GENDER-RELATED ALTERATIONS IN FREE FATTY ACIDS AND OXIDATIVE STRESS IN HYPERTENSION CO-MORBIDLY OCCURRING WITH TYPE 2 DIABETES MELLITUS

Increase in plasma free fatty acids (FFAs) concentrations may cause cellular damage via the induction of oxidative stress. The aim of this present study was to investigate FFAs and oxidative stress in hypertension co-morbidly occurring with Type 2 Diabetes Mellitus (T2DM). Age and sex matched control subjects (n=150) and patients (n=470) [hypertensive nondiabetics (HND, n=179), normotensive diabetics (ND, n=132), hypertensive diabetics (HD, n=159)] presenting at the Medical Out-Patient Clinic of the State Hospital, Abeokuta, Nigeria were recruited. Fasting plasma glucose, creatinine, urea, FFAs, thiobarbituric acid reactive substances (TBARS) were determined spectrophotometrically. The presence of either or both diseases resulted in significant increase (p<0.05) in the plasma FFAs and oxidative stress marker-TBARS in different compartments (plasma, erythrocytes andlipoproteins) for both male and female patients when compared with their control counterparts. The increase in FFAs was more marked in comorbidity female when compared with other female patients. There was significant (p<0.05) difference in gender FFAs concentrations. In both controls and patients, FFAs in plasma are significantly (p<0.05) higher in male when compared with their female counterparts. This research revealed biochemical variations in hypertension co-morbidly occurring with T2DMcharacterised by gender-related elevation in FFAs and enhanced oxidative stress. Plasma FFAs might be a good biomarker predicting the occurrence and development of hypertension and/or T2DM.

Abstract 024: Plasma Free Fatty Acids and Risk of Dementia: The Cardiovascular Health Study

Background: Plasma free fatty acids (FFAs) are a byproduct of lipolysis largely derived from adipose tissue. High plasma FFA levels have toxic effects on a variety of organs central to cardiometabolic disease. Whether FFAs associates with cognitive decline or dementia remains unknown. Objective: To assess the association of plasma FFAs with risk of cognitive decline and dementia. Methods: Plasma FFAs were measured in participant samples from the 1992-1993 study visit of the Cardiovascular Health Study (CHS) (mean age 74 yrs; 59% female; 14% African American [AA]; 24% APOE4 carriers). A total of 3,242 non-demented participants were followed with adjudication for dementia through 1998-1999 (n=456 cases). In addition, cognitive decline was assessed annually among all CHS participants using the 100-point modified Mini-Mental State examination (3MSE; n=4,417) and the Digital Symbol Substitution Test (DSST; n=4,254). Results: Higher plasma FFAs levels were positively associated with risk of dementia (Figure: Log-rank p-value=0.04). In Cox regression analysis adjusted for demographics, FFAs remained associated with risk of dementia (HR per SD [0.20 mEq/L] =1.10; 95% CI, 1.00-1.22). In fully adjusted models that included lifestyle factors, medical history, C-reactive protein, LDL-C, HDL-C, and 3MSE at baseline, the hazard ratio per SD was 1.13 (1.01-1.26), with a particularly strong association observed among AA participants (HR=1.49 [1.10-2.01], p interaction=0.08). FFA levels did not differ by APOE genotype, and adjustment for genotype did not influence results. FFA levels from the baseline visit were also associated with a decline in both cognitive assessments over 6 years of follow-up, with significant time x FFA interactions (p=0.04 for 3MSE and p=0.002 for DSST). We observed no interactions with APOE genotype or race for cognitive outcomes. Conclusions: In non-demented older men and women, higher plasma FFA levels are associated with faster cognitive decline and higher risk of dementia over the subsequent years.

Short-term oral α-lipoic acid does not prevent lipid-induced dysregulation of glucose homeostasis in obese and overweight nondiabetic men

Prolonged elevation of plasma free fatty acids (FFAs) induces insulin resistance and impairs pancreatic β-cell adaptation to insulin resistance. The mechanisms whereby lipid induces these impairments are not fully defined but may involve oxidative stress, inflammation, and endoplasmic reticulum stress. α-Lipoic acid (ALA), a commonly used health supplement with antioxidant, anti-inflammatory, and AMPK-activating properties, has been shown to have therapeutic value in type 2 diabetes and its complications. Here we examined the effects of ALA on insulin sensitivity and secretion in humans under the conditions of 24-h iv lipid infusion to elevate plasma FFAs. Eight overweight and obese male subjects underwent four randomized studies each, 4–6 wk apart: 1) SAL, 2-wk oral placebo followed by 24-h iv infusion of saline; 2) IH, 2-wk placebo followed by 24-h iv infusion of intralipid plus heparin to raise plasma FFAs approximately twofold; 3) IH + ALA, 2-wk ALA (1,800 mg/day) followed by 24-h infusion of intralipid plus heparin; and 4) ALA, 2-wk ALA followed by 24-h infusion of saline. Insulin secretion rates (ISR) and insulin sensitivity were assessed with a 2-h, 20-mmol/l hyperglycemic clamp and a hyperinsulinemic euglycemic clamp, respectively. ISR was not significantly different between treatments. Lipid infusion impaired insulin sensitivity with and without ALA pretreatment. These results indicate that ALA, administered orally at this dose for 2 wk, does not protect against lipid-induced insulin resistance in overweight and obese humans.

Peroxisome Proliferator-Activated Receptor β/δ (PPARβ/δ) but Not PPARα Serves as a Plasma Free Fatty Acid Sensor in Liver

ABSTRACT Peroxisome proliferator-activated receptor α (PPARα) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous genes were commonly regulated in liver between the two treatments, including many classical PPARα target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARα, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARβ/δ during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARα. Binding of PPARβ/δ to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARβ/δ by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulant of adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expression levels via PPARβ/δ. In contrast, the data did not support activation of PPARα by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARβ/δ target genes and show that upregulation of gene expression by PPARβ/δ is sensitive to plasma FFA levels. In contrast, this is not the case for PPARα, revealing a novel mechanism for functional differentiation between PPARs.

Abstract 2834: A Short-Term High Fat Diet Impairs Cardiac High Energy Phosphate Metabolism, Exercise Capacity and Cognitive Function

Background: Heart failure patients have low cardiac phosphocreatine/ATP (PCr/ATP) ratios, abnormal exercise tolerance and impaired cognitive function, which may be related to elevated circulating free fatty acids (FFAs). We tested whether briefly raising plasma FFAs, using diet, causes abnormalities in heart, brain and skeletal muscle in healthy subjects Methods and Results: Healthy males (n = 16, age 22 ± 1 years), recruited from the University of Oxford, were randomised to five days of a high fat diet containing 75 ± 1% of calorie intake through fat consumption, or an isocaloric control diet, providing 23 ± 1% of calorie intake as fat. In a cross-over design, subjects undertook the alternate diet after a two week wash out period. Cardiac 31 P magnetic resonance (MR) spectroscopy and MR imaging, echocardiography, exhaustive cycling for 1 h, and CDR computerised cognitive tests were used to assess cardiac PCr/ATP, cardiac function, exercise capacity and cognitive function, respectively, before and after the diets. Subjects on the HFD had a two-fold elevation in plasma FFAs, 12% lower cardiac PCr/ATP with no change in cardiac function, and a 12% lower maximal exercise performance (see Figure ). They also had impaired attention and speed (2.2 vs. 1.9 s, p < 0.001, and 1.10 vs. 1.05 s, respectively, p < 0.01) Discussion: We have shown a short term, high fat diet raised plasma FFA concentrations, impaired myocardial energetics, exercise capacity and cognition. Therefore high plasma FFAs may be detrimental for heart, skeletal muscle and brain in normal subjects and suggests a potential mechanism of impairment in heart failure patients.

Short-Term Manipulation of Plasma Free Fatty Acids Does Not Change Skeletal Muscle Concentrations of Ceramide and Glucosylceramide in Lean and Overweight Subjects

Context: Increased plasma free fatty acid (FFA) concentrations may be in part responsible for the increased levels of ceramide in skeletal muscle of obese subjects. Objective: We studied the effect of lowering and increasing plasma FFA levels on muscle ceramide and glucosylceramide concentrations in lean and obese subjects. Design: Plasma FFAs were either increased or decreased for 6 h by infusing a lipid emulsion or using Acipimox, respectively. Muscle biopsies were performed before and after the intervention for measurements of ceramide and glucosylceramide. Study Subjects: Eight lean [body mass index 21.9 (range, 19.6–24.6) kg/m2] and six overweight/obese [body mass index 34.4 (27.8–42.5) kg/m2] subjects without type 2 diabetes mellitus participated in the study. Main Outcome Measure: Differences in muscle ceramide and glucosylceramide upon manipulation of plasma FFAs were measured. Results: There were no differences in muscle ceramide and glucosylceramide between lean and obese subjects, respectively. Increasing or decreasing plasma FFAs for 6 h had no effect on ceramide [high FFAs: 24 (19–25) vs. 24 (22–27) pmol/mg muscle, P = 0.46; and 22 (20–28) vs. 24 (18–26) pmol/mg muscle, P = 0.89 in lean and obese, respectively; low FFAs: 26 (24–35) vs. 23 (18–27) pmol/mg muscle, P = 0.17 and 24 (15–44) vs. 24 (19–42) pmol/mg muscle, P = 0.6 in lean and obese, respectively] and glucosylceramide [high FFAs: 2.0 (1.7–4.3) vs. 3.4 (2.1–4.6) pmol/mg muscle, P = 0.17; and 3.0 (1.3–6.7) vs. 2.6 (1.2–3.9) pmol/mg muscle, P = 0.89 in lean and obese, respectively; low FFAs: 2.2 (1.0–4.4) vs. 1.7 (1.4–3.0) pmol/mg muscle, P = 0.92; and 6.6 (1.0–25.0) vs. 4.3 (1.3–7.6) pmol/mg muscle, P = 0.7 in lean and obese, respectively] concentrations in skeletal muscle. Conclusion: Short-term manipulation of plasma FFAs has no effect on ceramide and glucosylceramide concentrations in skeletal muscle from lean and obese subjects.

Elevated plasma free fatty acids decrease basal protein synthesis, but not the anabolic effect of leucine, in skeletal muscle

Elevations in free fatty acids (FFAs) impair glucose uptake in skeletal muscle. However, there is no information pertaining to the effect of elevated circulating lipids on either basal protein synthesis or the anabolic effects of leucine and insulin-like growth factor I (IGF-I). In chronically catheterized conscious rats, the short-term elevation of plasma FFAs by the 5-h infusion of heparin plus Intralipid decreased muscle protein synthesis by ∼25% under basal conditions. Lipid infusion was associated with a redistribution of eukaryotic initiation factor (eIF)4E from the active eIF4E·eIF4G complex to the inactive eIF4E·4E-BP1 complex. This shift was associated with a decreased phosphorylation of eIF4G but not 4E-BP1. Lipid infusion did not significantly alter either the total amount or phosphorylation state of mTOR, TSC2, S6K1, or the ribosomal protein S6 under basal conditions. In control rats, oral leucine increased muscle protein synthesis. This anabolic response was not impaired by lipid infusion, and no defects in signal transduction pathways regulating translation initiation were detected. In separate rats that received a bolus injection of IGF-I, lipid infusion attenuated the normal redistribution of eIF4E from the active to inactive complex and largely prevented the increased phosphorylation of 4E-BP1, eIF4G, S6K1, and S6. This IGF-I resistance was associated with enhanced Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1). These data indicate that the short-term elevation of plasma FFAs impairs basal protein synthesis in muscle by altering eIF4E availability, and this defect may be related to impaired phosphorylation of eIF4G, not 4E-BP1. Moreover, hyperlipidemia impairs IGF-I action but does not produce leucine resistance in skeletal muscle.

Prevention of in Vitro Lipolysis by Tetrahydrolipstatin

Background: Metabolic effects of free fatty acids (FFAs) frequently are tested using combined infusion of triglycerides and heparin, which stimulates lipolysis in vivo. Ongoing in vitro lipolysis, however, probably produces falsely high plasma FFA concentrations under these conditions. Therefore, this study aims to assess the efficacy of tetrahydrolipstatin (THL) in inhibiting plasma lipolytic activity and to improve plasma FFA determination. Methods: Plasma concentrations of FFAs and glycerol were measured in five healthy subjects in the presence and absence of THL. Blood was drawn at baseline, during infusion of a triglyceride emulsion (1.5 mL/min), and during infusion of triglycerides plus heparin (0.2 IU · kg−1 · min−1). In addition, the effects of storage temperature of the samples were analyzed. Results: In samples frozen immediately after collection, plasma FFAs were 28% lower in the presence of THL than in its absence (P = 0.008). When THL-free plasma was incubated for 3 h on ice or at room temperature, plasma FFAs were 22% (P = 0.02) and 91% (P = 0.0004) higher, respectively, than in samples frozen immediately. The addition of THL blunted temperature-dependent in vitro lipolysis by 88% (P &lt;0.01) and 89% (P &lt;0.001) after incubation on ice and at room temperature, respectively. Changes in plasma glycerol concentrations exhibited similar behavior. Conclusions: THL, which is safe and easy to handle, is a potent inhibitor of in vitro lipolysis and could, therefore, be added to blood samples drawn during triglyceride/heparin infusions to allow more accurate determination of plasma FFA concentrations.