Demetrio Ciociaro

Acute elevation of free fatty acid levels leads to hepatic insulin resistance in obese subjects

Raised levels of free fatty acids (FFA) compete with glucose for utilization by insulin-sensitive tissues, and, therefore, they may induce insulin resistance in the normal subject. The influence of experimental elevations in FFA levels on glucose metabolism in native insulin-resistant states is not known. We studied seven women with moderate obesity (63% above their ideal body weight) but normal glucose tolerance with the use of the insulin clamp technique with or without an infusion of Intralipid + heparin. Upon raising plasma insulin levels to approximately 60 microU/mL while maintaining euglycemia, whole body glucose utilization (3H-3-glucose) rose similarly without (from 66 +/- 7 to 113 +/- 11 mg/min m2, P less than .02) or with (from 70 +/- 7 to 137 +/- 19 mg/min m2, P less than .02) concomitant lipid infusion. In contrast, endogenous glucose production was considerably (73%) suppressed (from 66 +/- 7 to 15 +/- 8 mg/min m2, P less than .001) during the clamp without lipid, but declined only marginally (from 70 +/- 7 to 48 +/- 7 mg/min m2, NS) with lipid administration. The difference between the control and the lipid study was highly significant (P less than .02), and amounted to an average of 3.8 g of relative glucose overproduction during the second hour of the clamp. Blood levels of lactate rose by 34 +/- 15% (.1 greater than P greater than .05) in the control study but only by 17 +/- 10% (NS) during lipid infusion. Blood pyruvate concentrations fell in both sets of experiments (by approximately 45% at the end of the study) with similar time courses.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Insulin on Renal Sodium and Uric Acid Handling in Essential Hypertension

In normal subjects, insulin decreases the urinary excretion of sodium, potassium, and uric acid. We tested whether these renal effects of insulin are altered in insulin resistant hypertension. In 37 patients with essential hypertension, we measured the changes in urinary excretion of sodium, potassium, and uric acid in response to physiological euglycemic hyperinsulinemia (by using the insulin clamp technique at an insulin infusion rate of 6 pmol/min/kg). Glucose disposal rate averaged 26.6 +/- 1.5 mumol/min/kg, i.e., 20% lower than in normotensive controls (33.1 +/- 2.1 mumol/min/kg, P = .015) In the basal state, fasting plasma uric acid concentrations were higher in men than women (P < .001), were positively related to body mass index (r = 0.38, P = .02), waist/hip ratio (r = 0.35, P < .05), and serum triglyceride levels (r = 0.59, P = .0001), and negatively related to HDL cholesterol concentrations (r = -0.59, P = .0001) and glucose disposal rate (r = 0.42, P < .01). Uric acid clearance, on the other hand, was inversely related to body mass index (r = 0.41, P = .01), plasma uric acid (r = 0.65, P < .0001) and triglyceride concentrations (r = 0.39, P < .02), and directly related to HDL cholesterol levels (r = 0.52, P < .001). During insulin infusion, blood pressure, plasma uric acid and sodium concentration, and creatinine clearance did not change. In contrast, hyperinsulinemia caused a significant decrease in the urinary excretion of uric acid (2.67 +/- 0.12 to 1.86 +/- .14 mumol/min/1.73 m2, P = .0001), sodium (184 +/- 12 to 137 +/- 14 mumol/min/1.73 m2, P = .0001), and potassium (81 +/- 7 to 48 +/- 4 mumol/ min/1.73 m2, P = .0001). Both in absolute terms (clearance and fractional excretion rates) and percentagewise, these changes were similar to those found in normotensive subjects. Insulin-induced changes in urate excretion were coupled (r = 0.55, P < .0001) to the respective changes in sodium excretion. In hypertensive patients, higher uric acid levels and lower renal urate clearance rates cluster with insulin resistance and dyslipidemia. Despite insulin resistance of glucose metabolism, acute physiological hyperinsulinemia causes normal antinatriuresis, antikaliuresis, and antiuricosuria in these patients.

Visceral Fat in Hypertension: Influence on Insulin Resistance and β-Cell Function

Preferential visceral adipose tissue (VAT) deposition has been associated with the presence of insulin resistance in obese and diabetic subjects. The independent association of VAT accumulation with hypertension and its impact on insulin sensitivity and β-cell function have not been assessed. We measured VAT and subcutaneous fat depots by multiscan MRI in 13 nondiabetic men with newly detected, untreated essential hypertension (blood pressure=151±2/94±2 mm Hg, age=47±2 years, body mass index [BMI]=28.4±0.7 kg · m−2) and 26 age-matched and BMI-matched normotensive men (blood pressure=123±1/69±2 mm Hg). Insulin secretion was measured by deconvolution of C-peptide data obtained during an oral glucose tolerance test, and dynamic indices of β-cell function were calculated by mathematical modeling. For a similar fat mass in the scanned abdominal region (4.8±0.3 versus 3.9±0.3 kg, hypertensive subjects versus controls, P =0.06), hypertensive subjects had 60% more VAT than controls (1.6±0.2 versus 1.0±0.1 kg, P =0.003). Intrathoracic fat also was expanded in patients versus controls (45±5 versus 28±3 cm2, P =0.005). Insulin sensitivity was reduced (10.7±0.7 versus 12.9±0.4 mL · min−1 · kgffm −1, P =0.006), and total insulin output was proportionally increased (64 [21] versus 45 [24] nmol · m−2 · h, median [interquartile range], P =0.01), but dynamic indices of β-cell function (glucose sensitivity, rate sensitivity, and potentiation) were similar in the 2 groups. Abdominal VAT, insulin resistance, and blood pressure were quantitatively interrelated (ρ’s of 0.39 to 0.47, P <0.02 or less). In newly found, untreated men with essential hypertension, fat is preferentially accumulated intraabdominally and intrathoracically. Such visceral adiposity is quantitatively related to both height of blood pressure and severity of insulin resistance, but has no impact on the dynamics of β-cell function.

Early Hypertension Is Associated With Reduced Regional Cardiac Function, Insulin Resistance, Epicardial, and Visceral Fat

Mild-to-moderate hypertension is often associated with insulin resistance and visceral adiposity. Whether these metabolic abnormalities have an independent impact on regional cardiac function is not known. The goal of this study was to investigate the effects of increased blood pressure, insulin resistance, and ectopic fat accumulation on the changes in peak systolic circumferential strain. Thirty-five male subjects (age: 47±1 years; body mass index: 28.4±0.6 kg.m−2; mean±SEM) included 13 with normal blood pressure (BP: 113±5/67±2 mm Hg), 13 with prehypertension (BP: 130±1/76±2 mm Hg), and 9 newly diagnosed with essential hypertension (BP: 150±2/94±2 mm Hg) who underwent cardiac magnetic resonance tissue tagging (MRI) and MRI quantitation of abdominal visceral and epicardial fat. Glucose tolerance, on oral glucose tolerance test, and insulin resistance were assessed along with the serum lipid profile. All of the subjects had normal glucose tolerance, left- and right-ventricular volumes, and ejection fraction. Across the BP groups, left ventricular mass tended to increase, and circumferential shortening was progressively reduced at basal, midheart, and apical segments (on average, from −17.0±0.5% in normal blood pressure to −15.2±0.7% in prehypertension to −13.6±0.8% in those newly diagnosed with essential hypertension; P=0.004). Reduced circumferential strain was significantly associated with raised BP independent of age (r=0.41; P=0.01) and with epicardial and visceral fat, serum triglycerides, and insulin resistance independent of age and BP. In conclusion, regional left ventricular function is already reduced at the early stages of hypertension despite the normal global cardiac function. Insulin resistance, dyslipidemia, and ectopic fat accumulation are associated with reduced regional systolic function.

Insulin Resistance in Microalbuminuric Hypertension: Sites and Mechanisms

Microalbuminuria in patients with essential hypertension is a marker of incipient glomerular dysfunction and clusters with lipid and hemodynamic abnormalities. Recent evidence has shown that hypertensive patients with microalbuminuria have a hyperinsulinemic response to oral glucose, suggesting the presence of insulin resistance. To directly test this possibility we studied insulin action in two accurately matched groups (n = 10 each) of hypertensive patients with or without microalbuminuria (14 +/- 2 versus 52 +/- 7 mg/24 h-1, mean of three 24-hour collections). In response to glucose ingestion microalbuminuric patients showed slight hyperglycemia (area under the curve, 928 +/- 43 versus 784 +/-19 nmol/L-1/2h-1, P < .02) and a marked hyperinsulinemia (26.8 +/- 3.3 versus 49.8 +/- 3.7 nmol/L-1/2h-1, P < 0.01). Basal arterial blood pressure, heart rate, and forearm blood flow were similar in the two groups and did not change significantly during a 2-hour euglycemic insulin clamp. Insulin-stimulated wholebody glucose uptake was 25% lower in microalbuminuric patients (33.5 +/- 2.5 versus 25.2 +/- 2.1 mumol/min-1/kg-1, P < .02). This difference was entirely due to a 40% reduction in glycogen synthesis (12.9 +/- 1.8 versus 21.3 +/- 3.2 mumol/min-1/kg-1, P < .05) as glucose oxidation was similarly stimulated in the two groups. In contrast there was no difference in the ability of insulin to suppress hepatic glucose production (by approximately 100% at the end of the clamp), to decrease fractional sodium and potassium excretions (by 35%), to lower circulating free fatty acids (by 80%), and to reduce plasma potassium concentrations (by 10%).(ABSTRACT TRUNCATED AT 250 WORDS)

Long-Term Effects of Bariatric Surgery on Meal Disposal and β-Cell Function in Diabetic and Nondiabetic Patients

Gastric bypass surgery leads to marked improvements in glucose tolerance and insulin sensitivity in obese type 2 diabetes (T2D); the impact on glucose fluxes in response to a physiological stimulus, such as a mixed meal test (MTT), has not been determined. We administered an MTT to 12 obese T2D patients and 15 obese nondiabetic (ND) subjects before and 1 year after surgery (10 T2D and 11 ND) using the double-tracer technique and modeling of β-cell function. In both groups postsurgery, tracer-derived appearance of oral glucose was biphasic, a rapid increase followed by a sharp drop, a pattern that was mirrored by postprandial glucose levels and insulin secretion. In diabetic patients, surgery lowered fasting and postprandial glucose levels, peripheral insulin sensitivity increased in proportion to weight loss (∼30%), and β-cell glucose sensitivity doubled but did not normalize (compared with 21 nonsurgical obese and lean controls). Endogenous glucose production, however, was less suppressed during the MMT as the combined result of a relative hyperglucagonemia and the rapid fall in plasma glucose and insulin levels. We conclude that in T2D, bypass surgery changes the postprandial response to a dumping-like pattern and improves glucose tolerance, β-cell function, and peripheral insulin sensitivity but worsens endogenous glucose output in response to a physiological stimulus.

Impact of increased visceral and cardiac fat on cardiometabolic risk and disease

Diabet. Med. 29, 622–627 (2012)

Effect of pioglitazone on the metabolic and hormonal response to a mixed meal in type II diabetes.

We explored the mechanisms by which a 4‐month, placebo‐controlled pioglitazone treatment (45 mg/day) improves glycemic control in type II diabetic patients (T2D, n=27) using physiological testing (6‐h mixed meal) and a triple tracer technique ([6,6‐2H2]glucose infusion, 2H2O and [6‐3H]glucose ingestion) to measure endogenous glucose production (EGP), gluconeogenesis (GNG), insulin‐mediated glucose clearance and β‐cell glucose sensitivity (by c‐peptide modeling). Compared to sex/age/weight‐matched non‐diabetic controls, T2D patients showed inappropriately (for prevailing insulinemia) raised glucose production (1.05[0.53] vs 0.71[0.36]mmol min−1 kgffm−1 pM, P=0.03) because of enhanced GNG (73.1±2.4 vs 59.5±3.6%, P<0.01) persisting throughout the meal, reduced insulin‐mediated glucose clearance (6[5] vs 12[13]ml min−1 kgffm−1 nM−1, P<0.005), and impaired β‐cell glucose‐sensitivity (27[38] vs 71[37]pmol min−1 m−2 mM−1, P=0.002). Compared to placebo, pioglitazone improved glucose overproduction (P=0.0001), GNG and glucose underutilization (P=0.05) despite lower insulinemia. GNG improvement was quantitatively related to raised adiponectin. β‐cell glucose sensitivity was unchanged. In mild‐to‐moderate T2D, pioglitazone monotherapy decreased fasting and post‐prandial glycemia, principally via inhibition of gluconeogenesis, improved hepatic and peripheral insulin resistance.

Glucokinase links Krüppel-like factor 6 to the regulation of hepatic insulin sensitivity in nonalcoholic fatty liver disease

The polymorphism, KLF6‐IVS1‐27A, in the Krüppel‐like factor 6 (KLF6) transcription factor gene enhances its splicing into antagonistic isoforms and is associated with delayed histological progression of nonalcoholic fatty liver disease (NAFLD). To explore a potential role for KLF6 in the development of insulin resistance, central to NAFLD pathogenesis, we genotyped KLF6‐IVS1‐27 in healthy subjects and assayed fasting plasma glucose (FPG) and insulin sensitivities. Furthermore, we quantified messenger RNA (mRNA) expression of KLF6 and glucokinase (GCK), as an important mediator of insulin sensitivity, in human livers and in liver tissues derived from a murine Klf6 knockdown model (DeltaKlf6). Klf6 overexpression studies in a mouse hepatocyte line were utilized to mechanistically link KLF6 with Gck promoter activity. KLF6‐IVS1‐27Gwt (i.e., less KLF6 splicing) was associated with stepwise increases in FPG and insulin and reduced hepatic insulin sensitivity. KLF6 binds to the liver‐specific Gck promoter and activates a GCK promoter‐reporter, identifying GCK as a KLF6 direct transcriptional target. Accordingly, in DeltaKlf6 hepatocytes Gck expression was reduced and stable transfection of Klf6 led to up‐regulation of Gck. GCK and KLF6 mRNAs correlate directly in human NAFLD tissues and immunohistochemistry studies confirm falling levels of both KLF6 and GCK in fat‐laden hepatocytes. In contrast to full‐length KLF6, splice variant KLF6‐SV1 increases in NAFLD hepatocytes and inversely correlates with glucokinase regulatory protein, which negatively regulates GCK activity. Conclusion: KLF6 regulation of GCK contributes to the development of hepatic insulin resistance. The KLF6‐IVS1‐27A polymorphism, which generates more KLF6‐SV1, combats this, lowering hepatic insulin resistance and blood glucose. (HEPATOLOGY 2011)

Characterization of a reversed-phase high-performance liquid chromatographic system for the determination of blood amino acids

High-performance liquid chromatography was used to separate physiological amino acids in perchloric acid supernatants of blood samples. Precolumn derivatization with phenyl isothiocyanate was carried out, starting with 20 microliters of supernatant; 2-10 microliters were injected into a 30-cm Pico Tag column, which was eluted with a gradient of two eluents in 64 min. Stock amino acid solutions prepared in water, hydrochloric acid or perchloric acid showed comparable recoveries on serial dilution (parallelism test). The recovery of crystalline amino acids added to blood in amounts ranging from normal to six times normal was generally satisfactory. The within-assay relative standard deviations were less than 5% for many amino acids. The performance of the system was less than satisfactory for cysteine and methionine. Glutamine and asparagine are interconverted into glutamate and aspartate, respectively, in a time-dependent fashion; a separate measurement of one member of the pair is therefore required in order to assay the other starting from the sum of both chromatographic peaks. The method is suitable for the relatively rapid, sensitive and accurate measurement of blood amino acids in perchloric acid supernatants (in which other relevant metabolites are customarily assayed) over a wide range of physiological concentrations, on very small amounts of sample.