Characterization and effects of methyl-2- (4-aminophenyl)-1, 2-dihydro-1-oxo-7- (2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel potent inhibitor of cGMP-binding cGMP-specific phosphodiesterase (PDE5)

An isoquinolone derivative, methyl-2-(4-aminophenyl)-1, 2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), was found to be a novel potent inhibitor of cyclic GMP (cGMP)-binding cGMP-specific phosphodiesterase (PDE5). We investigated the inhibitory effects of T-1032 on six PDE isozymes isolated from canine tissues. T-1032 specifically inhibited the hydrolysis of cGMP by PDE5 partially purified from canine lung, at a low concentration (IC(50) = 1.0 nM, K(i) = 1.2 nM), in a competitive manner. In contrast, the IC(50) values of T-1032 for PDE1, PDE2, PDE3, and PDE4 were more than 1 microM. T-1032 also inhibited PDE6 from canine retina with an IC(50) of 28 nM, which is of the same order of magnitude as the IC(50) of sildenafil. cGMP hydrolytic activities of two alternative splice variants of canine PDE5 expressed in COS-7 cells were inhibited by this compound to a similar extent. T-1032 increased the intracellular concentration of cGMP in cultured rat vascular smooth muscle cells in the presence and absence of C-type natriuretic peptide, an activator of membrane-bound guanylate cyclase, whereas the compound did not change cyclic AMP levels. These data indicated that T-1032, which belongs to a new structural class of PDE5 inhibitors, is a potent and selective PDE5 inhibitor. This compound may be useful in pharmacological studies to examine the role of a cGMP/PDE5 pathway in tissues.     

Intravenous AICAR during hyperinsulinemia induces systemic hemodynamic changes but has no local metabolic effect

AMPK activation may stimulate glucose uptake in skeletal muscle, but the results in humans have so far been inconclusive. The authors investigated whether infusion of the AMPK activator, 5-aminoimidazole-4-carboxamide-riboside (AICAR), increased whole-body glucose infusion rate (GIR) and forearm skeletal muscle glucose uptake (FGU) during hyperin-sulinemia in vivo in healthy humans. Ten participants (paired data: n = 8) underwent 2 euglycemic hyperinsulinemic clamps (60 mU·m(-2)·min(-1), 120 minutes) with concomitant AICAR (67 mg·kg(-1)) or placebo (saline) administration over the last 60 minutes. The authors also measured forearm blood flow (FBF; plethysmography), heart rate, blood pressure, and AICAR and AICA-ribotide (ZMP) concentrations in plasma and erythrocytes. FGU and GIR (T = 95-120 min) did not differ between insulin + AICAR and insulin + placebo. Compared with insulin + placebo, insulin + AICAR raised heart rate more profoundly (T = 60-120 minutes: from 58 ± 3 to 70 ± 3 vs 60 ± 4 to 63 ± 4 bpm for placebo; P < .05 between treatments) and lowered blood pressure significantly. AICAR plasma concentrations increased significantly during AICAR infusion; AICAR was rapidly taken up by erythrocytes and phosphorylated to ZMP. In conclusion, AICAR does not seem to have a direct effect on systemic or local glucose uptake in humans. AICAR increases heart rate and decreases blood pressure, most likely by systemic vasodilation.     

Pharmacological profile of T-1032, a novel specific phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum

This study was designed to examine the pharmacological properties of T-1032 (methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate), a novel phosphodiesterase type 5 inhibitor, in isolated rat aorta and rabbit corpus cavernosum. T-1032 (3x10(-11) to 3x10(-7) M) caused an endothelium-dependent relaxation in the isolated rat aorta precontracted with phenylephrine, and the relaxation was accompanied by an increase in cGMP but not cAMP levels. The T-1032-induced relaxation was attenuated by N(G)-nitro-L-arginine methyl ester (L-NAME) (10(-3) M), a nitric oxide (NO) synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) (10(-5) M), a guanylyl cyclase inhibitor. T-1032 (10(-9), 10(-8) M) produced a potentiation of the relaxation induced by sodium nitroprusside, but not of the relaxation induced by isoproterenol. In the isolated rabbit corpus cavernosum precontracted with phenylephrine, the electrical field stimulation-induced relaxation was attenuated by treatment with tetrodotoxin (10(-6) M) as well as L-NAME (10(-4) M). The L-NAME-inhibited relaxation was restored by treatment with L-arginine (5x10(-4) M). T-1032 (10(-9) to 10(-6) M) and sildenafil (10(-9) to 10(-6) M) produced a potentiation of the electrical field stimulation-induced relaxation as well as a decrease in basal tension in a concentration-dependent manner. It was concluded that T-1032 had potentiating effects on the NO/cGMP signaling pathway in isolated tissues, probably through specific blockade of phosphodiesterase type 5. T-1032 would be a useful compound to examine the physiologic functions of phosphodiesterase type 5 in mammalian tissues.     

Role of alpha 1- and alpha 2-adrenoceptors in catecholamine-induced hyperglycaemia, lipolysis and insulin secretion in conscious fasted rabbits.

1. In conscious fasted rabbits an intravenous infusion of clonidine (2 micrograms kg-1 min-1) induced hyperglycaemia. The increase in blood glucose was accompanied by an inhibition of insulin secretion and basal lipolysis. 2. Yohimbine infused at a rate of 20 micrograms kg-1 min-1 suppressed clonidine-induced hyperglycaemia and blocked the inhibitory effect on insulin secretion mediated by the alpha 2-adrenoceptor agonist. 3. The intravenous infusion of amidephrine (10 micrograms kg-1 min-1) induced an increase in insulin secretion in the absence of patent hyperglycaemia. Prazosin, 0.3 mg kg-1 s.c. selectively antagonized the effect of amidephrine on insulin secretion. 4. Isoprenaline infusion (4.4 micrograms kg-1 min-1) evoked a significant increase in blood glycerol and immunoreactive insulin plasma levels. Both responses were clearly attenuated when alpha 2-adrenoceptors were simultaneously stimulated by selective (clonidine) and less selective (phenylephrine, 20 micrograms kg-1 min-1) agonists. 5. Amidephrine infusion did not induce appreciable changes in blood glycerol nor did it modify, isoprenaline-induced lipolytic response. 6. Simultaneous infusion of isoprenaline and amidephrine induced a remarkable increase in insulin secretion. 7. It is concluded that in normal fasted rabbits stimulation of alpha 2-adrenoceptors depresses basal and beta-adrenoceptor mediated lipolysis and insulin secretion. On the other hand, selective stimulation of alpha 1-adrenoceptors does not affect lipolysis but induces insulin release. Simultaneous stimulation of alpha 1- and beta-adrenoceptors potentiates the insulin secretory response.     

Alpha-adrenoceptor involvement in catecholamine-induced hyperglycaemia in conscious fasted rabbits.

In conscious fasted rabbits an intravenous infusion of phenylephrine (20 micrograms kg-1 min-1) induced hyperglycaemia. The increase in blood glucose was accompanied by a modest increase in insulin secretion and a reduction of liver glycogen. Muscle glycogen and blood lactate levels were not altered by treatment with phenylephrine. Prazosin, 1 mg kg-1 s.c., partially attenuated phenylephrine-induced hyperglycaemia. Phenoxybenzamine infusion (16.6 micrograms kg-1 min-1) for 15 min suppressed the increase in blood glucose and the reduction in liver glycogen evoked by phenylephrine. This alpha-adrenoceptor blocker also clearly attenuated the blood glucose elevation observed on infusing adrenaline at 0.3 microgram kg-1 min-1. Blockade by phenoxybenzamine of phenylephrine- and adrenaline-induced hyperglycaemia was not accompanied by a significant increase in immunoreactive insulin plasma levels. Yohimbine infused at a rate of 20 micrograms kg-1 min-1, also completely blocked phenylephrine-induced hyperglycaemia. This suppressor effect was accompanied by a marked rebound in insulin secretion. It is concluded that in normal fasted rabbits stimulation of alpha-adrenoceptors induces hyperglycaemia. The increase in blood glucose depends mainly on liver glycogenolysis and inhibition of insulin secretion. Separate blockade of each component suffices to reduce alpha-adrenoceptor-mediated hyperglycaemia.     

Vascular effects of metabolic inhibition by 2-deoxy-D-glucose in humans

2-Deoxy-D-glucose (2DG) is a structural analogue of glucose that inhibits the glycolytic pathway. In vitro 2DG induced vasodilation, which was inhibited by glibenclamide (blocker of ATP-dependent potassium channels). The vasodilation induced by 2DG may be an interesting model for metabolic vasodilation. In this study, we investigated whether 2DG induces vasodilation in the human skeletal muscle vascular bed and whether this vasodilation was inhibited by glibenclamide. We measured forearm blood flow (FBF, venous occlusion plethysmography) in response to intra-arterial 2DG (0.4 and 0.8 mg/min x dl of forearm volume, each dose for 30 min), either alone or with co-infusion of insulin to increase the cellular 2DG uptake, and compared the results with insulin infusion alone. Glibenclamide infusion (1.0 microg/min x dl) was added in a subgroup. 2DG alone did not change FBF ratio (experimental/control arm, 11 +/- 9%, p = 0.34), but 2DG with insulin significantly increased FBF ratio (32 +/- 17%, p = 0.04). The increase in FBF by 2DG and insulin was significantly more pronounced than the vasodilation induced by insulin alone (p = 0.03). Co-infusion of glibenclamide had no effect on the vasodilation induced by 2DG and insulin (percentage increase in FBF ratio 11 +/- 18% without and 17 +/- 12% with glibenclamide, p = 0.85). In conclusion, 2DG induces manifest vasodilation in humans when infused in combination with insulin. This vasodilation is not mediated by ATP-dependent potassium channels.     

NO-dependent and -independent elevation of plasma levels of insulin and glucose in rats by L-arginine.

1. L-Arginine elevates plasma insulin in man. Recent in vitro data indicate that this is based on stimulation of endogenous nitric oxide (NO) with subsequent pancreatic release of insulin by L-arginine. L-Arginine also raises plasma glucose. 2. We studied plasma levels of insulin, glucose and NO metabolites, as well as systemic blood pressure, in anaesthetized rats during i.v. infusion of L-arginine (25-200 mg kg-1 min-1) or glucose (55 mg kg-1 min-1), before and after administration of the NO synthesis inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME, 50 mg kg-1). 3. Before L-NAME, L-arginine elevated plasma insulin from about 15 to 65 ul-1 and glucose from 5.2 to 6.7 mmol l-1. These effects of L-arginine were not dose-related. 4. L-NAME alone had no effect on plasma insulin and glucose levels, but diminished the effects of a low dose (25 mg kg-1 min-1) of L-arginine on plasma insulin by about 40%, and that on plasma glucose by more than 90%. In contrast, the effects of a high dose (200 mg kg-1 min-1) of L-arginine on plasma insulin and glucose levels were not affected by L-NAME. 5. L-NAME elevated systemic blood pressure by about 35 mmHg. L-Arginine (25-100 mg kg-1 min-1) had no effect on systemic blood pressure, either before or after L-NAME. L-Arginine (200 mg kg-1 min-1) lowered systemic blood pressure, both before and after L-NAME. 6. Glucose infusion elevated plasma glucose from about 5.5 to 6.8 mmol l-1, and plasma insulin from about 18 to 26 ul-1. 7. The basal plasma levels of the NO metabolite nitrate (18 +/- 4 mumol l-1) were not affected by L-arginine (200 mg kg-1 min-1). Plasma nitrosohaemoglobin was likewise unaffected by L-arginine (200 mg kg-1 min-1). 8. We conclude that L-arginine separately elevates plasma insulin and glucose levels, both by NO-dependent and -independent mechanisms.     

Long-term treatment with a phosphodiesterase type 5 inhibitor improves pulmonary hypertension secondary to heart failure through enhancing the natriuretic peptides-cGMP pathway

In advanced heart failure (HF), the compensatory pulmonary vasodilation is attenuated due to the relative insufficiency of cGMP despite increased secretion of natriuretic peptides (NPs). Phosphodiesterase type 5 (PDE5) inhibitors prevent cGMP degradation, and thus may potentiate the effect of the NPs-cGMP pathway. We orally administered a specific PDE5 inhibitor, T-1032 (1 mg/kg; twice a day, n = 7) or placebo (n = 7) for 2 weeks in dogs with HF induced by rapid pacing (270 bpm, 3 weeks) and examined the plasma levels of atrial natriuretic peptide (ANP), cGMP, and hemodynamic parameters. We also examined the hemodynamic changes after injection of a specific NPs receptor antagonist, HS-142-1 (3 mg/kg), under treatment with T-1032. T-1032 significantly increased plasma cGMP levels compared with the vehicle group despite low plasma ANP levels associated with improvement in cardiopulmonary hemodynamics. HS-142-1 significantly decreased plasma cGMP levels in both groups, whereas it did not change all hemodynamic parameters in the vehicle group. In contrast, in the T-1032 group, HS-142-1 significantly increased pulmonary arterial pressure and pulmonary vascular resistance. These results indicated that long-term treatment with a PDE5 inhibitor improved pulmonary hypertension secondary to HF and the NPs-cGMP pathway contributed to this therapeutic effect.     

静注卡介苗建立免疫性胰岛素抵抗模型

目的建立并考察卡介苗诱导的胰岛素抵抗动物模型。方法用正糖钳实验方法及其他生化检验方法测定卡介苗每鼠10 mg iv后大鼠血糖、葡萄糖耐量、血清TNF-α、胰岛素刺激的葡萄糖代谢速率及其他生化指标的变化。结果卡介苗iv后第2,4及8周,动物的葡萄糖耐量均受到损伤,胰岛素刺激的葡萄糖代谢速率显著下降,血清TNF-α含量升高。结论卡介苗iv可造成稳定的胰岛素抵抗模型,其造模机理可能与升高大鼠血清TNF-α含量有关。     

水杨酸钠对胰岛素抵抗大鼠氧化应激水平的影响

目的探讨水杨酸钠对胰岛素抵抗大鼠胰岛素敏感性的影响及作用机制。方法分别给大鼠静脉输注脂肪乳＋肝素,脂肪乳＋肝素＋水杨酸钠和生理盐水7h,部分大鼠在输液的最后2h同时行清醒状态高胰岛素一正血糖钳夹试验,测定血浆葡萄糖、游离脂肪酸（FFA）、胰岛素、C肽、丙二醛（MDA）水平,检测肝脏、肌肉中MI）A含量及谷胱甘肽过氧化物酶（GSH—Px）活性。结果脂肪乳输注组葡萄糖输注率（GIR）是生理盐水输注组的45％,输注水杨酸钠可使GIR提高1．3倍（P〈0．01）。与生理盐水输注组比较,脂肪乳输注组血浆、肝脏、肌肉中MDA水平增加了2～4倍（P〈0．01）;GSH—PX活性降低45％～50％（P〈0．01）。水杨酸钠输注使MDA水平较脂肪乳输注组降低了62％-66％（P〈0．01）,GSH-PX活性升高35％～38％（P〈0．05）。结论FFA增高引起机体氧化应激增强,可能是导致胰岛素抵抗发生的机制之一。应用水杨酸钠后大鼠氧化应激减弱,胰岛素抵抗改善,故水杨酸钠可能通过降低氧化应激途径而发挥改善胰岛素抵抗的作用。     

Sildenafil and T-1032, phosphodiesterase type 5 inhibitors, showed a different vasorelaxant property in the isolated rat aorta

The vasorelaxant effects of sildenafil and T-1032 [methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate], two phosphodiesterase type 5 inhibitors, were examined in the isolated rat aorta. Sildenafil and T-1032, both of which have almost the same potency and selectivity regarding phosphodiesterase type 5 inhibitory activity, produced a similar, moderate, relaxation at 10(-10) to 10(-7) M (sildenafil: 66.8 +/- 13.7%; T-1032: 77.9 +/- 10.8% at 10(-7) M). However, sildenafil, but not T-1032, produced further relaxation at the higher concentrations (sildenafil: 102.0 +/- 0.6%; T-1032: 81.0 +/- 7.2% at 10(-4) M, P < 0.05). Sildenafil also produced a more potent relaxation than did T-1032 at the high concentrations (10(-5) and 10(-4) M) in endothelium-denuded aortic rings and in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor (3 x 10(-4) M). Moreover, the high concentrations of sildenafil, but not of T-1032, caused a rightward shift of the concentration-response curve for calcium chloride in K(+)-depolarized endothelium-denuded preparations. In the ligand binding assay for the L-type Ca(2+) channels, the affinities of sildenafil at 10(-5) M for binding sites of nitrendipine and (--)-desmethoxyverapamil [(--)- D888] (35.2 +/- 3.3% and 35.8 +/- 1.9%, respectively) were higher than those of T-1032 (11.8 +/- 4.0% and -13.1 +/- 1.3%, respectively, P < 0.05). Regarding cyclic nucleotide levels, both phosphodiesterase type 5 inhibitors increased cGMP levels at 10(-6) M. However, sildenafil, but not T-1032, further increased cGMP levels at the higher concentrations (sildenafil: 15.7 +/- 2.7 pmol/mg protein; T-1032: 5.6 +/- 0.6 pmol/mg protein at 10(-4) M, P < 0.05). These results suggested that high concentrations of sildenafil had additional vasorelaxant properties through mechanisms other than phosphodiesterase type 5 inhibition. Sildenafil-induced relaxation appears to be due to inhibition of the external Ca(2+)-dependent cascade for contraction and/or to an increase in cGMP levels. In contrast, T-1032 only showed a vasorelaxant property due to phosphodiesterase type 5 inhibition. In conclusion, T-1032 appears to be a specific phosphodiesterase type 5 inhibitor compared with sildenafil and a useful compound to examine the physiological function of phosphodiesterase type 5.     

8例中国病人茶碱代谢物的药物动力学

目的：研究病人常规剂量茶碱治疗后代谢物的动力学。方法：病人静滴茶碱（６．６μｍｏｌ·ｋｇ＾－１）。ＨＰＬＣ法测定给药前后２４ｈ茶碱及其代谢物：１，３－二甲基尿酸（ＤＭＵＡ），３－甲基黄嘌呤（３－ＭＸ），１－甲基尿酸（ＭＵＡ），中间代谢产物１－甲基黄嘌呤（１－ＭＸ）的浓度。结果：ＤＭＵＡ是代谢物中浓度最高的。３－ＭＸ的清除速率最低。１－ＭＸ很快转化成ＭＵＡ，体内浓度很低，但是，翌晨，１－ＭＸ又回升到     

Correction of hyperglycemia and insulin sensitivity by T-1095, an inhibitor of renal Na+-glucose cotransporters, in streptozotocin-induced diabetic rats

We investigated the effects of T-1095 (3-(benzo[b]furan-5-yl)-2',6'-dihydroxy-4'-methylpropiophenone 2'-O-(6-O-methoxycarbonyl)-beta-D-glucopyranoside), an orally active inhibitor of Na+-glucose cotransporter, on hyperglycemia and insulin resistance in skeletal muscle of streptozotocin (STZ)-induced diabetic rats. Chronic (4 weeks) administration of T-1095 as food admixture (0.01 -0.1% wt/wt) suppressed the blood glucose level without affecting the food intake and body weight. In addition, the reduced 2-deoxyglucose uptake and lactate release in the soleus muscle of STZ rat was ameliorated by chronic treatment of T-1095. These data suggest that T-1095 improves insulin sensitivity in skeletal muscle through correction of hyperglycemia and has novel therapeutic potential for treatment of diabetes mellitus through removing glucose toxicity.     

Extrahepatic metabolism and distribution of aspirin in vascular beds of sheep

The dispositions of aspirin, its metabolite, salicylic acid, and its subsequent metabolite, salicyluric acid, were studied in eight anesthetized sheep infused with aspirin (61 and 485 microgram X min-1 X kg-1) for 75 min. Plasma samples were withdrawn from the portal vein, hepatic vein, pulmonary artery, left ventricle, left femoral vein, and left femoral artery. Significant extraction of aspirin occurred across the liver and hind leg, with mean availabilities of 0.75, 0.98, and 0.82 observed across the liver, lung, and hind leg, respectively. The extraction of aspirin was not affected by coadministration of sodium salicylate (30-1200 mg equiv salicylic acid). This extraction reflected hydrolysis of aspirin to salicylic acid; the metabolism of aspirin in the hind leg, lung, and liver being confirmed with tissue homogenate studies. The metabolism of aspirin in the extrahepatic tissues is significant in relation to the proposed selective presystemic acetylation of platelet cyclooxygenase by aspirin and the use of low-dose aspirin for thrombotic indications.     

Dopamine and dobutamine reduce myocardial d-propoxyphene content in experimentally intoxicated rats.

The effect of sympathomimetic intervention with dopamine or dobutamine on the myocardial uptake of d-propoxyphene was investigated experimentally in rats. The d-propoxyphene (19 mg kg-1 h-1) was continuously infused, intravenously, over 45 min. After 20 min of infusion the rats were given either dopamine (12.5 micrograms kg-1 min-1 or 25 micrograms kg-1 min-1), dobutamine (25 micrograms kg-1 min-1 or 45 micrograms kg-1 min-1) or normal saline (control). Each group consisted of eight rats. The myocardial d-propoxyphene content was significantly lower in the two groups given dopamine and in the group given dobutamine 45 micrograms kg-1 min-1 than in the control group (P less than 0.05). This finding indicates the benefit of early sympathomimetic intervention with either dopamine or dobutamine in d-propoxyphene intoxication.     

T-1032, a novel specific phosphodiesterase type 5 inhibitor, increases venous compliance in anesthetized rats.

Nitric oxide (NO) donors including organic nitrates dilate capacitance vessels. As inhibition of phosphodiesterase type 5 results in the accumulation of guanosine 3'5'-cyclic monophosphate (cGMP), specific phosphodiesterase type 5 inhibitors are expected to have a vasodilator property similar to that of NO donors. To test this hypothesis, we examined the effect of methyl2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel specific phosphodiesterase type 5 inhibitor, on mean arterial pressure and mean circulatory filling pressure (an index of venodilation) compared with that of nitroglycerin and diltiazem in mecamylamine- and noradrenaline-treated anesthetized rats. Intravenous infusion of T-1032 (0.1, 1, 10 microg/kg/min) dose-dependently decreased mean arterial pressure (-3.8+/-0.3%, -9.1+/-0.8%, -16.8+/-1.5% at doses of 0.1, 1 and 10 microg/kg/min, respectively) and mean circulatory filling pressure (-6.1+/-0.9%, -12.5+/-0.7%, -18.6+/-3.0% at doses of 0.1, 1 and 10 microg/kg/min, respectively). The mean circulatory filling pressure-mean arterial pressure relationship revealed that T-1032 had a selective action on the mean circulatory filling pressure compared with diltiazem (10, 100 microg/kg/min) and a similar or more selective effect than nitroglycerin (0.3, 3 and 30 microg/kg/min). In the next study, we calculated venous compliance and unstressed volume from the mean circulatory filling pressure-volume relationship. Intravenous infusion of T-1032 (3 microg/kg/min) increased venous compliance (3.35+/-0.40 in T-1032 vs. 2.31+/-0.15 ml/kg/mm Hg in vehicle, P<0.05) without changing the unstressed volume (37.2+/-2.80 in T-1032 vs. 42.6+/-2.37 ml/kg in vehicle, P>0.05). It was concluded that T-1032 increased venous capacitance by increasing venous compliance, and that this selective phosphodiesterase type 5 inhibitor appeared to have a different vasodilator action from that of an NO donor and a Ca(2+) channel antagonist in that it had a selective action on the mean circulatory filling pressure.     

Influence of inhibition of extraneuronal uptake and of O-methylation on the hyperglycaemia caused by sympathomimetic amines in depancreatized rats.

This study aimed at testing whether the O-methylating system (extraneuronal uptake + O-methylation) modulates, in-vivo, beta-adrenoceptor-mediated responses. The influences of U-0521 (3,4-dihydroxymethylpropiophenone, an inhibitor of catechol-O-methyl transferase (COMT)) and hydrocortisone (an inhibitor of extraneuronal uptake) on the hyperglycaemia evoked by isoprenaline and adrenaline were compared. Both inhibitors enhanced the increase of the plasma glucose level induced by either isoprenaline (0.36 nmol kg-1 min-1) or adrenaline (0.55 nmol kg-1 min-1). The enhancement caused by U-0521 developed faster than that caused by hydrocortisone, but was of the same magnitude. This is the first report of supersensitivity to sympathomimetic amines caused by inhibition of either COMT or extraneuronal uptake in-vivo and for a response not involving smooth muscle cells.     

Lack of inducible nitric oxide synthase prevents lipid-induced skeletal muscle insulin resistance without attenuating cytokine level

We examined whether deletion of inducible nitric oxide synthase (iNOS) could prevent lipid infusion-induced insulin resistance in iNOS-knockout and wild-type mice with the in vivo euglycemic-hyperinsulinemic clamp technique. Plasma NO metabolites were increased in lipid-infused wild-type mice, while they were not increased in iNOS-knockout mice. Plasma tumor necrosis factor-α levels were increased in both wild-type and iNOS-knockout by lipid-infusion. Lipid infusion reduced glucose infusion rate (GIR) and whole body glucose uptake in wild-type mice, whereas iNOS-knockout mice displayed comparable GIR and whole body glucose uptake compared with the control. In the gastrocnemius, lipid infusion decreased glucose uptake and glycolysis that were accompanied with increased phosphorylation of c-Jun N-terminal kinase and reduced phosphorylation of phosphoinositide 3-kinases and serine/threonine kinase Akt. However, lipid infusion did not affect glucose uptake or phosphorylation of these proteins in iNOS-knockout mice. The mRNA levels of inflammatory cytokines were also increased in the gastrocnemis of wild-type and iNOS-knockout mice by lipid infusion. Nitrotyrosine level in the gastrocnemius was increased in lipid-infused wild-type mice but it was not increased in iNOS-knockout mice. These results suggest that lack of iNOS prevents lipid infusion-induced skeletal muscle insulin resistance without attenuating cytokine levels.     

P2y purinoceptor responses of beta cells and vascular bed are preserved in diabetic rat pancreas.

1. To investigate the effect of experimental diabetes on the P2y purinoceptor responses of pancreatic beta-cells and vascular bed, we used adenosine-5'-O-(2-thiodiphosphate) (ADP beta S), a potent and stable P2y agonist. This work was performed in the isolated perfused pancreas of the rat. 2. Diabetes was induced by streptozotocin (66 mg kg-1, i.p.). Five weeks after the induction of diabetes, on the day of pancreas isolation, the animals displayed marked hyperglycaemia (37.6 +/- 2.7 mM). Age-matched rats were used as controls. 3. Insulin response to a glucose stimulation from 5 to 10 mM was completely lost and stimulation of insulin release by the sulphonylurea, tolbutamide (185 microM), was drastically impaired in the diabetic pancreas (maximum responses were 1.5 +/- 0.4 and 7.0 +/- 1.4 ng min-1 for diabetic and age-matched rats respectively). 4. In contrast, in the diabetic pancreas ADP beta S (15 microM), infused in the presence of glucose 5 mM, elicited an immediate and significant insulin release similar to that observed in the age-matched pancreas (maximum responses were 7.6 +/- 1.5 and 6.7 +/- 1.3 ng min-1 respectively). This ADP beta S stimulating effect occurred independently of the glucose concentration (5, 8.3 and 28 mM) in the diabetic pancreas. On pancreatic vascular resistance, ADP beta S induced a similar vasodilatation in diabetic and age-matched rats. 5. In conclusion, ADP beta S retains its insulin stimulatory and vasodilator effects in experimental diabetes; P2y purinoceptors could therefore be considered as a new target for the development of antidiabetic drugs.     

C-peptide has no effect on forearm blood flow during local hyperinsulinaemia in healthy humans

BACKGROUND: C-peptide increases forearm blood flow (FBF) in patients with Type 1 diabetes, probably by interaction with insulin, but not in healthy subjects. It is unclear if the vasodilating effect is sealed at normal fasting insulin concentrations. METHODS: The effects of C-peptide alone and during local hyperinsulinaemia were studied in healthy young men. Subjects received intra-arterial insulin at 6 pmol min-1 (low dose) or placebo for 60 min with subsequent coinfusion of C-peptide at increasing doses of 2-60 pmol min-1 in a double-blind crossover study (n = 8). In control experiments insulin at 30 pmol min-1 (high dose) was coinfused with C-peptide (n = 3). FBF was measured by strain-gauge plethysmography. RESULTS: Placebo had no effect on FBF (mean percentage change from baseline at 50 min -3.1%, 95% confidence interval [CI]-14.9, + 8.7). Insulin infusion slightly enhanced FBF by + 10.2% (95% CI -6.8, + 27.2; low dose) and + 17.6% (95% CI -38.8, + 74.0; high dose), respectively. The mean individual difference of the change in FBF between low-dose insulin and placebo was + 13.3% (95% CI -6.0, + 32.7; P = NS). Infusion of C-peptide increased local C-peptide concentrations from 1.8 +/- 0.1 ng ml-1 to 6.1 +/- 2.8 ng ml-1, but had no effect on FBF during placebo or hyperinsulinaemia (mean difference vs low dose insulin -16.0%, 95% CI -38.9, + 6.9). CONCLUSION: The vasodilating effect of C-peptide seen in Type 1 diabetes is not detectable during fasting or hyperinsulinaemia in the forearm vasculature of healthy subjects. This suggests saturation of its vasodilating potency at insulin concentrations within the normal or in the supraphysiological range.