Cellular stretch increases superoxide production in the thick ascending limb

Superoxide (O(2)(-)) is an important regulator of kidney function. We have recently shown that luminal flow stimulates O(2)(-) production in the thick ascending limb (TAL), attributable in part to mechanical factors. Stretch, pressure and shear stress all change when flow increases in the TAL. We hypothesized that stretch rather than shear stress or pressure per se stimulates O(2)(-) production by TALs. We measured O(2)(-) production in isolated perfused rat TALs using fluorescence microscopy and dihydroethidium. Tubules were perfused with a Na-free solution to eliminate the confounding effect of Na transport. Flow induced an increase in O(2)(-) production from 29+/-4 to 90+/-8 AU/s (P<0.002; n=5). The response to flow is rapidly reversible. O(2)(-) production by TALs perfused at 10 nL/min decreased from 113+/-6 to 25+/-10 AU/s (P<0.003; n=4) 15 minutes after flow was stopped. Increasing pressure and stretch in the absence of shear stress caused a significant increase in O(2)(-) production (40+/-6 to 118+/-17 AU/s; P<0.02; n=5). In contrast, eliminating shear stress had no effect (107+/-9 versus 108+/-10 AU/s; n=5). Increasing stretch by 27+/-2% in the presence of flow while reducing pressure stimulated O(2)(-) production from 66+/-7 to 84+/-9 AU/s (29+/-8%; P<0.02; n=5). Tempol inhibited this increase (n=5). We conclude that increasing stretch rather than pressure or shear stress accounts for the mechanical aspect of flow-induced O(2)(-) production in the TAL. Stretch of the TAL during hypertension, diabetes, and salt loading may contribute to renal damage.     

Enhanced vascular production of superoxide in OLETF rat after the onset of hyperglycemia

This study was aimed to characterize the vascular production of superoxide in Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model of type 2 diabetes. The nitroblue tetrazolium staining in the aorta from old (30 weeks) OLETF rat was more prominent than that of age-matched control (LETO) rat, which was significantly inhibited by diphenyleneiodonium (10 micromol/l), but not by inhibitors for other oxidases such as xanthine oxidase, mitochondrial oxidase, nitric oxide synthase, and cyclooxygenase. In the aorta from old OLETF rat with hyperglycemia, the enhanced NADH oxidase activity in association with upregulated expression of p22phox and gp91phox was observed, but not in both LETO and young (10 weeks) OLETF rats without hyperglycemia. Furthermore, there was a positive correlation (P<0.01) between elevation of blood glucose level and increase in vascular NADH oxidase activity. Based on these results, it was suggested that the enhanced NADH oxidase activity in the aorta from OLETF rat occurred after the onset of hyperglycemia, thereby resulting in the increased vascular production of superoxide.     

Lipoic acid prevents hypertension,hyperglycemia, and the increase in heart mitochondrial superoxide production

BACKGROUND: The present study was designed to investigate whether the effects of dietary supplementation with alpha-lipoic acid could prevent the increase in mitochondrial superoxide production in the heart as well as the enhanced formation of advanced glycation end-products (AGE) that are associated with the development of hypertension and insulin resistance in chronically glucose-fed rats. METHODS: Sprague Dawley rats were either given or not given a 10% D-glucose solution to drink during 4 weeks, combined either with a normal chow diet or with alpha-lipoic acid supplemented diet. The oxidative stress was evaluated by measuring the heart mitochondrial superoxide production using the lucigenin chemiluminescence method. The formation of AGE was also assessed in plasma and aorta. RESULTS: Chronic administration of glucose resulted in a 29% increase in blood pressure, 30% increase in glycemia, 286% increase in insulinemia, and 408% increase in insulin resistance index. Chronic glucose feeding also resulted in a 22% greater mitochondrial superoxide anion production in heart and in an increase of 63% in AGE content in aorta. Increases in blood pressure, aorta AGE content and heart mitochondrial superoxide production were prevented in the rats fed glucose supplemented with lipoic acid. The simultaneous treatment with lipoic acid also attenuated the rise in insulin levels as well as in insulin resistance in the glucose fed rats. CONCLUSIONS: These findings demonstrate that alpha-lipoic acid supplementation prevents development of hypertension and hyperglycemia, presumably through its antioxidative properties, as reflected by prevention of an increase in heart mitochondrial superoxide anion production and in AGE formation in the aorta of chronically glucose treated rats.     

Short-term dehydroepiandrosterone treatment increases platelet cGMP production in elderly male subjects

OBJECTIVE: Several clinical and population-based studies suggest that dehydroepiandrosterone (DHEA) and its sulphate (DHEA-S) play a protective role against atherosclerosis and coronary artery disease in human. However, the mechanisms underlying this action are still unknown. It has recently been suggested that DHEA-S could delay atheroma formation through an increase in nitric oxide (NO) production. STUDY DESIGN AND METHODS: Twenty-four aged male subjects [age (mean +/- SEM): 65.4 +/- 0.7 year; range: 58.2-67.6 years] underwent a blinded placebo controlled study receiving DHEA (50 mg p.o. daily at bedtime) or placebo for 2 months. Platelet cyclic guanosine-monophosphate (cGMP) concentration (as marker of NO production) and serum levels of DHEA-S, DHEA, IGF-I, insulin, glucose, oestradiol (E(2)), testosterone, plasminogen activator inhibitor (PAI)-1 antigen (PAI-1 Ag), homocysteine and lipid profile were evaluated before and after the 2-month treatment with DHEA or placebo. RESULTS: At the baseline, all variables in the two groups were overlapping. All parameters were unchanged after treatment with placebo. Conversely, treatment with DHEA (a) increased (P < 0.001 vs. baseline) platelet cGMP (111.9 +/- 7.1 vs. 50.1 +/- 4.1 fmol/10(6) plts), DHEA-S (13.6 +/- 0.8 vs. 3.0 +/- 0.3 micromol/l), DHEA (23.6 +/- 1.7 vs. 15.3 +/- 1.4 nmol/l), testosterone (23.6 +/- 1.0 vs. 17.7 +/- 1.0 nmol/l) and E(2) (72.0 +/- 5.0 vs. 60.0 +/- 4.0 pmol/l); and (b) decreased (P < 0.05 vs. baseline) PAI-1 Ag (27.4 +/- 3.8 vs. 21.5 +/- 2.5 ng/ml) and low-density lipoprotein (LDL) cholesterol (3.4 +/- 0.2 vs. 3.0 +/- 0.2 mmol/l). IGF-I, insulin, glucose, triglycerides, total cholesterol, HDL cholesterol, HDL2 cholesterol, HDL3 cholesterol, apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB) and homocysteine levels were not modified by DHEA treatment. CONCLUSIONS: This study shows that short-term treatment with DHEA increased platelet cGMP production, a marker of NO production, in healthy elderly subjects. This effect is coupled with a decrease in PAI-1 and LDL cholesterol levels as well as an increase in testosterone and E(2) levels. These findings, therefore, suggest that chronic DHEA supplementation would exert antiatherogenic effects, particularly in elderly subjects who display low circulating levels of this hormone.     

Nitric oxide, endothelin-1, and superoxide production in arterial bypass grafts

In this study, basal and thrombin-stimulated release of nitric oxide and endothelin-1 in the internal mammary artery and the radial artery were measured, together with superoxide radicals generated after anoxia and reoxygenation. Arterial segments were obtained from patients undergoing coronary bypass operations. Quantification of nitric oxide was performed by measuring the stable oxidation products of nitric oxide. Endothelin levels were measured by an enzyme immunoassay kit, and the superoxides were measured by lucigenin-enhanced chemiluminescence. Basal and stimulated release of nitric oxide from the internal mammary artery is significantly higher than that in the radial artery. On the other hand, basal release of endothelin-1 is less in the internal mammary artery than in the radial artery, but similar after stimulation. In our study, the quantity of superoxide radicals produced by the internal mammary artery was greater than that produced by the radial artery. Our results show that there are differences between these 2 arteries in regard to production of nitric oxide, endothelin-1, and superoxide radicals. These differences may have a role in the process of atherogenesis and may contribute to long-term patency of arterial bypass grafts. These results may also explain the mechanism of radial artery graft spasm in coronary artery surgery and may constitute a basis for future pharmacological and clinical improvements for successful surgical application.     

Ouabain treatment increases nitric oxide bioavailability and decreases superoxide anion production in cerebral vessels

OBJECTIVE: Chronic administration of ouabain induces hypertension and increases the contribution of nitric oxide to vasoconstrictor responses in peripheral arteries. The aim of this study was to analyse whether ouabain treatment alters the nitric oxide bioavailability in cerebral arteries. METHODS: Basilar arteries from control and ouabain-treated rats ( approximately 8.0 microg/day, 5 weeks) were used. Vascular reactivity was analysed by isometric tension recording, protein expression by western blot, nitric oxide levels by diaminofluorescein-induced fluorescence, superoxide anion (O2) production by ethidium fluorescence and lucigenin chemiluminescence and plasma total antioxidant status by a commercial kit. RESULTS: The relaxations induced by bradykinin (1 nmol/l-10 micromol/l) and L-arginine (0.01-300 micromol/l) and the contractile responses induced by both N-nitro-L-arginine methyl ester (0.1-100 micromol/l) and oxyhaemoglobin (0.01-10 micromol/l) were greater in arteries from ouabain-treated than control rats. However, the relaxation to diethylamine NONOate-nitric oxide (0.1 nmol/l-10 micromol/l) and the contractions to KCl (7.5-120 mmol/l) and 5-hydroxytryptamine (0.01-10 micromol/l) were similar in arteries from both groups. Ouabain treatment increased basal nitric oxide levels but did not modify endothelial and neuronal nitric oxide synthase protein expression. O2 production was lower in cerebral arteries from ouabain-treated rats; however, plasma total antioxidant status and vascular protein expression of Cu/Zn-superoxide dismutase, Mn-superoxide dismutase and extracellular superoxide dismutase were similar in both groups. CONCLUSION: Chronic ouabain treatment increased nitric oxide basal levels in basilar arteries probably due to the decreased O2 levels. This might be an adaptive mechanism of the cerebral vasculature to the increase in blood pressure.     

Pancreatic iron and glucose dysregulation in thalassemia major

Pancreatic iron overload and diabetes mellitus (DM) are common in thalassemia major patients. However, the relationship between iron stores and glucose disturbances is not well defined. We used a frequently sampled oral glucose tolerance test (OGTT), coupled with mathematical modeling, and magnetic resonance imaging (MRI) to examine the impact of pancreatic, cardiac, and hepatic iron overload on glucose regulation in 59 patients with thalassemia major. According to OGTT results, 11 patients had DM, 12 had impaired glucose tolerance (IGT), 8 had isolated impaired fasting glucose (IFG), and 28 patients had normal glucose tolerance (NGT). Patients with DM had significantly impaired insulin sensitivity and insulin release. Insulin resistance was most strongly associated with markers of inflammation and somatic iron overload, while disposition index (DI) (a measure of beta cell function) was most strongly correlated with pancreas R2*. Patients with DM and IGT had significantly worse DI than those with NGT or IFG, suggesting significant beta cell toxicity. One‐third of patients having elevated pancreas R2* had normal glucose regulation (preclinical iron burden), but these patients were younger and had lower hepatic iron burdens. Our study indicates that pancreatic iron is the strongest predictor of beta cell toxicity, but total body iron burden, age, and body habitus also influence glucose regulation. We also demonstrate that MRI and fasting glucose/insulin are complementary screening tools, reducing the need for oral glucose tolerance testing, and identify high‐risk patients before irreversible pancreatic damage. Am. J. Hematol., 2011. © 2011 Wiley Periodicals, Inc.     

Investigation into the sources of superoxide in human blood vessels: angiotensin II increases superoxide production in human internal mammary arteries

BACKGROUND: Increased vascular superoxide anion (.O(2)(-)) production contributes to endothelial dysfunction and hypertension in animal models of cardiovascular disease. Observations in experimental animals suggest that angiotensin II (Ang II) increases.O(2)(-) production by activation of vascular NAD(P)H oxidase. We studied the sources of.O(2)(-) production in human blood vessels and investigated whether, and by what mechanism, Ang II might alter vascular.O(2)(-) production. METHODS AND RESULTS: Internal mammary arteries (IMAs) and saphenous veins (SVs) were collected at the time of cardiac surgery. Vessels were incubated in Krebs buffer at 37 degrees C.O(2)(-) was measured by lucigenin chemiluminescence. Basal. O(2)(-) concentrations were greater in IMAs than SVs. Inhibitors of NAD(P)H oxidase (10 micromol/L to 200 micromol/L diphenyleneiodonium) and xanthine oxidase (1 mmol/L allopurinol) caused reductions in.O(2)(-) concentrations in both IMAs and SVs. Western blotting of superoxide dismutase proteins demonstrated similar expression in IMAs and SVs. Vessels were also incubated in the presence or absence of Ang II (1 pmol/L to 1 micromol/L). Ang II increased.O(2)(-) production in IMAs at 4 hours of incubation (control, 978+/-117 pmol. min(-1). mg(-1); 1 micromol/L of Ang II, 1690+/-213 pmol. min(-1). mg(-1); n=27, P=0.0001, 95% CI 336, 925) but not in SVs. This effect was completely inhibited by coincubation of IMAs with DPI (100 micromol/L), a nonspecific Ang II antagonist ([sar(1), thre(8)]-Ang II, 1 micromol/L) and a specific Ang II type 1 (AT(1)) receptor antagonist (losartan, 1 micromol/L). Conclusions-. O(2)(-) production is greater in human IMAs than in SVs. NAD(P)H oxidase and xanthine oxidase are sources of.O(2)(-) production in these vessels. The vasoactive peptide Ang II increases.O(2)(-) production in human arteries by an AT(1) receptor-dependent mechanism.     

Chlamydia and atherosclerotic coronary arterial disease in Turkey

OBJECTIVE: Chlamydia pneumoniae, which is a Gram(-) intracellular bacteria, besides being a respiratory pathogen, is thought to play an active role in the progress of acute myocardial infarction and chronic coronary artery disease. In this study we aim to determine the frequency of C. pneumoniae in coronary artery lesions of Turkish people. METHODS AND RESULTS: The atherosclerotic material taken from 8 cases by directional atherectomy and from 23 cases by surgical endarterectomy and examined by indirect immunofluorescence (IIFA) test and polymerase chain reaction (PCR). C. pneumoniae positivity was 32.3% (10/31) by IIFA and 29.0% (9/31) by PCR while the evaluation of the methods together yield a positivity of 35.5% (11/31). CONCLUSIONS: A statistically significant difference could not be established between C. pneumoniae positive and negative groups according to age and the classical atherosclerotic risk factors such as diabetes mellitus, smoking, hypercholesterolaemia, hypertension, family history; besides, a statistically significant difference could not be found between the presence of C. pneumoniae and the severity and clinical picture of coronary artery disease.     

Augmented responses to vasoconstrictor stimuli in hypercholesterolemic and atherosclerotic monkeys

We examined effects of hypercholesterolemia and atherosclerosis on vasoconstrictor responses to norepinephrine and serotonin. Responses were compared in normal, atherosclerotic, and hypercholesterolemic but non-atherosclerotic cynomolgus monkeys. The hindlimb was perfused at constant flow so that changes in perfusion pressure indicated changes in vascular resistance. We measured the pressure gradient from the iliac to the dorsal pedal artery so that responses of the large artery segment could be determined. Serotonin decreased total hindlimb resistance in normal and hypercholesterolemic monkeys, but increased total resistance in atherosclerotic monkeys. There was a greater than 10-fold increase in constrictor responses of large arteries to serotonin in atherosclerotic monkeys, compared with normal and hypercholesterolemic monkeys. In contrast, we found that vasoconstrictor responses to norepinephrine are normal in atherosclerotic monkeys and increased in hypercholesterolemic monkeys prior to development of atherosclerosis. Hypercholesterolemia augmented responses of small vessels to norepinephrine. We conclude that, during early stages of hypercholesterolemia in cynomolgus monkeys, vasoconstrictor responses to norepinephrine are increased in small vessels. At a later stage, as atherosclerosis develops, responses to norepinephrine return to normal, but vasoconstrictor effects of large arteries to serotonin are greatly potentiated.     

Angiotensin II type 1 receptor blocker telmisartan suppresses superoxide production and reduces atherosclerotic lesion formation in apolipoprotein E-deficient mice

Angiotensin II is involved in the process of atherosclerosis and stimulates superoxide production from cardiovascular cells. We examined the effect of telmisartan, an angiotensin II type 1 receptor blocker, on atherosclerosis. We chronically treated apolipoprotein E-deficient mice with two different doses of telmisartan dissolved in drinking water (0.3 and 3 mg/kg) starting from 4 weeks of age for 12 weeks. Lipid contents were not different in both telmisartan-treated groups compared with control group. Systolic blood pressure was significantly reduced with 3 mg/kg, but unchanged with 0.3 mg/kg. The total atherosclerotic lesion size at the aortic sinus was reduced with 0.3 mg/kg compared with control, and additional reduction was proved with 3 mg/kg. The fibrotic change was not different among three groups, but MOMA-2-, malondialdehyde-, 4-hydroxy-2-nonenal-immunostained areas were reduced by telmisartan. As the mechanism, we revealed that both doses of telmisartan markedly reduced superoxide production from in situ vessels assessed by lucigenin-enhanced chemiluminescence and dihydroethidium staining. And NAD(P)H dependent oxidase activity in vessels was reduced by telmisartan. Further, 8-iso-prostaglandin F2alpha level, a systemic oxidative stress marker, obtained from urine and plasma samples were significantly reduced by telmisartan. Telmisartan reduced atherosclerosis in apolipoprotein E-deficient mice at least partly via the suppression of oxidative stress.     

Oxidized low-density lipoprotein increases superoxide production by endothelial nitric oxide synthase by inhibiting PKCalpha

OBJECTIVE: Oxidized low-density lipoprotein (ox-LDL) increases superoxide anion (O(2)(-)) production by the endothelial nitric oxide (NO) synthase (eNOS). We assessed whether the uncoupling of eNOS was associated with alterations in eNOS phosphorylation and/or the assembly of the eNOS signaling complex. METHODS AND RESULTS: In unstimulated human endothelial cells, eNOS Thr(495) was constitutively phosphorylated. ox-LDL, but not native LDL, enhanced the production of O(2)(-) by endothelial cells, an effect that was partially sensitive to NOS inhibition. ox-LDL, but not native LDL, induced a time- and concentration-dependent decrease in the phosphorylation of eNOS on Thr(495). Protein kinase C (PKC) has been reported to phosphorylate this residue, and the increase in the phosphorylation of Thr(495) induced by phorbol 12-myristate 13-acetate was attenuated in cells pretreated with ox-LDL. Moreover, the phosphorylation and activity of PKCalpha was attenuated by ox-LDL and paralleled the changes in eNOS phosphorylation. ox-LDL also induced the dissociation of eNOS from the plasma and Golgi membranes. In COS-7 cells, a T495A eNOS mutant generated significantly more O(2)(-) than a T495D mutant did, indicating that the dephosphorylation of Thr(495) alone can increase O(2)(-) production by eNOS. However, although the dephosphorylation of Thr(495) in histamine-stimulated endothelial cells enhanced the binding of calmodulin to eNOS, calmodulin no longer bound to eNOS from ox-LDL-treated endothelial cells. CONCLUSIONS: These results indicate that a decrease in the activity of PKCalpha in ox-LDL-treated endothelial cells is associated with the dephosphorylation of eNOS, dissociation of the eNOS signaling complex, and the enhanced production of eNOS-derived O(2)(-).     

Diffusional support of the thoracic aorta in atherosclerotic monkeys

We have demonstrated previously that there is increased blood flow through vasa vasorum in the aorta of atherosclerotic monkeys. In this study, a new method was used to examine diffusional support to the wall of blood vessels. Our goal was to determine whether proliferation of vasa vasorum in the outer media contributes to increased diffusional support of the atherosclerotic aorta and whether there are focal or homogeneous increases in diffusion to the aortic wall. Cynomolgus monkeys were fed normal or atherogenic diet for 1.5 or 4 years. Diffusional support of the thoracic aorta was determined by measuring the concentration of iodo[14C]antipyrine in the aortic media. The findings suggest that: 1) vasa vasorum provide only minimal nutritional support to the thoracic aorta of normal monkeys and monkeys with moderately severe atherosclerosis, and 2) in monkeys with severe atherosclerosis, diffusional support to the thoracic aorta is strikingly heterogeneous, ranging from minimal to large. We speculate that minimal diffusional support to the outer layers of the thickened atherosclerotic aorta may contribute to development of focal medial necrosis.     

Regression of atherosclerosis in monkeys reduces vascular superoxide levels

Superoxide (O2*-) in arteries may contribute to atherosclerosis in part by inactivation of nitric oxide. We hypothesized that regression of atherosclerosis in nonhuman primates is associated with a decrease in vascular NAD(P)H oxidase, decreased O2*- levels, and improved endothelium-dependent relaxation. Cynomolgus monkeys (n=28) were fed an atherogenic diet for 47+/-10 (mean+/-SE) months. In carotid arteries (containing advanced lesions), femoral arteries (moderate lesions), and saphena arteries (minimal lesions), we examined O2*- levels and vasomotor function. Compared with vessels from normal monkeys (n=8), O2*- levels (measured by lucigenin-enhanced chemiluminescence) were 3.3-fold higher in carotid, 1.7-fold higher in femoral, and not different in saphena arteries from atherosclerotic monkeys. Dihydroethidium staining also demonstrated increased O2*- levels throughout the vessel wall in femoral and carotid arteries from atherosclerotic monkeys. Components of the NAD(P)H oxidase (p22(phox) and p47(phox)) were increased in atherosclerotic arteries, and immunohistochemistry demonstrated colocalization primarily to areas of macrophage infiltration. Relaxation to acetylcholine was impaired in carotid and femoral, but not saphena, arteries from atherosclerotic monkeys. After 8 months of regression diet (n=9), serum cholesterol decreased to normal, and O2*- levels (basal and NAD(P)H-stimulated), as well as expression of NAD(P)H oxidase, returned toward normal. Relaxation to acetylcholine improved in femoral arteries, but not in the more diseased carotid arteries. We conclude that, in a primate model of moderately severe atherosclerosis and regression of atherosclerosis, changes in endothelial function are inversely related to O2*- and NAD(P)H oxidase levels. Reduction in vascular O2*- during regression of atherosclerosis may contribute to improvement in vasomotor function.     

Nuclear prelamin a recognition factor and iron dysregulation in multiple sclerosis

Metabolic Brain Disease - Dysregulation of iron metabolism and aberrant iron deposition has been associated with multiple sclerosis. However, the factors that contribute to this pathological state...     

Gene transfer of endothelial NO synthase and manganese superoxide dismutase on arterial vascular cell adhesion molecule-1 expression and superoxide production in deoxycorticosterone acetate-salt hypertension

Enhanced vascular cell adhesion molecule-1 (VCAM-1) expression directly contributes to vascular dysfunction in hypertension. Decreased NO and/or increased superoxide are causative factors for such an event in the vessel wall. The present study was undertaken to determine whether gene transfer of endothelial NO synthase (eNOS) or manganese superoxide dismutase (MnSOD) affects VCAM-1 levels in arteries from hypertensive rats. Isolated carotid and femoral arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats were transduced for 4 hours with adenoviral vectors encoding eNOS, MnSOD, or beta-galactosidase reporter genes. Recombinant eNOS or MnSOD expression was evident morphologically and quantitatively 24 hours after gene transfer. Immunohistochemistry, ELISA, and Western blot techniques were used to determine VCAM-1 expression and levels. In addition, endogenous eNOS and MnSOD and in situ superoxide levels were analyzed by immunoblotting and fluorescence confocal microscopy, respectively. Arterial VCAM-1 expression was significantly higher in DOCA-salt hypertensive rats than in sham-operated rats; this expression was accompanied by decreased MnSOD but unaltered endogenous eNOS levels. VCAM-1 expression was significantly lower in MnSOD- and eNOS-transduced hypertensive arteries, with a concomitant reduction of superoxide level. These results suggest that gene transfer of MnSOD or eNOS suppresses arterial VCAM-1 expression in DOCA-salt hypertension by reducing the superoxide level.     

The role of alpha-cell dysregulation in fasting and postprandial hyperglycemia in type 2 diabetes and therapeutic implications

The hyperglycemic activity of pancreatic extracts was encountered some 80 yr ago during efforts to optimize methods for the purification of insulin. The hyperglycemic substance was named "glucagon," and it was subsequently determined that glucagon is a 29-amino acid peptide synthesized and released from pancreatic alpha-cells. This article begins with a brief overview of the discovery of glucagon and the contributions that somatostatin and a sensitive and selective assay for pancreatic (vs. gut) glucagon made to understanding the physiological and pathophysiological roles of glucagon. Studies utilizing these tools to establish the function of glucagon in normal nutrient homeostasis and to document a relative glucagon excess in type 2 diabetes mellitus (T2DM) and precursors thereof are then discussed. The evidence that glucagon excess contributes to the development and maintenance of fasting hyperglycemia and that failure to suppress glucagon secretion contributes to postprandial hyperglycemia is then reviewed. Although key human studies are emphasized, salient animal studies highlighting the importance of glucagon in normal and defective glucoregulation are also described. The past eight decades of research in this area have led to development of new therapeutic approaches to treating T2DM that have been shown to, or are expected to, improve glycemic control in patients with T2DM in part by improving alpha-cell function or by blocking glucagon action. Accordingly, this review ends with a discussion of the status and therapeutic potential of glucagon receptor antagonists, alpha-cell selective somatostatin agonists, glucagon-like peptide-1 agonists, and dipeptidyl peptidase-IV inhibitors. Our overall conclusions are that there is considerable evidence that relative hyperglucagonemia contributes to fasting and postprandial hyperglycemia in patients with T2DM, and there are several new and emerging pharmacotherapies that may improve glycemic control in part by ameliorating the hyperglycemic effects of this relative glucagon excess.