Loss of alpha2B-adrenoceptors increases magnitude of hypertension following nitric oxide synthase inhibition

Vascular alpha(2B)-adrenoceptors (alpha(2B)-AR) may mediate vasoconstriction and contribute to the development of hypertension. Therefore, we hypothesized that blood pressure would not increase as much in mice with mutated alpha(2B)-AR as in wild-type (WT) mice following nitric oxide (NO) synthase (NOS) inhibition with N(omega)-nitro-l-arginine (l-NNA, 250 mg/l in drinking water). Mean arterial pressure (MAP) was recorded in heterozygous (HET) alpha(2B)-AR knockout mice and WT littermates using telemetry devices for 7 control and 14 l-NNA treatment days. MAP in HET mice was increased significantly on treatment days 1 and 4 to 14, whereas MAP did not change in WT mice (days 0 and 14 = 113 +/- 3 and 114 +/- 4 mmHg in WT, 108 +/- 0.3 and 135 +/- 13 mmHg in HET, P < 0.05). MAP was significantly higher in HET than in WT mice days 10 through 14 (P < 0.05). Thus blood pressure increased more rather than less in mice with decreased alpha(2B)-AR expression. We therefore examined constrictor responses to phenylephrine (PE, 10(-9) to 10(-4) M) with and without NOS inhibition to determine basal NO contributions to arterial tone. In small pressurized mesenteric arteries (inner diameter = 177 +/- 5 microm), PE constriction was decreased in untreated HET arteries compared with WT (P < 0.05). l-NNA (100 microM) augmented PE constriction more in HET arteries than in WT arteries, and responses were not different between groups in the presence of l-NNA. Acetylcholine dilated preconstricted arteries from HET mice more than arteries from WT mice. Endothelial NOS expression was increased in HET compared with WT mesenteric arteries by Western analysis. Griess assay showed increased NO(x) concentrations in HET plasma compared with those in WT plasma. These data demonstrate that diminished alpha(2B)-AR expression increases the dependence of arterial pressure and vascular tone on NO production and that vascular alpha(2B)-AR either directly or indirectly regulates vascular endothelial NOS function.     

Increased pulmonary responses to acute ozone exposure in obese db/db mice

Epidemiological studies indicate the incidence of asthma is increased in obese and overweight humans. Responses to ozone (O(3)), an asthma trigger, are increased in obese (ob/ob) mice lacking the satiety hormone leptin. The long form of leptin receptor (Ob-R(b)) is required for satiety; mice lacking this receptor (db/db mice) are also substantially obese. Here, wild-type (WT) and db/db mice were exposed to air or O(3) (2 ppm) for 3 h. Airway responsiveness, measured by the forced oscillation technique, was greater in db/db than WT mice after air exposure. O(3)-induced increases in pulmonary resistance and airway responsiveness were also greater in db/db mice. BALF eotaxin, IL-6, KC, and MIP-2 increased 4 h after O(3) exposure and subsided by 24 h, whereas protein and neutrophils continued to increase through 24 h. For each outcome, the effect of O(3) was significantly greater in db/db than WT mice. Previously published results obtained in ob/ob mice were similar except for O(3)-induced neutrophils and MIP-2, which were not different from WT mice. O(3) also induced pulmonary IL-1beta and TNF-alpha mRNA expression in db/db but not ob/ob mice. Leptin was increased in serum of db/db mice, and pulmonary mRNA expression of short form of leptin receptor (Ob-R(a)) was similar in db/db and WT mice. These data confirm obese mice have innate airway hyperresponsiveness and increased pulmonary responses to O(3). Differences between ob/ob mice, which lack leptin, and db/db mice, which lack Ob-R(b) but not Ob-R(a), suggest leptin, acting through Ob-R(a), can modify some pulmonary responses to O(3).     

Modulation of single-nephron GFR in the db/db mouse model of type 2 diabetes mellitus. II. Effects of renal mass reduction

This study examines for the first time the effects of uninephrectomy (Nx) on modulation of whole kidney glomerular filtration rate (GFR), single-nephron GFR (SNGFR), and progression of diabetic nephropathy in the db/db mouse model of type 2 diabetes mellitus. To characterize SNGFR and tubuloglomerular feedback (TGF) responses to Nx and chronic neuronal nitric oxide synthase inhibition in the db/db mouse, we studied the effects of Nx on whole kidney GFR, SNGFR, and TGF characteristics in db/db and wild-type (WT) mice after Nx or sham Nx. We also documented progression of glomerular changes over a 6-mo period. Whole kidney GFR and SNGFR were significantly higher in db/db Nx than db/db sham mice, without change in proximal tubule reabsorptive rates. The TGF responses, determined as proximal-distal SNGFR differences, were brisk: 12.1 +/- 1.0 vs. 8.4 +/- 0.6 nl/min in WT sham (P < 0.05), 15.7 +/- 1.0 vs. 12.0 +/- 1.0 nl/min in WT Nx (P < 0.05), and 17.8 +/- 1.3 vs. 14.3 +/- 1.0 nl/min in db/db Nx (P < 0.05) mice. Chronic ingestion of the neuronal nitric oxide synthase inhibitor S-methylthiocitrulline for 2-3 wk after Nx had no effect on SNGFR or the TGF response. These studies show further elevations in whole kidney GFR and SNGFR in these hyperglycemic morbidly obese db/db mice, with an intact TGF system after Nx. In addition, in the db/db Nx mice, 4-6 mo after Nx, there was an exacerbation of the lesions of diabetic nephropathy, as quantified by a significant increase in the ratio of mesangial surface area to total glomerular surface area.     

Exercise restores coronary vascular function independent of myogenic tone or hyperglycemic status in db/db mice

Regulation of coronary function in diabetic hearts is an important component in preventing ischemic cardiac events but remains poorly studied. Exercise is recommended in the management of diabetes, but its effects on diabetic coronary function are relatively unknown. We investigated coronary artery myogenic tone and endothelial function, essential elements in maintaining vascular fluid dynamics in the myocardium. We hypothesized that exercise reduces pressure-induced myogenic constriction of coronary arteries while improving endothelial function in db/db mice, a model of type 2 diabetes. We used pressurized mouse coronary arteries isolated from hearts of control and db/db mice that were sedentary or exercised for 1 h/day on a motorized exercise-wheel system (set at 5.2 m/day, 5 days/wk). Exercise caused a approximately 10% weight loss in db/db mice and decreased whole body oxidative stress, as measured by plasma 8-isoprostane levels, but failed to improve hyperglycemia or plasma insulin levels. Exercise did not alter myogenic regulation of arterial diameter stimulated by increased transmural pressure, nor did it alter smooth muscle responses to U-46619 (a thromboxane agonist) or sodium nitroprusside (an endothelium-independent dilator). Moderate levels of exercise restored ACh-simulated, endothelium-dependent coronary artery vasodilation in db/db mice and increased expression of Mn SOD and decreased nitrotyrosine levels in hearts of db/db mice. We conclude that the vascular benefits of moderate levels of exercise were independent of changes in myogenic tone or hyperglycemic status and primarily involved increased nitric oxide bioavailability in the coronary microcirculation.     

Modulation of single-nephron GFR in the db/db mouse model of type 2 diabetes mellitus

Hyperfiltration has been implicated in the progression toward diabetic nephropathy in type 2 diabetes mellitus (DM2). This study focuses for the first time on the in vivo modulation of single-nephron GFR (SNGFR) in the classic B6.Cg-m(+/+)Lepr(db)/J (db/db) mouse model of DM2. To obtain stable preparations, it was necessary to use a sustaining infusion of 3.3 ml.100 g body wt(-1) x h(-1), or higher. SNGFR (measured both proximally and distally) was greater in db/db vs. heterozygote (db/m) mice (P < 0.05) but not vs. the wild-type (WT) mice. The tubuloglomerular feedback (TGF) responses, determined as free-flow proximal vs. distal SNGFR differences, were significant in db/db mice (11.6 +/- 0.8 vs. 9.3 +/- 1.0 nl/min, P < 0.01), in db/m mice (8.0 +/- 0.8 vs. 7.2 +/- 0.6 nl/min, P < 0.02), and WT mice (9.9 +/- 0.6 vs. 8.9 +/- 0.7 nl/min, P < 0.05). After increasing the sustaining infusion in the db/db mice, to offset glycosuric urine losses, the SNGFR increased significantly, and the TGF response was abolished. In these volume-replete db/db mice, absolute fluid reabsorption measured both at the late proximal and distal tubular sites were significantly increased vs. db/m mice infused at 3.3 ml.100 g body wt(-1) x h(-1). After infusion of the neuronal nitric oxide synthase (nNOS) inhibitor S-methylthiocitrulline, SNGFR fell in both db/db and db/m mice. These studies show that SNGFR is elevated in this mouse model of DM2, is suppressed by nNOS inhibition, and is modulated by TGF influences that are altered by the diabetic state and responsive to changes in extracellular fluid volume.     

Indirect effects of leptin receptor deficiency on lymphocyte populations and immune response in db/db mice

Leptin-deficient ob/ob and leptin receptor (Ob-rb)-deficient db/db mice display a marked thymic atrophy and exhibit defective immune responses. Lymphocytes express leptin receptors and leptin exerts direct effects on T cells in vitro. In addition, ob/ob and db/db mice display multiple neuroendocrine and metabolic defects, through which leptin deficiency may indirectly affect the immune system in vivo. To study the relative contributions of direct and indirect effects of leptin on the immune system in a normal environment, we generated bone marrow chimeras (BMCs) by transplantation of leptin receptor-deficient db/db, or control db/+, bone marrow cells into wild-type (WT) recipients. The size and cellularity of the thymus, as well as cellular and humoral immune responses, were similar in db/db to WT and db/+ to WT BMCs. The immune phenotype of db/db mice is thus not explained by a cell autonomous defect of db/db lymphocytes. Conversely, thymus weight and cell number were decreased in the reverse graft setting in WT to db/db BMCs, indicating that expression of the leptin receptor in the environment is important for T cell development. Finally, normal thymocyte development occurred in fetal db/db thymi transplanted into WT hosts, indicating that direct effects of leptin are not required locally in the thymic microenvironment. In conclusion, direct effects of leptin on bone marrow-derived cells and on thymic stromal cells are not necessary for T lymphocyte maturation in normal mice. In contrast, leptin receptor deficiency affects the immune system indirectly via changes in the systemic environment.     

Short-term exercise worsens cardiac oxidative stress and fibrosis in 8-month-old db/db mice by depleting cardiac glutathione

Abstract

Moderate exercise improves cardiac antioxidant status in young humans and animals with Type-2 diabetes (T2D). Given that both diabetes and advancing age synergistically decrease antioxidant expression in most tissues, it is unclear whether exercise can upregulate cardiac antioxidants in chronic animal models of T2D. To this end, 8-month-old T2D and normoglycemic mice were exercised for 3 weeks, and cardiac redox status was evaluated. As expected, moderate exercise increased cardiac antioxidants and attenuated oxidative damage in normoglycemic mice. In contrast, similar exercise protocol in 8-month-old db/db mice worsened cardiac oxidative damage, which was associated with a specific dysregulation of glutathione (GSH) homeostasis. Expression of enzymes for GSH biosynthesis [γ-glutamylcysteine synthase, glutathione reductase] as well as for GSH-mediated detoxification (glutathione peroxidase, glutathione-S-transferase) was lower, while toxic metabolites dependent on GSH for clearance (4-hydroxynonenal) were increased in exercised diabetic mice hearts. To validate GSH loss as an important factor for such aggravated damage, daily administration of GSH restored cardiac GSH levels in exercised diabetic mice. Such supplementation attenuated both oxidative damage and fibrotic changes in the myocardium. Expression of transforming growth factor beta (TGF-β) and its regulated genes which are responsible for such profibrotic changes were also attenuated with GSH supplementation. These novel findings in a long-term T2D animal model demonstrate that short-term exercise by itself can deplete cardiac GSH and aggravate cardiac oxidative stress. As GSH administration conferred protection in 8-month-old diabetic mice undergoing exercise, supplementation with GSH-enhancing agents may be beneficial in elderly diabetic patients undergoing exercise.     

Exercise training does not correct abnormal cardiac glycogen accumulation in the db/db mouse model of type 2 diabetes

Substrate imbalance is a well-recognized feature of diabetic cardiomyopathy. Insulin resistance effectively limits carbohydrate oxidation, resulting in abnormal cardiac glycogen accumulation. Aims of the present study were to 1) characterize the role of glycogen-associated proteins involved in excessive glycogen accumulation in type 2 diabetic hearts and 2) determine if exercise training can attenuate abnormal cardiac glycogen accumulation. Control (db(+)) and genetically diabetic (db/db) C57BL/KsJ-lepr(db)/lepr(db) mice were subjected to sedentary or treadmill exercise regimens. Exercise training consisted of high-intensity/short-duration (10 days) and low-intensity/long-duration (6 wk) protocols. Glycogen levels were elevated by 35-50% in db/db hearts. Exercise training further increased (2- to 3-fold) glycogen levels in db/db hearts. Analysis of soluble and insoluble glycogen pools revealed no differential accumulation of one glycogen subspecies. Phosphorylation (Ser(640)) of glycogen synthase, an indicator of enzymatic fractional activity, was greater in db/db mice subjected to sedentary and exercise regimens. Elevated glycogen levels were accompanied by decreased phosphorylation (Thr(172)) of 5'-AMP-activated kinase and phosphorylation (Ser(79)) of its downstream substrate acetyl-CoA carboxylase. Glycogen concentration was not associated with increases in other glycogen-associated proteins, including malin and laforin. Novel observations show that exercise training does not correct diabetes-induced elevations in cardiac glycogen but, rather, precipitates further accumulation.     

Paracrine effects of endothelial cells in a diabetic mouse model: capacitative calcium entry stimulated thromboxane release

Augmented vasoconstriction contributes to arterial stiffness associated with diabetes. It has been shown that capacitative calcium entry induced by sarcoplasmic-endoplasmic reticulum calcium ATPase blocker cyclopiazonic acid (CPA) in endothelial cells stimulates production of constrictor prostaglandins, which causes contractions of vascular smooth muscle cells. The aim of the work was to study the effect of diabetes on the vasoconstrictor response induced by calcium entry into endothelial and smooth muscle cells. Force was measured in isolated aortae of diabetic ob/ob and control C57BL/6J mice under isometric conditions. Contractions caused by 10 mumol/l CPA in diabetic mouse aortae featured higher amplitudes and longer durations in comparison with nondiabetic aortae. These contractions were abolished by a COX inhibitor indomethacin (10 mumol/l) or a specific thromboxane A2 receptor blocker SQ 29548 (1 mumol/l) and were not observed in denuded aortae. The contractions were sensitive to extracellular Ca (2+) and store-operated channel blockers. All together this suggests that vasoconstriction was caused by thromboxane A2 synthesis in endothelial cells induced by Ca (2+) entry through store-operated channels. Higher concentrations of CPA (30 mumol/l) or thapsigargin (1 mumol/l) elicited indomethacin-resistant tonic contractions of aortae with 2-fold amplitude in diabetic mice compared to their nondiabetic littermates, which were sensitive to store-operated channel blockers, but not to indomethacin, SQ 29548, or denudation. In conclusions, increases in intracellular Ca (2+) cause augmented vasoconstriction in diabetic vasculature through endothelial synthesis of contractile prostaglandins. In addition capacitative Ca (2+) entry is enhanced in diabetic vascular smooth muscle. These mechanisms indicate possible targets for clinical applications.     

RhoA-Rho kinase mediates synergistic ET-1 and phenylephrine contraction of rat corpus cavernosum

Maintenance of the detumescent state of the penis is believed to involve the actions of several vasoconstrictors. However, our mechanistic understanding of any synergistic vasoconstrictor influences is extremely limited. We tested the hypothesis that a vasoconstrictor combination of endothelin (ET-1) and phenylephrine (PE) augments the constrictor responses in rat corporal cavernosal tissues by a mechanism involving the RhoA-Rho kinase pathway. Independently, ET-1 (1 nM-30 microM) and PE (100 nM-100 microM) both caused dose-dependent contractions of isolated rat cavernosal tissues. In combination, ET-1 (30 nM) augmented the contractile effect of PE and shifted the calculated EC50 for PE (90 +/- 12 to 45 +/- 5 microM). The active stress generated by cavernosal strips during the ET-1 + PE combined stimulation (4.9 +/- 0.2 mN/mm2) was greater than the combined stress generated with ET-1 (0.4 +/- 0.1 mN/mm2) or PE (3.3 +/- 0.2 mN/mm2) stimulations alone. Blockade of ETA receptors (30 nM; A-127722) reversed the augmented stress generation and the Rho-kinase inhibitor Y-27632 differentially and dose-dependently relaxed the tissue. The combined constrictor effect was associated with a fourfold increase of RhoA in the membrane faction of the tissue homogenates. We conclude that the ET-1 + PE combination potentiate vasoconstriction through mutual activation of the RhoA-Rho kinase pathway. The interactions of these agonists likely play important roles in the maintenance of the flaccid state and contribute to some forms of erectile dysfunction.     

Abolition of arteriolar dilation but not constriction to histamine in cremaster muscle of eNOS-/- mice

Histamine increases the permeability of capillaries and venules but little is known of its precapillary actions on the control of tissue perfusion. Using gene ablation and pharmacological interventions, we tested whether histamine could increase muscle blood flow through stimulating nitric oxide (NO) release from microvascular endothelium. Vasomotor responses to topical histamine were investigated in second-order arterioles in the superfused cremaster muscle of anesthetized C57BL6 mice and null platelet endothelial cell adhesion molecule-1 (PECAM-1-/-) and null endothelial NO synthase (eNOS-/-) mice aged 8-12 wk. Neither resting (17 +/- 1 microm) nor maximum diameters (36 +/- 2 microm) were different between groups, nor was the constrictor response (approximately 5 +/- 1 microm) to elevating superfusate oxygen from 0 to 21%. For arterioles of C57BL6 and PECAM-1-/- mice, cumulative addition of histamine to the superfusate produced vasodilation (1 nM-1 microM; peak response, 9 +/- 1 microm) and then vasoconstriction (10-100 microM; peak response, 12 +/- 2 microm). In eNOS-/- mice, histamine produced only vasoconstriction. In C57BL6 and PECAM-1-/- mice, vasodilation was abolished with Nomega-nitro-l-arginine (30 microM); in all mice, vasoconstriction was abolished with nifedipine (1 microM). Vasomotor responses were eliminated with pyrilamine (1 microM; H1 receptor antagonist) yet remained intact with cimetidine (1 microM; H2 receptor antagonist). These findings illustrate that the biphasic vasomotor response of mouse cremaster arterioles to histamine is mediated through H1 receptors on endothelium (NO-dependent vasodilation) as well as smooth muscle (Ca2+ entry and constriction). Thus histamine can increase as well as decrease muscle blood flow, according to local concentration. However, when NO production is compromised, only vasoconstriction and flow reduction occur.     

Role of PKC in the novel synergistic action of urotensin II and angiotensin II and in urotensin II-induced vasoconstriction

The intracellular signaling of human urotensin II (hU-II) and its interaction with other vasoconstrictors such as ANG II are poorly understood. In endothelium-denuded rat aorta, coadministration of hU-II (1 nM) and ANG II (2 nM) exerted a significant contractile effect that was associated with increased protein kinase C (PKC) activity and phosphorylation of PKC-alpha/betaII and myosin light chain, whereas either hU-II or ANG II administered alone at these concentrations had no statistically significant effect. This synergistic effect was abrogated by the PKC inhibitor chelerythrine (10 and 30 microM), the selective PKC-alpha/betaII inhibitor G?-6976 (0.1 and 1 microM), the hU-II receptor ligand urantide (30 nM and 1 microM), or the ANG II antagonist losartan (1 microM). Moreover, in endothelium-intact rat aorta, the synergistic effect of hU-II and ANG II was not exerted any longer, and this synergistic effect was unmasked by pretreatment of the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester. hU-II (10 nM) alone caused a long-lasting increase in phospho-PKC-theta, phospho-myosin light chain, and PKC activity, which was associated with long-lasting vasoconstriction. These changes were prevented by chelerythrine. Methoxyverapamil-thapsigargin treatment reduced the hU-II-induced vasoconstriction by approximately 50%. The methoxyverapamil-thapsigargin-resistant component of hU-II-induced vasoconstriction was dose-dependently inhibited by chelerythrine. In conclusion, hU-II induces a novel PKC-dependent synergistic action with ANG II in inducing vasoconstriction. PKC-alpha/betaII is probably the PKC isoform involved in this synergistic action. Nitric oxide produced in the endothelium probably masks this synergistic action. The long-lasting vasoconstriction induced by hU-II alone is PKC dependent and associated with PKC-theta phosphorylation.     

IL-1beta-mediated innate immunity is amplified in the db/db mouse model of type 2 diabetes

Chronic inflammation appears to play a critical role in type 2 diabetes and its complications. Here we tested the hypothesis that this inflammatory dysregulation affects the IL-1beta system and has functional consequences in the brain. Diabetic, db/db, and nondiabetic, db/+, mice were administered i.p. LPS, a potent cytokine inducer, at a dose of 100 microg/kg/mouse. db/db mouse innate immune-associated sickness behavior was 14.8, 33, 44.7, and 34% greater than that of db/+ mice at 2, 4, 8, and 12 h, respectively. When a fixed dose of LPS was used (5 microg/mouse), db/db mouse sickness was again enhanced 18.4, 22.2, and 14.5% at 4, 8, and 12 h as compared with db/+ mice. In diabetic mice, peritoneal macrophages produced more IL-1beta in response to LPS, and peritoneal levels of IL-1beta induced by LPS were increased. Importantly, IL-1R antagonist and type 2 IL-1 receptor (IL-1R2) failed to up-regulate in response to LPS in db/db mice. Finally, both peripheral and central administration of IL-1beta, itself, induced sickness in db/db mice that mimicked the effects of peripheral LPS and was significantly greater than that seen in db/+ mice. Taken together, these results indicate that IL-1beta-mediated innate immunity is augmented in db/db mice both at the periphery and in the brain, and the mechanism is due to diabetes-associated loss of IL-1beta counterregulation.     

Exacerbation of endothelial dysfunction during the progression of diabetes: role of oxidative stress

To test the deterioration of endothelial function during the progression of diabetes, shear stress-induced dilation (SSID; 10, 20, and 40 dyn/cm(2)) was determined in isolated mesenteric arteries (80-120 μm in diameter) of 6-wk (6W), 3-mo (3M), and 9-mo (9M)-old male db/db mice and their wild-type (WT) controls. Nitric oxide (NO)-mediated SSID was comparable in 6W WT and db/db mice, but the dilation was significantly reduced in 3M db/db mice and declined further in 9M db/db mice. Vascular superoxide production was progressively increased in 3M and 9M db/db mice, associated with an increased expression of NADPH oxidase. Inhibition of NADPH oxidase significantly improved NO-mediated SSID in arteries of 3M, but not in 9M, db/db mice. Although endothelial nitric oxide synthase (eNOS) expression was comparable in all groups, a progressive reduction in shear stress-induced eNOS phosphorylation existed in vessels of 3M and 9M db/db mice. Moreover, inducible NOS (iNOS) that was not detected in WT, nor in 6W and 3M db/db mice, was expressed in vessels of 9M db/db mice. A significantly increased expression of nitrotyrosine in total protein and immunoprecipitated eNOS was also found in vessels of 9M db/db mice. Thus, impaired NO bioavailability plays an essential role in the endothelial dysfunction of diabetic mice, which becomes aggravated when endothelial nitrosative stress is further activated via perhaps, an additional iNOS-mediated pathway during the progression of diabetes.     

Impaired response of UCP family to cold exposure in diabetic (db/db) mice

Impaired activity of the uncoupling protein (UCP) family has been proposed to promote obesity development. The present study examined differences in UCP responses to cold exposure between leptin-resistance obese (db/db) mice and their lean (C57Ksj) littermates. Basal UCP1 and UCP3 mRNA expression in brown adipose tissue was lower in obese mice compared with lean mice, but UCP2 expression in white adipose tissue (WAT) was higher. Basal skeletal muscle UCP3 did not change remarkably. The UCP family mRNAs, which were upregulated 12 and 24 h after cold exposure (4 degrees C), were returned to prior levels 12 h after rewarming exposure (21 degrees C) in lean mice. The accelerating effects of cold exposure on the UCP family were impaired in db/db obese mice. Together with these changes, WAT lipoprotein lipase mRNA was downregulated, and the concentration of serum free fatty acid was increased in response to cold exposure in the lean mice but not in db/db obese littermates. The impaired function of the UCP family and diminished lipolysis in response to cold exposure indicate that the reduced lipolytic activity may contribute to the inactivation of the UCP family in db/db obese mice.     

Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes.

Activation of protein kinase C (PKC) is implicated as an important mechanism by which diabetes causes vascular complications. We have recently shown that a PKC beta inhibitor ameliorates not only early diabetes-induced glomerular dysfunction such as glomerular hyperfiltration and albuminuria, but also overexpression of glomerular mRNA for transforming growth factor beta1 (TGF-beta1) and extracellular matrix (ECM) proteins in streptozotocin-induced diabetic rats, a model for type 1 diabetes. In this study, we examined the long-term effects of a PKC beta inhibitor on glomerular histology as well as on biochemical and functional abnormalities in glomeruli of db/db mice, a model for type 2 diabetes. Administration of a PKC beta inhibitor reduced urinary albumin excretion rates and inhibited glomerular PKC activation in diabetic db/db mice. Administration of a PKC beta inhibitor also prevented the mesangial expansion observed in diabetic db/db mice, possibly through attenuation of glomerular expression of TGF-beta and ECM proteins such as fibronectin and type IV collagen. These findings provide the first in vivo evidence that the long-term inhibition of PKC activation in the renal glomeruli can ameliorate glomerular pathologies in diabetic state, and thus suggest that a PKC beta inhibitor might be an useful therapeutic strategy for the treatment of diabetic nephropathy.     

Hepatic glycogen metabolism in the db/db mouse

Summary Knowledge of the metabolic changes that occur in insulin-resistant type 2 diabetes is relatively lacking compared to insulin-deficient type 1 diabetes. This paper summarizes the importance of the C57BL/KsJ-db/db mouse as a model of type 2 diabetes, and illustrates the effects that insulin-deficient and insulin-resistant states have on hepatic glycogen metabolism. A longitudinal study of db/db mice of ages 2–15 weeks revealed that significant changes in certain parameters of hepatic glycogen metabolism occur during this period. The liver glycogen levels were similar between diabetic and control mice. However, glycogen particles from db/db mice were on average smaller in mass and had shorter exterior and interior chain lengths. Total phosphorylase and phosphorylase a activities were elevated in the genetically diabetic mice. This was primarily due to an increase in the amount of enzymic protein apparently the result of a decreased rate of degradation. It was not possible to find a consistent alteration in glycogen synthase activity in the db/db mice. Glycogen synthase and phosphorylase from diabetic liver revealed some changes in kinetic properties in the form of a decrease in V_max, and altered sensitivity to inhibitors like ATP. The altered glycogen structure in db/db mice may have contributed to changes in the activities and properties of glycogen synthase and phosphorylase. The exact role played by hormones (insulin and glucagon) in these changes is not clear but further studies should reveal their contributions. The db/db mouse provides a good model for type 2 diabetes and for fluctuating insulin and glucagon ratios. Its use should clarify the regulation of hepatic glycogen metabolism and other metabolic processes known to be controlled by these hormones. The other animal models of type 2 diabetes, ob/ob mouse and fatty Zucker (fa/fa) rat, show similar impairment of hepatic glycogen metabolism. The concentrations of glycogen metabolizing enzymes are high and in vitro studies indicate enhanced rate of glycogen synthesis and breakdown. However, streptozotocin-induced diabetic animals and BB rats which resemble insulin-deficient type 1 diabetes are characterized by decreased glycogen turnover as a result of reduction in the levels of glycogen metabolizing enzymes.     

Eucapnic intermittent hypoxia augments endothelin-1 vasoconstriction in rats: role of PKCdelta

We reported previously that simulating sleep apnea by exposing rats to eucapnic intermittent hypoxia (E-IH) causes endothelin-dependent hypertension and increases constrictor sensitivity to endothelin-1 (ET-1). In addition, augmented ET-1-induced constriction in small mesenteric arteries (sMA) is mediated by increased Ca(2+) sensitization independent of Rho-associated kinase. We hypothesized that exposing rats to E-IH augments ET-1-mediated vasoconstriction by increasing protein kinase C (PKC)-dependent Ca(2+) sensitization. In sMA, the nonselective PKC inhibitor GF-109203x (3 microM) significantly inhibited ET-1-stimulated constriction in E-IH arteries but did not affect ET-1-stimulated constriction in sham arteries. Phospholipase C inhibitor U-73122 (1 microM) also inhibited constriction by ET-1 in E-IH but not sham sMA. In contrast, the classical PKC (cPKC) inhibitor G?-6976 (1 microM) had no effect on ET-1-mediated vasoconstriction in either group, but a PKCdelta-selective inhibitor (rottlerin, 3 microM) significantly decreased ET-1-mediated constriction in E-IH but not in sham sMA. ET-1 increased PKCdelta phosphorylation in E-IH but not sham sMA. In contrast, ET-1 constriction in thoracic aorta from both sham and E-IH rats was inhibited by G?-6976 but not by rottlerin. These observations support our hypothesis that E-IH exposure significantly increases ET-1-mediated constriction of sMA through PKCdelta activation and modestly augments ET-1 contraction in thoracic aorta through activation of one or more cPKC isoforms. Therefore, upregulation of a PKC pathway may contribute to elevated ET-1-dependent vascular resistance in this model of hypertension.     

Thymic dysfunction in the mutant diabetic (db/db) mouse

Thymic function has been explored in genetically diabetic homozygous C57BL/KsJ (db/db) mice by evaluating their serum thymic factor (FTS) levels with a rosette assay. As previously reported for other autoimmune mice (NZB or MRL/I mice), the age-dependent decline of FTS levels was significantly accelerated in diabetic mice when compared to heterozygous littermates. Furthermore, FTS inhibitory molecules were detected in db/db mouse sera (as early as 10 wk of age) as evaluated by their ability to absorb in vitro the activity of synthetic FTS in the rosette assay, and in vivo for their capacity to induce the disappearance of endogenous FTS when injected into normal mice. These inhibitors were shown to be immunoglobulins. Histologically, the thymus presented an accelerated involution starting with a cortical lymphocytic depletion and an increased number of Hassall's corpuscles. Ultrastructural studies showed alterations in thymic epithelial cells, mainly represented by an increasing number of cytoplasmic vacuoles. By means of indirect immunofluorescence with anti-FTS monoclonal antibodies, it was shown that the number of FTS+ cells was reduced in db/db mouse thymuses: at the age of 22 wk, diabetic mice had 10 times fewer FTS+ cells than heterozygotes of the same age. Taken together, these results indicate important abnormalities in the thymus of diabetic mice. It is possible that the associated lymphocyte dysfunction plays a role in the pathogenesis of the autoimmune disease presented by db/db mice.     

H2O2 increases production of constrictor prostaglandins in smooth muscle leading to enhanced arteriolar tone in Type 2 diabetic mice

Our previous study showed that arteriolar tone is enhanced in Type 2 diabetes mellitus (T2-DM) due to an increased level of constrictor prostaglandins. We hypothesized that, in mice with T2-DM, hydrogen peroxide (H(2)O(2)) is involved in the increased synthesis of constrictor prostaglandins, hence enhanced basal tone in skeletal muscle arterioles. Isolated, pressurized gracilis muscle arterioles ( approximately 100 microm in diameter) of mice with T2-DM (C57BL/KsJ-db(-)/db(-)) exhibited greater basal tone to increases in intraluminal pressure (20-120 mmHg) than that of control vessels (at 80 mmHg, control: 25 +/- 5%; db/db: 34 +/- 4%, P < 0.05), which was reduced back to control level by catalase (db/db: 24 +/- 4%). Correspondingly, in carotid arteries of db/db mice, the level of dichlorofluorescein-detectable and catalase-sensitive H(2)O(2) was significantly greater. In control arterioles, exogenous H(2)O(2) (0.1-100 micromol/l) elicited dilations (maximum, 58 +/- 10%), whereas in arterioles of db/db mice H(2)O(2) caused constrictions (-28 +/- 8%), which were converted to dilations (maximum, 16 +/- 5%) by the thromboxane A(2)/prostaglandin H(2) (TP) receptor antagonist SQ-29548. In addition, arteriolar constrictions in response to the TP receptor agonist U-46619 were not different between the two groups of vessels. Endothelium denudation did not significantly affect basal tone and H(2)O(2)-induced arteriolar responses in either control or db/db mice. Also, in arterioles of db/db mice, but not in controls, 3-nitrotyrosine staining was detected in the endothelial layer of vessels. Thus we propose that, in mice with T2-DM, arteriolar production of H(2)O(2) is enhanced, which leads to increased synthesis of the constrictor prostaglandins thromboxane A(2)/prostaglandin H(2) in the smooth muscle cells, which enhance basal arteriolar tone. These alterations may contribute to disturbed regulation of skeletal muscle blood flow in Type 2 diabetes mellitus.