Ho Seon Park

PPARγ Gene Transfer Sustains Apoptosis, Inhibits Vascular Smooth Muscle Cell Proliferation, and Reduces Neointima Formation After Balloon Injury in Rats

Objective—There is still debate as to whether antiatherosclerotic effect of PPARγ ligands is dependant on PPARγ gene itself or some other pathway. Methods and Results—To investigate the effect of PPARγ gene modulation on neointima formation after balloon injury, we delivered adenoviral vectors expressing the wild-type (WT) dominant negative (DN) PPARγ, or a control gene (β-galactosidase [BG]) into carotid artery after balloon injury in rosiglitazone (a PPARγ ligand)-treated (R+) (3 mg/kg/d) and nontreated (R−) rats. Two weeks after gene delivery, in both R+ and R− animals, the PPARγ-WT gene transfer showed a significantly lower intima-media ratio (IMR) than control group. Moreover, the delivery of a PPARγ-DN form showed the highest IMR (in R+WT, 0.51±0.15; R+BG, 0.89±0.14; R+DN, 1.20±0.18, P<0.05 and in R−WT, 0.91±0.21; R−BG, 1.44±0.23; R−DN, 1.74±0.29, P<0.05). Proliferation and migration showed same result pattern as IMR. In addition, apoptotic indices were significantly higher in the PPARγ-WT gene transferred group than in the PPARγ-DN group. Conclusions—In vivo transfer of the PPARγ-WT gene was found to inhibit smooth muscle proliferation, sustain apoptosis, and reduce neointima formation after balloon injury irrespective of rosiglitazone treatment. These results indicate that PPARγ overexpression itself has a protective role against restenosis after balloon injury.

Lysophosphatidylcholine Activates Adipocyte Glucose Uptake and Lowers Blood Glucose Levels in Murine Models of Diabetes

Glucose homeostasis is maintained by the orchestration of peripheral glucose utilization and hepatic glucose production, mainly by insulin. In this study, we found by utilizing a combined parallel chromatography mass profiling approach that lysophosphatidylcholine (LPC) regulates glucose levels. LPC was found to stimulate glucose uptake in 3T3-L1 adipocytes dose- and time-dependently, and this activity was found to be sensitive to variations in acyl chain lengths and to polar head group types in LPC. Treatment with LPC resulted in a significant increase in the level of GLUT4 at the plasma membranes of 3T3-L1 adipocytes. Moreover, LPC did not affect IRS-1 and AKT2 phosphorylations, and LPC-induced glucose uptake was not influenced by pretreatment with the PI 3-kinase inhibitor LY294002. However, glucose uptake stimulation by LPC was abrogated both by rottlerin (a protein kinase Cδ inhibitor) and by the adenoviral expression of dominant negative protein kinase Cδ. In line with its determined cellular functions, LPC was found to lower blood glucose levels in normal mice. Furthermore, LPC improved blood glucose levels in mouse models of type 1 and 2 diabetes. These results suggest that an understanding of the mode of action of LPC may provide a new perspective of glucose homeostasis.

Effect of ginsam, a vinegar extract from Panax ginseng, on body weight and glucose homeostasis in an obese insulin-resistant rat model

Extracts of ginseng species show antihyperglycemic activity. We evaluated the antihyperglycemic and antiobesity effects of ginsam, a component of Panax ginseng produced by vinegar extraction, which is enriched in the ginsenoside Rg3. Otsuka Long-Evans Tokushima Fatty rats, an obese insulin-resistant rat model, were assigned into 1 of 3 groups (n = 8 each): controls (isotonic sodium chloride solution, 5 mL/d), rats given 300 mg/(kg d) ginsam, and rats given 500 mg/(kg d) ginsam. An intraperitoneal 2-hour glucose tolerance test was performed at the end of the 6-week treatment. After 8 weeks, body and liver weights, visceral fat measured by computed tomography, and fasting glucose and insulin concentrations and lipid profiles were recorded. Insulin-resistant rats treated with ginsam had lower fasting and postprandial glucose concentrations compared with vehicle-treated rats. Importantly, overall glucose excursion during the intraperitoneal 2-hour glucose tolerance test decreased by 21.5% (P < .01) in the treated rats, indicating improved glucose tolerance. Plasma insulin concentration was significantly lower in ginsam-treated rats. These changes may be related to increased glucose transporter 4 expression in skeletal muscle. Interestingly, when the data from both ginsam-treated groups were combined, body weight was 60% lower in the ginsam-treated rats than in the controls (P < .01). Liver weight and serum alanine aminotransferase concentrations were also lower in the ginsam-treated rats. These effects were associated with increased peroxisome proliferator-activated receptor gamma expression and adenosine monophosphate-activated protein kinase phosphorylation in liver and muscle. Our data suggest that ginsam has distinct beneficial effects on glucose metabolism and body weight control in an obese animal model of insulin resistance by changing the expression of genes involved in glucose and fatty acid metabolism.

Effect of a dipeptidyl peptidase-IV inhibitor, des-fluoro-sitagliptin, on neointimal formation after balloon injury in rats.

Background Recently, it has been suggested that enhancement of incretin effect improves cardiac function. We investigated the effect of a DPP-IV inhibitor, des-fluoro-sitagliptin, in reducing occurrence of restenosis in carotid artery in response to balloon injury and the related mechanisms. Methods and Findings Otsuka Long-Evans Tokushima Fatty rats were grouped into four: control (normal saline) and sitagliptin 100, 250 and 500 mg/kg per day (n = 10 per group). Sitagliptin or normal saline were given orally from 1 week before to 2 weeks after carotid injury. After 3 weeks of treatment, sitagliptin treatment caused a significant and dose-dependent reduction in intima-media ratio (IMR) in obese diabetic rats. This effect was accompanied by improved glucose homeostasis, decreased circulating levels of high-sensitivity C-reactive protein (hsCRP) and increased adiponectin level. Moreover, decreased IMR was correlated significantly with reduced hsCRP, tumor necrosis factor-α and monocyte chemoattractant protein-1 levels and plasminogen activator inhibitor-1 activity. In vitro evidence with vascular smooth muscle cells (VSMCs) demonstrated that proliferation and migration were decreased significantly after sitagliptin treatment. In addition, sitagliptin increased caspase-3 activity and decreased monocyte adhesion and NFκB activation in VSMCs. Conclusions Sitagliptin has protective properties against restenosis after carotid injury and therapeutic implications for treating macrovascular complications of diabetes.

Control of Adipogenesis by the SUMO-Specific Protease SENP2

ABSTRACT Here, we demonstrate that SENP2, a desumoylating enzyme, plays a critical role in the control of adipogenesis. SENP2 expression was markedly increased upon the induction of adipocyte differentiation, and this increase was dependent on protein kinase A activation. Remarkably, knockdown of SENP2 led to a dramatic attenuation of adipogenesis with a marked decrease in PPARγ and C/EBPα mRNA levels. Knockdown of SENP2 also caused a marked reduction in the level of C/EBPβ protein but not in that of C/EBPβ mRNA. Interestingly, sumoylation of C/EBPβ dramatically increased its ubiquitination and destabilization, and this increase could be reversed by SENP2. In addition, overexpression of C/EBPβ could overcome the inhibitory effect of SENP2 knockdown on adipogenesis. Furthermore, SENP2 was absolutely required for adipogenesis of preadipocytes implanted into mice. These results establish a critical role for SENP2 in the regulation of adipogenesis by desumoylation and stabilization of C/EBPβ and in turn by promoting the expression of its downstream effectors, such as PPARγ and C/EBPα.

Endothelial Progenitor Cell Cotransplantation Enhances Islet Engraftment by Rapid Revascularization

Impaired revascularization of transplanted islets is a critical problem that leads to progressive islet loss. Since endothelial progenitor cells (EPCs) are known to aid neovascularization, we aimed to enhance islet engraftment by cotransplanting EPCs with islets. Porcine islets, with (islet-EPC group) or without (islet-only group) human cord blood–derived EPCs, were transplanted into diabetic nude mice. The islet-EPC group reached euglycemia by ∼11 days posttransplantation, whereas the islet-only group did not. Also, the islet-EPC group had a higher serum porcine insulin level than the islet-only group. Islets from the islet-EPC group were more rapidly revascularized at the early period of transplantation without increment of final capillary density at the fully revascularized graft. Enhanced revascularization rate in the islet-EPC group was mainly attributed to stimulating vascular endothelial growth factor-A production from the graft. The rapid revascularization by EPC cotransplantation led to better graft perfusion and recovery from hypoxia. EPC cotransplantation was also associated with greater β-cell proliferation, probably by more basement membrane production and hepatocyte growth factor secretion. In conclusion, cotransplantation of EPCs and islets induces better islet engraftment by enhancing the rate of graft revascularization. These findings might provide a directly applicable tool to enhance the efficacy of islet transplantation in clinical practice.

Lysophosphatidic acid regulates blood glucose by stimulating myotube and adipocyte glucose uptake

Lysophosphatidic acid (LPA) is known to have diverse cellular effects, but although LPA is present in many biological fluids, including blood, its effects on glucose metabolism have not been elucidated. In this study, we investigated whether LPA stimulation is related to glucose regulation. LPA was found to enhance glucose uptake in a dose-dependent manner both in L6 GLUT4myc myotubes and 3T3-L1 adipocytes by triggering GLUT4 translocation to the plasma membrane. Moreover, the effect of LPA on glucose uptake was completely inhibited by pretreating both cells with LPA receptor antagonist Ki16425 and Gi inhibitor pertussis toxin. In addition, LPA increased the phosphorylation of AKT-1 with no effects on IRS-1, and LPA-induced glucose uptake was abrogated by pretreatment with the PI 3-kinase inhibitor LY294002. When low concentration of insulin and LPA were treated simultaneously, an additive effect on glucose uptake was observed in both cell types. In line with its cellular functions, LPA significantly lowered blood glucose levels in normal mice but did not affect insulin secretion. LPA also had a glucose-lowering effect in streptozotocin-treated type 1 diabetic mice. In combination, these results suggest that LPA is involved in the regulation of glucose homeostasis in muscle and adipose tissues.

EGb761, a Ginkgo Biloba Extract, Is Effective Against Atherosclerosis In Vitro, and in a Rat Model of Type 2 Diabetes

Background EGb761, a standardized Ginkgo biloba extract, has antioxidant and antiplatelet aggregation and thus might protect against atherosclerosis. However, molecular and functional properties of EGb761 and its major subcomponents have not been well characterized. We investigated the effect of EGb761 and its major subcomponents (bilobalide, kaemferol, and quercetin) on preventing atherosclerosis in vitro, and in a rat model of type 2 diabetes. Methods and Results EGb761 (100 and 200 mg/kg) or normal saline (control) were administered to Otsuka Long-Evans Tokushima Fatty rats, an obese insulin-resistant rat model, for 6 weeks (from 3 weeks before to 3 weeks after carotid artery injury). Immunohistochemical staining was performed to investigate cell proliferation and apoptosis in the injured arteries. Cell migration, caspase-3 activity and DNA fragmentation, monocyte adhesion, and ICAM-1/VCAM-1 levels were explored in vitro. Treatment with EGb761 dose-dependently reduced intima-media ratio, proliferation of vascular smooth muscle cells (VSMCs) and induced greater apoptosis than the controls. Proliferation and migration of VSMCs in vitro were also decreased by the treatment of EGb761. Glucose homeostasis and circulating adiponectin levels were improved, and plasma hsCRP concentrations were decreased in the treatment groups. Caspase-3 activity and DNA fragmentation increased while monocyte adhesion and ICAM-1/VCAM-1 levels decreased significantly. Among subcomponents of EGb761, kaemferol and quercetin reduced VSMC migration and increased caspase activity. Conclusions EGb761 has a protective role in the development of atherosclerosis and is a potential therapeutic agent for preventing atherosclerosis.

Effect of a peroxisome proliferator-activated receptor γ sumoylation mutant on neointimal formation after balloon injury in rats

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor regulating inflammation, atherosclerosis, insulin sensitivity and adipogenesis. Recently, it has been discovered that modification by the small ubiquitin-like modifier (SUMO) plays an important role in PPARgamma activity. In the present study, we investigated the effect of sumoylation on the antiatherogenic property of PPARgamma. PPARgamma-K107R sumoylation mutant, PPARgamma-wild type (WT) and control genes were transfected on vascular smooth muscle cells (VSMCs) to compare their effect on the proliferation and migration. Adenoviral vectors expressing the PPARgamma-K107R, PPARgamma-WT or control gene were delivered into the carotid arteries of rats after balloon injury. The PPARgamma-K107R increased the transcriptional activity of peroxisome proliferator response element (PPRE) and had a more potent transcriptional repression activity on the inducible nitric oxide synthase (iNOS) promoter as compared to the other sumoylation mutants or WT. PPARgamma-K107R or WT gene transfer inhibited VSMCs proliferation and migration to a greater extent than the control. The PPARgamma-K107R had more potent activity than PPARgamma-WT in this regard. PPARgamma-K107R or WT transfer showed a significantly lower intima-media ratio (IMR) than the control after balloon injury in rats. Again, the delivery of the PPARgamma-K107R decreased IMR further compared to PPARgamma-WT. In addition, the PPARgamma-K107R transfer showed a lower proliferation index and a higher apoptotic index than PPARgamma-WT. In conclusion, the PPARgamma sumoylation mutant K107R strongly inhibited VSMCs proliferation and migration, sustained apoptosis, and reduced neointimal formation after balloon injury. These results indicate that desumoylation at K107 in PPARgamma might play an important role against atherosclerosis.

Anti-diabetic efficacy of KICG1338, a novel glycogen synthase kinase-3β inhibitor, and its molecular characterization in animal models of type 2 diabetes and insulin resistance

Selective inhibition of glycogen synthase kinase-3 (GSK3) has been targeted as a novel therapeutic strategy for diabetes mellitus. We investigated the anti-diabetic efficacy and molecular mechanisms of KICG1338 (2-(4-fluoro-phenyl)-3H-imidazo[4,5-b]pyridine-7-carboxylic acid(4-methyl-pyridin-3-yl)-amide), a GSK3β inhibitor, in three animal models: Otsuka Long-Evans Tokushima Fatty (OLETF) rats, leptin receptors-deficient db/db mice, and diet-induced obese (DIO) mice. Biochemical parameters including glucose tolerance tests and gene expressions associated with glucose metabolism were investigated. Glucose excursion decreased significantly by KICG1338-treated OLETF rats, accompanied by increase in insulin receptor substrate-1 and glucose transporter (GLUT)-4 expressions in muscle and decreased GLUT-2 expression in liver. Glucose-lowering effects were similarly observed in KICG1338-treated db/db and DIO mice. KICG1338 treatment increased adiponectin levels and decreased TNF-α levels. KICG1338 therapy also led to greater β-cell preservation and less hepatic fat infiltration with decreased expressions of genes involved in inflammation and endoplasmic reticulum stress. These data demonstrate anti-diabetic efficacy of KICG1338, a novel GSK3β inhibitor.