Giuseppe Paolisso

Oxidative Stress and Diabetic Vascular Complications

Long-term vascular complications still represent the main cause of morbidity and mortality in diabetic patients. Although prospective randomized long-term clinical studies comparing the effects of conventional and intensive therapy have demonstrated a clear link between diabetic hyperglycemia and the development of secondary complications of diabetes, they have not defined the mechanism through which excess glucose results in tissue damage. Evidence has accumulated indicating that the generation of reactive oxygen species (oxidative stress) may play an important role in the etiology of diabetic complications. This hypothesis is supported by evidence that many biochemical pathways strictly associated with hyperglycemia (glucose autoxidation, polyol pathway, prostanoid synthesis, protein glycation) can increase the production of free radicals. Furthermore, exposure of endothelial cells to high glucose leads to augmented production of superoxide anion, which may quench nitric oxide, a potent endothelium-derived vasodilator that participates in the general homeostasis of the vasculature. In further support of the consequential injurious role of oxidative stress, many of the adverse effects of high glucose on endothelial functions, such as reduced endothelial-dependent relaxation and delayed cell replication, are reversed by antioxidants. A rational extension of this proposed role for oxidative stress is the suggestion that the different susceptibility of diabetic patients to microvascular and macrovascular complications may be a function of the endogenous antioxidant status.

Postprandial endothelial activation in healthy subjects and in type 2 diabetic patients: role of fat and carbohydrate meals.

OBJECTIVES To compare the effect of a high-fat meal and a high-carbohydrate meal (pizza), with and without antioxidant vitamins, on endothelial activation in healthy subjects and in patients with type 2 diabetes mellitus. BACKGROUND The postprandial state is becoming increasingly acknowledged to affect some early events of atherogenesis. METHODS In a randomized, observer-blinded, crossover study, 20 newly diagnosed type 2 diabetic patients and 20 age- and gender-matched healthy subjects received two meals at one-week intervals: a high-fat meal (760 calories) and an isoenergetic high-carbohydrate meal (non-cheese pizza). In all subjects, the same meals were repeated immediately following ingestion of vitamin E, 800 IU, and ascorbic acid, 1,000 mg. RESULTS In normal subjects, the high-fat meal increased the plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which were prevented by vitamins. No change in these parameters occurred after pizza ingestion or pizza ingestion with vitamins. In diabetic patients, basal concentrations of glucose, cytokines and adhesion molecules were significantly higher than in nondiabetic controls. Both meals significantly increased cytokine and adhesion molecule levels, but the increase was more sustained following the high-fat meal. There was no significant change from baseline when vitamin supplementation accompanied each meal. There was a relationship between changes in serum triglycerides and changes in TNF-alpha (r = 0.39, p < 0.01), IL-6 (r = 0.28, p < 0.05) and VCAM-1 (r = 0.25, p < 0.05), and between changes in plasma glucose and changes in IL-6 (r = 0.36, p < 0.01) and ICAM-1 (r = 0.31, p < 0.02). CONCLUSIONS An oxidative mechanism mediates endothelial activation induced by post-meal hyperlipidemia and hyperglycemia.

Diabetes mellitus, hypertension, and cardiovascular disease: Which role for oxidative stress?

Accelerated atherosclerotic vascular disease is the leading cause of mortality in patients with diabetes mellitus. Endothelium-derived nitric oxide (NO) is a potent endogenous nitrovasodilator and plays a major role in modulation of vascular tone. Selective impairment of endothelium-dependent relaxation has been demonstrated in aortas of both nondiabetic animals exposed to elevated concentrations of glucose in vitro and insulin-dependent diabetic animals. The impaired NO release in experimentally induced diabetes may be prevented by a number of antioxidants. It has been hypothesized that oxygen-derived free radicals (OFR) generated during both glucose autoxidation and formation of advanced glycosylation end products may interfere with NO action and attenuate its vasodilatory activity. The oxidative injury may also be increased in diabetes mellitus because of a weakened defense due to reduced endogenous antioxidants (vitamin E, reduced glutathione [GSH]). A defective endothelium-dependent vascular relaxation has been found in animal models of hypertension and in hypertensive patients. An imbalance due to reduced production of NO or increased production of free radicals, mainly superoxide anion, may facilitate the development of an arterial functional spasm. Treatment with different antioxidants increases blood flow in the forearm and decreases blood pressure and viscosity in normal humans; vitamin E inhibits nonenzymatic glycosylation, oxidative stress, and red blood cell microviscosity in diabetic patients. Long-term randomized clinical trials of adequate size in secondary and primary prevention could support the free-radical hypothesis for diabetic diabetic vascular complications and the use of antioxidants to reduce the risk of coronary heart disease.

A genome-wide association study identifies protein quantitative trait loci (pQTLs)

There is considerable evidence that human genetic variation influences gene expression. Genome-wide studies have revealed that mRNA levels are associated with genetic variation in or close to the gene coding for those mRNA transcripts – cis effects, and elsewhere in the genome – trans effects. The role of genetic variation in determining protein levels has not been systematically assessed. Using a genome-wide association approach we show that common genetic variation influences levels of clinically relevant proteins in human serum and plasma. We evaluated the role of 496,032 polymorphisms on levels of 42 proteins measured in 1200 fasting individuals from the population based InCHIANTI study. Proteins included insulin, several interleukins, adipokines, chemokines, and liver function markers that are implicated in many common diseases including metabolic, inflammatory, and infectious conditions. We identified eight Cis effects, including variants in or near the IL6R (p = 1.8×10−57), CCL4L1 (p = 3.9×10−21), IL18 (p = 6.8×10−13), LPA (p = 4.4×10−10), GGT1 (p = 1.5×10−7), SHBG (p = 3.1×10−7), CRP (p = 6.4×10−6) and IL1RN (p = 7.3×10−6) genes, all associated with their respective protein products with effect sizes ranging from 0.19 to 0.69 standard deviations per allele. Mechanisms implicated include altered rates of cleavage of bound to unbound soluble receptor (IL6R), altered secretion rates of different sized proteins (LPA), variation in gene copy number (CCL4L1) and altered transcription (GGT1). We identified one novel trans effect that was an association between ABO blood group and tumour necrosis factor alpha (TNF-alpha) levels (p = 6.8×10−40), but this finding was not present when TNF-alpha was measured using a different assay , or in a second study, suggesting an assay-specific association. Our results show that protein levels share some of the features of the genetics of gene expression. These include the presence of strong genetic effects in cis locations. The identification of protein quantitative trait loci (pQTLs) may be a powerful complementary method of improving our understanding of disease pathways.

Role of magnesium in insulin action, diabetes and cardio-metabolic syndrome X

Magnesium (Mg) is one of the most abundant ions present in living cells and its plasma concentration is remarkably constant in healthy subjects. Plasma and intracellular Mg concentrations are tightly regulated by several factors. Among them, insulin seems to be one of the most important. In vitro and in vivo studies have demonstrated that insulin may modulate the shift of Mg from extracellular to intracellular space. Intracellular Mg concentration has also been shown to be effective in modulating insulin action (mainly oxidative glucose metabolism), offset calcium-related excitation-contraction coupling, and decrease smooth cell responsiveness to depolarizing stimuli. A poor intracellular Mg concentration, as found in noninsulin-dependent diabetes mellitus (NIDDM) and in hypertensive patients, may result in a defective tyrosine-kinase activity at the insulin receptor level and exaggerated intracellular calcium concentration. Both events are responsible for the impairment in insulin action and a worsening of insulin resistance in noninsulin-dependent diabetic and hypertensive patients. By contrast, in NIDDM patients daily Mg administration, restoring a more appropriate intracellular Mg concentration, contributes to improve insulin-mediated glucose uptake. The benefits deriving- from daily Mg supplementation in NIDDM patients are further supported by epidemiological studies showing that high daily Mg intake are predictive of a lower incidence of NIDDM. In conclusion, a growing body of studies suggest that intracellular Mg may play a key role in modulating insulin-mediated glucose uptake and vascular tone. We further suggest that a reduced intracellular Mg concentration might be the missing link helping to explain the epidemiological association between NIDDM and hypertension.

Common variation in the FTO gene alters diabetes-related metabolic traits to the extent expected, given its effect on BMI

OBJECTIVE—Common variation in the FTO gene is associated with BMI and type 2 diabetes. Increased BMI is associated with diabetes risk factors, including raised insulin, glucose, and triglycerides. We aimed to test whether FTO genotype is associated with variation in these metabolic traits. RESEARCH DESIGN AND METHODS—We tested the association between FTO genotype and 10 metabolic traits using data from 17,037 white European individuals. We compared the observed effect of FTO genotype on each trait to that expected given the FTO-BMI and BMI-trait associations. RESULTS—Each copy of the FTO rs9939609 A allele was associated with higher fasting insulin (0.039 SD [95% CI 0.013–0.064]; P = 0.003), glucose (0.024 [0.001–0.048]; P = 0.044), and triglycerides (0.028 [0.003–0.052]; P = 0.025) and lower HDL cholesterol (0.032 [0.008–0.057]; P = 0.009). There was no evidence of these associations when adjusting for BMI. Associations with fasting alanine aminotransferase, γ-glutamyl-transferase, LDL cholesterol, A1C, and systolic and diastolic blood pressure were in the expected direction but did not reach P < 0.05. For all metabolic traits, effect sizes were consistent with those expected for the per allele change in BMI. FTO genotype was associated with a higher odds of metabolic syndrome (odds ratio 1.17 [95% CI 1.10–1.25]; P = 3 × 10−6). CONCLUSIONS—FTO genotype is associated with metabolic traits to an extent entirely consistent with its effect on BMI. Sample sizes of >12,000 individuals were needed to detect associations at P < 0.05. Our findings highlight the importance of using appropriately powered studies to assess the effects of a known diabetes or obesity variant on secondary traits correlated with these conditions.

A gender--dependent genetic predisposition to produce high levels of IL-6 is detrimental for longevity.

Current literature indicates that elevated IL‐6 serum levels are associated with diseases, disability and mortality in the elderly. In this paper, we studied the IL‐6 promoter genetic variability at –174 C/G locus and its effect on IL‐6 serum levels in a total of 700 people from 60 to 110 years of age, including 323 centenarians. We found that the proportion of homozygotes for the G allele at –174 locus decreases in centenarian males, but not in centenarian females. Moreover, we found that, only among males, homozygotes for the G allele at –174 locus have higher IL‐6 serum levels in comparison with carriers of the C allele. On the whole, our data suggest that those individuals who are genetically predisposed to produce high levels of IL‐6 during aging, i.e. –174 locus GG homozygous men, are disadvantaged for longevity.

Hypertension, diabetes mellitus, and insulin resistance : The role of intracellular magnesium

Magnesium is one of the most abundant ions present in living cells and its plasma concentration is remarkably constant in healthy subjects. Plasma and intracellular magnesium concentrations are tightly regulated by several factors. Among them, insulin seems to be one of the most important. In fact, in vitro and in vivo studies have demonstrated that insulin may modulate the shift of magnesium from extracellular to intracellular space. Intracellular magnesium concentration has also been shown to be effective on modulating insulin action (mainly oxidative glucose metabolism), offset calcium-related excitation-contraction coupling, and decrease smooth cell responsiveness to depolarizing stimuli, by stimulating Ca2+-dependent K+ channels. A poor intracellular magnesium concentration, as found in non-insulin-dependent diabetes mellitus (NIDDM) and in hypertensive (HP) patients, may result in a defective tyrosine-kinase activity at the insulin receptor level and exaggerated intracellular calcium concentration. Both events are responsible for the impairment in insulin action and a worsening of insulin resistance in non-insulin-dependent diabetic and hypertensive patients. By contrast, in NIDDM patients daily magnesium administration, restoring a more appropriate intracellular magnesium concentration, contributes to improve insulin-mediated glucose uptake. Similarly, in HP patients magnesium administration may be useful in decreasing arterial blood pressure and improving insulin-mediated glucose uptake. The benefits deriving from daily magnesium supplementation in NIDDM and HP patients are further supported by epidemiological studies showing that high daily magnesium intake to be predictive of a lower incidence of NIDDM and HP. In conclusion, a growing body of studies suggest that intracellular magnesium may play a key role on modulating insulin-mediated glucose uptake and vascular tone. We further suggest that a reduced intracellular magnesium concentration might be the missing link helping to explain the epidemiological association between NIDDM and hypertension.

MAGNESIUM AND GLUCOSE HOMEOSTASIS

SummaryMagnesium is an important ion in all living cells being a cofactor of many enzymes, especially those utilising high energy phosphate bounds. The relationship between insulin and magnesium has been recently studied. In particular it has been shown that magnesium plays the role of a second messenger for insulin action; on the other hand, insulin itself has been demonstrated to be an important regulatory factor of intracellular magnesium accumulation. Conditions associated with insulin resistance, such as hypertension or aging, are also associated with low intracellular magnesium contents. In diabetes mellitus, it is suggested that low intracellular magnesium levels result from both increased urinary losses and insulin resistance. The extent to which such a low intracellular magnesium content contributes to the development of macro- and microangiopathy remains to be established. A reduced intracellular magnesium content might contribute to the impaired insulin response and action which occurs in Type 2 (non-insulin-dependent) diabetes mellitus. Chronic magnesium supplementation can contribute to an improvement in both islet Beta-cell response and insulin action in non-insulin-dependent diabetic subjects.

Opposite effects of short- and long-term fatty acid infusion on insulin secretion in healthy subjects

SummaryOur study investigates short- and long-term effects of infusion of non-esterified fatty acids (NEFA) on insulin secretion in healthy subjects. Twelve healthy individuals underwent a 24-h Intralipid (10% triglyceride emulsion) infusion at a rate of 0.4 ml/min with a simultaneous infusion of heparin (a bolus of 200 U followed by 0.2 U/min per kg body weight). After an overnight fast (baseline), at 6 and at 24 h of Intralipid infusion and 24 h after Intralipid discontinuation (recovery test), all subjects underwent an intravenous glucose tolerance test (iv-GTT) (25 g of glucose/min). Intralipid infusion caused a threefold rise in plasma NEFA concentrations with no difference between the 6- and the 24-h concentrations. Compared to baseline acute insulin response (AIR) (AIR=63±8 mU/l), short-term (6-h) Intralipid infusion was associated with a significant increase in AIR (86±12 mU/l p<0.01); in contrast, long-term (24-h) Intralipid delivery was associated with inhibition of AIR (31±5 mU/l) compared to baseline (p<0.001) and to the 6-h (p<0.03) triglyceride emulsion infusion. Intralipid infusion was associated with a progressive and significant decline in respiratory quotient (RQ). A positive correlation between changes in fasting plasma NEFA concentrations and AIR at the 6-h infusion (r=0.89 p<0.001) was found. In contrast, at the end of the Intralipid infusion period, changes in plasma NEFA concentrations and AIR were negatively correlated (r=−0.87 p<0.001). The recovery test showed that fasting plasma NEFA concentrations, RQ and AIR had returned to baseline values. In the control study (n=8) 0.9% NaCl infusion did not mimick the effect of Intralipid. In conclusion, our study demonstrates that short- and long-term exposures of beta cells to high plasma NEFA concentrations have opposite effects on glucose-induced insulin secretion.