Sadanori Okada

Adipose Tissue–Specific Regulation of Angiotensinogen in Obese Humans and Mice: Impact of Nutritional Status and Adipocyte Hypertrophy

BACKGROUND The adipose tissue renin-angiotensin system (RAS) has been implicated in the pathophysiology of obesity and dysfunction of adipose tissue. However, neither regulation of angiotensinogen (AGT) expression in adipose tissue nor secretion of adipose tissue-derived AGT has been fully elucidated in humans. METHODS Human subcutaneous abdominal adipose tissue (SAT) biopsies were performed for 46 subjects with a wide range of body mass index (BMI). Considering the mRNA level of AGT and indices of body fat mass, the amount of adipose tissue-derived AGT secretion (A-AGT-S) was estimated. Using a mouse model of obesity and weight reduction, plasma AGT levels were measured with a newly developed enzyme-linked immunosorbent assay (ELISA), and the contribution of A-AGT-S to plasma AGT levels was assessed. RESULTS A-AGT-S was substantially increased in obese humans and the value was correlated with the plasma AGT level in mice. A-AGT-S and plasma AGT were higher in obese mice, whereas lower in mice with weight reduction. However, the AGT mRNA levels in the liver, kidney, and aorta were not altered in the mouse models. In both humans and mice, the AGT mRNA levels in mature adipocytes (MAs) were comparable to those in stromal-vascular cells. Coulter Multisizer analyses revealed that AGT mRNA levels in the MAs were inversely correlated with the average size of mature adipocytes. CONCLUSIONS This study demonstrates that adipose tissue-derived AGT is substantially augmented in obese humans, which may contribute considerably to elevated levels of circulating AGT. Adipose tissue-specific regulation of AGT provides a novel insight into the clinical implications of adipose tissue RAS in human obesity.

Glucocorticoid reamplification within cells intensifies NF-κB and MAPK signaling and reinforces inflammation in activated preadipocytes

Increased expression and activity of the intracellular glucocorticoid-reactivating enzyme 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) contribute to dysfunction of adipose tissue. Although the pathophysiological role of 11 beta-HSD1 in mature adipocytes has long been investigated, its potential role in preadipocytes still remains obscure. The present study demonstrates that the expression of 11 beta-HSD1 in preadipocyte-rich stromal vascular fraction (SVF) cells in fat depots from ob/ob and diet-induced obese mice was markedly elevated compared with lean control. In 3T3-L1 preadipocytes, the level of mRNA and reductase activity of 11 beta-HSD1 was augmented by TNF-alpha, IL-1 beta, and LPS, with a concomitant increase in inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), or IL-6 secretion. Pharmacological inhibition of 11 beta-HSD1 and RNA interference against 11 beta-HSD1 reduced the mRNA and protein levels of iNOS, MCP-1, and IL-6. In contrast, overexpression of 11 beta-HSD1 further augmented TNF-alpha-induced iNOS, IL-6, and MCP-1 expression. Moreover, 11 beta-HSD1 inhibitors attenuated TNF-alpha-induced phosphorylation of NF-kappaB p65 and p38-, JNK-, and ERK1/2-MAPK. Collectively, the present study provides novel evidence that inflammatory stimuli-induced 11 beta-HSD1 in activated preadipocytes intensifies NF-kappaB and MAPK signaling pathways and results in further induction of proinflammatory molecules. Not limited to 3T3-L1 preadipocytes, we also demonstrated that the notion was reproducible in the primary SVF cells from obese mice. These findings highlight an unexpected, proinflammatory role of reamplified glucocorticoids within preadipocytes in obese adipose tissue.

Decreased circulating CD34+ cells are associated with progression of diabetic nephropathy

Aims  Circulating progenitor cells such as CD34+ cells play a key role in maintenance of vascular endothelial function and neovascularization, and a decrease in the number of CD34+ cells is associated with cardiovascular disease. However, the contribution of circulating progenitor cells to microvascular disease, such as diabetic nephropathy, is unclear. This study was therefore designed to clarify the association between diabetic nephropathy and circulating CD34+ cells.

Adipose tissue–specific dysregulation of angiotensinogen by oxidative stress in obesity

Adipose tissue expresses all components of the renin-angiotensin system including angiotensinogen (AGT). Recent studies have highlighted a potential role of AGT in adipose tissue function and homeostasis. However, some controversies surround the regulatory mechanisms of AGT in obese adipose tissue. In this context, we here demonstrated that the AGT messenger RNA (mRNA) level in human subcutaneous adipose tissue was significantly reduced in obese subjects as compared with nonobese subjects. Adipose tissue AGT mRNA level in obese mice was also lower as compared with their lean littermates; however, the hepatic AGT mRNA level remained unchanged. When 3T3-L1 adipocytes were cultured for a long period, the adipocytes became hypertrophic with a marked increase in the production of reactive oxygen species. Expression and secretion of AGT continued to decrease during the course of adipocyte hypertrophy. Treatment of the 3T3-L1 and primary adipocytes with reactive oxygen species (hydrogen peroxide) or tumor necrosis factor alpha caused a significant decrease in the expression and secretion of AGT. On the other hand, treatment with the antioxidant N-acetyl cysteine suppressed the decrease in the expression and secretion of AGT in the hypertrophied 3T3-L1 adipocytes. Finally, treatment of obese db/db mice with N-acetyl cysteine augmented the expression of AGT in the adipose tissue, but not in the liver. The present study demonstrates for the first time that oxidative stress dysregulates AGT in obese adipose tissue, providing a novel insight into the adipose tissue-specific interaction between the regulation of AGT and oxidative stress in the pathophysiology of obesity.

Low-Dose Aspirin Therapy in Patients With Type 2 Diabetes and Reduced Glomerular Filtration Rate Subanalysis from the JPAD trial

OBJECTIVE Type 2 diabetes accompanied by renal damage is a strong risk factor for atherosclerotic events. The purpose of this study was to investigate the efficacy of low-dose aspirin therapy on primary prevention of atherosclerotic events in patients with type 2 diabetes and coexisting renal dysfunction. RESEARCH DESIGN AND METHODS The Japanese Primary Prevention of Atherosclerosis With Aspirin for Diabetes (JPAD) trial was a prospective, randomized, open-label trial conducted throughout Japan that enrolled 2,539 type 2 diabetic patients without a history of atherosclerotic diseases. Patients were assigned to the aspirin group (81 mg/day or 100 mg/day) or the nonaspirin group and followed for a median of 4.37 years. The primary end points were atherosclerotic events of fatal and nonfatal ischemic heart disease, stroke, and peripheral arterial disease. RESULTS The analysis included 2,523 patients who had serum creatinine measured. In 1,373 patients with baseline estimated glomerular filtration rate (eGFR) 60–89 mL/min/1.73 m2, the incidence of primary end points was significantly lower in the aspirin group than in the nonaspirin group (aspirin, 30/661; nonaspirin, 55/712; hazard ratio 0.57 [95% CI 0.36–0.88]; P = 0.011). Low-dose aspirin therapy did not reduce primary end points in patients with eGFR ≥90 mL/min/1.73 m2 (aspirin, 9/248; nonaspirin, 11/270; 0.94 [0.38–2.3]) or those with eGFR <60 mL/min/1.73 m2 (aspirin, 29/342; nonaspirin, 19/290; 1.3 [0.76–2.4]). The Cox proportional hazard model demonstrated a significant interaction between mild renal dysfunction (eGFR 60–89 mL/min/1.73 m2) and aspirin (P = 0.02). CONCLUSIONS These results suggest a differential effect of low-dose aspirin therapy in diabetic patients with eGFR 60–89 mL/min/1.73 m2.

Pioglitazone treatment stimulates circulating CD34-positive cells in type 2 diabetes patients

Circulating bone marrow derived immature cells, including CD34-positive (CD34(+)) cells, contribute to maintenance of the vasculature, not only as a pool of endothelial progenitor cells (EPCs), but also as a source of growth/angiogenesis factor. We hypothesized that the thiazolidineone compound pioglitazone could stimulate the circulating CD34(+) cells in diabetic patients. Thirty-four patients with type 2 diabetes received 15-30 mg pioglitazone for 24 weeks. The number of circulating CD34(+) cells significantly increased at 12 and continued this effect for 24 weeks (1.08+/-0.39, 1.34+/-0.34 and 1.32+/-0.28cells/microl at 0, 12 and 24 weeks, respectively). The change of CD34(+) cell levels (DeltaCD34(+) cells) between 0 and 12 weeks was significantly correlated with the change of high sensitive C reactive protein levels (Deltahs-CRP) and change in adiponectin levels (Deltaadiponectin) (r=-0.412, r=0.359, respectively). Our study demonstrated that pioglitazone treatment increased circulating CD34(+) cells, suggesting that this effect may at least partly contribute to the anti-atherosclerotic action of pioglitazone.

Low-Dose Aspirin for Primary Prevention of Cardiovascular Events in Patients With Type 2 Diabetes MellitusClinical Perspective: 10-Year Follow-Up of a Randomized Controlled Trial

Background: The long-term efficacy and safety of low-dose aspirin for primary prevention of cardiovascular events in patients with type 2 diabetes mellitus are still inconclusive. Methods: The JPAD trial (Japanese Primary Prevention of Atherosclerosis With Aspirin for Diabetes) was a randomized, open-label, standard care–controlled trial examining whether low-dose aspirin affected cardiovascular events in 2539 Japanese patients with type 2 diabetes mellitus and without preexisting cardiovascular disease. Patients were randomly allocated to receive aspirin (81 or 100 mg daily; aspirin group) or no aspirin (no-aspirin group) in the JPAD trial. After that trial ended in 2008, we followed up with the patients until 2015, with no attempt to change the previously assigned therapy. Primary end points were cardiovascular events, including sudden death, fatal or nonfatal coronary artery disease, fatal or nonfatal stroke, and peripheral vascular disease. For the safety analysis, hemorrhagic events, consisting of gastrointestinal bleeding, hemorrhagic stroke, and bleeding from any other sites, were also analyzed. The primary analysis was conducted for cardiovascular events among patients who retained their original allocation (a per-protocol cohort). Analyses on an intention-to-treat cohort were conducted for hemorrhagic events and statistical sensitivity. Results: The median follow-up period was 10.3 years; 1621 patients (64%) were followed up throughout the study; and 2160 patients (85%) retained their original allocation. Low-dose aspirin did not reduce cardiovascular events in the per-protocol cohort (hazard ratio, 1.14; 95% confidence interval, 0.91–1.42). Multivariable Cox proportional hazard model adjusted for age, sex, glycemic control, kidney function, smoking status, hypertension, and dyslipidemia showed similar results (hazard ratio, 1.04; 95% confidence interval, 0.83–1.30), with no heterogeneity of efficacy in subgroup analyses stratified by each of these factors (all interaction P>0.05). Sensitivity analyses on the intention-to-treat cohort yielded consistent results (hazard ratio, 1.01; 95% confidence interval, 0.82–1.25). Gastrointestinal bleeding occurred in 25 patients (2%) in the aspirin group and 12 (0.9%) in the no-aspirin group (P=0.03), and the incidence of hemorrhagic stroke was not different between groups. Conclusions: Low-dose aspirin did not affect the risk for cardiovascular events but increased risk for gastrointestinal bleeding in patients with type 2 diabetes mellitus in a primary prevention setting. Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00110448.

Differential Effect of Low-Dose Aspirin for Primary Prevention of Atherosclerotic Events in Diabetes Management: A subanalysis of the JPAD trial

OBJECTIVE Recent reports showed that low-dose aspirin was ineffective in the primary prevention of cardiovascular events in diabetic patients overall. We hypothesized that low-dose aspirin would be beneficial in patients receiving insulin therapy, as a high-risk group. RESEARCH DESIGN AND METHODS This study is a subanalysis of the Japanese Primary Prevention of Atherosclerosis With Aspirin for Diabetes (JPAD) trial—a randomized, controlled, open-label trial. We randomly assigned 2,539 patients with type 2 diabetes and no previous cardiovascular disease to the low-dose aspirin group (81 or 100 mg daily) or to the no-aspirin group. The median follow-up period was 4.4 years. We investigated the effect of low-dose aspirin on preventing atherosclerotic events in groups receiving different diabetes management. RESULTS At baseline, 326 patients were treated with insulin, 1,750 with oral hypoglycemic agents (OHAs), and 463 with diet alone. The insulin group had the longest history of diabetes, the worst glycemic control, and the highest prevalence of diabetic microangiopathies. The diet-alone group had the opposite characteristics. The incidence of atherosclerotic events was 26.6, 14.6, and 10.4 cases per 1,000 person-years in the insulin, OHA, and diet-alone groups, respectively. In the insulin and OHA groups, low-dose aspirin did not affect atherosclerotic events (insulin: hazard ratio [HR] 1.19 [95% CI 0.60−2.40]; OHA: HR 0.84 [0.57−1.24]). In the diet-alone group, low-dose aspirin significantly reduced atherosclerotic events, despite the lowest event rates (HR 0.21 [0.05−0.64]). CONCLUSIONS Low-dose aspirin reduced atherosclerotic events predominantly in the diet-alone group and not in the insulin or OHA groups.

Aspirin possibly reduces cerebrovascular events in type 2 diabetic patients with higher C-reactive protein level: Subanalysis from the JPAD Trial

BACKGROUND AND PURPOSE There are few data that demonstrate a significant effect of aspirin therapy for diabetic patients as primary prevention for cardiovascular events. A guideline recommends the use of aspirin as a primary prevention strategy in patients with diabetes who are at increased cardiovascular risk including those who have additional risk factors. To clarify the effect of primary prevention with aspirin therapy on diabetic patients, the relationship between C-reactive protein (CRP) and the incidence of atherosclerotic events was investigated in participants in the Japanese primary prevention of atherosclerosis with aspirin for diabetes (JPAD) trial. METHODS AND SUBJECTS We divided the JPAD participants according to the CRP level at enrollment; CRP ≥0.1mg/dl: high CRP group, CRP <0.1mg/dl: low CRP group. The high CRP group consisted of 1131 patients and the low CRP group consisted of 398 patients. ESSENTIAL RESULTS There was no significant difference in the incidence of primary atherosclerotic events between the high CRP group and the low CRP group. Of the atherosclerotic events, the incidence of cerebrovascular events, however, was significantly higher in the high CRP group than in the low CRP group. The incidence of cerebrovascular events was higher in the high CRP group than in the low CRP group in patients without aspirin therapy, although there was no significant difference in the incidence of the cerebrovascular events between the high CRP group and the low CRP group in patients undergoing aspirin therapy. PRINCIPAL CONCLUSIONS Aspirin therapy may reduce cerebrovascular events in diabetic patients with higher CRP. Aspirin therapy could be an additional strategy as primary prevention for diabetic patients with higher CRP.

Is long-term low-dose aspirin therapy associated with renal dysfunction in patients with type 2 diabetes? JPAD2 cohort study

Background Low-dose aspirin is widely recommended for patients at high risk for cardiovascular disease (CVD); however, it remains uncertain whether long-term treatment adversely affects renal function in patients with diabetes. We investigated whether long-term low-dose aspirin affects renal dysfunction in patients with diabetes. Methods We conducted a randomized controlled trial (RCT), the Japanese Primary Prevention of Atherosclerosis with Aspirin for Diabetes (JPAD) trial, to evaluate low-dose aspirin as primary prevention for CVD in patients with type 2 diabetes. We followed the patients with negative urine dipstick albumin of the JPAD trial in a cohort study after the RCT period was completed. Patients were randomly allocated to receive aspirin (81 mg or 100 mg daily, aspirin group) or no aspirin (no aspirin group). After the RCT, the treating physician decided whether to administer aspirin. We evaluated the incidence of positive urine dipstick albumin and annual changes in estimated glomerular filtration rate (eGFR). Results Positive urine dipstick albumin developed in 297 patients in the aspirin group (n = 1,075) and 270 patients in the no aspirin group (n = 1,098) during follow-up (median, 8.5 years). Intention-to-treat analysis showed low-dose aspirin did not increase the incidence of positive urine dipstick albumin (hazard ratio [HR], 1.17; 95% confidence interval [CI], 0.995–1.38). On-treatment analysis yielded similar results (HR, 1.08; 95% CI, 0.92–1.28). Multivariable analysis showed the incidence of positive urine dipstick albumin was higher among the elderly and those with elevated serum creatinine, high hemoglobin A1c, or high blood pressure; however, low-dose aspirin did not increase the risk of positive urine dipstick albumin. There were no significant differences in annual changes in eGFR between the groups (aspirin, −0.8 ± 2.9; no aspirin, −0.9 ± 2.5 ml/min/1.73m2/year). Conclusion Long-term low-dose aspirin does not affect eGFR and positive urine dipstick albumin in patients with type 2 diabetes.