Tomoko Asagami

Impaired nitric oxide synthase pathway in diabetes mellitus: Role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase

Background—An endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), is elevated in patients with type 2 diabetes mellitus (DM). This study explored the mechanisms by which ADMA becomes elevated in DM. Methods and Results—Male Sprague-Dawley rats were fed normal chow or high-fat diet (n=5 in each) with moderate streptozotocin injection to induce type 2 DM. Plasma ADMA was elevated in diabetic rats (1.33±0.31 versus 0.48±0.08 &mgr;mol/L;P <0.05). The activity, but not the expression, of dimethylarginine dimethylaminohydrolase (DDAH) was reduced in diabetic rats and negatively correlated with their plasma ADMA levels (P <0.05). DDAH activity was significantly reduced in vascular smooth muscle cells and human endothelial cells (HMEC-1) exposed to high glucose (25.5 mmol/L). The impairment of DDAH activity in vascular cells was associated with an accumulation of ADMA and a reduction in generation of cGMP. In human endothelial cells, coincubation with the antioxidant polyethylene glycol–conjugated superoxide dismutase (22 U/mL) reversed the effects of the high-glucose condition on DDAH activity, ADMA accumulation, and cGMP synthesis. Conclusions—A glucose-induced impairment of DDAH causes ADMA accumulation and may contribute to endothelial vasodilator dysfunction in DM.

Apelin is necessary for the maintenance of insulin sensitivity

The recently discovered peptide apelin is known to be involved in the maintenance of insulin sensitivity. However, questions persist regarding its precise role in the chronic setting. Fasting glucose, insulin, and adiponectin levels were determined on mice with generalized deficiency of apelin (APKO). Additionally, insulin (ITT) and glucose tolerance tests (GTT) were performed. To assess the impact of exogenously delivered apelin on insulin sensitivity, osmotic pumps containing pyroglutamated apelin-13 or saline were implanted in APKO mice for 4 wk. Following the infusion, ITT/GTTs were repeated and the animals euthanized. Soleus muscles were harvested and homogenized in lysis buffer, and insulin-induced Akt phosphorylation was determined by Western blotting. Apelin-13 infusion and ITTs/GTTs were also performed in obese diabetic db/db mice. To probe the underlying mechanism for apelins effects, apelin-13 was also delivered to cultured C2C12 myotubes. 2-[3H]deoxyglucose uptake and Akt phosphorylation were assessed in the presence of various inhibitors. APKO mice had diminished insulin sensitivity, were hyperinsulinemic, and had decreased adiponectin levels. Soleus lysates had decreased insulin-induced Akt phosphorylation. Administration of apelin to APKO and db/db mice resulted in improved insulin sensitivity. In C2C12 myotubes, apelin increased glucose uptake and Akt phosphorylation. These events were fully abrogated by pertussis toxin, compound C, and siRNA knockdown of AMPKalpha1 but only partially diminished by LY-294002 and not at all by L-NAME. We conclude that apelin is necessary for the maintenance of insulin sensitivity in vivo. Apelins effects on glucose uptake and Akt phosphorylation are in part mediated by a G(i) and AMPK-dependent pathway.

Hyperglycemia limits experimental aortic aneurysm progression.

OBJECTIVE Diabetes mellitus (DM) is associated with reduced progression of abdominal aortic aneurysm (AAA) disease. Mechanisms responsible for this negative association remain unknown. We created AAAs in hyperglycemic mice to examine the influence of serum glucose concentration on experimental aneurysm progression. METHODS Aortic aneurysms were induced in hyperglycemic (DM) and normoglycemic models by using intra-aortic porcine pancreatic elastase (PPE) infusion in C57BL/6 mice or by systemic infusion of angiotensin II (ANG) in apolipoprotein E-deficient (ApoE(-/-)) mice, respectively. In an additional DM cohort, insulin therapy was initiated after aneurysm induction. Aneurysmal aortic enlargement progression was monitored with serial transabdominal ultrasound measurements. At sacrifice, AAA cellularity and proteolytic activity were evaluated by immunohistochemistry and substrate zymography, respectively. Influences of serum glucose levels on macrophage migration were examined in separate models of thioglycollate-induced murine peritonitis. RESULTS At 14 days after PPE infusion, AAA enlargement in hyperglycemic mice (serum glucose ≥ 300 mg/dL) was less than that in euglycemic mice (PPE-DM: 54% ± 19% vs PPE: 84% ± 24%, P < .0001). PPE-DM mice also demonstrated reduced aortic mural macrophage infiltration (145 ± 87 vs 253 ± 119 cells/cross-sectional area, P = .0325), elastolysis (% residual elastin: 20% ± 7% vs 12% ± 6%, P = .0209), and neovascularization (12 ± 8 vs 20 ± 6 vessels/high powered field, P = .0229) compared with PPE mice. Hyperglycemia limited AAA enlargement after ANG infusion in ApoE(-/-) mice (ANG-DM: 38% ± 12% vs ANG: 61% ± 37% at day 28). Peritoneal macrophage production was reduced in response to thioglycollate stimulation in hyperglycemic mice, with limited augmentation noted in response to vascular endothelial growth factor administration. Insulin therapy reduced serum glucose levels and was associated with AAA enlargement rates intermediate between euglycemic and hyperglycemic mice (PPE: 1.21 ± 0.14 mm vs PPE-DM: 1.00 ± 0.04 mm vs PPE-DM + insulin: 1.14 ± 0.05 mm). CONCLUSIONS Hyperglycemia reduces progression of experimental AAA disease; lowering of serum glucose levels with insulin treatment diminishes this protective effect. Identifying mechanisms of hyperglycemic aneurysm inhibition may accelerate development of novel clinical therapies for AAA disease.

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of abdominal aortic aneurysm (AAA) ever since it was first described in rats by Anidjar and colleagues.(1) The rationale for its development was based on the disrupted nature of elastin observed in AAAs. This rat model has been modified to produce AAAs in the infrarenal aortic region of mice.(2) The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Moreover, it is a viable platform to test potential therapeutic agents for AAA. In this video, we demonstrate the elastase infusion AAA procedure used in our laboratory. Mice are anesthetized using 2.5% isoflurane, and a laparotomy is performed under sterile conditions. The abdominal aortais isolated with the assistance of an operating stereomicroscope (Leica). After placing temporary ligatures around the proximal and distal aorta, an aortotomy is created at the bifurcation with the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing is introduced through the aortotomy and secured. The aortic lumen is subsequently perfused for 5-15 minutes at 100 mm Hg with saline containing type I porcine pancreatic elastase (4.5 U/mL; Sigma Chemical Co.). After removing the perfusion catheter, the aortotomy is repaired without constriction of the lumen.

Selective Glucocorticoid Receptor (GR-II) Antagonist Reduces Body Weight Gain in Mice

Previous research has shown that mifepristone can prevent and reverse weight gain in animals and human subjects taking antipsychotic medications. This proof-of-concept study tested whether a more potent and selective glucocorticoid receptor antagonist could block dietary-induced weight gain and increase insulin sensitivity in mice. Ten-week-old, male, C57BL/6J mice were fed a diet containing 60% fat calories and water supplemented with 11% sucrose for 4 weeks. Groups (n = 8) received one of the following: CORT 108297 (80 mg/kg QD), CORT 108297 (40 mg/kg BID), mifepristone (30 mg/kg BID), rosiglitazone (10 mg/kg QD), or vehicle. Compared to mice receiving a high-fat, high-sugar diet plus vehicle, mice receiving a high-fat, high-sugar diet plus either mifepristone or CORT 108297 gained significantly less weight. At the end of the four week treatment period, mice receiving CORT 108297 40 mg/kg BID or CORT 108297 80 mg/kg QD also had significantly lower steady plasma glucose than mice receiving vehicle. However, steady state plasma glucose after treatment was not highly correlated with reduced weight gain, suggesting that the effect of the glucocorticoid receptor antagonist on insulin sensitivity may be independent of its mitigating effect on weight gain.

Differential effects of insulin sensitivity on androgens in obese women with polycystic ovary syndrome or normal ovulation

The differential effects of insulin sensitivity and adiposity on androgen concentrations in women with polycystic ovary syndrome (PCOS) are unclear. To address this issue, we divided 43 overweight women into 4 groups based on both their clinical classification (PCOS or normal) and whether they were insulin resistant (IR) or insulin sensitive (IS) by their steady-state plasma glucose concentrations. Total testosterone concentrations were significantly increased as a function of both clinical classification (PCOS vs normal, P < .0001) and steady-state plasma glucose concentration (IR vs IS, P = .002). Mean testosterone concentrations were higher in PCOS-IR compared with PCOS-IS, normal-IR, or normal-IS women (P < .005). In addition, there was a statistically significant interaction (P = .03) between clinical classification (PCOS vs normal) and insulin sensitivity (IR vs IS) for testosterone concentrations. In contrast, androstenedione concentrations were higher in women with PCOS (P = .001), irrespective of whether they were IR or IS (P = .31); and no interaction between clinical classification and insulin sensitivity was discerned (P = .34). These results indicate that both PCOS and insulin resistance independently contributed to increased total testosterone concentrations within a group of overweight/obese women. These findings are consistent with the hypothesis that the ovaries of women with PCOS are hypersensitive to the ability of insulin to increase testosterone production and that the more insulin resistant the patient, the higher the testosterone concentration. In contrast, androstenedione concentrations seem to be independent of differences in insulin resistance. Our findings emphasize the need to increase understanding of the factors that modulate ovarian androgen secretion.

Enhanced monocyte adherence to thoracic aortae from rats with two forms of experimental hypertension

To extend our previous observation that thoracic aortae from rats with spontaneous hypertension (SHR) bind monocytoid cells with enhanced avidity, we isolated thoracic aortae from two different forms of rodent hypertension: Dahl salt-sensitive (Dahl-S) rats fed a high salt diet and Sprague-Dawley (S-D) rats fed a fructose-enriched diet. Blood pressure was determined 14 days after feeding normal chow or chow containing 8% NaCl to Dahl-S and Dahl salt-resistant (Dahl-R) rats, and either chow or a fructose-enriched diet to S-D and Fischer 344 (F-344) rats. Blood pressure was similar in Dahl-S and Dahl-R rats on the chow diet, but higher in Dahl-S rats in response to the 8% NaCl diet (188 +/- 7 v 137 +/- 3 mm Hg, P < .001). Blood pressure also increased when S-D rats consumed fructose as compared with chow (149 +/- 4 v 128 +/-2, P < .05), whereas blood pressure did not change with diet in F-344. Thoracic aortae were removed from rats in each experimental group, and their ability to bind murine monocytoid cells quantified. Measurements of monocyte binding were performed on one experimental and one control rat simultaneously, and results presented as the ratio of cells bound by thoracic aortae from the experimental compared with the control rat. With this approach, the ratio of monocyte binding (8% NaCl/chow) was increased in Dahl-S versus Dahl-R rats (1.7 +/- 0.1 v 1.3 +/- 0.1, P < .05), as well as in S-D as compared with F-344 rats (1.7 +/- 0.2 v 1.1 +/-0.1, P < .05). These results provide evidence that hypertension in Dahl-S and fructose-fed S-D rats was associated with changes in the endothelium that favor atherogenesis.