E. Buzzigoli

Non-Alcoholic Fatty Liver Disease (NAFLD) and Its Connection with Insulin Resistance, Dyslipidemia, Atherosclerosis and Coronary Heart Disease

Non-alcoholic fatty liver disease is marked by hepatic fat accumulation not due to alcohol abuse. Several studies have demonstrated that NAFLD is associated with insulin resistance leading to a resistance in the antilipolytic effect of insulin in the adipose tissue with an increase of free fatty acids (FFAs). The increase of FFAs induces mitochondrial dysfunction and development of lipotoxicity. Moreover, in subjects with NAFLD, ectopic fat also accumulates as cardiac and pancreatic fat. In this review we analyzed the mechanisms that relate NAFLD with metabolic syndrome and dyslipidemia and its association with the development and progression of cardiovascular disease.

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.

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

Diabet. Med. 29, 622–627 (2012)

The Effect of Pioglitazone on the Liver Role of adiponectin

OBJECTIVE—Diabetic hyperglycemia results from insulin resistance of peripheral tissues and glucose overproduction due to increased gluconeogenesis (GNG). Thiazolidinediones (TZDs) improve peripheral insulin sensitivity, but the effect on the liver is less clear. The goal of this study was to examine the effect of TZDs on GNG. RESEARCH DESIGN AND METHODS—Twenty sulfonylurea-treated type 2 diabetic subjects were randomly assigned (double-blind study) to receive pioglitazone (PIO group; 45 mg/day) or placebo (Plc group) for 4 months to assess endogenous glucose production (EGP) (3-3H-glucose infusion), GNG (D2O technique), and insulin sensitivity by two-step hyperinsulinemic-euglycemic clamp (240 and 960 pmol/min per m2). RESULTS—Fasting plasma glucose (FPG) (10.0 ± 0.8 to 7.7 ± 0.7 mmol/l) and HbA1c (9.0 ± 0.4 to 7.3 ± 0.6%) decreased in the PIO and increased in Plc group (P < 0.05 PIO vs. Plc). Insulin sensitivity increased ∼40% during high insulin clamp after pioglitazone (P < 0.01) and remained unchanged in the Plc group (P < 0.05 PIO vs. Plc). EGP did not change, while GNG decreased in the PIO group (9.6 ± 0.7 to 8.7 ± 0.6 μmol · min−1 · kgffm−1) and increased in the Plc group (8.0 ± 0.5 to 9.6 ± 0.8) (P < 0.05 PIO vs. Plc). Change in FPG correlated with change in GNG flux (r = 0.63, P < 0.003) and in insulin sensitivity (r = 0.59, P < 0.01). Plasma adiponectin increased after pioglitazone (P < 0.001) and correlated with ΔFPG (r = −0.54, P < 0.03), ΔGNG flux (r = −0.47, P < 0.05), and Δinsulin sensitivity (r = 0.65, P < 0.005). Plasma free fatty acids decreased after pioglitazone and correlated with ΔGNG flux (r = 0.54, P < 0.02). From stepwise regression analysis, the strongest determinant of change in FPG was change in GNG flux. CONCLUSIONS—Pioglitazone improves FPG, primarily by reducing GNG flux in type 2 diabetic subjects.

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.

Effects of adding exercise to a 16-week very low-calorie diet in obese, insulin-dependent type 2 diabetes mellitus patients.

CONTEXT Reduction of 50% excess body weight, using a very low-calorie diet (VLCD; 450 kcal/d) improves insulin sensitivity in obese type 2 diabetes mellitus patients. OBJECTIVE The objective of the study was to evaluate whether adding exercise to the VLCD has additional benefits. DESIGN This was a randomized intervention study. SETTING The study was conducted at a clinical research center in an academic medical center. SUBJECTS Twenty-seven obese [body mass index 37.2 ± 0.9 kg/m(2) (mean ± sem)] insulin-treated type 2 diabetes mellitus patients. INTERVENTION Patients followed a 16-wk VLCD. Thirteen of them simultaneously participated in an exercise program (E) consisting of 1-h, in-hospital training and four 30-min training sessions on a cycloergometer weekly. OUTCOME MEASURES Insulin resistance was measured by a hyperinsulinemic euglycemic clamp. Insulin signaling, mitochondrial DNA (mtDNA) content, and intramyocellular lipid content was measured in skeletal muscle biopsies. RESULTS Baseline characteristics were identical in both groups. Substantial weight loss occurred (-23.7 ± 1.7 kg VLCD-only vs. -27.2 ± 1.9 kg VLCD+E, P = NS within groups). The exercise group lost more fat mass. Insulin-stimulated glucose disposal increased similarly in both study groups [15.0 ± 0.9 to 39.2 ± 4.7 μmol/min(-1) · kg lean body mass (LBM(-1)) VLCD-only vs. 17.0 ± 1.0 to 37.5 ± 3.5 μmol/min(-1) · kg LBM(-1) in VLCD+E], as did phosphorylation of the phosphatidylinositol 3-kinase-protein kinase B/AKT insulin signaling pathway. In contrast, skeletal muscle mtDNA content increased only in the VLCD+E group (1211 ± 185 to 2288 ± 358, arbitrary units, P = 0.016 vs. 1397 ± 240 to 1196 ± 179, P = NS, VLCD-only group). Maximum aerobic capacity also only increased significantly in the VLCD+E group (+6.6 ± 1.7 ml/min(-1) · kg LBM(-1) vs. +0.7 ± 1.5 ml/min(-1) · kg LBM(-1) VLCD-only, P = 0.017). CONCLUSION Addition of exercise to a 16-wk VLCD induces more fat loss. Exercise augments maximum aerobic capacity and skeletal muscle mtDNA content. These changes are, however, not reflected in a higher insulin-stimulated glucose disposal rate.

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)

Peripheral insulin resistance predicts liver damage in nondiabetic subjects with nonalcoholic fatty liver disease

Surrogate indexes of insulin resistance and insulin sensitivity are widely used in nonalcoholic fatty liver disease (NAFLD), although they have never been validated in this population. We aimed to validate the available indexes in NAFLD subjects and to test their ability to predict liver damage also in comparison with the NAFLD fibrosis score. Surrogate indexes were validated by the tracer technique (6,6‐D2‐glucose and U‐13C‐glucose) in the basal state and during an oral glucose tolerance test. The best‐performing indexes were used in an independent cohort of 145 nondiabetic NAFLD subjects to identify liver damage (fibrosis and nonalcoholic steatohepatitis). In the validation NAFLD cohort, homeostasis model assessment of insulin resistance, insulin to glucose ratio, and insulin sensitivity index Stumvoll had the best association with hepatic insulin resistance, while peripheral insulin sensitivity was most significantly related to oral glucose insulin sensitivity index (OGIS), insulin sensitivity index Stumvoll, and metabolic clearance rate estimation without demographic parameters. In the independent cohort, only oral glucose tolerance test‐derived indexes were associated with liver damage and OGIS was the best predictor of significant (≥F2) fibrosis (odds ratio = 0.76, 95% confidence interval 0.61‐0.96, P = 0.0233) and of nonalcoholic steatohepatitis (odds ratio = 0.75, 95% confidence interval 0.63‐0.90, P = 0.0021). Both OGIS and NAFLD fibrosis score identified advanced (F3/F4) fibrosis, but OGIS predicted it better than NAFLD fibrosis score (odds ratio = 0.57, 95% confidence interval 0.45‐0.72, P < 0.001) and was also able to discriminate F2 from F3/F4 (P < 0.003). Conclusion: OGIS is associated with peripheral insulin sensitivity in NAFLD and inversely associated with an increased risk of significant/advanced liver damage in nondiabetic subjects with NAFLD. (Hepatology 2016;63:107–116)

Decreased whole body lipolysis as a mechanism of the lipid-lowering effect of pioglitazone in type 2 diabetic patients

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 [(2)H(5)]glycerol rate of appearance (R(a))] 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 beta-hydroxybutyrate, and a decreased suppression of glycerol R(a) (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 R(a) was more suppressed [-40 (137) vs. +7 (202) mumol/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.

Altered amino acid concentrations in NAFLD: Impact of obesity and insulin resistance

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).