Resistant Starch Production and Glucose Release from Pre-Prepared Chilled Food: The SPUD Project.

With an increasing prevalence of diabetes worldwide, effective dietary strategies for blood glucose control are crucial. As carbohydrates make up approximately 50% of the diet, it is neither practical nor advisable to avoid them altogether. Most of the carbohydrate in the diet is derived from starch, found in potatoes, pasta, rice and bread. These foods are often processed in some way before consumption, yet little is known about the effects processing, such as chilling and reheating, has on the glycaemic response, particularly when the food is consumed in the context of a mixed meal. This article introduces the SPUD project, a BBSRC DRINC-funded initiative. Taking the potato as the model carbohydrate, this project will investigate, via in vitro and in vivo studies, the effects of domestic food processing techniques on the glycaemic response. A final study, utilising intrinsically labelled potato and a dual stable isotope methodology, will model glucose flux data to determine the underlying mechanisms of action.

Antiangiogenic and tumour inhibitory effects of downregulating tumour endothelial FABP4.

Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte differentiation. Previously we have shown that FABP4 in endothelial cells is induced by the NOTCH1 signalling pathway, the latter of which is involved in mechanisms of resistance to antiangiogenic tumour therapy. Here, we investigated the role of FABP4 in endothelial fatty acid metabolism and tumour angiogenesis. We analysed the effect of transient FABP4 knockdown in human umbilical vein endothelial cells on fatty acid metabolism, viability and angiogenesis. Through therapeutic delivery of siRNA targeting mouse FABP4, we investigated the effect of endothelial FABP4 knockdown on tumour growth and blood vessel formation. In vitro, siRNA-mediated FABP4 knockdown in endothelial cells led to a marked increase of endothelial fatty acid oxidation, an increase of reactive oxygen species and decreased angiogenesis. In vivo, we found that increased NOTCH1 signalling in tumour xenografts led to increased expression of endothelial FABP4 that decreased when NOTCH1 and VEGFA inhibitors were used in combination. Angiogenesis, growth and metastasis in ovarian tumour xenografts were markedly inhibited by therapeutic siRNA delivery targeting mouse endothelial FABP4. Therapeutic targeting of endothelial FABP4 by siRNA in vivo has antiangiogenic and antitumour effects with minimal toxicity and should be investigated further.

Extracellular Signal-Regulated Kinase 2 and CHOP Restrict the Expression of the Growth Arrest-Specific p20K Lipocalin Gene to G0.

The activation of the growth arrest-specific (gas) p20K gene depends on the interaction of C/EBPß with two elements of a 48-bp promoter region termed the quiescence-responsive unit (QRU). Here we identify extracellular signal-related kinase 2 (ERK2) as a transcriptional repressor of the p20K QRU in cycling chicken embryo fibroblasts (CEF). ERK2 binds to repeated GAAAG sequences overlapping the C/EBPß sites of the QRU. The recruitment of ERK2 and C/EBPß is mutually exclusive and dictates the expression of p20K. C/EBP homologous protein (CHOP) was associated with C/EBPß under conditions promoting endoplasmic reticulum (ER) stress and, to a lesser extent, in cycling CEF but was not detectable when C/EBPß was immunoprecipitated from contact-inhibited cells. During ER stress, overexpression of CHOP inhibited p20K, while its downregulation promoted p20K, indicating that CHOP is also a potent inhibitor of p20K. Transcriptome analyses revealed that hypoxia-responsive genes are strongly induced in contact-inhibited but not serum-starved CEF, and elevated levels of nitroreductase activity, a marker of hypoxia, were detected at confluence. Conditions of hypoxia (2% O2) induced growth arrest in subconfluent CEF and markedly stimulated p20K expression, suggesting that the control of proliferation and gas gene expression is closely linked to limiting oxygen concentrations associated with high cell densities.

Micro-techniques for analysis of human adipose tissue fatty acid composition in dietary studies.

BACKGROUND AND AIMS: Adipose tissue (AT) fatty acid (FA) composition is considered to be the gold standard long-term biomarker of dietary fatty acid intake. Typically this measurement is made directly from samples collected via large-needle-biopsy or incision. However, with growing interest in the role of AT in relation to health, ideally the fatty acid composition would be analysed along with other measurements, such as gene expression or histology, on a single AT sample. Here we assess alternative ways of obtaining AT for measuring FA composition, in some cases in conjunction with other measurements. METHODS AND RESULTS: The FA composition of tissue obtained via different methods was compared to that of tissue collected via large-needle or surgical biopsy. Fatty acid composition was not significantly different in AT collected by small-needle mini-biopsy (n = 10), from an RNA 'lipid layer' (obtained during RNA extraction, 2 sites, n = 6 for each), or from cryosectioned tissue prepared for histology (n = 10). We also assessed the usefulness of the composition of plasma NEFA as a surrogate marker of subcutaneous AT (n = 58-80). Most FAs in plasma NEFA correlated strongly with those in AT (P < 0.05). CONCLUSION: It is feasible to measure the FA composition of AT on very small amounts of tissue. Additionally, it is possible to measure FA composition on the lipid rich 'by-product' of AT samples undergoing RNA extraction for gene expression. Samples sectioned for histology are also suitable. This provides further opportunities for multidisciplinary collaborations that may lead to a better application of dietary biomarkers.

Failure to increase postprandial blood flow in subcutaneous adipose tissue is associated with tissue resistance to adrenergic stimulation.

AIMS: Adequate adipose tissue blood flow (ATBF) is essential for its metabolic and endocrine functions. From a metabolic point of view, sufficient increases in ATBF after meals permits full storage of excess energy into fat, thus protecting other tissues against the toxic effects of fatty acids and glucose spillover. It was previously shown that postprandial increases in ATBF are blunted in obese and insulin-resistant subjects, and that much of the postprandial ATBF response is the result of ß-adrenergic activation. Examination of previously recorded data on postprandial ATBF responses revealed an underlying heterogeneity, with postprandial ATBF being largely unresponsive to food stimuli in a substantial proportion of normal weight healthy people (low responders). Our study tests the hypothesis that this unresponsive pattern is due to resistance to ß-adrenergic stimulation in adipose tissue. METHODS: Five responders and five low responders were selected from a previously studied cohort and matched for BMI (20.5±0.7 vs 22±1 kg/m(2), respectively), gender (male/female: 2/3) and age (30±3 vs 37±6 years). Subcutaneous adipose tissue microinfusions of stepwise increasing doses of isoproterenol were performed with concomitant monitoring of blood flow, using the (133)Xenon washout technique. RESULTS: Although BMI was similar between responders and low responders, there were significant differences in fat mass (9.9±1.6 vs 14.4±1.6 kg; P<0.05) and four-point skinfold thickness (33±4 vs 52±16 mm; P<0.05). Lack of ATBF response to oral glucose was confirmed in the low responder group. In responders, ATBF was higher at baseline (5.4±1 vs 3.4±1 mL/min/100 g of tissue) and responded more distinctly to increasing isoproterenol doses (10(-8) M: 7.6±1.4 vs 4.9±1; 10(-6) M: 12.5±1.7 vs 7.5±1.6; and 10(-4) M: 20 ±1.7 vs 9±0.9 mL/min/100 g of tissue). CONCLUSION: These data suggest that the lack of glucose-stimulated ATBF is associated with resistance to sympathetic activation in adipose tissue.

Adipose tissue fatty acid metabolism in insulin-resistant men.

AIMS/HYPOTHESIS: Increased NEFA production and concentrations may underlie insulin resistance. We examined systemic and adipose tissue NEFA metabolism in insulin-resistant overweight men (BMI 25-35 kg/m2). METHODS: In a cohort study we examined NEFA concentrations in men in the upper quartile of fasting insulin (n = 124) and in men with fasting insulin below the median (n = 159). In a metabolic study we examined NEFA metabolism in the fasting and postprandial states, in ten insulin-resistant men and ten controls. RESULTS: In the cohort study, fasting NEFA concentrations were not significantly different between the two groups (median values: insulin-resistant men, 410 micromol/l; controls, 445 micromol/l). However, triacylglycerol concentrations differed markedly (1.84 vs 1.18 mmol/l respectively, p < 0.001). In the metabolic study, arterial NEFA concentrations again did not differ between groups, whereas triacylglycerol concentrations were significantly higher in insulin-resistant men. Systemic NEFA production and the release of NEFA from subcutaneous adipose tissue, expressed per unit of fat mass, were both reduced in insulin-resistant men compared with controls (fasting values by 32%, p = 0.02, and 44%, p = 0.04 respectively). 3-Hydroxybutyrate concentrations, an index of hepatic fat oxidation and ketogenesis, were lower (p = 0.03). CONCLUSIONS/INTERPRETATION: Adipose tissue NEFA output is not increased (per unit weight of tissue) in insulin resistance. On the contrary, it appears to be suppressed by high fasting insulin concentrations. Alterations in triacylglycerol metabolism are more marked than those in NEFA metabolism and are indicative of altered metabolic partitioning of fatty acids (decreased oxidation, increased esterification) in the liver.

Removal of triacylglycerols from chylomicrons and VLDL by capillary beds: the basis of lipoprotein remnant formation.

The triacylglycerol content of chylomicrons and VLDL (very-low-density lipoprotein) compete for the same lipolytic pathway in the capillary beds. Although chylomicron triacylglycerols appear to be the favoured substrate for lipoprotein lipase, VLDL particles compete in numbers. Methods to quantify the specific triacylglycerol removal from VLDL and chylomicrons may involve endogenous labelling of the triacylglycerol substrate with stable isotopes in combination with arteriovenous blood sampling in humans. Arteriovenous quantification of remnant lipoproteins suggests that adipose tissue with its high lipoprotein lipase activity is a principal site for generation of remnant lipoproteins. Under circumstances of reduced efficiency in the removal of triacylglycerols from lipoproteins, there is accumulation of remnant lipoproteins, which are potentially atherogenic.

The in vivo effects of the Pro12Ala PPARgamma2 polymorphism on adipose tissue NEFA metabolism: the first use of the Oxford Biobank.

AIMS/HYPOTHESIS: To investigate the phenotypic effects of common polymorphisms on adipose tissue metabolism and cardiovascular risk factors, we set out to establish a biobank with the unique feature of allowing a prospective recruit-by-genotype approach. The first use of this biobank investigates the effects of the peroxisome proliferator-activated receptor (PPAR) Pro12Ala polymorphism on integrative tissue-specific physiology. We hypothesised that Ala12 allele carriers demonstrate greater adipose tissue metabolic flexibility and insulin sensitivity. MATERIALS AND METHODS: From a comprehensive population register, subjects were recruited into a biobank, which was genotyped for the Pro12Ala polymorphism. Twelve healthy male Ala12 carriers and 12 matched Pro12 homozygotes underwent detailed physiological phenotyping using stable isotope techniques, and measurements of blood flow and arteriovenous differences in adipose tissue and muscle in response to a mixed meal containing [1,1,1-(13)C]tripalmitin. RESULTS: Of 6,148 invited subjects, 1,072 were suitable for inclusion in the biobank. Among Pro12 homozygotes, insulin sensitivity correlated with HDL-cholesterol concentrations, and inversely correlated with blood pressure, apolipoprotein B, triglyceride and total cholesterol concentrations. Ala12 carriers showed no such correlations. In the meal study, Ala12 carriers had lower plasma NEFA concentrations, higher adipose tissue and muscle blood flow, and greater insulin-mediated postprandial hormone-sensitive lipase suppression along with greater insulin sensitivity than Pro12 homozygotes. CONCLUSIONS/INTERPRETATION: This study shows that a recruit-by-genotype approach is feasible and describes the biobank's first application, providing tissue-specific physiological findings consistent with the epidemiological observation that the PPAR Ala12 allele protects against the development of type 2 diabetes.

Adverse physicochemical properties of tripalmitin in beta cells lead to morphological changes and lipotoxicity in vitro.

AIMS/HYPOTHESIS: Long-term exposure of beta cells to lipids, particularly saturated fatty acids in vitro, results in cellular dysfunction and apoptosis (lipotoxicity); this could contribute to obesity-related diabetes. Our aims were to relate cell death to intracellular triglyceride concentration, composition and localisation following incubation of INS1 cells in saturated and unsaturated NEFA in high and low glucose concentrations. MATERIALS AND METHODS: Insulin-producing INS1 cells were cultured (24 h; 3 and 20 mmol/l glucose) with palmitic, oleic or linoleic acids and the resulting intracellular lipids were analysed by gas chromatography and microscopy. Cell death was determined by quantitative microscopy and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and glucose-stimulated insulin secretion by ELISA. RESULTS: All NEFA (0.5 mmol/l, 0.5% albumin) inhibited glucose-stimulated (20 mmol/l) insulin secretion. Cytotoxicity was evident only with palmitic acid (p<0.05), in which case intracellular triglyceride consisted largely of tripalmitin in angular-shaped dilated endoplasmic reticulum. Cytotoxicity and morphological disruption were reduced by addition of unsaturated NEFA. Triglyceride content (control cells; 14.5 ng/mug protein) increased up to 10-fold following incubation in NEFA (oleic acid 153.2 ng/mug protein; p<0.05) and triglyceride and phospholipid fractions were both enriched with the specific fatty acid added to the medium (p<0.05). CONCLUSIONS/INTERPRETATION: In INS1 cells, palmitic acid is converted in the endoplasmic reticulum to solid tripalmitin (melting point >65 degrees C), which could induce endoplasmic reticulum stress proteins and signal apoptosis; lipid-induced apoptosis would therefore be a consequence of the physicochemical properties of these triglycerides. Since cellular triglycerides composed of single species of fatty acid are not likely to occur in vivo, destruction of beta cells by saturated fatty acids could be predominantly an in vitro scenario.

The effects of rosiglitazone on fatty acid and triglyceride metabolism in type 2 diabetes.

AIMS/HYPOTHESIS: We investigated the effects of rosiglitazone on NEFA and triglyceride metabolism in type 2 diabetes. METHODS: In a double-blind, placebo-controlled, cross-over study of rosiglitazone in diet-treated type 2 diabetic subjects, we measured arteriovenous differences and tissue blood flow in forearm muscle and subcutaneous abdominal adipose tissue, used stable isotope techniques, and analysed gene expression. Responses to a mixed meal containing [1,1,1-(13)C]tripalmitin were assessed. RESULTS: Rosiglitazone induced insulin sensitisation without altering fasting NEFA concentrations (-6.6%, p=0.16). Postprandial NEFA concentrations were lowered by rosiglitazone compared with placebo (-21%, p=0.04). Adipose tissue NEFA release was not decreased in the fasting state by rosiglitazone treatment (+24%, p=0.17) and was associated with an increased fasting hormone-sensitive lipase rate of action (+118%, p=0.01). Postprandial triglyceride concentrations were decreased by rosiglitazone treatment (-26%, p<0.01) despite unchanged fasting concentrations. Rosiglitazone did not change concentrations of triglyceride-rich lipoprotein remnants. Adipose tissue blood flow increased with rosiglitazone (+32%, p=0.03). Postprandial triglyceride [(13)C]palmitic acid concentrations were unchanged, whilst NEFA [(13)C]palmitic acid concentrations were decreased (p=0.04). In muscle, hexokinase II mRNA expression was increased by rosiglitazone (+166%, p=0.001) whilst the expression of genes involved in insulin signalling was unchanged. Adipose tissue expression of FABP4, LPL and FAT/CD36 was increased. CONCLUSIONS/INTERPRETATION: Rosiglitazone decreases postprandial NEFA and triglyceride concentrations. This may represent decreased spillover of NEFAs from adipose tissue depots. Decreased delivery of NEFAs to the liver may lead to lowered postprandial triglyceride concentrations. Upregulation of hexokinase II expression in muscle may contribute to insulin sensitisation by rosiglitazone.

Subcutaneous adipose tissue blood flow varies between superior and inferior levels of the anterior abdominal wall.

OBJECTIVE: Blood flow regulation is thought to mediate the metabolic functions of adipose tissue. Different depots, and even different layers within the subcutaneous adipose tissue, may vary in metabolic activity and blood flow. Therefore, we investigated if any differences in subcutaneous adipose tissue blood flow (ATBF) exist at different locations of the anterior abdominal wall. METHODS: ATBF was measured 8-10 cm above or below the umbilicus, at 8-10 cm (both sides) from the midline, in 18 healthy subjects (BMI range 18-33 kg/m(2)). Measurements of ATBF were performed using (133)xenon washout, during a stable baseline period and after ingestion of 75 g of glucose. RESULTS: At baseline, ATBF was greater at the upper level compared to the lower level (4.4+/-0.3 vs 3.8+/-0.2 ml min(-1) 100 g tissue(-1), P=0.005), but was not different between the right and the left sides at either level. ATBF increased in response to oral glucose at all sites. The mean increase at the superior level was also greater than the inferior level (3.5+/-0.7 vs 2.2+/-0.6 ml min(-1) 100 g tissue(-1), P=0.001). CONCLUSIONS: Even at a constant depth and with only 16-20 cm difference between sites, there are significant differences in function of the same adipose depot. These findings have physiological and methodological implications for in vivo metabolic studies of human adipose tissue.

Integrative physiology of human adipose tissue.

Adipose tissue is now recognised as a highly active metabolic and endocrine organ. Great strides have been made in uncovering the multiple functions of the adipocyte in cellular and molecular detail, but it is essential to remember that adipose tissue normally operates as a structured whole. Its functions are regulated by multiple external influences such as autonomic nervous system activity, the rate of blood flow and the delivery of a complex mix of substrates and hormones in the plasma. Attempting to understand how all these factors converge and regulate adipose tissue function is a prime example of integrative physiology. Adipose tissue metabolism is extremely dynamic, and the supply of and removal of substrates in the blood is acutely regulated according to the nutritional state. Adipose tissue possesses the ability to a very large extent to modulate its own metabolic activities, including differentiation of new adipocytes and production of blood vessels as necessary to accommodate increasing fat stores. At the same time, adipocytes signal to other tissues to regulate their energy metabolism in accordance with the body's nutritional state. Ultimately adipocyte fat stores have to match the body's overall surplus or deficit of energy. This implies the existence of one (or more) signal(s) to the adipose tissue that reflects the body's energy status, and points once again to the need for an integrative view of adipose tissue function.

Achievement of dietary fatty acid intakes in long-term controlled intervention studies: approach and methodology.

OBJECTIVE: To describe the calculations and approaches used to design experimental diets of differing saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) compositions for use in a long-term dietary intervention study, and to evaluate the degree to which the dietary targets were met. DESIGN, SETTING AND SUBJECTS: Fifty-one students living in a university hall of residence consumed a reference (SFA) diet for 8 weeks followed by either a moderate MUFA (MM) diet or a high MUFA (HM) diet for 16 weeks. The three diets were designed to differ only in their proportions of SFA and MUFA, while keeping total fat, polyunsaturated fatty acids (PUFA), trans-fatty acids, and the ratio of palmitic to stearic acid, and n-6 to n-3 PUFA, unchanged. RESULTS: Using habitual diet records and a standardised database for food fatty acid compositions, a sequential process of theoretical fat substitutions enabled suitable fat sources for use in the three diets to be identified, and experimental margarines for baking, spreading and the manufacture of snack foods to be designed. The dietary intervention was largely successful in achieving the fatty acid targets of the three diets, although unintended differences between the original target and the analysed fatty acid composition of the experimental margarines resulted in a lower than anticipated MUFA intake on the HM diet, and a lower ratio of palmitic to stearic acid compared with the reference or MM diet. CONCLUSIONS: This study has revealed important theoretical considerations that should be taken into account when designing diets of specific fatty acid composition, as well as practical issues of implementation.

Acute effects of meal fatty acid composition on insulin sensitivity in healthy post-menopausal women.

Postprandial plasma insulin concentrations after a single high-fat meal may be modified by the presence of specific fatty acids although the effects of sequential meal ingestion are unknown. The aim of the present study was to examine the effects of altering the fatty acid composition in a single mixed fat-carbohydrate meal on glucose metabolism and insulin sensitivity of a second meal eaten 5 h later. Insulin sensitivity was assessed using a minimal model approach. Ten healthy post-menopausal women underwent four two-meal studies in random order. A high-fat breakfast (40 g fat) where the fatty acid composition was predominantly saturated fatty acids (SFA), n-6 polyunsaturated fatty acids (PUFA), long-chain n-3 PUFA or monounsaturated fatty acids (MUFA) was followed 5 h later by a low-fat, high-carbohydrate lunch (5.7 g fat), which was identical in all four studies. The plasma insulin response was significantly higher following the SFA meal than the other meals after both breakfast and lunch (P<0.006) although there was no effect of breakfast fatty acid composition on plasma glucose concentrations. Postprandial insulin sensitivity (SI(Oral)) was assessed for 180 min after each meal. SI(Oral) was significantly lower after lunch than after breakfast for all four test meals (P=0.019) following the same rank order (SFA < n-6 PUFA < n-3 PUFA < MUFA) for each meal. The present study demonstrates that a single meal rich in SFA reduces postprandial insulin sensitivity with 'carry-over' effects for the next meal.

Interaction between specific fatty acids, GLP-1 and insulin secretion in humans.

AIMS/HYPOTHESIS: Fatty acids affect insulin secretion in vivo, but little is known about the effects of specific fatty acids. Our aim was to investigate differential effects of acutely increased plasma monounsaturated, polyunsaturated and saturated fatty acids on glucose-stimulated insulin secretion in healthy humans. METHODS: A new experimental protocol was used to increase plasma monounsaturated (MUFA test), polyunsaturated (PUFA test) or saturated (SFA test) non-esterified fatty acids for 2 h by repeated oral fat feeding and continuous intravenous heparin infusion. This was followed by a hyperglycaemic clamp (10 mmol/l) to test insulin secretion in response to a prior plasma NEFA increase. RESULTS: Total plasma NEFA concentrations were increased during the fat tests compared to the control visit (1.7-fold increase for MUFA and SFA tests and 1.4-fold increase for PUFA test; p<0.001). Exaggerated responses in plasma insulin, C-peptide and proinsulin concentrations were seen during the hyperglycaemic clamp after increasing plasma NEFA concentrations compared with the control (p<0.01). The effects were greatest for the MUFA test followed by the PUFA test and SFA test (p<0.01). Plasma GLP-1 concentrations increased during fat feeding, with a higher response during the MUFA test compared to PUFA and SFA tests (p<0.01). CONCLUSION/INTERPRETATION: Increasing plasma NEFA concentrations by oral fat feeding with heparin infusion augments glucose-stimulated insulin secretion with the greatest effect for monounsaturated fatty acids and the lowest effect for saturated fatty acids. Monounsaturated fatty acids also increase GLP-1 more than saturated fatty acids. Therefore, the exaggerated insulin concentrations could be due to both NEFA and GLP-1.

Substituting dietary saturated fat with polyunsaturated fat changes abdominal fat distribution and improves insulin sensitivity.

AIMS/HYPOTHESIS: British dietary recommendations are to decrease total fat intake to less than 30 % of daily energy intake and saturated fat to less than 10 %. In practice, it is difficult for people to make these changes. It may be easier to encourage people to switch from a diet rich in saturated fatty acids to one rich in polyunsaturated fatty acids. METHODS: A total of 17 subjects - six people with Type II (non-insulin-dependent) diabetes mellitus, six non-obese and five obese people without diabetes - were randomised to spend two 5-week periods on a diet rich in saturated or in polyunsaturated fatty acids, in a crossover design. At the start of the study and after each dietary period, we assessed abdominal fat distribution using magnetic resonance imaging, insulin sensitivity using hyperinsulinaemic-euglycaemic clamps and fasting lipid parameters. RESULTS: Dietary compliance, assessed by weekly 3-day dietary records and measurement of biochemical markers, was good. Energy and fat intake appeared to be reduced on the diet rich in polyunsaturated fatty acids although body weights did not change. Insulin sensitivity and plasma low density lipoprotein cholesterol concentrations improved with the diet rich in polyunsaturated fatty acids compared with the diet rich in saturated fatty acids. There was also a decrease in abdominal subcutaneous fat area. CONCLUSION/INTERPRETATION: If this result is confirmed in longer-term studies, this dietary manipulation would be more readily achieved by the general population than the current recommendations and could result in considerable improvement in insulin sensitivity, reducing the risk of developing Type II diabetes.

Monitoring adipose tissue blood flow in man: a comparison between the (133)xenon washout method and microdialysis.

INTRODUCTION: Adipose tissue blood flow (ATBF) increases after meal intake and a failure to regulate ATBF in the postprandial period seems to be a feature of insulin resistance and obesity. ATBF can be measured quantitatively by the (133)Xe washout technique, but the microdialysis ethanol escape method has also been employed to detect relative changes in ATBF. METHODS: We compared (133)Xe washout and the recovery of exogenous ethanol and endogenous urea by microdialysis in abdominal subcutaneous adipose tissue, after physiological stimulation of ATBF by ingestion of oral glucose (75 g) in eight healthy people (age 23-52 y, body mass index (BMI) 19.4-29.6 kg/m(2)). RESULTS: The ATBF response was heterogeneous. In subjects responding vigorously to the stimulus as measured by (133)Xe washout, the microdialysis ethanol escape was increased (indicating an increase in ATBF). An increased recovery of urea was observed, also indicating an increase in ATBF. The recovery of both small molecules was delayed compared with increased blood flow and failed to return to baseline in response to a rapid decline in ATBF. CONCLUSION: We conclude that the (133)Xe washout technique is more responsive to physiological change in ATBF than ethanol escape or urea recovery by microdialysis.

Identification, sequence analysis, and phylogeny of the immediate early gene 1 of the Trichoplusia ni single nucleocapsid polyhedrosis virus.

Substantial research has been conducted on the immediate early I (ie-1) genes from the prototype baculovirus Auographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). In both cases ie-1 gene products have been implicated in transcriptional activation and repression. In this study an ie-1 homolog was identified from Trichoplusia ni single nucleocapsid polyhedrosis virus (TniSNPV). Nucleotide sequence analysis indicated that the TniSNPV ie-1 gene consists of a 2,217 nucleotide open reading frame (ORF), encoding a protein with a molecular mass of 84.464 kDa. This represents the largest baculovirus ie-1 gene characterised to date. Of the seven ie-1 homologs identified to date, the TniSNPV ie-1 shared most sequence similarity with the ie-1 gene of Spodoptera exigua MNPV (SeMNPV) (41%). At the nucleotide level, expected TATA and CAGT motifs were found to precede each ie-1 ORE. At the protein level, it was confirmed that the N-termini are poorly conserved, but share the characteristic of having a high proportion of acidic amino acids. In addition it was found that N-terminal regions significantly matched the SET domain in the Swiss-Prot prosite database. The C-terminal regions of the deduced IE-1 sequences were found to be substantially more conserved than the N-termini. Several conserved motifs were identified in the C-terminal sequences. A phylogenetic tree of nine baculovirus IE-1 proteins was constructed using maximum parsimony analysis. The phylogenetic estimation of the ie-1 genes shows that TniSNPV is a member of the previously described lepidopteran NPV group II and it is most closely related to SeMNPV.

Second meal effect: modified sham feeding does not provoke the release of stored triacylglycerol from a previous high-fat meal.

The present study was carried out to determine whether cephalic stimulation, associated with eating a meal, was sufficient stimulus to provoke the release of stored triacylglycerol (TAG) from a previous high-fat meal. Ten subjects were studied on three separate occasions. Following a 12 h overnight fast, subjects were given a standard mixed test meal which contained 56 g fat. Blood samples were taken before the meal and for 5 h after the meal when the subjects were randomly allocated to receive either water (control) or were modified sham fed a low-fat (6 g fat) or moderate-fat (38 g fat) meal. Blood samples were collected for a further 3 h. Compared with the control, modified sham feeding a low- or moderate-fat meal did not provoke an early entry of TAG, analysed in either plasma or TAG-rich lipoprotein (TRL) fraction (density <1.006 kg/l). The TRL-retinyl ester data showed similar findings. A cephalic phase secretion of pancreatic polypeptide, without a significant increase in cholecystokinin levels, was observed on modified sham feeding. Although these data indicate that modified sham feeding was carried out successfully, analysis of the fat content of the expectorant showed that our subjects may have accidentally ingested a small amount of fat (0.7 g for the low-fat meal and 2.4 g for the moderate-fat meal). Nevertheless, an early TAG peak following modified sham feeding was not demonstrated in the present study, suggesting that significant ingestion of food, and not just oro-sensory stimulation, is necessary to provoke the release of any TAG stored from a previous meal.