFD-fed lean mice, neither luseogliflozin treatment or PVAT removal attenuated the intima-to-media (I/M) ratio of injured arteries. However, in HFD-fed mice, luseogliflozin or PVAT removal reduced the I/M ratio, whereas their combination showed no additive reduction. In PVAT surrounding injured femoral arteries of HFD-fed mice, luseogliflozin treatment decreased the adipocyte sizes. Furthermore, luseogliflozin reduced accumulation of macrophages expressing platelet-derived growth factor-B (PDGF-B) and increased adiponectin gene expression. Gene expression levels of Pdgf-b in PVAT were correlated with the I/M ratio. CONCLUSIONS: Our present study suggests that luseogliflozin could attenuate neointimal hyperplasia after wire injury in HFD-fed mice partly via suppression of macrophage PDGF-B expression in PVAT. Inhibition of PVAT remodeling by luseogliflozin may be a novel therapeutic target for vascular remodeling after angioplasty.

Chemerin-9, a potent agonist of chemerin receptor (ChemR23), prevents atherogenesis.

Plasma levels of chemerin, an adipocytokine produced from the adipose tissues and liver, are associated with metabolic syndrome and coronary artery disease (CAD). Chemerin and its analog, chemerin-9, are known to bind to their receptor, ChemR23. However, whether chemerin and chemerin-9 affect atherogenesis remains to be elucidated. We investigated the expression of chemerin and ChemR23 in human coronary arteries and cultured human vascular cells. The effects of chemerin and chemerin-9 on atheroprone phenomena were assessed in human THP1 monocytes, human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs) and aortic lesions in Apoe-/- mice. In patients with CAD, a small amount of ChemR23, but not chemerin, was expressed within atheromatous plaques in coronary arteries. Chemerin and ChemR23 were expressed at high levels in THP1 monocytes, THP1-derived macrophages, and HUVECs; however, their expression in HASMCs was weak. Chemerin and chemerin-9 significantly suppressed the tumor necrosis factor-α (TNF-α)-induced mRNA expression of adhesion and pro-inflammatory molecules in HUVECs. Chemerin and chemerin-9 significantly attenuated the TNF-α-induced adhesion of THP1 monocytes to HUVECs and macrophage inflammatory phenotype. Chemerin and chemerin-9 suppressed oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation associated with down-regulation of CD36 and up-regulation of ATP-binding cassette transporter A1 (ABCA1). In HASMCs, chemerin and chemerin-9 significantly suppressed migration and proliferation without inducing apoptosis. In the Apoe-/- mice, a 4-week infusion of chemerin-9 significantly decreased the areas of aortic atherosclerotic lesions by reducing intraplaque macrophage and SMC contents. Our results indicate that chemerin-9 prevents atherosclerosis. Therefore, the development of chemerin analogs/ChemR23 agonists may serve as a novel therapeutic target for atherosclerotic diseases.

Association of glucose and blood pressure variability on oxidative stress in patients with type 2 diabetes mellitus and hypertension: a cross-sectional study.

BACKGROUND: The present study evaluated the effects of glucose and blood pressure (BP) variability on oxidative stress in patients with type 2 diabetes mellitus (T2DM) and hypertension. METHODS: A total of 60 inpatients with T2DM underwent continuous glucose monitoring (CGM) and ambulatory BP monitoring (ABPM). Oxidative stress was estimated using the diacron-reactive oxygen metabolites (d-ROMs) test. Glucose variability, mean glucose level, percentage coefficient of variation for glucose, mean amplitude of glycemic excursions (MAGE), and area under the postprandial plasma glucose curve were determined through CGM. BP variability was assessed by measuring average BP, standard deviation (SD) of systolic and diastolic BP, and coefficient of variation (CV) of systolic and diastolic BP during daytime and nighttime ABPM. RESULTS: Participants had a mean age of 64.5 ± 13.3 years with the duration of the disease 13.9 ± 12.4 years and HbA1c of 8.5 ± 1.2%. Univariate analysis showed that MAGE, nighttime SDs of systolic and diastolic BP, and nighttime CV of systolic BP were significantly correlated with d-ROMs. Further, stepwise multiple regression analysis identified MAGE, nighttime SD and CV of diastolic BP, estimated glomerular filtration rate, and smoking as independent contributors to d-ROMs. CONCLUSIONS: Oxidative stress was associated with daily glucose and nighttime diastolic BP variability in patients with T2DM and hypertension.Trial registration UMIN Clinical Trial Registry UMIN000035615, Registered January 22, 2019-retrospectively registered.

Legumain Promotes Atherosclerotic Vascular Remodeling.

Legumain, a recently discovered cysteine protease, is increased in both carotid plaques and plasma of patients with carotid atherosclerosis. Legumain increases the migration of human monocytes and human umbilical vein endothelial cells (HUVECs). However, the causal relationship between legumain and atherosclerosis formation is not clear. We assessed the expression of legumain in aortic atheromatous plaques and after wire-injury-induced femoral artery neointimal thickening and investigated the effect of chronic legumain infusion on atherogenesis in Apoe-/- mice. We also investigated the associated cellular and molecular mechanisms in vitro, by assessing the effects of legumain on inflammatory responses in HUVECs and THP-1 monocyte-derived macrophages; macrophage foam cell formation; and migration, proliferation, and extracellular matrix protein expression in human aortic smooth muscle cells (HASMCs). Legumain was expressed at high levels in atheromatous plaques and wire injury-induced neointimal lesions in Apoe-/- mice. Legumain was also expressed abundantly in THP-1 monocytes, THP-1 monocyte-derived macrophages, HASMCs, and HUVECs. Legumain suppressed lipopolysaccharide-induced mRNA expression of vascular cell adhesion molecule-1 (VCAM1), but potentiated the expression of interleukin-6 (IL6) and E-selectin (SELE) in HUVECs. Legumain enhanced the inflammatory M1 phenotype and oxidized low-density lipoprotein-induced foam cell formation in macrophages. Legumain did not alter the proliferation or apoptosis of HASMCs, but it increased their migration. Moreover, legumain increased the expression of collagen-3, fibronectin, and elastin, but not collagen-1, in HASMCs. Chronic infusion of legumain into Apoe-/- mice potentiated the development of atherosclerotic lesions, accompanied by vascular remodeling, an increase in the number of macrophages and ASMCs, and increased collagen-3 expression in plaques. Our study provides the first evidence that legumain contributes to the induction of atherosclerotic vascular remodeling.

Antiatherogenic effects of liraglutide in hyperglycemic apolipoprotein E-null mice via AMP-activated protein kinase-independent mechanisms.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) exert potent glucose-lowering effects without increasing risks for hypoglycemia and weight gain. Preclinical studies have demonstrated direct antiatherogenic effects of GLP-1RAs in normoglycemic animal models; however, the underlying mechanisms in hyperglycemic conditions have not been fully clarified. Here we aimed to elucidate the role of AMP-activated protein kinase (AMPK) in antiatherogenic effects of GLP-1RAs in hyperglycemic mice. Streptozotocin-induced hyperglycemic apolipoprotein E-null mice were treated with vehicle, low-dose liraglutide (17 nmol·kg-1·day-1), or high-dose liraglutide (107 nmol·kg-1·day-1) in experiment 1 and the AMPK inhibitor dorsomorphin, dorsomorphin + low-dose liraglutide, or dorsomorphin + high-dose liraglutide in experiment 2. Four weeks after treatment, aortas were collected to assess atherosclerosis. In experiment 1, metabolic parameters were similar among the groups. Assessment of atherosclerosis revealed that high-dose liraglutide treatments reduced lipid deposition on the aortic surface and plaque volume and intraplaque macrophage accumulation at the aortic sinus. In experiment 2, liraglutide-induced AMPK phosphorylation in the aorta was abolished by dorsomorphin; however, the antiatherogenic effects of high-dose liraglutide were preserved. In cultured human umbilical vein endothelial cells, liraglutide suppressed tumor necrosis factor-induced expression of proatherogenic molecules; these effects were maintained under small interfering RNA-mediated knockdown of AMPKα1 and in the presence of dorsomorphin. Conversely, in human monocytic U937 cells, the anti-inflammatory effects of liraglutide were abolished by dorsomorphin. In conclusion, liraglutide exerted AMPK-independent antiatherogenic effects in hyperlipidemic mice with streptozotocin-induced hyperglycemia, with the possible involvement of AMPK-independent suppression of proatherogenic molecules in vascular endothelial cells.

Pheochromocytoma presenting with severe hyperglycemia and metabolic acidosis following intra-articular glucocorticoid administration: a case report.

BACKGROUND: There are several reports of pheochromocytoma crisis triggered by systemic glucocorticoid administration. However, pheochromocytoma crisis after intra-articular glucocorticoid administration has been rarely reported. CASE PRESENTATION: A 45-year-old Japanese man presented to our hospital with a sudden, severe headache. He had no history of diabetes. He had received an intra-articular injection of betamethasone (2 mg) for joint pain, 2 days prior to his admission. On examination, his blood pressure was 240/126 mmHg and pulse was 120 beats/minute. The possibility of cerebrovascular events was ruled out by imaging studies and lumbar puncture. Blood tests revealed severe hyperglycemia (523 mg/dL) and metabolic acidosis (pH 7.21, anion gap 26.2 mEq/L, lactate 11.75 mmol/L) with a glycosylated hemoglobin level of 5.7%. Although a urine sample could not be obtained, fulminant type 1 diabetes mellitus and diabetic ketoacidosis were suspected based on these findings. However, after the initial treatment for diabetic ketoacidosis, his insulin secretion was found to be normal and the plasma levels of ketones were not elevated. This excluded the possibility of fulminant type 1 diabetes mellitus and diabetic ketoacidosis. Subsequently, a left adrenal gland tumor and elevated levels of serum catecholamine and urinary catecholamine metabolites were detected, while his other hormone levels were normal. Serum catecholamine levels did not decrease following the clonidine test, and a functional scintigraphy using iodine-131 metaiodobenzylguanidine showed strong uptake in the region of the left adrenal gland. Although no signs of pheochromocytoma crisis, such as paroxysmal hyperglycemia and hypertension, had been observed since admission, a pheochromocytoma was diagnosed based on the investigations. After controlling his blood pressure, a left adrenalectomy was performed. CONCLUSIONS: This case illustrates that intra-articular glucocorticoid administration can induce a pheochromocytoma crisis and an increase in hyperglycemia, and that pheochromocytoma crisis can resemble the clinical picture of fulminant type 1 diabetes mellitus owing to severe hyperglycemia with metabolic acidosis and normal glycosylated hemoglobin levels, especially under the influence of glucocorticoid.

Anti-Atherogenic Effects of Vaspin on Human Aortic Smooth Muscle Cell/Macrophage Responses and Hyperlipidemic Mouse Plaque Phenotype.

Vaspin (visceral adipose tissue-derived serine protease inhibitor) was recently identified as a novel adipocytokine with insulin-sensitizing effects. Serum vaspin levels are reported either increased or decreased in patients with coronary artery disease. Our translational research was performed to evaluate the expression of vaspin in human coronary atherosclerotic lesions, and its effects on atherogenic responses in human macrophages and human aortic smooth muscle cells (HASMC), as well as aortic atherosclerotic lesion development in spontaneously hyperlipidemic Apoe−/− mice, an animal model of atherosclerosis. Vaspin was expressed at high levels in macrophages/vascular smooth muscle cells (VSMCs) within human coronary atheromatous plaques. Vaspin significantly suppressed inflammatory phenotypes with nuclear factor κB down-regulation in human macrophages. Vaspin significantly suppressed oxidized low-density lipoprotein-induced foam cell formation with CD36 and acyl-coenzyme A: cholesterol acyltransferase-1 down-regulation and ATP-binding cassette transporters A1 and G1, and scavenger receptor class B type 1 up-regulation in human macrophages. Vaspin significantly suppressed angiotensin II-induced migration and proliferation with ERK1/2 and JNK down-regulation, and increased collagen production with phosphoinositide 3-kinase and Akt up-regulation in HASMCs. Chronic infusion of vaspin into Apoe−/− mice significantly suppressed the development of aortic atherosclerotic lesions, with significant reductions of intraplaque inflammation and the macrophage/VSMC ratio, a marker of plaque instability. Our study indicates that vaspin prevents atherosclerotic plaque formation and instability, and may serve as a novel therapeutic target in atherosclerotic cardiovascular diseases.

Glucose-Dependent Insulinotropic Polypeptide Suppresses Peripheral Arterial Remodeling in Male Mice.

Glucose-dependent insulinotropic polypeptide (GIP) exhibits direct cardiovascular actions in addition to its well-known insulinotropic effect. However, the role of GIP in peripheral artery disease remains unclear. In this study, we evaluated the effects of GIP against peripheral arterial remodeling in mouse models. The genetic deletion of GIP receptor (GIPR) led to exaggerated neointimal hyperplasia after transluminal femoral artery wire injury. Conversely, chronic GIP infusion suppressed neointimal hyperplasia and facilitated endothelial regeneration. The beneficial effects of GIP were abrogated by inhibiting nitric oxide (NO) synthase, suggesting a possible mechanism mediated by NO. In cultured human umbilical vein endothelial cells (HUVECs), GIP elevated cytosolic calcium levels without affecting intracellular cAMP levels. Furthermore, GIP dose-dependently increased NO production, whereas this effect was abolished by inhibiting AMP-activated protein kinase (AMPK). GIP induced AMPK phosphorylation, which was abrogated by inhibiting phospholipase C and calcium-calmodulin-dependent protein kinase kinase but not by adenylate cyclase or liver kinase B1, suggesting the existence of a calcium-mediated GIPR signaling pathway. These effects of GIP were retained in severe hyperglycemic Leprdb/ Leprdb mice and in high-glucose-cultured HUVECs. Overall, we demonstrated the protective effects of GIP against peripheral arterial remodeling as well as the involvement of a calcium-mediated GIPR signaling pathway in vascular endothelial cells. Our findings imply the potential vascular benefits of multiple agonists targeting G protein-coupled receptors, including GIPR, which are under development for the treatment of type 2 diabetes.

Effects of insulin and analogues on carcinogen-induced mammary tumours in high-fat-fed rats.

It is not fully clarified whether insulin glargine, an analogue with a high affinity for insulin-like growth factor-1 receptor (IGF-1R), increases the risk for cancers that abundantly express IGF-1R such as breast cancer or some types of breast cancer. To gain insight into this issue, female Sprague-Dawley rats fed a high-fat diet were given the carcinogen N-methyl-N-nitrosourea and randomly assigned to vehicle (control), NPH (unmodified human insulin), glargine or detemir (n = 30 per treatment). Insulins were given subcutaneously (15 U/kg/day) 5 days a week. Mammary tumours were counted twice weekly, and after 6 weeks of treatment, extracted for analysis. None of the insulin-treated groups had increased mammary tumour incidence at any time compared with control. At 6 weeks, tumour multiplicity was increased with NPH or glargine (P < 0.05) and tended to be increased with detemir (P = 0.2); however, there was no difference among insulins (number of tumours per rat: control = 0.8 ± 0.1, NPH = 1.8 ± 0.3, glargine = 1.5 ± 0.4, detemir = 1.4 ± 0.4; number of tumours per tumour-bearing rat: control = 1.3 ± 0.1, NPH = 2.2 ± 0.4, glargine = 2.7 ± 0.5, detemir = 2.3 ± 0.5). IGF-1R expression in tumours was lower than that in Michigan Cancer Foundation-7 (MCF-7) cells, a cell line that shows greater proliferation with glargine than unmodified insulin. In rats, glargine was rapidly metabolised to M1 that does not have greater affinity for IGF-1R. In conclusion, in this model of oestrogen-dependent breast cancer in insulin-resistant rats, insulin and insulin analogues increased tumour multiplicity with no difference between insulin types.

Adropin Contributes to Anti-Atherosclerosis by Suppressing Monocyte-Endothelial Cell Adhesion and Smooth Muscle Cell Proliferation.

Adropin, a peptide hormone expressed in liver and brain, is known to improve insulin resistance and endothelial dysfunction. Serum levels of adropin are negatively associated with the severity of coronary artery disease. However, it remains unknown whether adropin could modulate atherogenesis. We assessed the effects of adropin on inflammatory molecule expression and human THP1 monocyte adhesion in human umbilical vein endothelial cells (HUVECs), foam cell formation in THP1 monocyte-derived macrophages, and the migration and proliferation of human aortic smooth muscle cells (HASMCs) in vitro and atherogenesis in Apoe-/- mice in vivo. Adropin was expressed in THP1 monocytes, their derived macrophages, HASMCs, and HUVECs. Adropin suppressed tumor necrosis factor α-induced THP1 monocyte adhesion to HUVECs, which was associated with vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 downregulation in HUVECs. Adropin shifted the phenotype to anti-inflammatory M2 rather than pro-inflammatory M1 via peroxisome proliferator-activated receptor γ upregulation during monocyte differentiation into macrophages. Adropin had no significant effects on oxidized low-density lipoprotein-induced foam cell formation in macrophages. In HASMCs, adropin suppressed the migration and proliferation without inducing apoptosis via ERK1/2 and Bax downregulation and phosphoinositide 3-kinase/Akt/Bcl2 upregulation. Chronic administration of adropin to Apoe-/- mice attenuated the development of atherosclerotic lesions in the aorta, with reduced the intra-plaque monocyte/macrophage infiltration and smooth muscle cell content. Thus, adropin could serve as a novel therapeutic target in atherosclerosis and related diseases.

Catestatin Prevents Macrophage-Driven Atherosclerosis but Not Arterial Injury-Induced Neointimal Hyperplasia.

Catestatin, a catecholamine-release inhibitory peptide, has multiple cardiovascular activities. Conflicting results have been recently reported by increased or decreased plasma levels of catestatin in patients with coronary artery disease (CAD). However, there have been no previous reports regarding the effects of catestatin on arteriosclerosis. This study evaluated the vasoprotective effects of catestatin on human macrophages, human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs) in vitro, and aortic atherosclerosis and wire injury-induced femoral artery neointimal hyperplasia in apolipoprotein E-deficient (ApoE-/-) mice fed with a high-cholesterol diet. Histological expression of catestatin in coronary artery lesions and its plasma level were compared between CAD and non-CAD patients. Catestatin was abundantly expressed in cultured human monocytes, macrophages, HASMCs and HUVECs. Catestatin significantly suppressed lipopolysaccharide-induced upregulation of tumour necrosis factor-α, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in HUVECs. Catestatin significantly suppressed inflammatory responses and oxidized low-density lipoprotein-induced foam cell formation associated with acyl-CoA:cholesterol acyltransferase-1 downregulation and ATP-binding cassette transporter A1 upregulation in human macrophages. Catestatin significantly suppressed migration, proliferation and collagen-1 expression without inducing apoptosis, and increased elastin and fibronectin expression in HASMCs. Administration of catestatin into ApoE-/- mice significantly retarded entire aortic atherosclerotic lesions with declined contents of macrophages, SMCs and collagen fibres in atheromatous plaques, but not the femoral artery injury-induced neointimal hyperplasia. In CAD patients, catestatin levels were significantly decreased in plasma but increased in coronary atheromatous plaques. This study provided the first evidence that catestatin could prevent macrophage-driven atherosclerosis, but not SMC-derived neointimal hyperplasia after vascular injury.

Caveolin-1, a binding protein of CD26, is essential for the anti-inflammatory effects of dipeptidyl peptidase-4 inhibitors on human and mouse macrophages.

We previously reported that inhibition of dipeptidyl peptidase (DPP)-4, the catalytic site of CD26, prevents atherosclerosis in animal models through suppression of inflammation; however, the underlying molecular mechanisms have not been fully elucidated. Caveolin-1 (Cav-1), a major structural protein of caveolae located on the surface of the cellular membrane, has been reported to modulate inflammatory responses by binding to CD26 in T cells. In this study, we investigated the role of Cav-1 in the suppression of inflammation mediated by the DPP-4 inhibitor, teneligliptin, using mouse and human macrophages. Mouse peritoneal macrophages were isolated from Cav-1+/+ and Cav-1-/- mice after stimulation with 3% thioglycolate. Inflammation was induced by the toll-like receptor (TLR)4 agonist, lipopolysaccharide (LPS), isolated from Escherichia coli. The expression of pro-inflammatory cytokines was determined using reverse transcription-polymerase chain reaction. Co-expression of Cav-1 and CD26 was detected using immunohistochemistry in both mouse and human macrophages. Teneligliptin treatment (10 nmol/L) suppressed the LPS-induced expression of interleukin (IL)-6 (70%) and tumor necrosis factor-α (37%) in peritoneal macrophages isolated from Cav-1+/+ mice. However, teneligliptin did not have any effect on the macrophages from Cav-1-/- mice. In human monocyte/macrophage U937 cells, teneligliptin treatment suppressed LPS-induced expression of pro-inflammatory cytokines in a dose-dependent manner (1-10 nmol/L). These anti-inflammatory effects of teneligliptin were mimicked by gene knockdown of Cav-1 or CD26 using small interfering RNA transfection. Furthermore, neutralization of these molecules using an antibody against CD26 or Cav-1 also showed similar suppression. Teneligliptin treatment specifically inhibited TLR4 and TLR5 agonist-mediated inflammatory responses, and suppressed LPS-induced phosphorylation of IL-1 receptor-associated kinase 4, a downstream molecule of TLR4. Next, we determined whether teneligliptin could directly inhibit the physical interaction between Cav-1 and CD26 using the Biacore system. Binding of CD26 to Cav-1 protein was detected. Unexpectedly, teneligliptin also bound to Cav-1, but did not interfere with CD26-Cav-1 binding, suggesting that teneligliptin competes with CD26 for binding to Cav-1. In conclusion, we demonstrated that Cav-1 is a target molecule for DPP-4 inhibitors in the suppression of TLR4-mediated inflammation in mouse and human macrophages.