Mototsugu Nagao

Comparison of three α‐glucosidase inhibitors for glycemic control and bodyweight reduction in Japanese patients with obese type 2 diabetes

α‐Glucosidase inhibitors (αGIs) are widely used for the primary treatment of type 2 diabetes. We compared the clinical effects of three αGIs (miglitol, acarbose and voglibose) in patients with obese type 2 diabetes.

Liquid chromatography-tandem mass spectrometry determination of human plasma 1-palmitoyl-2-hydroperoxyoctadecadienoyl-phosphatidylcholine isomers via promotion of sodium adduct formation.

Accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product of phosphatidylcholine, in blood plasma has been observed in various pathological conditions, including atherosclerosis. In this study, we investigated the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to develop a method for accurate quantification of PCOOH (1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine, 16:0/HpODE PC), focusing on isomers such as 16:0/13-HpODE PC and 16:0/9-HpODE PC. Sodiated PCOOH ([M+Na](+), m/z 812) provided not only a known product ion (m/z 147) but also characteristic product ions (m/z 541 for 16:0/13-HpODE PC and m/z 388 for 16:0/9-HpODE PC). Thus, three multiple reaction monitorings (MRMs) could be performed. MRM (812/147) enabled determination of 16:0/HpODE PC, and MRM (812/541) and MRM (812/388) allowed specific measurement of 16:0/13-HpODE PC and 16:0/9-HpODE PC, respectively. By using this method, we could determine plasma PCOOH concentrations in healthy subjects and patients with angiographically significant stenosis. In healthy subject and patient plasma, the concentration of 16:0/HpODE PC was close to the sum of the concentrations of 16:0/13-HpODE PC and 16:0/9-HpODE PC. This finding shows that radical and/or enzymatic oxidation, rather than singlet oxygen oxidation, is recognized to cause peroxidation of PC. The newly developed LC-MS/MS method appears to be a powerful tool for developing a better understanding of in vivo lipid peroxidation and its involvement in human diseases.

Characterization of pancreatic islets in two selectively bred mouse lines with different susceptibilities to high-fat diet-induced glucose intolerance.

Hereditary predisposition to diet-induced type 2 diabetes has not yet been fully elucidated. We recently established 2 mouse lines with different susceptibilities (resistant and prone) to high-fat diet (HFD)-induced glucose intolerance by selective breeding (designated selectively bred diet-induced glucose intolerance-resistant [SDG-R] and -prone [SDG-P], respectively). To investigate the predisposition to HFD-induced glucose intolerance in pancreatic islets, we examined the islet morphological features and functions in these novel mouse lines. Male SDG-P and SDG-R mice were fed a HFD for 5 weeks. Before and after HFD feeding, glucose tolerance was evaluated by oral glucose tolerance test (OGTT). Morphometry and functional analyses of the pancreatic islets were also performed before and after the feeding period. Before HFD feeding, SDG-P mice showed modestly higher postchallenge blood glucose levels and lower insulin increments in OGTT than SDG-R mice. Although SDG-P mice showed greater β cell proliferation than SDG-R mice under HFD feeding, SDG-P mice developed overt glucose intolerance, whereas SDG-R mice maintained normal glucose tolerance. Regardless of whether it was before or after HFD feeding, the isolated islets from SDG-P mice showed impaired glucose- and KCl-stimulated insulin secretion relative to those from SDG-R mice; accordingly, the expression levels of the insulin secretion-related genes in SDG-P islets were significantly lower than those in SDG-R islets. These findings suggest that the innate predispositions in pancreatic islets may determine the susceptibility to diet-induced diabetes. SDG-R and SDG-P mice may therefore be useful polygenic animal models to study the gene-environment interactions in the development of type 2 diabetes.

Elevated miR-130a/miR130b/miR-152 expression reduces intracellular ATP levels in the pancreatic beta cell

MicroRNAs have emerged as important players of gene regulation with significant impact in diverse disease processes. In type-2 diabetes, in which impaired insulin secretion is a major factor in disease progression, dysregulated microRNA expression in the insulin-secreting pancreatic beta cell has been widely-implicated. Here, we show that miR-130a-3p, miR-130b-3p, and miR-152-3p levels are elevated in the pancreatic islets of hyperglycaemic donors, corroborating previous findings about their upregulation in the islets of type-2 diabetes model Goto-Kakizaki rats. We demonstrated negative regulatory effects of the three microRNAs on pyruvate dehydrogenase E1 alpha (PDHA1) and on glucokinase (GCK) proteins, which are both involved in ATP production. Consequently, we found both proteins to be downregulated in the Goto-Kakizaki rat islets, while GCK mRNA expression showed reduced trend in the islets of type-2 diabetes donors. Overexpression of any of the three microRNAs in the insulin-secreting INS-1 832/13 cell line resulted in altered dynamics of intracellular ATP/ADP ratio ultimately perturbing fundamental ATP-requiring beta cell processes such as glucose-stimulated insulin secretion, insulin biosynthesis and processing. The data further strengthen the wide-ranging influence of microRNAs in pancreatic beta cell function, and hence their potential as therapeutic targets in type-2 diabetes.

Repetitive Glucose Spikes Accelerate Atherosclerotic Lesion Formation in C57BL/6 Mice

Background A number of epidemiological studies demonstrated that postprandial hyperglycemia is a risk factor for cardiovascular disease in individuals with impaired glucose tolerance. Although several laboratory studies have addressed the plausible causal role of postprandial acute hyperglycemia (glucose spikes) in the development of atherosclerosis, there is little convincing evidence in vivo whether the atherosclerotic lesion formation can be accelerated solely by glucose spikes. Here, we assessed the effect of repetitive glucose spikes on atherosclerotic lesion formation in mice. Methods Female C57BL/6 mice were fed an atherogenic diet from 8 to 28 weeks of age. During the atherogenic diet feeding period, the mice orally received a glucose solution (50 mg glucose/mouse; G group) or water (W group) twice daily, 6 days a week. Atherosclerotic lesion formation in the aortic sinus was quantitatively analyzed in serial cross-sections by oil red O staining. Results G group mice showed transient increases in blood glucose level (~5 mmol/L above W group), and the levels returned to levels similar to those in W group mice within 60 min. No significant differences in glucose tolerance, insulin sensitivity, and plasma lipid profiles were observed after the 20-week repetitive administration between the 2 groups. G group mice showed an approximately 4-fold greater atherosclerotic lesion size in the aortic sinus than W group mice. Gene expression levels of Cd68 and Icam1 in the thoracic aorta were higher in G group mice than in W group mice. Conclusions These results indicate that glucose spikes can accelerate atherosclerotic lesion formation, with little influence on other metabolic disorders. Repetitive glucose administration in wild-type mice may serve as a simple and useful approach to better understanding the causal role of glycemic spikes in the development of atherosclerosis.

Involvement of Rac GTPase activation in phosphatidylcholine hydroperoxide-induced THP-1 cell adhesion to ICAM-1.

Increasing evidence indicates that phospholipid oxidation plays important roles in atherosclerosis. Here, we investigated the involvement of Rho-family GTPases inphosphatidylcholine hydroperoxide (PCOOH)-induced THP-1 cell adhesion to ICAM-1. Isoprenoid depletion by fluvastatin and geranylgeranyltransferase inhibition by GGTI-286 suppressed PCOOH-induced cell adhesion to ICAM-1 and F-actin-rich membrane protrusion formation. Pull-down assays demonstrated the activation of Rac1 and Rac2 in PCOOH-treated cells. Pan-Rho-family GTPase inhibitor Clostridium difficile toxin B, Rac-specific inhibitor NSC23776, and RNA interference of the Rac isoforms suppressed the cell adhesion. These findings indicate the involvement of Rac GTPase activation in PCOOH-induced cell adhesion to ICAM-1 via actin reorganization.

Transgenerational changes of metabolic phenotypes in two selectively bred mouse colonies for different susceptibilities to diet-induced glucose intolerance

We recently established 2 mouse lines with different susceptibilities (prone and resistant) to high-fat diet (HFD)-induced glucose intolerance by selective breeding (designated selectively bred diet-induced glucose intolerance-prone [SDG-P] and -resistant [SDG-R], respectively). In the present study, we analyzed transgenerational changes in metabolic phenotypes in these 2 mouse colonies to explore how the distinct phenotypes have emerged through the repetitive selection. Using C57BL/6, C3H, and AKR as background strains, mice showing inferior and superior glucose tolerance after HFD feeding were selected and bred repetitively over 20 generations to produce SDG-P and SDG-R, respectively. In addition to the blood glucose levels, HFD intake and body weight were also measured over the selective breeding period. As the generations proceeded, SDG-P mice became more susceptible to HFD-induced glucose intolerance and body weight gain, whereas SDG-R mice had gradually reduced HFD intake. The differences in fasting and post-glucose challenge blood glucose levels, body weight, and HFD intake became more evident between the 2 colonies through the selective breeding, mainly due to the HFD-induced glucose metabolism impairment and body weight gain in SDG-P mice and the reduction of HFD intake in SDG-R mice. These transgenerational changes in the metabolic phenotypes suggest that the genetic loci associated with the quantitative traits have been selectively enriched in SDG-P and SDG-R.

Fat intake and the development of type 2 diabetes [Review]

The increase in the number of patients with diabetes has become a worldwide healthcare issue, with numbers predicted to reach approximately 600 million by 2035. In Asia-Pacific region, the prevalence of type 2 diabetes has increased dramatically in recent decades, of which the major causes are believed to be modern lifestyle changes, e.g., Western dietary pattern and reduced physical activity, on their genetic basis of lower insulin secretory capacity. Particularly, in East Asian countries, the amount of fat intake has increased nearly three-fold over this half of century; dietary fat appears to be the major culprit of type 2 diabetes pandemic in East Asia. However, convincing evidence has not yet been provided as to whether high-fat diet causes type 2 diabetes in epidemiological cohort studies. Here, we summarize clinical studies regarding fat intake and type 2 diabetes, and animal studies on high-fat diet-induced diabetes including our recent works on the novel mouse lines (selectively bred diet-induced glucose intolerance-prone [SDG-P] and -resistant [SDG-R]) to address the etiology of high-fat diet-induced diabetes. These epidemiological and experimental findings would provide further insight into the etiology of type 2 diabetes under the modern nutritional environment, namely in the context of increased fat intake.

Effect of impaired glucose tolerance on atherosclerotic lesion formation: An evaluation in selectively bred mice with different susceptibilities to glucose intolerance

OBJECTIVE Impaired glucose tolerance (IGT) is an independent risk factor for atherosclerotic cardiovascular disease. However, due to the lack of appropriate animal models, the underlying mechanisms for IGT-induced atherosclerosis remain to be elucidated in vivo. We recently used selective breeding to establish 2 mouse lines with distinctively different susceptibilities to diet-induced glucose intolerance, designated selectively bred diet-induced glucose intolerance-resistant (SDG-R) and SDG-prone (SDG-P), respectively. Here, we assessed atherosclerotic lesion formation in these mice. METHODS Female SDG-R and SDG-P mice were fed an atherogenic diet (AD; 1.25% cholesterol, 0.5% sodium cholate, and 36% energy as fat) for 20 weeks (8-28 weeks of age). Oral glucose tolerance tests were performed during the AD-feeding period. Atherosclerotic lesion formation was quantitatively analyzed in serial aortic sinus sections by oil red O staining. Plasma lipids were measured after the AD-feeding period. RESULTS Glucose tolerance was impaired in SDG-P mice as compared to SDG-R mice over the 20-week AD-feeding period. No significant differences were observed in any plasma lipid measurement between the 2 mouse lines. Aortic sinus atherosclerotic lesion formation in SDG-P mice was approximately 4-fold greater than that in SDG-R mice. CONCLUSION In 2 mouse lines with different susceptibilities to diet-induced glucose intolerance, IGT accelerated atherosclerotic lesion formation. These mice may therefore serve as useful in vivo models for investigating the causal role of IGT in the pathogenesis of atherosclerosis.

Selective breeding of mice for different susceptibilities to high fat diet-induced glucose intolerance : Development of two novel mouse lines, Selectively bred Diet-induced Glucose intolerance-Prone and -Resistant

Aims/Introduction:  The development of type 2 diabetes is primarily due to lifestyle and environmental factors, as well as genetics, as shown by familial clustering. To establish mouse lines for evaluating heritable factors determining susceptibility to diet‐induced diabetes, we performed selective breeding for differences in high fat diet (HFD)‐induced glucose intolerance.