Lipid accumulation impairs lysosomal acid lipase activity in hepatocytes: Evidence in NAFLD patients and cell cultures.

AIMS: It has been hypothesized that the activity of lysosomal acid lipase (LAL), a key enzyme involved in lipid metabolism, is involved in the NAFLD phenotype. To clarify the role of LAL in NAFLD, we studied 164 consecutive patients with biopsy-proven NAFLD and fat-loaded HepG2 cells. METHODS: LAL activity was measured (i) on dried blood spots (DBS) from NAFLD patients and dyslipidemic subjects without fatty liver and (ii) on liver biopsies from NAFLD patients. LAL activity and expression were evaluated in HepG2 cells cultured in the presence of free fatty acids (FAs), with or without a PPAR-alpha agonist. RESULTS: LAL activity was significantly reduced in patients with NAFLD compared to dyslipidemic subjects. LAL activity measured in liver biopsies from NAFLD patients was highly correlated to that measured on DBS and was independent of LAL expression in the liver. In a fully adjusted model, LAL activity on DBS was associated only with platelets and, when normalized by platelet count, it did not differ according to fibrosis stage. In vitro, FA loading of HepG2 fully replicated the impairment of LAL activity observed in NALFD patients. In these cells, the activation of PPAR-alpha receptors prevented and corrected FA-induced LAL impairment, by stimulating FA oxidation and LAL expression. CONCLUSIONS: LAL activity is reduced in NAFLD patients, independently from disease progression. In vitro, impaired LAL activity induced by FA loading was rescued by PPAR-alpha activation. These data suggest that the pharmacological modulation of LAL should be explored in the management of NAFLD patients.

Treatment with fibrates is associated with higher LAL activity in dyslipidemic patients.

Lysosomal acid lipase (LAL) is responsible for the hydrolysis of cholesteryl esters (CE) and triglycerides (TG) within the lysosomes; generated cholesterol and free fatty acids (FFA) are released in the cytosol where they can regulate their own synthesis and metabolism. When LAL is not active, as in case of genetic mutations, CE and TG accumulate in the lysosomal compartment, while the lack of release of cholesterol and FFA in the cytosol leads to an upregulation of their synthesis. Thus, LAL plays a central role in the intracellular homeostasis of lipids. Since there are no indications about the effect of different lipid-lowering agents on LAL activity, aim of the study was to address the relationship between LAL activity and the type of lipid-lowering therapy in a cohort of dyslipidemic patients. LAL activity was measured on dried blood spot from 120 patients with hypercholesterolemia or mixed dyslipidemia and was negatively correlated to LDL-cholesterol levels. Among enrolled patients, ninety-one were taking one or more lipid-lowering drugs, as statins, fibrates, ezetimibe and omega-3 polyunsaturated fatty acids. When patients were stratified according to the type of lipid-lowering treatment, i.e. untreated, taking statins or taking fibrates, LAL activity was significantly higher in those with fibrates, even after adjustment for sex, age, BMI, lipid parameters, liver function, metabolic syndrome, diabetes and statin use. In a subset of patients tested after 3 months of treatment with micronized fenofibrate, LAL activity raised by 21%; the increase was negatively correlated with baseline LAL activity. Thus, the use of fibrates is independently associated with higher LAL activity in dyslipidemic patients, suggesting that the positive effects of PPAR-α activation on cellular and systemic lipid homeostasis can also include an improved LAL activity.

Is thyroid-stimulating hormone within the normal reference range a risk factor for atherosclerosis in women?

The relationship between overt hypothyroidism and cardiovascular risk has been well documented and some data also suggest an association between cardiovascular risk and subclinical hypothyroidism. The aim of our study was to investigate, in a large cohort of euthyroid women, the association of thyroid stimulating hormone (TSH) within the normal reference range with cardiovascular risk factors. The study was carried out on 744 women with normal thyroid function (TSH 0.3-4.9 µU/mL). Women with TSH above the median (≥2.1 µU/mL) were more obese, had greater waist girth, were more hypertensive and had higher levels of total cholesterol (TC), serum triglycerides (TG), blood sugar (BG) and lower levels of HDL-cholesterol (HDL-C) than women with TSH below the median. TSH was significantly correlated with body mass index (BMI), waist circumference, BG, TG, TC, HDL-C and hypertension. Multiple backward stepwise regression analysis with age, waist circumference and TSH as independent variables confirmed the strong association of TSH with BG, TG, HDL-C and hypertension. A total of 205 patients (28%) fulfilled the definition criteria of the metabolic syndrome and the prevalence of metabolic syndrome was significantly greater in patients with TSH above than in patients with TSH below the median. Results of logistic analysis, including age and TSH as predictor variables, confirmed the association of TSH with metabolic syndrome.The results of this study suggest that TSH in the upper limits of the reference range (above 2.1 µU/ml) is associated with a less favourable cardiometabolic profile and consequently with a higher risk of developing cardiovascular diseases.

Changes in serum lipids and blood glucose in non diabetic patients with metabolic syndrome after mixed meals of different composition.

Aims. To investigate the postprandial changes in serum lipoproteins and blood glucose and to verify whether different nutrient composition of the meal elicits different response in patients with (MetS+) and without (MetS-) metabolic syndrome. Research Design and Methods. 50 MetS+ patients and 50 age- and sex-matched MetS- consumed a regular lunch chosen among those more similar to their usual diet. Blood was drawn in the morning after 12-hour fasting and 2 and 4:30 hours after the meal. Results. Serum triglycerides increased more in MetS+ (35%, 4:30 hours after the meal) than in MetS- (29%), HDL-cholesterol decreased 2 hours after the meal in both groups (-4% and -5%, resp.). Blood sugar similarly increased in both groups (19%, 2 hours after the meal in MetS+ and 17% in MetS-) and plasma insulin increased more and remained high longer in MetS+ (73.5 and 52.3 µU/mL, 2 and 4:30 hours after the meal) than in MetS- (46.7 and 21.6 µU/mL). Difference in nutrient composition of the meal (carbohydrate 57%, fat 28% versus carbohydrate 45%, fat 35%) was not associated with differences in postprandial levels of triglycerides, HDL-cholesterol, glucose, and insulin within each group. Conclusions. As compared with MetS-, MetS+ patients show a greater hypertriglyceridemic and hyperinsulinemic response to a regular lunch whatever the carbohydrate or fat content of the meal.

Hypercoagulability in patients with type 2 diabetes mellitus detected by a thrombin generation assay.

Diabetes is a risk factor for the development of atherothrombosis and venous thromboembolism (VTE). We investigated whether plasma from patients with type 2 diabetes has an imbalance of pro- versus anti-coagulation resulting in hypercoagulability despite normal conventional coagulation tests. We analyzed blood samples from 60 patients with type 2 diabetes and 60 gender- and age-matched healthy subjects (controls) for the levels of pro- and anti-coagulant factors, for thrombin generation and for the numbers of cell-derived circulating microparticles bearing such pro-coagulant triggers as tissue factor and negatively charged phospholipids. The levels of pro- or anti-coagulants as measured with conventional coagulation tests or single factor measurements were similar to those of the control population. In contrast, the median (range) of the height of the thrombin peak (taken as an index of thrombin generation) was higher in patients [205 nM (126-352)] than controls [151 nM (41-289)], P < 0.001. The median numbers of circulating microparticles were higher for patients [5,041/µl (1,821-13,132)] than for controls [1,753/µl (554-13,308)], P < 0.001 and their values were correlated with the height of the thrombin peak (ρ = 0.66, P < 0.001). In conclusion, plasma from patients with type 2 diabetes possesses an imbalance of pro- versus anti-coagulation resulting in hypercoagulability that can be detected by thrombin generation tests, but not by the measurement of the single pro- or anti-coagulant factors. This hypercoagulability is associated with increased numbers of circulating microparticles bearing endogenous pro-coagulant triggers. These findings might explain the relatively high risk of atherothrombosis and VTE described in these patients.

Effects of moderate variations in the macronutrient content of the diet on cardiovascular disease risk factors in obese patients with the metabolic syndrome.

BACKGROUND: The metabolic syndrome is a cluster of abnormalities that is accompanied by a 2-fold increase in the risk of cardiovascular disease. Even if there is full agreement that lifestyle changes to induce weight loss are the first-line approach, the ideal diet for the treatment of the metabolic syndrome remains uncertain. OBJECTIVE: The objective was to compare the effects of 2 diets on cardiovascular disease risk factors in obese patients with the metabolic syndrome. DESIGN: The study was carried out in 100 patients randomly assigned to either a diet relatively rich in carbohydrate [65% of energy as carbohydrate, 13% as protein, and 22% as fat (17% as unsaturated fat)] or a diet that was low in carbohydrate and high in protein and in monounsaturated fat [48% of energy as carbohydrate, 19% as protein, and 33% as fat (24% as unsaturated fat)]. RESULTS: All 100 patients completed the 5-mo study. At the end of the study, all the components of the metabolic syndrome (except HDL, which did not change) decreased significantly in both groups. With the high-carbohydrate diet, a significant decrease in LDL-cholesterol concentrations was also observed. Although the extent of the resolution of the metabolic syndrome was not different between groups, the low-carbohydrate diet was associated with a greater decrease in the prevalence of hypertension (P < 0.05) and of hypertriacylglycerolemia (P < 0.001). CONCLUSION: Tailoring diet interventions to the specific presentation of the metabolic syndrome may be the best way of reducing the risk factors for cardiovascular disease.

Changes in serum triglycerides and high-density lipoprotein concentration and composition after a low-fat mixed meal. Effects of gender and insulin resistance.

OBJECTIVE: Postprandial lipemia is generally studied after a test meal that provides most of the calories as fat and that does not reflect the common food intake. We investigated postprandial changes in serum triglycerides (TG) and in high-density lipoprotein (HDL) concentration and composition after a regular meal poor in fat (30% of calories). METHODS: Fifty-four women and 54 men had breakfast at 8:00 a.m. (12% of daily calories) and lunch at 12:30 p.m. (53% of daily calories). RESULTS: With respect to fasting values, TG increased more in men (24% at 2:30 p.m. and 30% at 5:00 p.m.) than in women (19% and 23%, respectively). HDL cholesterol decreased by 4% both in men and women at 2:30 p.m., and in both genders levels returned towards baseline levels at 5:00 p.m. Apolipoprotein A-I (apo A-I) significantly decreased in men (-3% at 2:30 p.m.), but did not change in women. The apo A-I/HDL cholesterol ratio significantly increased by 3% in men at 2:30 p.m. and by 5% both in men and women at 5:00 p.m. Postprandial serum TG were higher and HDL cholesterol and apo A-I were lower in subjects of both genders with insulin resistance (high HOMA(IR)) than in those with low HOMA(IR). The greatest increase in serum TG (39%) was observed in men with high HOMA(IR). HDL cholesterol and apo A-I significantly decreased and the apo A-I/HDL-C ratio significantly increased only in this subgroup of subjects. CONCLUSIONS: Ingestion of low doses of fat in a mixed meal is followed by variable increases of serum TG, and the greatest response is found in insulin-resistant men. In this subset of subjects, postprandial hypertriglyceridaemia is associated with alterations in HDL that might be consistent with an increased risk of cardiovascular disease.

Effects of simvastatin on blood pressure in hypercholesterolemic patients: An open-label study in patients with hypertension or normotension.

BACKGROUND: Simvastatin has been reported to improve endotheliumdependent vascular relaxation in patients with hypercholesterolemia. The consequent decrease in arterial stiffness might be associated with a decrease in blood pressure (BP). OBJECTIVE: The aim of this study was to determine whether simvastatin 20 and 40 mg/d have an effect on systolic and diastolic blood pressure (SBP and DBP, respectively) in patients with hypercholesterolemia, and, if so, whether the effect is dose dependent and/or is related to the changes in the serum lipid profile. METHODS: This 6-month, open-label study was conducted at the Lipid Clinics of the Department of Internal Medicine, University of Milan, Maggiore Hospital IRCCS, and of the Department of Internal Medicine 1, G. Salvini Hospital, Garbagnate Milanese (Milan, Italy). Patients aged 18 to 80 years with primary hypercholesterolemia who were following a low-fat, low-cholesterol diet for >2 months before the study were enrolled. Patients at high risk for cardiovascular disease (CVD), according to the National Cholesterol Education Program Adult Treatment Panel II guidelines, were given simvastatin 20 mg (tablet) QD for 3 months, and those at low risk for CVD continued with diet only for 3 months (controls). Efficacy variables included body weight, SBP, DBP, and serum lipid levels (total cholesterol [TC], low-density lipoprotein cholesterol [LDL-C], high density lipoprotein cholesterol [HDL-C], and triglycerides [TG]). At 3 months, patients in the simvastatin + diet group who reached their therapeutic goal continued to receive simvastatin 20 mg/d for 3 additional months. In simvastatintreated patients who were normotensive at baseline or who became normotensive at 3 months but who did not reach the therapeutic goal, the simvastatin dosage was increased to 40 mg/d. Patients in both groups who remained hypertensive at 3 months were switched to hypotensive therapy. In the diet-only group, patients who were formerly normotensive or who became normotensive at 3 months but who did not reach their therapeutic goal continued with diet only or started lipid-lowering therapy. All other patients in the diet-only group continued to be treated with diet only, for 3 additional months. Efficacy variables were measured again at 6 months. Tolerability of simvastatin was assessed at each visit using patient interview and measurement of serum aminotransferase and creatine phosphokinase levels. RESULTS: The study population comprised 222 patients (132 women, 90 men; mean [SEM] age, 53.9 [0.95] years [range, 23-76 years]); 115 high-risk patients (57 with untreated stage 1 hypertension) were assigned to the simvastatin + diet group, and 107 low-risk patients (29 with untreated stage 1 hypertension) were assigned to the diet-only group. In the simvastatin group, after 3 months of therapy, mean SBP was decreased by 3.9 (1.49) mm Hg (change, -2.9%), mean DBP decreased by 3.0 (0.87) mm Hg (change, -3.7%), mean TC decreased by 90.6 (3.98) mg/dL (change, -27.0%), mean LDL-C decreased by 88.9 (3.88) mg/dL (change, -35.6%), and mean TG decreased by 26.3 (7.34) mg/dL (change, -15.8%) (all, P < 0.001). Mean HDL-C increased by 3.6 (1.16) mg/dL (change, 6.9%; P < 0.001). The BP-lowering effect was found only in patients with hypertension at baseline (n = 57); in these patients, mean SBP decreased by 7.2 (2.44) mm Hg (change, -4.8%; P < 0.005 vs baseline) and DBP decreased by 4.8 (1.29) mm Hg (change, -5.6%; P < 0.001 vs baseline). Also in the simvastatin group, 26 patients (22.6%) achieved their target SBP/DBP. In patients with normotension at baseline (n = 58), neither SBP nor DBP was changed significantly (changes, -0.8 [1.65] and -1.4 [1.15] mm Hg, respectively [-0.6% and -1.8%, respectively]). The changes in serum lipid levels were similar between hypertensive and normotensive patients in the simvastatin group. Forty-one patients (18 hypertensive and 23 normotensive at baseline) were treated with simvastatin 40 mg/d plus diet between months 3 and 6. At 6 months, no further significant decrease was observed in mean BP. In contrast, the expected dose-dependent response was observed for TC and LDL-C levels. In the diet-only group, no significant changes occurred in BP or serum lipid levels. Changes in BP, TC, LDL-C, TG, and HDL-C were significantly greater in the simvastatin + diet group than in the diet-only group (all, P < 0.001). Body weight did not change significantly in either group. CONCLUSIONS: In this group of patients with hypercholesterolemia, the starting dosage of simvastatin (20 mg/d) was associated with reductions in SBP and DBP within 3 months of treatment in patients with hypertension, and this effect was independent of the lipid-lowering properties of the drug. Although the decrease in BP was modest, it is likely clinically relevant. Further studies on this topic are advisable.

Tumor necrosis factor alpha promoter polymorphisms and insulin resistance in nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease, which can range from fatty liver alone to nonalcoholic steatohepatitis and cirrhosis, is related to insulin resistance. Tumor necrosis factor alpha (TNF-alpha) may induce insulin resistance, and polymorphisms of its promoter have been associated with an increased release of this cytokine. We analyzed (1) the prevalence of insulin resistance, (2) the prevalence of the 238 and 308 TNF-alpha polymorphisms, and (3) the relationship among TNF-alpha polymorphisms, insulin resistance, and the occurrence of steatohepatitis in 99 patients with nonalcoholic fatty liver diagnosed by ultrasonography and confirmed by histologic analysis in the 53 who underwent biopsy. METHODS: Insulin resistance was evaluated by the homeostatic metabolic assessment insulin resistance indices and TNF-alpha polymorphisms by polymerase chain reaction and restriction fragment length polymorphism analysis. RESULTS: Insulin resistance was detected in almost all of the patients and was more severe in those with steatohepatitis. The prevalence of the 238, but not of the 308, TNF-alpha polymorphism was higher in subjects with nonalcoholic fatty liver than in controls (31% vs. 15%; P < 0.0001), and patients positive for TNF-alpha polymorphisms had higher insulin resistance indices, a higher prevalence of impaired glucose tolerance, and a lower number of associated risk factors for steatosis. CONCLUSIONS: TNF-alpha polymorphisms could represent a susceptibility genotype for insulin resistance, nonalcoholic fatty liver, and steatohepatitis.