Markers of de novo lipogenesis in adipose tissue: associations with small adipocytes and insulin sensitivity in humans.

AIMS/HYPOTHESIS: Previous studies have shown relationships between fatty acid ratios in adipose tissue triacylglycerol (TG), adipocyte size and measures of insulin sensitivity. We hypothesised that variations in adipose tissue de novo lipogenesis (DNL) in relation to adiposity might explain some of these observations. METHODS: In a cross-sectional study, subcutaneous abdominal adipose tissue biopsies from 59 people were examined in relation to fasting and post-glucose insulin sensitivity. Adipocyte size, TG fatty acid composition and mRNA expression of lipogenic genes were determined. RESULTS: We found strong positive relationships between adipose tissue TG content of the fatty acids myristic acid (14:0) and stearic acid (18:0) with insulin sensitivity (HOMA model) (p < 0.01 for each), and inverse relationships with adipocyte size (p < 0.01, p < 0.05, respectively). Variation in 18:0 content was the determinant of the adipose tissue TG 18:1 n-9/18:0 ratio, which correlated negatively with insulin sensitivity (p < 0.01), as observed previously. Adipose tissue 18:0 content correlated positively with the mRNA expression of lipogenic genes (e.g. FASN, p < 0.01). Lipogenic gene expression (a composite measure derived from principal components analysis) was inversely correlated with adipocyte cell size (p < 0.001). There was no relationship between dietary saturated fatty acid intake and adipose tissue 18:0 content. CONCLUSIONS/INTERPRETATION: Our data suggest a physiological mechanism whereby DNL is downregulated as adipocytes expand. Taken together with other data, they also suggest that hepatic and adipose tissue DNL are not regulated in parallel. We also confirm a strong relationship between small adipocytes and insulin sensitivity, which is independent of BMI.

Enhanced metabolic cycling in subjects after colonic resection for ulcerative colitis.

Colonic resection leads to insulin resistance, but the mechanisms are unknown. We used an integrated approach to examine adipose tissue and skeletal muscle metabolism in patients lacking a colon. Ten healthy colectomized patients having undergone surgery for ulcerative colitis and 10 matched control subjects were studied with a hyperinsulinemic-euglycemic clamp to measure insulin sensitivity, an arteriovenous sampling meal tolerance study to measure postprandial substrate flux across adipose tissue and skeletal muscle, and adipose tissue and skeletal muscle biopsies to quantify the expression of genes involved in glucose and lipid metabolism. Colectomized subjects exhibited lower insulin sensitivity (homeostatic model assessment model, 33% reduction, P = 0.03; minimal model, 29% reduction, P = 0.05), elevated aldosterone (9-fold, P = 0.003), leptin (2.2-fold, P = 0.03), and an increased rate of nonesterified fatty acid and glycerol release from adipose tissue (P = 0.02) especially in the late postprandial period. The uptake of fatty acids into muscle was also significantly increased (P = 0.007), as were muscle CD36 and LPL mRNA expression compared with controls. In adipose tissue, hormone-sensitive lipase mRNA expression was increased (P = 0.015), whereas peroxisome proliferator-activated receptor-gamma expression was decreased (P = 0.02), as was that of CD36 (P = 0.001). In this study, alterations in fatty acid metabolism after colonic resection altered may have contributed to the impairment of insulin sensitivity.

Soybean sterol composition and utilization by Phytophthora sojae.

The sterol fraction of Glycine max (soybean) was found to contain a mixture of 13 major sterols which differed dramatically in composition between seeds and shoots. Typical C4-desmethyl Delta(5)-sterols, including sitosterol, predominate the sterol mixture of shoots, whereas C4-methyl sterol intermediates, cycloartenol and 24(28)-methylene cycloartanol, accumulate in seeds. The significance of modified sterol profile of shoot compared to seed was relevant to the physiology of Phytophthora sojae, a phytopathogen of soybean shown to be auxotrophic for sterol. Sterols native to the host plant containing a C4-methyl group, such as cycloartenol, were not utilized by the fungus. Alternatively, all Delta(5)-sterols added to the culture media of P. sojae supported normal growth and promoted viable oospore production. The results demonstrate the importance of sterols in plant-fungal interactions and offer the possibility of bioengineering the phytosterol pathway for resistance to phytopathogens which scavenge specific sterols of the host plant to complete the life cycle.

The formation of a 1-5 phosphodiester linkage in the spontaneous breakdown of 5-phosphoribosyl-alpha-1-pyrophosphate.

The decomposition of 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) in the presence of Mg2+ at pH=7.8 yields a combination of products including ribose 5-phosphate, ribose 1-phosphate, 5-phosphoribosyl 1,2 cyclic phosphate, inorganic phosphate, and pyrophosphate. Hydrogen decoupled 31P NMR analysis of the product mixture also exhibits a sharp peak (+2.6 ppm from phosphocreatine) in a chemical shift region which includes phosphodiester bonds. Alkaline phosphatase treatment of the product mixture results in cleavage of monophosphate esters such as ribose 1-phosphate and ribose 5-phosphate, but does not affect the unidentified peak. Homonuclear (1H) correlation spectroscopy (COSY) of a partially purified sample was successful in identifying the hydrogen spectra of this compound. Combined with results from the splitting patterns of selectively decoupled 31P spectra, the COSY data indicate that several hydrogens are directly coupled to the unknown phosphate group with J value matches to the hydrogen on carbon one and to the two hydrogens on carbon five. Heteronuclear (1H-31P) chemical shift correlation studies confirm these couplings and further substantiate the formation of a ribose 1-5 phosphate linkage during the degradation of PRPP under these conditions. It is presently unknown whether this is an intramolecular or intermolecular phosphodiester linkage, although some spectroscopic evidence suggest the intramolecular bond formation, i.e. a ribose 1,5-cyclic phosphate (R-1,5cP). The formation of R-1,5cP helps explain the observation that the 5-phosphate group from PRPP becomes labile during the spontaneous degradation of PRPP.

Does the DASH diet lower blood pressure by altering peripheral vascular function?

We tested whether lowering of blood pressure (BP) on the dietary approaches to stop hypertension (DASH) diet was associated with changes in peripheral vascular function: endothelial function, assessed by flow-mediated vasodilatation (FMD) of the brachial artery, and subcutaneous adipose tissue blood flow (ATBF). We also assessed effects on heart rate variability (HRV) as a measure of autonomic control of the heart. We allocated 27 men and women to DASH diet and control groups. We measured FMD, ATBF and HRV on fasting and after ingestion of 75 g glucose, before and after 30 days on dietary intervention, aiming for weight maintenance. The control group did not change their diet. The DASH-diet group complied with the diet as shown by significant reductions in systolic (P<0.001) and diastolic (P=0.005) BP, and in plasma C-reactive protein (P<0.01), LDL-cholesterol (P<0.01) and apolipoprotein B (P=0.001), a novel finding. Body weight changed by <1 kg. There were no changes in the control group. We found no changes in FMD, or in ATBF, in the DASH-diet group, although heart rate fell (P<0.05). Glucose and insulin concentrations did not change. In this small-scale study, the DASH diet lowered BP independently of peripheral mechanisms.

Angiotensin II: a major regulator of subcutaneous adipose tissue blood flow in humans.

We investigated the functional roles of circulating and locally produced angiotensin II (Ang II) in fasting and postprandial adipose tissue blood flow (ATBF) regulation and examined the interaction between Ang II and nitric oxide (NO) in ATBF regulation. Local effects of the pharmacological agents (or contralateral saline) on ATBF, measured with 133Xe wash-out, were assessed using the recently developed microinfusion technique. Fasting and postprandial (75 g glucose challenge) ATBF regulation was investigated in nine lean healthy subjects (age, 29 +/- 3 years; BMI, 23.4 +/- 0.7 kg m(-2)) using local Ang II stimulation, Ang II type 1 (AT1) receptor blockade, and angiotensin-converting enzyme (ACE) inhibition. Furthermore, NO synthase (NOS) blockade alone and in combination with AT1 receptor blockade was used to examine the interaction between Ang II and NO. Ang II induced a dose-dependent decrease in ATBF (10(-9)m: -16%, P = 0.04; 10(-7)m: -33%, P < 0.01; 10(-5)m: -53%P < 0.01). Fasting ATBF was not affected by ACE inhibition, but was increased by approximately 55% (P < 0.01) by AT(1) receptor blockade. NOS blockade induced a approximately 30% (P = 0.001) decrease in fasting ATBF. Combined AT1 receptor and NOS blockade increased ATBF by approximately 40% (P = 0.003). ACE inhibition and AT1 receptor blockade did not affect the postprandial increase in ATBF. We therefore conclude that circulating Ang II is a major regulator of fasting ATBF, and a major proportion of the Ang II-induced decrease in ATBF is NO independent. Locally produced Ang II does not appear to regulate ATBF. Ang II appears to have no major effect on the postprandial enhancement of ATBF.

Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 alpha-demethylase: implications for sterol biosynthesis.

Sterol 14 alpha-demethylase (14DM) is a cytochrome P-450 involved in sterol biosynthesis in eukaryotes. It was reported that Mycobacterium smegmatis also makes cholesterol and that cholesterol is essential to Mycobacterium tuberculosis (MT) infection, although the origin of the cholesterol is unknown. A protein product from MT having about 30% sequence identity with eukaryotic 14 alpha-demethylases has been found to convert sterols to their 14-demethyl products indicating that a sterol pathway might exist in MT. To determine the optimal sterol structure recognized by MT 14DM, binding of 28 sterol and sterol-like (triterpenoids) molecules to the purified recombinant 14 alpha-demethylase was examined. Like eukaryotic forms, a 3 beta-hydroxy group and a 14 alpha-methyl group are essential for substrate acceptability by the bacterial 14 alpha-demethylase. The high affinity binding of 31-norcycloartenol without detectable activity indicates that the Delta(8)-bond is required for activity but not for binding. As for plant 14 alpha-demethylases, 31-nor-sterols show a binding preference for MT 14DM. Similar to enzymes from mammals and yeast, a C24-alkyl group is not required for MT 14DM binding and activity, whereas it is for plant 14 alpha-demethylases.Thus, substrate binding to MT 14DM seems to share common features with all eukaryotic 14 alpha-demethylases, the MT form seemingly having the broadest substrate recognition of all forms of 14 alpha-demethylase studied so far. - Bellamine, A., A. T. Mangla, A. L. Dennis, W. D. Nes, and M. R. Waterman. Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 alpha-demethylase: implications for sterol biosynthesis. J. Lipid Res. 2001. 42: 128;-136.