Yusaku Mori

Effects of PKF275-055, a dipeptidyl peptidase–4 inhibitor, on the development of atherosclerotic lesions in apolipoprotein E–null mice

We recently discovered that glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide can both prevent the development of atherosclerosis in apolipoprotein E-null (Apoe(-/-)) mice. In the present study, we attempted to extend these findings to orally administered dipeptidyl peptidase (DPP)-4 inhibitor. Seventeen-week-old Apoe(-/-) mice fed an atherogenic diet were administered a DPP-4 inhibitor, vildagliptin analogue (PKF275-055 [PKF], 100 µm/[kg d]), in drinking water over a period of 4 weeks. Aortic atherosclerosis and oxidized low-density lipoprotein-induced foam cell formation were determined. Orally administered PKF increased plasma levels of active glucagon-like peptide-1 by 3.5-fold, increased total glucose-dependent insulinotropic polypeptide levels by 2-fold, reduced body weight by 13%, and reduced plasma cholesterol levels by 30%. Compared with drinking water controls, PKF significantly suppressed total aortic atherosclerotic lesions, atheromatous plaque in the aortic root, and macrophage accumulation in the aortic wall by 30% to 40% (P < .001). None of these changes were associated with the PKF-induced reductions in body weight and plasma cholesterol levels. Foam cell formation was suppressed by 40% in the exudate peritoneal macrophages obtained from the PKF-treated mice. The DPP-4 inhibitor prevents the development of atherosclerotic lesions by suppressing macrophage foam cell formation.

FFA-induced hepatic insulin resistance in vivo is mediated by PKCδ, NADPH oxidase, and oxidative stress

Fat-induced hepatic insulin resistance plays a key role in the pathogenesis of type 2 diabetes in obese individuals. Although PKC and inflammatory pathways have been implicated in fat-induced hepatic insulin resistance, the sequence of events leading to impaired insulin signaling is unknown. We used Wistar rats to investigate whether PKCδ and oxidative stress play causal roles in this process and whether this occurs via IKKβ- and JNK-dependent pathways. Rats received a 7-h infusion of Intralipid plus heparin (IH) to elevate circulating free fatty acids (FFA). During the last 2 h of the infusion, a hyperinsulinemic-euglycemic clamp with tracer was performed to assess hepatic and peripheral insulin sensitivity. An antioxidant, N-acetyl-L-cysteine (NAC), prevented IH-induced hepatic insulin resistance in parallel with prevention of decreased IκBα content, increased JNK phosphorylation (markers of IKKβ and JNK activation, respectively), increased serine phosphorylation of IRS-1 and IRS-2, and impaired insulin signaling in the liver without affecting IH-induced hepatic PKCδ activation. Furthermore, an antisense oligonucleotide against PKCδ prevented IH-induced phosphorylation of p47(phox) (marker of NADPH oxidase activation) and hepatic insulin resistance. Apocynin, an NADPH oxidase inhibitor, prevented IH-induced hepatic and peripheral insulin resistance similarly to NAC. These results demonstrate that PKCδ, NADPH oxidase, and oxidative stress play a causal role in FFA-induced hepatic insulin resistance in vivo and suggest that the pathway of FFA-induced hepatic insulin resistance is FFA → PKCδ → NADPH oxidase and oxidative stress → IKKβ/JNK → impaired hepatic insulin signaling.

Amelioration of Hyperglycemia with a Sodium-Glucose Cotransporter 2 Inhibitor Prevents Macrophage-Driven Atherosclerosis through Macrophage Foam Cell Formation Suppression in Type 1 and Type 2 Diabetic Mice.

Direct associations between hyperglycemia and atherosclerosis remain unclear. We investigated the association between the amelioration of glycemia by sodium-glucose cotransporter 2 inhibitors (SGLT2is) and macrophage-driven atherosclerosis in diabetic mice. We administered dapagliflozin or ipragliflozin (1.0 mg/kg/day) for 4-weeks to apolipoprotein E-null (Apoe −/−) mice, streptozotocin-induced diabetic Apoe −/− mice, and diabetic db/db mice. We then determined aortic atherosclerosis, oxidized low-density lipoprotein (LDL)-induced foam cell formation, and related gene expression in exudate peritoneal macrophages. Dapagliflozin substantially decreased glycated hemoglobin (HbA1c) and glucose tolerance without affecting body weight, blood pressure, plasma insulin, and lipids in diabetic Apoe −/− mice. Aortic atherosclerotic lesions, atheromatous plaque size, and macrophage infiltration in the aortic root increased in diabetic Apoe −/− mice; dapagliflozin attenuated these changes by 33%, 27%, and 20%, respectively. Atherosclerotic lesions or foam cell formation highly correlated with HbA1c. Dapagliflozin did not affect atherosclerosis or plasma parameters in non-diabetic Apoe −/− mice. In db/db mice, foam cell formation increased by 4-fold compared with C57/BL6 mice, whereas ipragliflozin decreased it by 31%. Foam cell formation exhibited a strong correlation with HbA1c. Gene expression of lectin-like ox-LDL receptor-1 and acyl-coenzyme A:cholesterol acyltransferase 1 was upregulated, whereas that of ATP-binding cassette transporter A1 was downregulated in the peritoneal macrophages of both types of diabetic mice. SGLT2i normalized these gene expressions. Our study is the first to demonstrate that SGLT2i exerts anti-atherogenic effects by pure glucose lowering independent of insulin action in diabetic mice through suppressing macrophage foam cell formation, suggesting that foam cell formation is highly sensitive to glycemia ex vivo.

Anagliptin, a dipeptidyl peptidase-4 inhibitor, decreases macrophage infiltration and suppresses atherosclerosis in aortic and coronary arteries in cholesterol-fed rabbits.

INTRODUCTION Several studies have demonstrated suppression of aortic atherosclerosis by dipeptidyl peptidase-4 (DPP-4) inhibitors in hypercholesterolemic mice. However, it remains unknown whether DPP-4 inhibitors also exert anti-atherogenic effects in coronary arteries. We examined the effect of anagliptin, a DPP-4 inhibitor, on atherosclerosis development in the aorta and coronary arteries in a high-cholesterol diet-fed rabbits. METHODS Japanese white rabbits were fed either normal chow (n=8) or a diet containing 0.5% cholesterol (n=34) for 14weeks. Cholesterol-fed rabbits were given 0.3% anagliptin or not in drinking water (each n=16 and 18) for 12weeks. RESULTS Dietary cholesterol intake markedly increased serum total cholesterol (TC) levels (1464±150mg/dL, mean±SE), and the most striking increase was observed among the major lipoproteins in very low-density lipoprotein (VLDL) as determined by high-performance liquid chromatography. No significant changes were observed in body weight, water intake, hemoglobin A1c, or glucose response to intravenous glucose loading following anagliptin administration. Anagliptin decreased TC and VLDL-cholesterol as well as cholesterol absorption markers sitosterol and campesterol slightly, although not significantly. Serum DPP-4 activity was suppressed by 82%, and active glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide levels were increased 2- to 3-fold by anagliptin treatment. Severe hypercholesterolemia resulted in the development of atherosclerosis in the aorta, and the ratio of atherosclerotic lesions to the total aortic surface area was 22±2%. Anagliptin suppressed the lesion ratio to 9±2% (p<0.001). Atherosclerotic lesions were clearly observed in the coronary arteries, where the mean intima-media area was enlarged, and intimal formation was developed. Anagliptin treatment attenuated the intima-media area and the intimal area by 43%. Alpha-smooth muscle actin-positive and macrophage-positive areas in the coronary arteries were suppressed by 66 and 75%, respectively, after anagliptin treatment. The aortic lesion ratio and the coronary intima area were correlated with each other (r=0.506, p<0.01), and each lesion correlated with TC in the whole cholesterol-fed rabbits. Gene expression of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 in the carotid arteries was markedly reduced by approximately 90%, and vascular DPP-4 activity was reduced by 66% after anagliptin treatment. CONCLUSIONS We demonstrated for the first time that a DPP-4 inhibitor can substantially suppress plaque formation in coronary arteries with a marked reduction in macrophage accumulation likely via its anti-inflammatory properties.

A Dipeptidyl Peptidase-4 Inhibitor but not Incretins Suppresses Abdominal Aortic Aneurysms in Angiotensin II-Infused Apolipoprotein E-Null Mice

AIM The main pathophysiology of abdominal aortic aneurysm (AAA) considerably overlaps with that of atherosclerosis. We reported that incretins [glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic polypeptide (GIP)] or a dipeptidyl peptidase-4 inhibitor (DPP-4I) suppressed atherosclerosis in apolipoprotein E-null (Apoe－/－) mice. Here we investigated the effects of incretin-related agents on AAA in a mouse model. METHODS Apoe－/－ mice maintained on an atherogenic diet were subcutaneously infused with saline, Ang II (2000 ng/kg/min), Ang II, and native GLP-1 (2.16 nmol/kg/day) or Ang II and native GIP (25 nmol/kg/day) for 4 weeks. DPP-4I (MK0626, 6 mg/kg/day) was provided in the diet to the Ang II-infused mice with or without incretin receptor antagonists [(Pro3) GIP and exendin (9-39)]. RESULTS AAA occurred in 70% of the animals receiving Ang II. DPP-4I reduced this rate to 40% and significantly suppressed AAA dilatation, fibrosis, and thrombosis. In contrast, incretins failed to attenuate AAA. Incretin receptor blockers did not reverse the suppressive effects of DPP-4I on AAA. In the aorta, DPP-4I significantly reduced the expression of Interleukin-1β and increased that of tissue inhibitor of metalloproteinase (TIMP)-2. In addition, DPP-4I increased the ratio of TIMP-2 to matrix metalloproteinases-9. CONCLUSIONS DPP-4I, MK0626, but not native incretins has protective effects against AAA in Ang II-infused Apoe－/－ mice via suppression of inflammation, proteolysis, and fibrosis in the aortic wall.

Anti‐atherogenic and anti‐inflammatory properties of glucagon‐like peptide‐1, glucose‐dependent insulinotropic polypepide, and dipeptidyl peptidase‐4 inhibitors in experimental animals

We reported that native incretins, liraglutide and dipeptidyl peptidase‐4 inhibitors (DPP‐4i) all confer an anti‐atherosclerotic effect in apolipoprotein E‐null (Apoe−/−) mice. We confirmed the anti‐atherogenic property of incretin‐related agents in the mouse wire injury model, in which the neointimal formation in the femoral artery is remarkably suppressed. Furthermore, we showed that DPP‐4i substantially suppresses plaque formation in coronary arteries with a marked reduction in the accumulation of macrophages in cholesterol‐fed rabbits. DPP‐4i showed an anti‐atherosclerotic effect in Apoe−/− mice mainly through the actions of glucagon‐like peptide‐1 and glucose‐dependent insulinotropic polypepide. However, the dual incretin receptor antagonists partially attenuated the suppressive effect of DPP‐4i on atherosclerosis in diabetic Apoe−/− mice, suggesting an incretin‐independent mechanism. Exendin‐4 and glucose‐dependent insulinotropic polypepide elicited cyclic adenosine monophosphate generation, and suppressed the lipopolysaccharide‐induced gene expression of inflammatory molecules, such as interleukin‐1β, interleukin‐6 and tumor necrosis factor‐α, in U937 human monocytes. This suppressive effect, however, was attenuated by an inhibitor of adenylate cyclase and mimicked by 8‐bromo‐cyclic adenosine monophosphate or forskolin. DPP‐4i substantially suppressed the lipopolysaccharide‐induced expression of inflammatory cytokines without affecting cyclic adenosine monophosphate generation or cell proliferation. DPP‐4i more strongly suppressed the lipopolysaccharide‐induced gene expression of inflammatory molecules than incretins, most likely through inactivation of CD26. Glucagon‐like peptide‐1 and glucose‐dependent insulinotropic polypepide suppressed oxidized low‐density lipoprotein‐induced macrophage foam cell formation in a receptor‐dependent manner, which was associated with the downregulation of acyl‐coenzyme A cholesterol acyltransferase‐1 and CD36, as well as the up‐regulation of adenosine triphosphate‐binding cassette transporter A1. Our studies strongly suggest that incretin‐related agents have favorable effects on macrophage‐driven atherosclerosis in experimental animals.

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.

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.

Combination Therapy with a Sodium-Glucose Cotransporter 2 Inhibitor and a Dipeptidyl Peptidase-4 Inhibitor Additively Suppresses Macrophage Foam Cell Formation and Atherosclerosis in Diabetic Mice

Dipeptidyl peptidase-4 inhibitors (DPP-4is), in addition to their antihyperglycemic roles, have antiatherosclerotic effects. We reported that sodium-glucose cotransporter 2 inhibitors (SGLT2is) suppress atherosclerosis in a glucose-dependent manner in diabetic mice. Here, we investigated the effects of combination therapy with SGLT2i and DPP-4i on atherosclerosis in diabetic mice. SGLT2i (ipragliflozin, 1.0 mg/kg/day) and DPP-4i (alogliptin, 8.0 mg/kg/day), either alone or in combination, were administered to db/db mice or streptozotocin-induced diabetic apolipoprotein E-null (Apoe−/−) mice. Ipragliflozin and alogliptin monotherapies improved glucose intolerance; however, combination therapy did not show further improvement. The foam cell formation of peritoneal macrophages was suppressed by both the ipragliflozin and alogliptin monotherapies and was further enhanced by combination therapy. Although foam cell formation was closely associated with HbA1c levels in all groups, DPP-4i alone or the combination group showed further suppression of foam cell formation compared with the control or SGLT2i group at corresponding HbA1c levels. Both ipragliflozin and alogliptin monotherapies decreased scavenger receptors and increased cholesterol efflux regulatory genes in peritoneal macrophages, and combination therapy showed additive changes. In diabetic Apoe−/− mice, combination therapy showed the greatest suppression of plaque volume in the aortic root. In conclusion, combination therapy with SGLT2i and DPP4i synergistically suppresses macrophage foam cell formation and atherosclerosis in diabetic mice.

Increased blood pressure in nesfatin/nuclebindin-2-transgenic mice

Nesfatin/nucleobindin-2 (nesf/NUCB2), a precursor of the anorexigenic protein nesfatin-1, is selectively expressed in the hypothalamic nuclei, which are central to the regulation of the autonomic nervous system. The present study sought to investigate the involvement of nesf/NUCB2 in the regulation of blood pressure and ingestive behavior, by using nesf/NUCB2-transgenic (Tg) mice. Blood pressure and heart rates were measured under conscious and unconscious conditions. Twenty-four-hour water intake and urine volume of male nesf/NUCB2-Tg mice and their littermates in metabolic cages were measured. After killing, kidney weight was measured and the mRNA expression of epithelial sodium channel (ENaC)-α and ENaC-γ was measured in the hypothalamus and kidney with real-time PCR. Systolic, diastolic and mean blood pressure were significantly higher in nesf/NUCB2-Tg mice, but pulse rate was not affected in conscious mice. In contrast, isoflurane anesthesia prevented an increase in blood pressure in the nesf/NUCB2-Tg mice. Twenty-four-hour water intake and urine volume were significantly higher in the nesf/NUCB2-Tg mice than in their non-Tg littermates. Urine sodium concentration was significantly lower in the nesf/NUCB2-Tg mice, although the serum sodium concentration and urine sodium excretion were not different between the genotypes. Kidney weight was significantly higher in the nesf/NUCB2-Tg mice than their non-Tg littermates, although there were no clear differences in the kidney histological findings between genotypes. The mRNA expression of ENaC-γ, but not ENaC-α, was decreased in the hypothalami of nesf/NUCB2-Tg mice. Our data suggested that Nesf/NUCB2 is involved in the regulation of blood pressure in the brain.