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Phthalates and bisphenol A (BPA) are plasticizers used in many industrial products that can act as endocrine disruptors and lead to metabolic diseases. During the LIFE PERSUADED project, we measured the urinary concentrations of BPA and Di(2-ethylhexyl)phthalate (DEHP) metabolites in 900 Italian women representative of the Italian female adult population (living in the north, centre, and south of Italy in both rural and urban areas). The whole cohort was exposed to DEHP and BPA with measurable levels above limit of detection in more than 99% and 95% of the samples, respectively. The exposure patterns differed for the two chemicals in the three macro-areas with the highest urinary levels for DEHP in south compared to central and northern Italy and for BPA in northern compared to central and southern Italy. BPA levels were higher in women living in urban areas, whereas no difference between areas was observed for DEHP. The estimated daily intake of BPA was 0.11 μg/kg per day, about 36-fold below the current temporary tolerable daily intake of 4 μg/kg per day established by the EFSA in 2015. The analysis of cumulative exposure showed a positive correlation between DEHP and BPA. Further, the reduction of exposure to DEHP and BPA, through specific legislative measures, is necessary to limit the harmfulness of these substances.

Although the effect of glucagon on glucose metabolism has been well characterized, its effect on lipid metabolism, particularly lipolysis and de novo lipogenesis, in humans in vivo has been poorly studied with controversial results. We examined the effect of short-term hyperglucagonemia on de novo lipogenesis (DNL) and endogenous glucose production (EGP) in 8 healthy normal glucose tolerant subjects (5M/3F, age=35±5, BMI= 24±1) who received a 12-hour (6PM to 6AM) glucagon infusion (6ng/kg/min) infusion with measurement of EGP (3-3H-glucose) on the following morning 6-AM. Subjects received deuterated water (D2O, 5g/kg fat free mass) at PM on the night prior to study. 8 weeks later subjects returned for a repeat study with 12-hour infusion of normal saline. DNL was quantified by measuring the incorporation of deuterated water into circulating triglycerides. Gluconeogenic rate was measured as (EGP) x (plasma C5/C2 glucose ratio) (EGP) x (plasma C5/C2 glucose ratio) . Plasma esterified and non-esterified FFA levels were measured with gas chromatography mass spectrometry (GC-MS) . Plasma glucagon increased from 57±3 to 219±21 pg/ml within one hour and remained elevated throughout the study. DNL was significantly decreased (1.64 ± 0.58% vs. 3.± 0.98%, p Disclosure X.Chen: None. F.Carli: None. S.Pezzica: None. G.Mocciaro: None. D.Ciociaro: None. A.Gastaldelli: Advisory Panel; Boehringer Ingelheim International GmbH, Novo Nordisk Global Business Services, Consultant; Boehringer Ingelheim International GmbH, Inventiva Pharma, Other Relationship; Eli Lilly and Company, Gilead Sciences, Inc., Pfizer Inc., Speaker's Bureau; Novo Nordisk. R.A.Defronzo: Advisory Panel; AstraZeneca, Boehringer Ingelheim International GmbH, Intarcia Therapeutics, Inc., Novo Nordisk, Research Support; AstraZeneca, Boehringer Ingelheim International GmbH, Merck & Co., Inc., Speaker's Bureau; AstraZeneca. D.Tripathy: None. Funding EFSD Albert Renold Travel Fellowship; FAVHR

Plasma concentrations of amino acids (AAs), in particular, branched chain AAs (BCAAs), are often found increased in nonalcoholic fatty liver disease (NAFLD); however, if this is due to increased muscular protein catabolism, obesity, and/or increased insulin resistance (IR) or impaired tissue metabolism is unknown. Thus, we evaluated a) if subjects with NAFLD without obesity (NAFLD-NO) compared to those with obesity (NAFLD-Ob) display altered plasma AAs compared to controls (CTs); and b) if AA concentrations are associated with IR and liver histology. Glutamic acid, serine, and glycine concentrations are known to be altered in NAFLD. Because these AAs are involved in glutathione synthesis, we hypothesized they might be related to the severity of NAFLD. We therefore measured the AA profile of 44 subjects with NAFLD without diabetes and who had a liver biopsy (29 NAFLD-NO and 15 NAFLD-Ob) and 20 CTs without obesity, by gas chromatography-mass spectrometry, homeostasis model assessment of insulin resistance, hepatic IR (Hep-IR; Hep-IR = endogenous glucose production × insulin), and the new glutamate-serine-glycine (GSG) index (glutamate/[serine + glycine]) and tested for an association with liver histology. Most AAs were increased only in NAFLD-Ob subjects. Only alanine, glutamate, isoleucine, and valine, but not leucine, were increased in NAFLD-NO subjects compared to CTs. Glutamate, tyrosine, and the GSG-index were correlated with Hep-IR. The GSG-index correlated with liver enzymes, in particular, gamma-glutamyltransferase (R = 0.70), independent of body mass index. Ballooning and/or inflammation at liver biopsy were associated with increased plasma BCAAs and aromatic AAs and were mildly associated with the GSG-index, while only the new GSG-index was able to discriminate fibrosis F3-4 from F0-2 in this cohort. Conclusion Increased plasma AA concentrations were observed mainly in subjects with obesity and NAFLD, likely as a consequence of increased IR and protein catabolism. The GSG-index is a possible marker of severity of liver disease independent of body mass index. (Hepatology 2018;67:145-158).

Gastric bypass surgery (RYGB) leads to improvement or remission of type 2 diabetes (T2D). Long-term studies indicate that the remission rate is higher 2 than 10 years post-surgery; whether the positive effects of surgery on ß-cell function (ß-GS) and glucose metabolism are maintained long term after surgery has not been determined. We studied 21 patients (12 T2D and 9 nondiabetic (ND)) before (B), 1 (1y) and 7 years (7ys) after RYGB with the use of a mixed meal test (MTT) in combination with glucose tracers to measure postmeal glucose fluxes and a euglycemic insulin clamp for insulin sensitivity (M). ß-GS (insulin secretion and dose response curve) was assessed by mathematical modelling. Weight loss 1y after RYGB was 30% and 35% of initial weight for ND and T2D respectively, and 24% and 30% at 7ys. T2D was resolved 1y post surgery in T2D and this outcome was maintained at 7ys (HbA1c 58±4 vs. 35±1 vs. 41±2 mmol/mol; B, 1y and 7ys). M value improved at 1y in proportion to the weight lost (5.0±0.8 to 7.2±0.7 in ND, and 4.3±0.8 to 7.5±0.7 mg. kgffm-1. min-1 in T2D, p Conclusion: In both ND and T2D, RYGB induces marked improvements in glucose tolerance, insulin sensitivity (both muscle and hepatic) and ß-cell function that are maintained 7 years after surgery. Disclosure S. Camastra: None. B.D. Astiarraga: None. C. Barbieri: None. M. Gaggini: None. M. Palumbo: None. R. Berta: None. D. Ciociaro: None. M. Anselmino: None. A. Mari: None. E. Ferrannini: None. A. Gastaldelli: Advisory Panel; Self; Gilead Sciences, Inc.. Other Relationship; Self; European Foundation for the Study of Diabetes. Consultant; Self; INVENTIVA SA.

Glucagon-like peptide 1 receptors (GLP-1Rs) have been found in the brain, but whether GLP-1R agonists (GLP-1RAs) influence brain glucose metabolism is currently unknown. The study aim was to evaluate the effects of a single injection of the GLP-1RA exenatide on cerebral and peripheral glucose metabolism in response to a glucose load. In 15 male subjects with HbA(1c) of 5.7 ± 0.1%, fasting glucose of 114 ± 3 mg/dL, and 2-h glucose of 177 ± 11 mg/dL, exenatide (5 μg) or placebo was injected in double-blind, randomized fashion subcutaneously 30 min before an oral glucose tolerance test (OGTT). The cerebral glucose metabolic rate (CMR(glu)) was measured by positron emission tomography after an injection of [(18)F]2-fluoro-2-deoxy-d-glucose before the OGTT, and the rate of glucose absorption (RaO) and disposal was assessed using stable isotope tracers. Exenatide reduced RaO(0–60 min) (4.6 ± 1.4 vs. 13.1 ± 1.7 μmol/min ⋅ kg) and decreased the rise in mean glucose(0–60 min) (107 ± 6 vs. 138 ± 8 mg/dL) and insulin(0–60 min) (17.3 ± 3.1 vs. 24.7 ± 3.8 mU/L). Exenatide increased CMR(glu) in areas of the brain related to glucose homeostasis, appetite, and food reward, despite lower plasma insulin concentrations, but reduced glucose uptake in the hypothalamus. Decreased RaO(0–60 min) after exenatide was inversely correlated to CMR(glu). In conclusion, these results demonstrate, for the first time in man, a major effect of a GLP-1RA on regulation of brain glucose metabolism in the absorptive state.

Aims To investigate the effect of exenatide on glucose disposal, insulin secretion, s-cell function, lipolysis, and hormone concentrations in non-diabetic, morbidly obese subjects under physiological conditions. Materials and methods Patients were assigned to exenatide 10 µg twice daily (EXE, n = 15) or control (CT, n = 15) for 3 months. Patients received a meal test/tracer study (MTT) to measure endogenous glucose production (EGP), rate of oral glucose appearance (RaO), insulin secretion rate (ISR), s-cell function, hepatic (HIR) and adipose tissue insulin resistance (AT-IR) and insulin sensitivity (IS). Results Post-treatment the EXE group showed a significant reduction in body weight (p

Glucagon-like peptide 1 (GLP-1) receptor agonists (GLP-1-RAs) act on multiple tissues, in addition to the pancreas. Recent studies suggest that GLP-1-RAs act on liver and adipose tissue to reduce insulin resistance (IR). Thus, we evaluated the acute effects of exenatide (EX) on hepatic (Hep-IR) and adipose (Adipo-IR) insulin resistance and glucose uptake. Fifteen male subjects (age = 56 ± 8 years; body mass index = 29 ± 1 kg/m2 ; A1c = 5.7 ± 0.1%) were studied on two occasions, with a double-blind subcutaneous injection of EX (5 μg) or placebo (PLC) 30 minutes before a 75-g oral glucose tolerance test (OGTT). During OGTT, we measured hepatic (HGU) and adipose tissue (ATGU) glucose uptake with [18 F]2-fluoro-2-deoxy-D-glucose/positron emission tomography, lipolysis (RaGly) with [U-2 H5 ]-glycerol, oral glucose absorption (RaO) with [U-13 C6 ]-glucose, and hepatic glucose production (EGP) with [6,6-2 H2 ]-glucose. Adipo-IR and Hep-IR were calculated as (FFA0-120min ) × (Ins0-120min ) and (EGP0-120min ) × (Ins0-120min ), respectively. EX reduced RaO, resulting in reduced plasma glucose and insulin concentration from 0 to 120 minutes postglucose ingestion. EX decreased Hep-IR (197 ± 28 to 130 ± 37; P = 0.02) and increased HGU of orally administered glucose (23 ± 4 to 232 ± 89 [μmol/min/L]/[μmol/min/kg]; P = 0.003) despite lower insulin (23 ± 5 vs. 41 ± 5 mU/L; P < 0.02). EX enhanced insulin suppression of RaGly by decreasing Adipo-IR (23 ± 4 to 13 ± 3; P = 0.009). No significant effect of insulin was observed on ATGU (EX = 1.16 ± 0.15 vs. PLC = 1.36 ± 0.13 [μmol/min/L]/[μmol/min/kg]). CONCLUSION Acute EX administration (1) improves Hep-IR, decreases EGP, and enhances HGU and (2) reduces Adipo-IR, improves the antilipolytic effect of insulin, and reduces plasma free fatty acid levels during OGTT. (Hepatology 2016;64:2028-2037).

Diabet. Med. 29, 622–627 (2012) Abstract Objective Previous studies have highlighted the associations between abdominal, cardiac or total fat accumulation and cardiovascular disease. The aim of this study was to investigate the impact of different ectopic fat depots on measurements of metabolic dysfunction and cardiovascular disease risk. Methods Using magnetic resonance imaging in 113 subjects, we measured abdominal (visceral and subcutaneous) and cardiac (epicardial and extra-pericardial) fat depots and examined their association with overall (BMI) and abdominal obesity (waist circumference), dyslipidaemia (triglycerides, total and HDL cholesterol), glucose tolerance (by an oral glucose tolerance test) and insulin sensitivity, blood pressure and 10-year coronary heart disease risk by Framingham score. Results Fat accumulation was proportional to the degree of obesity, with body fat ranging from 14 to 33 kg, visceral fat from 0.8 to 1.8 kg and cardiac fat from 134 to 236 g. Most cardiac fat (70% on average) was extra-pericardial, with a wide variability for both cardiac depots (epicardial: 172–2008 mm2; extra-pericardial: 100–5056 mm2). Only visceral and extra-pericardial fat, but not epicardial or subcutaneous fat, could discriminate between subjects with three or more factors of the metabolic syndrome or medium-to-high coronary heart disease risk score. Controlling for gender and BMI by multivariable analysis, the best marker of reduced insulin sensitivity was visceral fat (partial r = −0.35); extra-pericardial fat was the closest associate of increased blood pressure (partial r = 0.26) and both extra-pericardial and visceral fat clustered with hypertriglyceridaemia (partial r = 0.29 and 0.24; both P

The polymorphism, KLF6-IVS1-27A, in the Kruppel-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)

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.

AIMS/HYPOTHESIS: Preferential visceral adipose tissue (VAT) accumulation has been clearly associated with insulin resistance. In contrast, the impact of visceral obesity on beta cell function is controversial. METHODS: In 62 non-diabetic women and men (age 24-69 years, BMI 21-39 kg/m2), we measured VAT and subcutaneous adipose tissue (SAT) fat mass by magnetic resonance imaging. We also measured insulin secretion and beta cell function by C-peptide deconvolution and physiological modelling of data from a frequently sampled, 75-g, 3-h OGTT, respectively. RESULTS: VAT (range 0.1-3.1 kg) was strongly related to sex, age and BMI; SAT was related to sex and BMI. Controlling for sex, age, BMI and SAT by multivariate analysis, excess VAT was associated with a clinical phenotype comprising higher plasma glucose levels, BP, heart rate and serum transaminases. The corresponding metabolic phenotype consisted of insulin resistance (partial r=-0.38) and hyperinsulinaemia (partial r=0.29). The latter, however, was appropriate for the degree of insulin resistance regardless of obesity and abdominal fat distribution. Moreover, none of the model-derived parameters describing beta cell function (glucose sensitivity, rate sensitivity and potentiation) was independently associated with excess VAT. CONCLUSIONS/INTERPRETATION: In non-diabetic Caucasian adults of either sex, preferential visceral fat deposition in itself is part of an insulin-resistant phenotype. The insulin secretory response to a physiological challenge is increased to fully compensate for the insulin resistance, but the dynamics of beta cell function (glucose sensitivity, rate sensitivity and potentiation) are largely preserved.

The disposal of a mixed meal was examined in 11 male subjects by multiple (splanchnic and femoral) catheterization combined with double-isotope technique (intravenous [2-3H]glucose plus oral U-[14C]starch). Glucose kinetics and organ substrate balance were measured basally and for 5 h after eating pizza (600 kcal) containing carbohydrates 75 g as starch, proteins 37 g, and lipids 17 g. The portal appearance of ingested carbohydrate was maximal (1.0 mmol/min) between 30 and 60 min after the meal and gradually declined thereafter, but was still incomplete at 300 min (0.46+/-0.08 mmol/min). The total amount of glucose absorbed by the gut over the 5 h of the study was 247+/-26 mmol (45+/-6 g), corresponding to 60+/-6% of the ingested starch. Net splanchnic glucose balance (-6.7+/-0.5 micromol x kg(-1) x min(-1), basal) rose by 250-300% between 30 and 60 min and then returned to baseline. Hepatic glucose production (HGP) was suppressed slightly and only tardily in response to meal ingestion (approximately 30% between 120 and 300 min). Splanchnic glucose uptake (3.7+/-0.6 micromol x kg(-1) x min(-1), basal) peaked to 9.8+/-2.0 micromol x kg(-1) x min(-1) (P30% of the ingested proteins to the systemic circulation; and 5) after meal ingestion, skeletal muscle takes up BCAA to replenish muscle protein stores.

We tested whether acute α2-blockade affects insulin secretion, glucose and fat metabolism, thermogenesis, and hemodynamics in humans. During a 5-h epinephrine infusion (50 ng ⋅ min−1 ⋅ kg−1) in five volunteers, deriglidole, a selective α2-receptor inhibitor, led to a more sustained rise in plasma insulin and C-peptide levels (+59 ± 14 vs. +28 ± 6, and +273 ± 18 vs. +53 ± 14 pM, P < 0.01 vs. placebo) despite a smaller rise in plasma glucose (+0.90 ± 0.4 vs. +1.5 ± 0.3 mM, P < 0.01). Another 10 subjects were studied in the postabsorptive state and during a 4-h hyperglycemic (+4 mM) clamp, coupled with the ingestion of 75 g of glucose at 2 h. In the postabsorptive state, hepatic glucose production, resting energy expenditure, and plasma insulin, free fatty acid (FFA), and potassium concentrations were not affected by acute α2-blockade. Hyperglycemia elicited a biphasic rise in plasma insulin (to a peak of 140 ± 24 pM), C-peptide levels (1,520 ± 344 pM), and insulin secretion (to 410 ± 22 pmol/min); superimposed glucose ingestion elicited a further twofold rise in insulin and C-peptide levels, and insulin secretion. However, α2-blockade failed to change these secretory responses. Fasting blood β-hydroxybutyrate and glycerol and plasma FFA and potassium concentrations all declined with hyperglycemia; time course and extent of these changes were not affected by α2-blockade. Resting energy expenditure (+25 vs. +16%, P < 0.01) and external cardiac work (+28% vs. +19%, P < 0.01) showed larger increments after α2-blockade. We conclude that acute α2-blockade in humans 1) prevents epinephrine-induced inhibition of insulin secretion, 2) does not potentiate basal or intravenous- or oral glucose-induced insulin release, 3) enhances thermogenesis, and 4) increases cardiac work.

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.

Abstract 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 mmol ·L −1 · 2 h −1 , P −1 · 2 h −1 , P −1 · kg −1 , P −1 ·kg −1 , P r =.51, P P

Although hyperuricemia is a frequent finding in insulin-resistant states, insulin's effect on renal uric acid (UA) handling is not known. In 20 healthy volunteers, diastolic blood pressure, body weight, and fasting plasma insulin were positively (and age was negatively) related to fasting plasma UA concentrations, together accounting for 53% of their variability. During an insulin clamp, urine flow was lower than during fasting conditions (1.01 +/- 0.12 vs. 1.56 +/- 0.32 ml/min, P = 0.04), whereas creatinine clearance was unchanged (129 +/- 7 and 131 +/- 9 ml/min, P = not significant). Hyperinsulinemia did not alter serum UA concentrations (303 +/- 13 vs. 304 +/- 12 microM) but caused a significant decrease in urinary UA excretion [whether expressed as absolute excretion rate (1.66 +/- 0.21 vs. 2.12 +/- 0.23 mumol/min, P = 0.03), clearance rate (5.6 +/- 0.8 vs. 7.3 +/- 0.8 ml/min, P = 0.03), or fractional excretion (4.48 +/- 0.80 ml/min vs. 6.06 +/- 0.64%, P < 0.03)]. Hyperinsulinemia was also associated with a 30% (P < 0.001) fall in urine Na excretion. Fractional UA excretion was related to Na fractional excretion under basal conditions (r = 0.59, P < 0.01) and during the insulin period (r = 0.53, P < 0.02). Furthermore, the insulin-induced changes in fractional UA and Na excretion correlated with one another (r = 0.66, P < 0.001). Physiological hyperinsulinemia acutely reduces urinary UA and Na excretion in a coupled fashion.

Enhanced release of triglycerides (TG) by the liver is implicated in the pathogenesis of the metabolic syndrome. The aim of the study was to evaluate whether a primary elevation in hepatic glucose utilization (HGU), as induced by an acute rise in circulating glucose values during physiological hyperinsulinemia, promotes TG synthesis in spite of the reduction in free fatty acids (FFA) levels.Glucose dose-response studies were conducted in anesthetized pigs using positron emission tomography (PET) to quantify HGU during fasting euglycemic conditions (EF), and under two-step hyperglycemic hyperinsulinemia (1st-HH +3.0, 2nd-HH +6.0 mmol/L over EF glucose values). Liver biopsies were obtained in three animals to evaluate the relationship between glucose exposure and hepatic fat content.Plasma glucose levels were progressively increased in the two-step studies, and otherwise stable within every hour of PET scanning. HGU increased almost fivefold with raising glucose levels, from 0.033 ± 0.009 in EF to 0.149 ± 0.043 in 1st-HH, p = 0.02, and to 0.138 ± 0.050 μmol/min/g in 2nd-HH, p = 0.03. Circulating TG concentrations increased by 50 and 100% in the two hyperglycemic conditions (p = 0.03 2nd-HH vs. EF), in spite of a 70% suppression in plasma FFA levels. The hepatic TG content paralleled the glucose loads. Plasma γ-glutamyl transferase (γ-GT) was increased by 17% (p0.05).A short-term elevation in circulating glucose levels within the physiological postprandial range was sufficient to increase HGU, resulting in a significant synthesis and release of TG by the liver, which was accompanied by an acute rise in γ-GT and liver fat content.

P840 ALTERATION IN LIPID METABOLISM AFTER AN ORAL FAT LOAD IN PATIENTS WITH NAFLD L. Mezzabotta, E. Vanni, C. Rosso, M. Gaggini, R. Gambino, E. Buzzigoli, C. Saponaro, D. Ciociaro, M.L. Abate, F. Saba, F. Salomone, G.P. Caviglia, S. Carenzi, A. Smedile, M. Rizzetto, M. Cassader, A. Gastaldelli, E. Bugianesi. Medical Sciences, University of Turin, Turin, Cardiometabolic Risk Unit, Institute of Clinical Physiology – CNR, Pisa, U.O.C. of Gastroenterology, Azienda Sanitaria Provinciale di Catania, Catania, Italy E-mail: laviniaemme@hotmail.com

Pioglitazone has been shown to reduce fasting triglyceride levels. The mechanisms of this effect have not been fully elucidated, but decreased lipolysis may contribute to blunt the hypertriglyceridemic response to a meal. To test this hypothesis, we studied 27 type 2 diabetes mellitus (T2DM) patients and 7 sex-, age-, and body mass index-matched nondiabetic controls. Patients were randomized to pioglitazone (45 mg/day) or placebo for 16 wk. Whole body lipolysis was measured [as the [2H5]glycerol rate of appearance (Ra)] in the fasting state and for 6 h following a mixed meal. Compared with controls, T2DM had higher postprandial profiles of plasma triglycerides, free fatty acid (FFA), and β-hydroxybutyrate, and a decreased suppression of glycerol Ra( P < 0.04) despite higher insulin levels [268 (156) vs. 190 (123) pmol/l, median (interquartile range)]. Following pioglitazone, triglycerides and FFA were reduced ( P = 0.05 and P < 0.04, respectively), and glycerol Rawas more suppressed [−40 (137) vs. +7 (202) μmol/min of placebo, P < 0.05] despite a greater fall in insulin [−85 (176) vs. −20 (58) pmol/l, P = 0.05]. We conclude that, in well-controlled T2DM patients, whole body lipolysis is insulin resistant, and pioglitazone improves the insulin sensitivity of lipolysis.

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-(2)H(2)]glucose infusion, (2)H(2)O and [6-(3)H]glucose ingestion) to measure endogenous glucose production (EGP), gluconeogenesis (GNG), insulin-mediated glucose clearance and beta-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) kg(ffm)(-1) pM, P=0.03) because of enhanced GNG (73.1+/-2.4 vs 59.5+/-3.6%, P

Under resting conditions, the failing heart shifts fuel use toward greater glucose and lower free fatty acid (FFA) oxidation. We hypothesized that chronic metabolic abnormalities in patients with dilated cardiomyopathy (DCM) are associated with the absence of the normal increase in myocardial glucose uptake and maintenance of cardiac mechanical efficiency in response to pacing stress. In 10 DCM patients and 6 control subjects, we measured coronary flow by intravascular ultrasonometry and sampled arterial and coronary sinus blood. Myocardial metabolism was determined at baseline, during atrial pacing at 130 beats/min, and at 15 min of recovery by infusion of [3H]oleate and [13C]lactate and measurement of transmyocardial arteriovenous differences of oxygen and metabolites. At baseline, DCM patients showed depressed coronary flow, reduced uptake and oxidation of FFA, and preferential utilization of carbohydrates. During pacing, glucose uptake increased by 106% in control subjects but did not change from baseline in DCM patients. Lactate release increased by 122% in DCM patients but not in control subjects. Cardiac mechanical efficiency in DCM patients was not different compared with control subjects at baseline but was 34% lower during stress. Fatty acid uptake and oxidation did not change with pacing in either group. Our results show that in DCM there is preferential utilization of carbohydrates, which is associated with reduced flow and oxygen consumption at rest and an impaired ability to increase glucose uptake during stress. These metabolic abnormalities might contribute to progressive cardiac deterioration and represent a target for therapeutic strategies aimed at modulating cardiac substrate utilization.

Diabetic hyperglycemia results from insulin resistance of peripheral tissues and glucose overproduction due to increased gluconeogenesis (GNG). Thiazolidinediones have been shown to improve glycemic control and increase peripheral insulin sensitivity. Whether chronic thiazolidinedione treatment is associated with a decrease in GNG has not been determined.We studied 26 diet-treated type 2 diabetic patients randomly assigned to rosiglitazone (RSG; 8 mg/d; n = 13) or placebo (n = 13) for 12 wk. At baseline and 12 wk, we measured endogenous glucose production (by [3H]glucose infusion) and GNG (by the [2H]2O technique) after a 15-h fast. Peripheral insulin sensitivity was evaluated by a two-step (240 and 960 pmol/min/m(-2)) euglycemic insulin clamp.Compared with placebo, RSG reduced fasting plasma glucose (9.7 +/- 0.7 to 7.4 +/- 0.3 mmol/liter; P0.001), fasting fractional GNG (-15 +/- 4%; P = 0.002), and fasting GNG flux (-3.9 +/- 1.2 micromol/min/kg fat-free mass; P = 0.004), with no effect on glycogenolytic flux. Changes in GNG flux and fasting glucose were tightly correlated (r = 0.83; P0.0001). During both clamp steps, RSG enhanced insulin-mediated glucose clearance (by 26% and 31%; P = 0.01 and P0.02, respectively). In a subgroup of patients studied with magnetic resonance imaging, the reduction in GNG flux was correlated (r = 0.65; P0.02) with the reduction in visceral fat area.RSG increases peripheral tissue insulin sensitivity and decreases endogenous glucose release via an inhibition of gluconeogenesis.

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 cm 2 , P =0.005). Insulin sensitivity was reduced (10.7±0.7 versus 12.9±0.4 mL · min −1 · kg ffm −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

Single-drug treatment of essential hypertension (HT) is often insufficient to normalize blood pressure (BP), and high doses of antihypertensive agents can have adverse effects on glucose tolerance (GT) and insulin sensitivity. This study tested whether aggressive BP lowering with combination treatment had any influence on GT or insulin action. In all, 29 nonobese (body mass index [BMI]

P840 ALTERATION IN LIPID METABOLISM AFTER AN ORAL FAT LOAD IN PATIENTS WITH NAFLD L. Mezzabotta, E. Vanni, C. Rosso, M. Gaggini, R. Gambino, E. Buzzigoli, C. Saponaro, D. Ciociaro, M.L. Abate, F. Saba, F. Salomone, G.P. Caviglia, S. Carenzi, A. Smedile, M. Rizzetto, M. Cassader, A. Gastaldelli, E. Bugianesi. Medical Sciences, University of Turin, Turin, Cardiometabolic Risk Unit, Institute of Clinical Physiology – CNR, Pisa, U.O.C. of Gastroenterology, Azienda Sanitaria Provinciale di Catania, Catania, Italy E-mail: laviniaemme@hotmail.com

Metabolic and hemodynamic abnormalities have been separately described in obesity, and weight reduction is known to lead to some improvement in each. Our aim was to simultaneously assess metabolic and cardiovascular function in normotensive, normotolerant patients with moderate obesity (body mass index = 32.6 +/- 1.1 kg/m2) before and after weight loss. The obese were insulin resistant [37.4 +/- 4.8 mumol/min.kg FFM; P0.02 vs. 12 lean controls (50.6 +/- 2.6), on a euglycemic insulin clamp], secreted more insulin both in the fasting state and after oral glucose (70 +/- 10 vs. 48 +/- 6 nmol/mmol.L plasma glucose; P0.05), and had higher resting energy expenditure (4.62 +/- 0.18 vs. 4.00 +/- 0.23 kJ/min), systolic and mean blood pressure, stroke volume (87 +/- 8 vs. 67 +/- 4 mL/min; P = 0.05), and cardiac output. There was, however, no relationship between the metabolic and hemodynamic abnormalities. After a weight loss of 11 +/- 1 kg (approximately 15%), insulin sensitivity improved in proportion to the weight reduction, whereas insulin hypersecretion and high energy expenditure persisted. In contrast, all hemodynamic changes reverted to normal. We conclude that in moderate obesity, the metabolic and cardiovascular abnormalities are largely independent of one another; accordingly, weight loss affects them differentially. Partial weight normalization may provide sufficient cardiovascular protection.

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.

OBJECTIVES To test whether cardiological syndrome X is an insulin-resistant state. SETTING, DESIGN AND SUBJECTS: The coronary care unit of a referral centre for angina pectoris in Pisa, Italy. A case-control study, involving 10 patients with unequivocal (angiographycally proven) cardiological syndrome X, but normal glucose tolerance, blood pressure and lipid levels, and 13 matched healthy subjects. MAIN OUTCOME MEASURES Insulin sensitivity and pattern of substrate oxidation (assessed by the euglycaemic insulin clamp technique in combination with indirect calorimetry). RESULTS Fasting plasma glucose and insulin levels were 5.05 +/- 0.11 versus 4.88 +/- 0.11 mmol l-1 and 68 +/- 10 versus 56 +/- 6 pmol l-1, respectively (controls versus patients, ns). During the insulin clamp, glucose disposal rate was nearly identical in patients and controls (25.9 +/- 1.8 and 27.2 +/- 1.8 mumol kg-1 min-1, respectively. P = 0.88). Non-oxidative glucose disposal accounted for similar proportions of total glucose uptake (59 versus 53%, patients versus controls, ns). Resting energy expenditure (13.7 +/- 0.6 versus 13.8 +/- 0.8 cal kg-1 min-1, ns) and insulin-induced thermogenesis were similar in the two groups. Fasting plasma NEFA concentrations (0.64 +/- 0.09 and 0.64 +/- 0.06 mmol l-1, patients and controls, ns) fell in a similar time-course and to virtually identical nadirs (0.13 +/- 0.02 and 0.14 +/- 0.02 mmol l-1) after insulin infusion. Fasting plasma potassium was similar in patients and controls (3.99 +/- 0.10 and 4.16 +/- 0.04 mmol l-1, ns), and insulin induced equivalent hypokalaemia (-14 versus -19%). CONCLUSIONS None of the in vivo actions of insulin were impaired in patients with 'pure' syndrome X when compared to matched controls. Therefore, we conclude that cardiological syndrome X is not an insulin resistant state per se, and that any decrease in insulin sensitivity found in this condition is likely to be secondary.

To test whether carnitine availability is rate-limiting for fat oxidation under conditions of augmented oxidative use of fatty substrates, two series of studies were performed. In study no. 1, L-carnitine (1 g + 0.5 g/h intravenously [i.v.]) or saline was given to eight volunteers during a 4-hour infusion of a 10% triglyceride emulsion, thereby increasing plasma free-carnitine levels from 38 +/- 4 to 415 +/- 55 mumol/L. Fat infusion increased plasma triglyceride levels (80%) and lipid oxidation (30%), and decreased (28%) carbohydrate oxidation (as measured by indirect calorimetry); hypercarnitinemia had no influence on these responses. In study no. 2 in 12 healthy subjects a bolus of L-carnitine (3 g) or saline was administered 40 minutes before aerobic exercise (bicycling for 40 minutes at 60 W), followed by 2 minutes of anaerobic exercise (250 W) and 50 minutes of recovery. Oxygen consumption (VO2), increased to 18.3 +/- 0.7 mL.min-1 x kg-1 during aerobic exercise, reached a maximum of 46.0 +/- 0.8 mL.min-1 x kg-1 during the anaerobic bout, and returned to baseline within a few minutes, with no difference between control and carnitine. At virtually identical mean energy expenditure rates (196 +/- 7 v 197 +/- 7 J.min-1 x kg-1, saline v carnitine), after carnitine administration the entire exercise protocol was sustained by a lower mean carbohydrate oxidation rate (42.1 +/- 3.6 v 36.5 +/- 2.3 mumol.min-1 x kg-1, P < .03) and a higher mean lipid oxidation rate (6.7 +/- 1.0 v 8.3 +/- 0.7 mumol.min-1 x kg-1, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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)

Metabolic abnormalities occur in biguanide-treated diabetic patients. We investigated the relationship between plasma metformin and phenformin concentrations and metabolic effects. Drug levels were measured in 37 type II diabetic patients by HPLC. The method was sensitive, specific, and linear over a wide range of drug concentrations. Metformin and phenformin values ranged from 236 to 718 ng/ml and from 28 to 114 ng/ml, respectively. The plasma metformin level was correlated with triglycerides (r = -0.55; P less than 0.05) but not with drug dosage, plasma glucose, HbA1, creatinine, creatinine clearance, lactate, pyruvate, lipid, and clinical parameters. Plasma phenformin concentrations correlated with lactate (r = 0.49; P less than 0.05) and HbA1 (r = 0.50; P less than 0.05) but not with drug dosage, parameters of diabetes control, creatinine, creatinine clearance, pyruvate, and clinical parameters. The clinical usefulness of this HPLC method, the evidence that the increase of lactate is related to the circulating phenformin levels, and the demonstration that the metformin effect on triglyceride metabolism is correlated to plasma drug levels are the positive findings of this work.