Initial hyperinsulinemia and subsequent ß-cell dysfunction is associated with elevated palmitate levels.

BACKGROUND: The prevalence of obesity-related diabetes in childhood is increasing and circulating levels of nonesterified fatty acids may constitute a link. Here, the association between palmitate and insulin secretion was investigated in vivo and in vitro. METHODS: Obese and lean children and adolescents (n = 80) were included. Palmitate was measured at fasting; insulin and glucose during an oral glucose tolerance test (OGTT). Human islets were cultured for 0 to 7 d in presence of 0.5 mmol/l palmitate. Glucose-stimulated insulin secretion (GSIS), insulin content and apoptosis were measured. RESULTS: Obese subjects had fasting palmitate levels between 0.10 and 0.33 mmol/l, with higher average levels compared to lean subjects. While obese children with elevated palmitate (>0.20 mmol/l) had accentuated insulin levels during OGTT, obese adolescents with high palmitate had delayed first-phase insulin response. In human islets exposed to palmitate for 2 d GSIS was twofold enhanced, but after 7 d attenuated. Intracellular insulin content decreased time-dependently in islets cultured in the presence of palmitate and cleaved caspase 3 increased. CONCLUSION: The rapid accentuated and delayed insulin secretory responses observed in obese children and adolescents, respectively, with high palmitate levels may reflect changes in islet secretory activity and integrity induced by extended exposure to the fatty acid.

Free fatty acid determination in plasma by GC-MS after conversion to Weinreb amides.

Circulating free fatty acids (FFAs) play important physiological roles as contributing components in cellular structure as well as energy utilization. Elevated levels of circulating FFAs are associated with metabolic aberrations in humans. FFAs differ in chain length and saturation and may be altered in metabolically dysregulated conditions, such as type 2 diabetes mellitus. Potentially, alterations in circulating levels of specific FFAs could also be important in terms of prognostic value. Various methods have been used to analyze FFAs. In this study, a straightforward and accurate method for the determination of FFAs in plasma has been established and evaluated, through conversion of plasma FFAs into acid fluorides followed by conversion to Weinreb amides (dimethylamide). The method is mild, efficient, selective, and quantitative for FFAs, when analyzed with capillary gas chromatography tandem mass spectrometry. Standard curves were linear over the range of 1,000-20,000 ng/mL with a correlation coefficient (r(2)) of 0.998, and coefficient of variation of triplicate analysis was <10 %. The gas chromatography-mass spectrometry (GC-MS) technique was reproducible and repeatable, and recoveries were above 90 %. From the generated MS spectra, five specific FFAs were identified. An explicit interest was the quantification of palmitate (C16:0) and palmitoleate (C16:1), which have been connected with detrimental and positive effects on the insulin-producing beta cells, respectively. The results demonstrate the suitability of Weinreb amides for efficient and rapid isolation of FFAs in plasma, prior to quantitative GC-MS analysis. We suggest that the method can be used as a routine standardized way of quantifying FFAs.

Palmitate-induced impairments of ß-cell function are linked with generation of specific ceramide species via acylation of sphingosine.

Prolonged exposure to palmitate impairs ß-cell function and mass. One of the proposed mechanisms is alteration in ceramide (Cer) generation. In the present study, exposure to palmitate induced the level of palmitoyl transferase and Cer synthases, enzymes of the Cer de novo and salvage pathways, and doubled total Cer levels, which was associated with decreased insulin secretion and augmented apoptosis in MIN6 cells and human islets. By inhibiting enzymes of the pathways pharmacologically with myriocin (ISP-1) or fumonisin B1 or by small interfering RNA (siRNA), we showed that Cer(14:0), Cer(16:0), Cer(20:1), and Cer(24:0) species, generated by the salvage pathway, are linked to the harmful effect of palmitate on ß-cells. Oleate attenuates negative effects of palmitate on ß-cells. When oleate was included during culture of MIN6 cells with palmitate, the palmitate-induced up-regulation of the enzymes of the de novo and salvage pathways was prevented resulting in normalized levels of all Cer species except Cer(20:1). Our data suggest that enhanced Cer generation in response to elevated palmitate levels involves both de novo and salvage pathways. However, the negative effects of palmitate on ß-cells are attributed to generation of Cer species Cer(14:0), Cer(16:0), and Cer(24:0) via acylation of sphingosine.

Lipids and lipid oxidation with emphasis on cholesterol oxides in some Indian sweets available in London.

Ghee (clarified butter oil), a major ingredient in Indian sweets, is an important source of saturated fatty acids, cholesterol and cholesterol oxidation products (COP) that are considered risk factors for atherosclerosis. The high frequency of atherosclerotic complications reported among the Indian immigrants in England prompted determination of lipids and lipid oxidation status of a ghee sample and 15 Indian sweets available in London supermarkets. The fatty acid profile of the samples shows saturated fats (about 73%), mainly composed of myristic, palmitic and stearic acids, except in two samples. There were large variations in thio-barbituric acid reacting substance values (19-260 microg/100 g) and total COP (1.4-51.2 microg/g lipids) among the sweet samples. Regular consumption of some of these sweets can be a source of considerable amounts of saturated fatty acids, cholesterol and COP in the diet and may contribute to atherosclerosis.