"Vasocrine" signalling from perivascular fat: a mechanism linking insulin resistance to vascular disease

Adipose tissue expresses cytokines that inhibit insulin signalling pathways in liver and muscle. Obesity also results in impairment of endothelium-dependent vasodilatation in response to insulin. We propose a vasoregulatory role for local deposits of fat around the origin of arterioles supplying skeletal muscle. Isolated first-order arterioles from rat cremaster muscle are under dual regulation by insulin, which activates both endothelin-1 mediated vasoconstriction and nitric-oxide-mediated vasodilatation. In obese rat arterioles, insulin-stimulated NO synthesis is impaired, resulting in unopposed vasoconstriction. We propose that this vasoconstriction is the consequence of production of the adipocytokine tumour necrosis factor alpha from the cuff of fat seen surrounding the origin of the arteriole in obese rats--a depot to which we ascribe a specialist vasoregulatory role. We suggest that this cytokine accesses the nutritive vascular tree to inhibit insulin-mediated capillary recruitment--a mechanism we term "vasocrine" signalling. We also suggest a homology between this vasoactive periarteriolar fat and both periarterial and visceral fat, which may explain relations between visceral fat, insulin resistance, and vascular disease.     

Vascular responses to IGF-I and insulin are impaired in aortae of hypertensive rats

OBJECTIVE: Insulin-like growth factor-I (IGF-I) and insulin are important vasoactive peptides but little is known about their effects in hypertension. DESIGN: We compared the responses of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rat aortae to IGF-I and insulin. METHODS: Aortae were removed from WKY and SHRSP, cut into 2-3 mm rings, and contractile responses to phenylephrine and endothelin-1 studied in organ chambers in the presence of vehicle, IGF-I (0.1 micromol/l) or insulin (0.1 micromol/l). In addition, the effects of nitric oxide synthase (NOS) inhibition, phosphatidylinositol 3-kinase (PI3-kinase) inhibition and superoxide scavenging on these responses were investigated. RESULTS: Incubation with IGF-I and insulin caused attenuation of phenylephrine-induced and endothelin-1-induced vasoconstriction in arteries from normotensive but not hypertensive animals. In the arteries from WKY rats, co-incubation with either wortmannin or LY294002, inhibitors of PI3-kinase, attenuated the effect of IGF-I. The vasorelaxant effect of IGF-I was also abolished by removal of the endothelium or addition of the NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME). Co-incubation with tiron, a superoxide scavenger, suggested that the attenuation of IGF-I vasodilation in SHRSP arteries was not due to excess superoxide production. CONCLUSION: In WKY, IGF-I/insulin attenuate phenylephrine-mediated constrictions via PI3-kinase/nitric oxide pathways. In contrast, in SHRSP these pathways are dysfunctional and IGF-I has little effect on vascular responses.     

Effects of insulin on the acetylcholine-induced hyperpolarization in the guinea pig mesenteric arterioles

Background: Insulin induces endothelium-dependent vasodilatation, which may be casually related to the insulin resistance and hypertension. Endothelium-derived nitric oxide (NO) is the most important mechanism of insulin-induced vasodilatation, and a possible contribution of endothelium-derived hyperpolarizing factor (EDHF) is also considered. Attempts were made to observe the effects of insulin on acetylcholine (ACh)-induced hyperpolarization in the submucosal arteriole of the guinea pig ileum, the objective being to investigate possible involvement of EDHF in the actions of insulin. Methods: Conventional microelectrode techniques were applied to measure the membrane potential of smooth muscle cells in the submucosal arteriole. EDHF-induced hyperpolarization was elicited by ACh in the presence of both N^ω-nitro- -arginine ( -NNA) (100 μM) and diclofenac (1 μM). Results: The resting membrane potential was −70.9 mV, and Ba²⁺ (0.5 mM) depolarized the membrane to −33.0 mV. Insulin (10 μU/ml to 100 mU/ml) did not change the membrane potential in the absence or presence of Ba²⁺. In the presence of Ba²⁺, ACh (3 μM) hyperpolarized the membrane with two components, an initial large hyperpolarization followed by a slow and small one. Low concentration of insulin (100 μU/ml) did not alter the ACh-induced hyperpolarization. High concentration of insulin (100 mU/ml) shortened the time required to reach the peak amplitude and tended to increase the peak amplitude of the ACh-induced hyperpolarization. Conclusions: The data show that insulin enhances the ACh-induced hyperpolarization in the submucosal arterioles of the guinea pig ileum. The results suggested that EDHF also accounts for one of the endothelial factors involved in the insulin-induced vasodilatation.     

Insulin inhibits glucagon secretion by the activation of PI3-kinase in In-R1-G9 cells.

Intracellular mechanisms through which insulin inhibits glucagon secretion remain to be elucidated in glucagon secreting cells. In this study, we confirmed that, in In-R1-G9 cells, a pancreatic alpha cell line, insulin stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and activated phosphatidylinositol 3-kinase (PI3-kinase). We further studied, using wortmannin, an inhibitor of PI3-kinase, whether the inhibitory effect of insulin on glucagon secretion was mediated through PI3-kinase pathway in these cells. In static incubation studies, insulin significantly inhibited glucagon secretion at 2, 6 and 12 h, which was completely abolished by pretreatment with wortmannin. In perifusion studies, insulin significantly suppressed glucagon secretion after 10 min, which was also blocked by wortmannin. Insulin also reduced glucagon mRNA at 6 and 12 h but not at 2 h. Wortmannin also abolished insulin-induced reduction of glucagon mRNA. Insulin increased the amount of 85 kDa subunit of PI3-kinase in plasma membrane fraction (PM), with a reciprocal decrease of the kinase in cytosol fraction (CY). Insulin also increased PI3-kinase activity in PM, but not in CY. Our results suggest that insulin suppressed glucagon secretion by inhibiting glucagon release and gene expression. Both actions were mediated by activation of PI3-kinase. Recruitment and activation of PI3-kinase in plasma membrane might be relevant at least in part to insulin-induced inhibition of glucagon release.     

Insulin administered at reoxygenation exerts a cardioprotective effect in myocytes by a possible anti-apoptotic mechanism

The metabolic cocktail of glucose-insulin-potassium (GIK) has been shown to reduce mortality in humans and reduce infarct size in the rat when administered from the onset of reperfusion following an ischemic insult. The mechanisms underlying GIK mediated cardioprotection are, however, still unclear. Recent data implicates insulin "alone" as the major protagonist of cardioprotection when administered at the time of reperfusion. We have therefore begun to investigate an insulin activated signalling pathway and the putative role of apoptosis in this insulin-induced cardioprotection. Simulated ischemia and reoxygenation were induced in rat neonatal cardiocyte experiments. The administration of insulin [0.3 mU/ml] at the moment of reoxygenation (Ins(R)) enhanced myocardial cell viablility as assessed by trypan blue exclusion compared to vehicle alone treated control myocytes (Ins(R)50+/-2%v controls 70+/-1%, P<0.001). This insulin-mediated cardioprotection was due, in part to a reduction in myocyte apoptosis as measured by TUNEL (Ins(R)29+/-2%v controls 49+/-3%, P<0.001) and Annexin V staining (Ins(R)34+/-2%v controls 65+/-3%, P<0.001). These cardioprotective and anti-apoptotic effects of insulin were completely abolished by the tyrosine kinase inhibitor lavendustin A and by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin. Thus, we conclude that the early administration of insulin appears to be an effective modality to reduce reoxgygenation injury in cardiocytes, in part, via the attenuation of ischemia/reoxygenation-induced apoptosis. Moreover, the cardioprotective and anti-apoptotic effects of insulin are mediated via tyrosine kinase and PI3-kinase signalling pathways.     

Ascorbate inhibits reduced arteriolar conducted vasoconstriction in septic mouse cremaster muscle

OBJECTIVE: The mechanism of neuronal nitric oxide synthase (nNOS)-dependent reduction in arteriolar conducted vasoconstriction in sepsis, and the possible protection by antioxidants, are unknown. The authors hypothesized that ascorbate inhibits the conduction deficit by reducing nNOS-derived NO production. METHODS: Using intravital microscopy and the cecal ligation and perforation (CLP) model of sepsis (24 h), arterioles in the cremaster muscle of male C57BL/6 wild-type mice were locally stimulated with KCl to initiate conducted vasoconstriction. The authors used the ratio of conducted constriction (500 microm upstream) to local constriction as an index of conduction (CR500). Cremaster muscle NOS enzymatic activity and protein expression, and plasma nitrite/nitrate levels were determined in control and septic mice. Intravenous ascorbate bolus (200 mg/kg in 0.1 ml of saline) was given early (0 h) or delayed at 23 h post CLP. RESULTS: Sepsis reduced CR500 from 0.73 +/- 0.03 to 0.21 +/- 0.03, increased nNOS activity from 87 +/- 9 to 220 +/- 29 pmol/mg/h and nitrite/nitrate from 16 +/- 1 to 39 +/- 3 microM, without affecting nNOS protein expression. Ascorbate at 0 and 23 h prevented/reversed the conduction deficit and the increases in nNOS activity and nitrite/nitrate level. NO donor SNAP (S-nitroso-N-acetylpenicillamine) reestablished the conduction deficit in ascorbate-treated septic mice. Superoxide scavenger MnTBAP (Mn(III)tetrakis(4-benzoic acid)porphyrin chloride) did not affect this deficit. CONCLUSION: These data indicate that early and delayed intravenous boluses of ascorbate prevent/reverse sepsis-induced deficit in arteriolar conducted vasoconstriction in the cremaster muscle by inhibiting nNOS-derived NO production.     

Direct assessment of splanchnic uptake and metabolic effects of human and porcine insulin

The hepatic vein catheterization technique was used to quantitate the splanchnic uptake and the metabolic effects of biosynthetic human insulin (BHI) and porcine insulin (PI) in normal man. BHI and PI were infused into a peripheral vein (0.9-1.3 mU kg-1 min-1) for 60 min together with SRIH (0.6 mg/h) to inhibit endogenous insulin secretion and glucose to induce moderate hyperglycemia (9-10 mmol/liter). During the infusion period, arterial-hepatic venous difference of plasma C-peptide as well as splanchnic C-peptide output fell by more than 98% indicating virtually complete cessation of endogenous insulin release. Under these conditions, the arterial-hepatic venous differences in plasma insulin concentrations represent a valid and direct measurement of splanchnic insulin uptake. During BHI infusion, arterial insulin levels rose to 82 +/- 11 (SE) microU/ml (range: 33-105 microU/ml). Splanchnic insulin uptake paralleled the rise of arterial insulin, reaching 430 +/- 72 microU kg-1 min-1 at 60 min. No appreciable difference between BHI and PI was demonstrable. A highly significant correlation between arterial insulin concentrations and splanchnic insulin uptake was found (r = 0.816; P less than 0.001). Accordingly, both fractional splanchnic insulin extraction and splanchnic insulin clearance remained unchanged throughout insulin infusion and averaged 70 +/- 4% and 5.3 +/- 2 ml kg-1 min-1, respectively. With BHI infusion, splanchnic glucose balance (-8.5 +/- 0.9 mumol kg-1 min-1, basal) became positive (7.3 +/- 1 mumol kg-1 min-1). In contrast, basal splanchnic lactate uptake was inhibited by BHI and there was lactate production (from 3.4 +/- 0.9 to -1.7 +/- 1.4 mumol kg-1 min-1). Similar changes in splanchnic glucose and lactate metabolism occurred during PI infusion. These studies indicate that: 1) A considerable amount of insulin (70 +/- 4%) is extracted by the splanchnic bed on a single passage, after exogenous administration of either human insulin or PI; 2) over a physiological range of insulin concentrations (33-105 microU/ml) a linear relationship exists between arterial insulin concentrations and splanchnic insulin removal; and 3) BHI and PI do not differ appreciably with respect to their uptake and metabolic effects at the splanchnic level.     

Reduced arteriolar conducted vasoconstriction in septic mouse cremaster muscle is mediated by nNOS-derived NO

OBJECTIVE: Increased nitric oxide (NO) production in sepsis precipitates microcirculatory dysfunction. We aimed (i) to determine if NO is the key water-soluble factor in the recently discovered sepsis-induced deficit in arteriolar conducted vasoconstriction, (ii) to identify which nitric oxide synthase (NOS) isoforms account for this deficit, and (iii) to examine the potential role of connexin37 (Cx37, a hypothesized signaling target of NO) in arteriolar conduction. METHODS: Using intravital microscopy and the cecal ligation and perforation 24-h model of sepsis, arterioles in the cremaster muscle of male C57BL/6 wild-type (WT), iNOS-/-, eNOS-/-, nNOS-/- and Cx37-/- mice were locally stimulated with KCl to initiate conducted vasoconstriction. We used the ratio of conducted constriction (500 microm upstream) to local constriction as an index of conduction (CR500). NOS enzymatic activity and protein expression were determined in control and septic cremaster muscles.RESULTS: Sepsis reduced CR500 in WT mice [from 0.77 +/- 0.05 to 0.20 +/- 0.02 (means +/- SE) independent of the site of stimulation along the arteriole], in iNOS-/- and eNOS-/- mice, but not in nNOS-/- mice. The nNOS inhibitor 7-nitroindazole or NO scavenger HbO2 restored CR500 in septic WT mice, but blockade of soluble guanylate cyclase had no effect. Sepsis increased cNOS (eNOS + nNOS) activity in WT mice (from 340 +/- 40 to 490 +/- 30 pmol/mg/h) and in eNOS-/-, but not in nNOS-/- mice (iNOS activity was negligible in all mice). Sepsis did not alter nNOS protein expression in WT mice. CR500 in non-septic Cx37-/- mice (0.15 +/- 0.1) was similar to that observed in septic WT mice. CONCLUSION: Increased nNOS activity and the resultant increased NO production in the septic mouse cremaster muscle are the key factors responsible for the deficit in conducted vasoconstriction along the arteriole. Deletion of Cx37 results in reduced CR500, which is consistent with the hypothesis that Cx37 in the arteriole could be a target of NO signaling.     

Modulation of insulin secretion by insulin and glucose in type II diabetes mellitus

We studied the dose-response characteristics of insulin's ability to modulate its own secretion in normal and type II diabetic (NIDDM) subjects by measuring suppression of serum C-peptide levels during insulin infusions with the plasma glucose level held constant. In normal subjects at euglycemia, primed continuous insulin infusion rates of 15, 40, 120, and 240 mU/M2 X min acutely raised serum insulin to steady state levels of 37 +/- 2 (+/- SE), 96 +/- 6, 286 +/- 17, and 871 +/- 93 microU/ml, respectively. During each infusion, maximal suppression of C-peptide to 30% of basal levels occurred by 130 min. At the higher insulin levels (greater than or equal to 100 microU/ml), C-peptide levels fell rapidly, with an apparent t1/2 of 13 min, which approximates estimates for the t1/2 of circulating C-peptide in man. This is consistent with an immediate 70% inhibition of the basal rate of insulin secretion. At the lower insulin level (37 +/- 2 microU/ml), C-peptide levels fell to 30% of basal values less rapidly (apparent t1/2, 33 min), suggesting that 70% inhibition of basal insulin secretion rates was achieved more slowly. In NIDDM subjects, primed continuous insulin infusion rates of 15, 40, 120, and 1200 mU/M2 X min acutely raised serum insulin to steady state levels of 49 +/- 7, 93 +/- 11,364 +/- 31, and 10,003 +/- 988 microU/ml. During studies at basal hyperglycemia, only minimal C-peptide suppression was found, even at pharmacological insulin levels (10,003 +/- 988 microU/ml). However, if plasma glucose was allowed to fall during the insulin infusions, there was a rapid decrease in serum C-peptide to 30% of basal levels, analogous to that in normal subjects. Three weeks of intensive insulin therapy did not alter C-peptide suppression under conditions of hyperinsulinemia and falling plasma glucose. The following conclusions were reached. 1) In normal subjects, insulin (40-1000 microU/ml) inhibits its own secretion in a dose-responsive manner; more time is required to achieve maximal 70% suppression at the lower insulin level (40 microU/ml). 2) In NIDDM studied at basal hyperglycemia, insulin has minimal ability to suppress its own secretion. Thus, impaired feedback inhibition could contribute to basal hyperinsulinemia. 3) Under conditions of hyperinsulinemia and falling plasma glucose, insulin secretion is rapidly suppressed in NIDDM (analogous to that in normal subjects studied during euglycemia.     

体外研究胰岛素及磷脂酰肌醇3-激酶途径对一氧化氮生成的影响

目的探讨胰岛素及磷脂酰肌醇3-激酶(PI3-K)途径对NO生成的影响。方法检测胰岛素、葡萄糖以及PI3-K活性不可逆的抑制剂(Wortmannin)对培养的人脐静脉内皮细胞(HUVECs)PI3-K表达以及NO、超氧阴离子(O_2~-)产生和内皮型一氧化氮合酶(eNOS)活性的影响。实验分为对照组、10 mU/L胰岛素组、100 mU/L胰岛素组、甘露醇组、5 mmol/L葡萄糖+10 mU/L胰岛素组(5 mmol/L G1组)、25 mmol/L葡萄糖+100 mU/L胰岛素组(25 mmol/L G2组)、50 nmol/L Wortmannin组(50 nmol/L W组)、50 nmol/L Wortmannin+10 mU/L胰岛素组(50 nmol/L W1组)和50 nmol/L Wortmannin+100 mU/L胰岛素组(50 nmol/L W2组)。结果与对照组比较,不同浓度胰岛素组eNOS活性及NO水平显著升高(P<0.01);25 mmol/L G2组、50 nmol/L W组、50 nmol/LW1组和50 nmol/L W2组eNOS活性及NO水平均显著降低,O_2~-生成明显增加(P<0.01);与对照组比较,不同浓度胰岛组、50 nmol/L W组、50 nmol/L W1组和50 nmol/L W2组PI3-K蛋白表达显著升高(P<0.05,P<0.01)。结论 PI3-K信号途径对于促进NO产生、维持血管内皮细胞的正常功能具有重要作用,在高糖、高胰岛素状态下该条途径受损并由此引发内皮功能障碍。     

Endothelin antagonism uncovers insulin-mediated vasorelaxation in vitro and in vivo

The endothelial actions of insulin remain an area of intense research because they relate to both insulin sensitivity and vascular tone. Physiological doses of insulin evoke endothelium-dependent vasorelaxation in humans; however, this remains a pharmacological phenomenon in rat aortas. Because insulin may stimulate the divergent production of both nitric oxide and endothelin-1, we hypothesized that the lack of insulin-induced vasorelaxation at low/subthreshold concentrations may be due to the concurrent production of endothelin-1, which in turn serves to inhibit nitric oxide-dependent, insulin-mediated dilation. To investigate this, we studied the effects of subthreshold concentrations of insulin (100 mU/L) on norepinephrine-induced contraction in rat aortas following short-term and long-term endothelin blockade. In addition, the effects of tetrahydrobiopterin inhibition (with diaminohydroxyprimidine) on norepinephrine-induced contraction in the presence of insulin and endothelin receptor blockade were investigated. Subthreshold concentrations of insulin failed to evoke vasorelaxation in rat aortas. Strikingly, short-term endothelin A/B receptor blockade with bosentan (10(-2) mmol/L) uncovered insulin-mediated dilation; the percent maximum contraction and sensitivity of aortas to norepinephrine were attenuated (% maximum relaxation: bosentan+insulin 74+/-4%* versus bosentan 92+/-3%, insulin 107+/-5% P:<0.002; pD(2) values: bosentan+insulin 6.87+/-0.14* versus bosentan 7.40+/-0.15, insulin 7.63+/-0.11, *P:<0.002). This effect was mediated through endothelin A receptors because bosentan and BQ-123 (10(-2) mmol/L) attenuated norepinephrine-induced contraction to a similar degree. In addition, insulin evoked vasorelaxation in aortas isolated from rats after long-term bosentan treatment (100 mg. kg(-1). d(-1), 3 weeks). The component of insulin-mediated vasorelaxation uncovered by endothelin receptor blockade was tetrahydrobiopterin-dependent because it was reversed by diaminohydroxyprimidine. These data demonstrate, for the first time, the functional interaction between insulin, endothelin-1, and tetrahydrobiopterin in modulating vascular tone in rat aortas in vitro and in vivo.     

Metabolism of insulin and immunoreactive insulin-like substances (proinsulin, HWIRI) in vivo in man

Immunoreactive insulin (IRI) and its three constituents as determined by gel filtration: high molecular weight substances with insulin immunoreactivity (HWIRI), proinsulin (PI) and insulin (I), were measured hourly during 28 hours in the portal, hepatic and peripheral venous systems of a patient operated for a nesidioblastoma. The PI levels were constant from one system to the other (7.1 +/- 0.8 microU/ml; 7.3 +/- 0.8 microU/ml; 9.2 +/- 1.2 microU/ml). The I levels decreased from the portal (53.3 +/- 5.2 microU/ml) to the hepatic venous system (28.0 +/- 1.0 microU/ml) and from the latter to the peripheral venous system (19.8 +/- 2.0 microU/ml). The HWIRI levels were stable between the portal and the hepatic venous systems (4.7 +/- 0.7 microU/ml; 4.8 +/- 0.6 microU/ml) and higher in the peripheral system (8.2 +/- 1.2 microU/ml). Our results demonstrate that: --I is essentially degraded by the liver. --There is no variation of PI in the three compartments. --HWIRI is formed in the circulation. --Variations of IRI levels between venous systems acquire physiological significance only when accompanied by the variations of the levels of each constituent.     

Impairment of PI3-K/Akt pathway underlies attenuated endothelial function in aorta of type 2 diabetic mouse model

The phosphatidylinositol 3-kinase (PI3-K) pathway, which activates serine/threonine protein kinase Akt, enhances endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production. We investigated the involvement of the PI3-K/Akt pathway in the relaxation responses to acetylcholine (ACh) and clonidine in a new type 2 diabetic model (streptozotocin plus nicotinamide-induced diabetic mice). Plasma glucose and insulin levels were significantly elevated in our model, and intravenous glucose tolerance tests revealed clear abnormalities in glucose tolerance and insulin responsiveness. Although in our model the ACh-induced relaxation and NOx- (NO2-+NO3-)/cGMP production were unchanged, the clonidine-induced and insulin-induced relaxations and NOx-/cGMP production were all greatly attenuated. In control mice, the clonidine-induced and insulin-induced relaxations were each abolished by LY294002 and by Wortmannin (inhibitors of PI3-K), and also by Akt-inhibitor treatment. The ACh-induced relaxation was unaffected by such treatments in either group of mice. The expression level of total Akt protein was significantly decreased in the diabetic mice aorta, but those for the p85 and p110gamma subunits of PI3-K were not. The clonidine-induced Ser-473 phosphorylation of Akt through PI3-K was significantly decreased in our model; however, that induced by ACh was not. These results suggest that relaxation responses and NO production mediated via the PI3-K/Akt pathway are decreased in this type 2 diabetic model. This may be a major cause of endothelial dysfunction (and the resulting hypertension) in type 2 diabetes.     

Insulin and glucose mediate opposite intracellular ionized magnesium variations in human lymphocytes

Insulin is capable of increasing intracellular magnesium, although very little is known about the effect of insulin on the biologically active fraction of magnesium, i.e. the ionized quota (Mg(i)(2+)), its interactions with glucose, and the cellular mechanisms involved in these processes. We studied the interactions of the effects of insulin and glucose on intracellular ionized magnesium in human lymphocytes. Mg(i)(2+) was measured using a fluorimetric method and the Mg(2+)-sensitive dye, furaptra. We found that insulin significantly increases the Mg(i)(2+)(without insulin 227 +/- 14 microM, with 10 microU/ml, insulin 301 +/- 30 microM, P<0.0001, n = 12) in a dose-dependent manner in all three glucose concentrations tested (5, 7 and 15 mmol/l). The half-maximal effect of insulin was approximately 0.8 microU/ml. Glucose and insulin showed opposite effects in their ability to modify Mg(i)(2+) in lymphocytes. Inhibitors of the membrane Na(+)- Mg(2+) transport system and of phosphatidylinositol (PI) 3-kinase abolish the insulin-mediated increase of Mg(i)(2+), thus suggesting that insulin is capable of increasing Mg(i)(2+) by modulating the activity of this transport system, possibly through the mediation of PI 3-kinase activation. Taking into account the relationship between insulin and glucose plasma levels and their opposing effects on Mg(i)(2+), this mechanism may represent the two limbs of a biphasic regulatory system of Mg(i)(2+) in both physiological and pathological conditions.     

Altered insulin-mediated and insulin-like growth factor-1-mediated vasorelaxation in aortas of obese Zucker rats

OBJECTIVE: Insulin and insulin-like growth factor-1 (IGF-1) have vasorelaxant effects in vivo, which is dependent on nitric oxide (NO) production. The aim of this study was to investigate the vasorelaxant responses mediated by insulin and/or IGF-1 in aortas of obese Zucker rats. METHODS: The thoracic aortas of eight lean and eight obese Zucker rats (6 months old) were isolated for vasorelaxation analysis. Insulin-induced and IGF-1-induced vasorelaxant responses were evaluated by the isometric tension of aortic rings in the organ bathes. The roles of phosphatidylinositol 3-kinase (PI3K) and nitric oxide synthase (NOS) in vasorelaxant responses were examined by treating selective inhibitors, such as wortmannin (an inhibitor of PI3K) and N (omega)-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor). In addition, the vascular responses to sodium nitroprusside (SNP), a direct vasodilator of vascular smooth muscle, were examined. RESULTS: The insulin-induced vasorelaxation in aortas of obese rats was significantly decreased, whereas the IGF-1-induced vasorelaxation was significantly increased, compared with that in lean rats. After the pre-administration of wortmannin or L-NAME, the altered insulin-induced or IGF-1-induced vasorelaxation was abolished. There was no significant difference in the SNP-induced vasorelaxation between lean and obese rats. CONCLUSION: Our findings suggested that the decreased insulin-mediated vasorelaxation in obese rats appeared to be counteracted by the increased IGF-1-mediated vasorelaxation. Furthermore, the NO-dependent pathway was involved in the altered vasorelaxant responses. However, the SNP-induced vasorelaxation was not changed in obese rats.     

Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase

AIMS: We have evaluated, in cultured human cavernosal smooth muscle cells, the expression and activity of calcium-dependent constitutive nitric oxide synthase (cNOS) and the ability of insulin to induce nitric oxide (NO) production and to increase intracellular cyclic nucleotides guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). METHODS: cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluorescence, cNOS activity as l-[3H]-citrulline production from l-[3H]-arginine and cyclic nucleotides by radioimmunoassay. RESULTS: cNOS mRNA and cNOS protein were found in cultured cells; cNOS activity was increased by 5-min exposure to 1 micro mol/l calcium ionophore ionomycin (from 0.1094+/-0.0229 to 0.2685+/-0.0560 pmol/min per mg cell protein, P=0.011) and to 2 nmol/l insulin (from 0.1214+/-0.0149 to 0.2045+/-0.0290 pmol/min per mg cell protein, P=0.041). Insulin increased both cGMP and cAMP in a dose- and time-dependent manner (i.e. with 2 nmol/l insulin, cGMP rose from 2.71+/-0.10 to 6.80+/-0.40 pmol/10(6) cells at 30 min, P=0.0001; cAMP from 1.26+/-0.06 to 3.02+/-0.30 pmol/10(6) cells at 60 min, P=0.0001). NOS inhibitor N(G)-monomethyl-l-arginine and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 blunted these effects of insulin. The action of insulin on cyclic nucleotides persisted in the presence of phosphodiesterase inhibition, guanylate cyclase activation by NO donors and adenylate cyclase activation by Iloprost or forskolin. CONCLUSION: Human cavernosal smooth muscle cells, by expressing cNOS activity, are a source of NO and not only its target; in these cells, insulin rapidly activates cNOS through a PI 3-kinase pathway, with a consequent increase of both cyclic nucleotides, thus directly influencing the mechanisms involved in penile vascular tone and interplaying with classical haemodynamic mediators.     

Inhibition of nitric oxide synthesis attenuates insulin-mediated sympathetic activation in rats

BACKGROUND AND OBJECTIVES : Infusion of insulin produces sympathoexcitation, nitric oxide (NO) generation and NO-mediated vasodilation. Because central nervous system NO may inhibit sympathetic outflow, the present study was designed to determine whether NO synthase blockade would enhance insulin-mediated sympathetic activation. We additionally aimed to determine whether augmented sympathoexcitation and reduced NO-mediated vasodilation, during combined NO synthase blockade and hyperinsulinemia, would result in a blood pressure increase. DESIGN AND METHODS : We infused vehicle (Control; n = 7) or insulin (10 mU/min) in anaesthetized rats receiving either no pretreatment (Insulin; n = 7) or after pretreatment with the NO blocker, NG-monomethyl-L-arginine (L-NMMA-insulin; 0.25 mg/kg per min; n = 7), while measuring mean arterial pressure (MAP), heart rate and lumbar sympathetic nerve activity (SNA) during euglycemic clamp. An additional control group received L-NMMA (L-NMMA; n = 7). RESULTS : Insulin rats had large SNA increases (190 +/- 22% from 100% baseline), contrasting with small increases in the Control (136 +/- 10%) and L-NMMA (135 +/- 20%) groups. Unexpectedly, NO blockade abolished insulin-induced SNA increases in the L-NMMA-insulin group (96 +/- 12%). In agreement with the SNA findings, Insulin rats had heart rate increases while no heart rate changes were observed in the L-NMMA-insulin, Control, or L-NMMA groups. In addition, there was an unexpected was a lack of MAP increase in L-NMMA-insulin rats. MAP also did not change in the Control, L-NMMA or Insulin groups. CONCLUSIONS : These findings suggest that NO is necessary for insulin to exert its sympathoexcitatory effects, and that insulin-induced NO release may play a role in activating increases in lumbar SNA.     

Carbon monoxide from heme oxygenase-2 Is a tonic regulator against NO-dependent vasodilatation in the adult rat cerebral microcirculation

Although the brain generates NO and carbon monoxide (CO), it is unknown how these gases and their enzyme systems interact with each other to regulate cerebrovascular function. We examined whether CO produced by heme oxygenase (HO) modulates generation and action of constitutive NO in the rat pial microcirculation. Immunohistochemical analyses indicated that HO-2 occurred in neurons and arachnoid trabecular cells, where NO synthase 1 (NOS1) was detectable, and also in vascular endothelium-expressing NOS3, suggesting colocalization of CO- and NO-generating sites. Intravital microscopy using a closed cranial window preparation revealed that blockade of the HO activity by zinc protoporphyrin IX significantly dilates arterioles. This vasodilatation depended on local NOS activities and was abolished by CO supplementation, suggesting that the gas derived from HO-2 tonically regulates NO-mediated vasodilatory response. Bioimaging of NO by laser-confocal microfluorography of diaminofluorescein indicated detectable amounts of NO at the microvascular wall, the subdural mesothelial cells, and arachnoid trabecular cells, which express NOS in and around the pial microvasculature. On CO inhibition by the HO inhibitor, regional NO formation was augmented in these cells. Such a pattern of accelerated NO formation depended on NOS activities and was again attenuated by the local CO supplementation. Studies using cultured porcine aortic endothelial cells suggested that the inhibitory action of CO on NOS could result from the photo-reversible gas binding to the prosthetic heme. Collectively, CO derived from HO-2 appears to serve as a tonic vasoregulator antagonizing NO-mediated vasodilatation in the rat cerebral microcirculation.     

Similar effects of pulsatile and constant intravenous insulin delivery

Effects of constant and pulsatile i.v. insulin delivery were examined in seven healthy subjects by means of euglycemic clamp technique. Each subject received constant insulin infusion (0.175 mU/kg.min) or insulin pulses at 12-min intervals (2.1 mU/kg) in randomized order for 8-h periods (08.00-16.00 h). Endogenous secretion of insulin was inhibited by concomitant administration of somatostatin (300 micrograms/h). Serum insulin concentrations during constant infusion (12 +/- 1 microU/ml) did not differ from basal values (11 +/- 1 microU/ml). Pulsatile insulin delivery resulted in oscillations of mean concentrations between values of about 10 and 20 microU/ml. Mean blood glucose concentrations during experiments were kept at 80 +/- 1 mg/dl, irrespective of the mode of insulin administration. Moreover, dextrose requirements for maintenance of these glucose concentrations did not differ over the hole periods of examination. We conclude that effects of constant and pulsatile delivery of basal amounts of insulin are not different. This at least applies to peripheral, short-term insulin administration in somatostatin-treated normal man, during an euglycemic clamp.