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Skin wounds derive therapeutic benefit from redeployment of dermal tissues, whether as split-thickness allo- and autografts or as biological dressings comprising cultured cells. However, the clinical outcome is strongly influenced by the techniques used for cell/tissue grafting and also the microbiological status of the wound. Here we report that microtopography incorporated into the surface of a novel polymeric material, derivatized with fibronectin to promote attachment and encourage motility, improved the efficiency of cell transfer onto de-epithelialized human skin ex vivo. The microtopography had two functions, first as a conduit for migrating cells to cross between the vehicle and recipient surface and second to shield adherent cells from destruction by mechanical shearing during handling and application. Quantitative analysis showed that topographic projections (columns) rather than recesses (pits) in the hydrogel surface achieved the highest efficiency of cell transfer. In order to address the crucial relevance of microbiological contamination to the success of wound grafting, the effect of iodine on several common bacterial pathogens was examined using an XTT+C(Q10) kinetic cell viability assay. Increasing concentrations of iodine initially stressed and after 0.5% v/v were subsequently bacteriocidal for Gram-negative Pseudomonas aeruginosa and Escherichia coli and Gram-positive Bacillus subtillis and Staphylococcus aureus. Slightly higher doses of iodine (approx 1-1.5% v/v) were required to kill HaCaT cells outright, but for both pro- and eukaryotes the major determinant of cytotoxicity was absolute dose rather than duration of exposure. Iodine delivered by the hydrogel at low concentration was bacteriostatic but not apparently cytotoxic to epithelial cells as measured by MTT end-point cell viability assay. Zone of inhibition studies confirmed that bacteriocidal quantities of neomycin, phenol red, and silver could also be delivered using the same hydrogel. This research suggests that grafting cell-based biological dressings to wounds using a topographically modified hydrogel dressing capable of simultaneous reducing the microbiological threat to a successful outcome may be a realistic clinical proposition.

Alkaline phosphatase (ALP) is expressed in 3T3-L1 preadipocytes, and its activity increases during adipogenesis. The purpose of this study was to determine whether ALP activity could be used as a measure of intracellular lipid accumulation in human preadipocytes and 3T3-L1 cells and which of the factors that induce adipogenesis are responsible for stimulating ALP activity. Adipogenesis was initiated in 3T3-L1 cells by incubation with differentiation medium containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. The effect of leaving out each of the differentiation medium components was studied. Adipogenesis was also assessed in human preadipocytes and 3T3-L1 cells in the presence of the ALP inhibitor histidine. ALP activity was measured using an automated colorimetric assay and intracellular lipid accumulation was measured using the lipid-specific dye oil red O. Removal of insulin or dexamethasone from the differentiation medium had little effect on either ALP activity or lipid accumulation in 3T3-L1 cells, while removal of IBMX blocked both. Histidine inhibited ALP activity and adipogenesis in human preadipocytes and 3T3-L1 cells. Pearson univariate correlation analysis demonstrated strong correlations between ALP activity and lipid accumulation in human preadipocytes (r=0.78, n=69) and in 3T3-L1 cells (r=0.92, n=27). These data suggest that ALP and fat storage are tightly linked during preadipocyte maturation and that the measurement of ALP activity may be a novel technique for the quantification of intracellular lipid accumulation that is more sensitive and rapid than currently used methods.

Background: A previous study has demonstrated that alkaline phosphatase (AP) may play a role in the control of intracellular lipid accumulation in the rodent preadipocyte cell line, 3T3-L1. The present study investigated whether AP may have a similar function in preadipocytes isolated from human mammary gland tissue., Methods: Preadipocyte maturation was induced in the presence or absence of the tissue non-specific AP inhibitors levamisole and histidine, and the tissue-specific AP inhibitor PheGlyGly. Cellular AP activity and adipogenesis were both assessed at 0 and 12 days post-induction of differentiation., Results: After differentiation, AP activity increased 5.1 +/- 1.3-fold in the absence and 8.9 +/- 2.8-fold (P < 0.05) in the presence of levamisole. However, adipogenesis increased 1.95 +/- 0.11-fold in the absence but only 1.36 +/- 0.06-fold (P < 0.001) in the presence of levamisole. There was a 4.2 +/- 2.2-fold increase in AP activity in the absence and a 0.51 +/- 0.46-fold (P < 0.05) decrease in the presence of histidine. Adipogenesis increased 2.09 +/- 0.35-fold in the absence of histidine but only 1.22 +/- 0.30-fold (P < 0.05) in the presence of histidine. PheGlyGly had no effects. Fluorescent microscopy showed AP activity was localized to the triglyceride-containing droplets of the cell., Conclusion: This is the first study to show that tissue non-specific AP inhibitors can block adipogenesis in human preadipocytes.

Objectives: Studies have shown clear associations of abdominal obesity with lipid and glucose metabolism and cytokine levels in a number of different population groups. However, no such studies have been performed in an African population in which visceral adipose tissue levels have been shown to be lower than in European subjects., Design and Patients: Cross-sectional analysis in 124 African women., Measurements: Fasting serum samples were taken from all subjects and anthropometric measurements obtained. Blood levels of glucose, insulin, total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, triglyceride, interleukin (IL)-6, IL-8 and IL-18 were measured. Subjects were separated into normal and abnormal glucose tolerant groups and into tertiles according to waist circumference (WC). Insulin resistance was assessed using the homeostasis model assessment (HOMA)., Results: Abnormal glucose-tolerant subjects had higher WC, glucose and HOMA levels than the normal glucose-tolerant group. Increased WC was associated with higher triglyceride, insulin and HOMA levels and lower HDL levels. Multiple regression analyses showed that WC associated positively with HOMA and serum triglyceride levels and negatively with HDL levels. IL18 was a positive but weak determinant of the HOMA level and BMI correlated positively with serum IL-6 concentrations., Conclusions: Although previous studies have shown that African subjects have a lower visceral adipose depot size than European subjects, abdominal obesity is still associated with insulin resistance and dyslipidaemia. The association between abdominal obesity and metabolic dysfunction within this population is not dependent upon IL-6, IL-8 or IL-18.

Administration of heparin during extracorporeal procedures increases the risk of haemorrhage. Various reactor designs, including the use of heparinase and poly-L-lysine. HBr hollow fiber, have been investigated for the removal of heparin prior to the blood being returned to the patient; however, none of them have been implemented clinically. In this paper it is proposed that beads made from poly-L-lysine/alginate can be used to remove the heparin. The aim of this work is to perform the necessary experiments in order to get the information required to design a heparin removal reactor that uses these beads. The experiments are aimed at measuring the removal rates of heparin by the beads, testing the efficiency of the beads to remove heparin, determining repeatability and identifying factors that could influence the removal rate. Batch rate experiments using poly-L-lysine/alginate beads in saline solutions were performed to investigate the removal rate of heparin. The results, which indicate that heparin is efficiently removed, may lead to improved bioreactor designs.

Reduced plasma adiponectin levels are associated with insulin resistance. Black South Africans, like African Americans, are more insulin-resistant than BMI-matched white subjects, as are Asian Indians. We investigated whether this interethnic variation in insulin resistance is due to differences in plasma adiponectin levels. Blood and anthropometric measurements were taken from black, white and Asian-Indian subjects. Serum adiponectin, lipids, glucose and insulin were measured; insulin sensitivity was calculated using HOMA. Black (HOMA = 2.62 +/- 0.99) and Asian-Indian subjects (HOMA = 3.41 +/- 2.85) were more insulin-resistant than BMI-matched white (HOMA = 1.76 +/- 0.63) subjects (p = 0.0001). Furthermore, the white subjects had higher adiponectin levels (8.11 +/- 4.39 microg/ml) compared to black (5.71 +/- 2.50 microg/ml) and Asian Indian (5.86 +/- 2.50 microg/ml) subjects (p = 0.003). When all ethnic groups were combined, multiple regression analysis demonstrated that serum adiponectin levels corrected for BMI and ethnicity did not correlate with HOMA, but did explain 10.0 % of the variance in HDL-cholesterol levels. Within each ethnic group, adiponectin only correlated inversely with HOMA in white subjects. Adiponectin may play a role in determining serum HDL-cholesterol levels, but ethnic variation in insulin sensitivity is not dependent on serum levels of this adipokine. The relationship between adiponectin and insulin resistance varies across ethnic groups.

Autoantibodies to insulin are often the first autoantibodies detected in young children with type 1 diabetes and can be present before the onset of clinical diabetes. These autoantibodies and their epitopes are, however, not well characterized. We explored the use of monoclonal antibodies and their recombinant Fab as reagents for epitope analysis. In this study we cloned and characterized the recombinant Fab of the insulin-specific monoclonal antibody CG7C7. We found the epitope of this antibody to be located predominantly at the A-chain loop of the insulin molecule. The recombinant Fab was then used to compete for insulin binding against insulin autoantibodies present in sera from patients with type 1 or type 1.5 diabetes. In competition experiments with sera positive for autoantibodies to insulin the recombinant Fab significantly reduced the binding to [125I]-insulin by sera of type 1 (n = 35) and type 1.5 diabetes [latent autoimmune diabetes in adults (LADA)] (n = 14) patients (P < 0.0001). We conclude that competition between insulin-specific monoclonal antibodies or their recombinant Fab and insulin autoantibodies should prove useful in the epitope analysis of autoantibodies to insulin.

Objective: As alkaline phosphatase may play a role in cell differentiation, our aim was to study the possible role of this enzyme in the differentiation of preadipocytes (3T3-L1 cells) into adipocytes., Research Methods and Procedures: 3T3-L1 cells were grown in medium containing insulin, dexamethasone and IBMX to induce adipogenesis. Adipogenesis was measured using the triglyceride-specific dye, oil red O at 0, 3, 7 and 11 days after initiation of adipogenesis in the presence or absence of the alkaline phosphatase inhibitors, levamisole, histidine and Phe-Gly-Gly. Intracellular localisation of the enzyme was detected using ELF-phosphatase, a fluorescent substrate and alkaline phosphatase gene expression was assessed using RT-PCR., Results: Alkaline phosphatase activity was detected in untransformed cells (1.91+/-0.62 mU/mg protein) and activity increased 11.5+/-1.4-fold after 11 days treatment with transformation medium and 5.3+/-0.3-fold in transformation medium containing levamisole (p<0.05). Triglyceride content of cells increased 3.1+/-0.2-fold after 11 days treatment with transformation medium and 2.1+/-0.3-fold in the presence of levamisole (p<0.005). Histidine inhibited adipogenesis and alkaline phosphatase to a greater extent than did levamisole, but Phe-Gly-Gly had no effect on these variables. Alkaline phosphatase was localised around the lipid droplets of the cells. Gene expression of alkaline phosphatase increased during adipogenesis., Discussion: This study demonstrates that tissue-nonspecific alkaline phosphatase is present in 3T3-L1 cells and that it may play a role in the control of adipogenesis.

Objective: Lower lipid and insulin levels are found during a glucose-tolerance test in obese black than obese white South African women. Therefore, beta-cell function and lipid metabolism were compared in these populations during a mixed meal., Research Methods and Procedures: Blood concentrations of glucose, free fatty acids (FFAs), insulin, lipograms, and in vivo FFA oxidation were determined at fasting and for 7 hours after oral administration of a mixed emulsion containing glucose-casein-sucrose-lipid and [1-(13)C] palmitic acid in 8 lean black women (LBW), 10 obese black women (OBW), 9 lean white women (LWW), and 10 obese white women (OWW). Subcutaneous and visceral fat mass was assessed by computerized tomography., Results: Visceral fat area was higher in OWW (152.7 +/- 17.0 cm(2)) than OBW (80.0 +/- 6.7 cm(2); p < 0.01). In OBW, 30-minute insulin levels were higher (604.3 +/- 117.6 pM) than OWW (311.0 +/- 42.9 pM; p < 0.05). Total triglyceride was higher in OWW (706.7 +/- 96.0 mM x 7 hours) than OBW (465.7 +/- 48.2 mM x 7 hours; p < 0.05) and correlated with visceral fat area (beta = 0.38, p = 0.05). Palmitate oxidation was higher in lean than obese women in both ethnic groups and correlated negatively with fat mass (beta = -0.58, p < 0.005)., Discussion: The higher 30-minute insulin response in OBW may reflect a higher insulinotropic effect of FFAs or glucose. The elevated triglyceride level of OWW may be due to their higher visceral fat mass and possibly reduced clearance by adipose tissue.

The traditional Zulu remedy impila (Callilepis laureola) can cause acute fatal hepatocellular necrosis, especially in children. We investigated the mechanism(s) of toxicity using HuH-7 hepatocytes. Impila tubers were extracted with boiling water and the aqueous extract was used at different concentrations to study the effects on the morphology of the cells. Flow cytometry and labelling with fluorescent antibodies to tubulin were also used. At high concentrations, necrosis occurred; however, at lower concentrations, the extracts gave rise to a variety of changes including hypercondensation of chromatin, multinucleate cells, nuclear fragmentation and apoptosis. In addition, we observed destruction of cytoplasmic tubulin. These findings give further insight into the mechanism of toxicity of herbal remedies containing atractyloside.

The aim of this study was to determine the contribution of birth weight and gestational age to glucose tolerance in premature neonates. The study group consisted of 100 premature and/or small-for-gestational age infants. Anthropometric measurements were performed both at birth and at the time of a standardized milk feed carried out at 19.6 +/- 12.1 d (range, 1-65 d) after birth. Fasting and postprandial glucose and insulin levels were measured. Birth weight, as a proxy mirror of the intrauterine environment, was found to influence the glucose concentration following a standardized milk feed (beta = -0.46; P = 0.01 for birth weight z-score with 60-min glucose level), whereas gestational age did not. Small-for-gestational age neonates had higher 60-min insulin levels than appropriate-for-gestational age neonates (115.4 +/- 9.5 vs. 68.4 +/- 14.2; P < 0.05) despite similar glucose levels. Neonates born of mothers who were on antihypertensive treatment were smaller and had a higher insulin secretory response than neonates from normotensive mothers. Postnatal growth velocity (kilograms per day) correlated with birth weight (beta = -0.65; P < 0.0001) and insulin resistance (beta = -0.31; P = 0.0004), independently of each other. This study shows that glucose tolerance of the neonate is determined by weight attained at birth irrespective of gestational age and that maternal blood pressure may influence insulin sensitivity of the newborn. Furthermore, catch-up growth in neonates is determined by birth weight and insulin sensitivity.

The effect of acetylation of potato starch on swelling, enzymatic degradation, and bovine serum albumin (BSA, molecular mass 68 kDa) release rate from polymer films was studied. Potato starch and potato starch acetates (SA), having a degree of substitution of 1.9 or 2.6, were investigated. Polymer films were incubated in phosphate buffer solution pH 7.4 in the absence and presence of enzymes (alpha-amylase, amyloglucosidase, esterase) or in human serum. The acetylation of potato starch decreased its swelling considerably. Increased acetylation of starch also considerably retarded its enzymatic degradation. Due to the decreased swelling and degradation of SA films, BSA was released much slower from SA films than from potato starch films, both in the presence and absence of enzymes.

A number of traditional remedies used in South Africa contain pyrrolizidine alkaloids, some of which are hepatotoxic. We investigated the effect on human HuH-7 cells of Senecio latifolius DC., a plant that is a component of some traditional remedies and which is known to contain toxic pyrrolizidine alkaloids. Cells were also treated with extracts of a standard pyrrolizidine, retrorsine. The changes in the gross morphology of the cells were studied using light microscopy after haematoxylin and eosin staining. The cytoskeleton was investigated using fluorescence-labelled anti-beta-tubulin antibody and the nuclear organisation was studied using fluorescence-labelled antinuclear antibodies. The plant extracts gave rise to dose-dependent gross morphological changes. At high doses, we observed necrosis and at lower doses, destruction of the cytoskeleton, nuclear fragmentation and apoptosis. Doses of less than the equivalent of 330 ng/ml retrorsine led to multinucleated cells with failure in spindle formation and clumping of nuclear chromatin. This latter finding suggests that chronic low-dose treatment with such traditional remedies could give rise to teratogenic and/or carcinogenic effects.

Abnormalities observed in intermediary metabolism may be related to the pathogenesis of obesity-related diseases such as type 2 diabetes. Glycerol and lactate production was estimated in the sc adipose tissue of two anatomical regions of 10 lean (LW), 10 obese (OW), and 10 matched diabetic (DW) black urban women. This was done with the sc microdialysis technique and combined with adipose tissue blood flow (ATBF) rates calculated from (133)Xe clearance. Biochemical measurements were made in the postabsorptive and postprandial state. Bioimpedance and computed tomography scans were used to define body composition. DW present with more visceral fat (DW, 138 +/- 5.0; OW, 66.6 +/- 5.0 cm; P < 0.01). This was associated with elevated free testosterone levels (DW, 1.21 +/- 0.1; OW, 0.75 +/- 0.1 nmol/L; P < 0.05). The fasting FFA, glycerol, and lactate levels increased across the three groups (LW < OW < DW). During the oral glucose tolerance test, glucose levels were elevated in DW, with higher insulin levels [0 h: DW, 207 +/- 8.6; OW, 100 +/- 7.2 pmol/L (P < 0.01); 1 h: DW, 410 +/- 15.2; OW, 320 +/- 10.9 pmol/L (P < 0.05)], but with a flat Cpeptide response (1 h: DW, 932 +/- 40; OW, 1764 +/- 40 pmol/L; P < 0.05). Plasma lactate levels increased significantly in LW and OW at 1 h (P < 0.001), but remained lower in LW vs. OW for all time points. ATBF was highest in LW [abdominal, 0 h: DW, 4.5 +/- 0.2; OW, 1.7 mL/100 g.min (P < 0.01); femoral, 0 h: DW, 3.4 +/- 0.2; OW, 1.8 +/- 0.3 mL/100 g.min (P < 0.01)]. ATBF did not increase in DW during the oral glucose tolerance test. Glycerol release (GR) was used to assess the lipolytic rate and was highest in LW in the abdominal area [0 h: LW, 1.7 +/- 0.2; OW, 1.1 +/- 0.2 micromol/kg.min (P < 0.05); DW, 0.78 +/- 0.05 micromol/kg.min (P < 0.05 vs. OW)]. By contrast, GR was higher in the femoral area of OW (0 h: OW, 1.6 +/- 0.2; LW, 1.15 +/- 0.1 micromol/kg.min; P < 0.05). Regional differences were observed for GR in both OW and DW (femoral > abdominal). Lactate release (LR) was low in DW [abdominal, 0 h: DW, 3.5 +/- 0.4; OW, 7.8 +/- 1.0 micromol/kg.min (P < 0.001); femoral, 0 h: DW, 3.1 +/- 0.3; OW, 9.0 +/- 0.9 micromol/kg.min (P < 0.001)]. LR was appropriately low for body fat mass in LW, with a brisk increase between 0 and 1.5 h. A negative correlation exists between GR (abdominal area) and insulin levels in the postabsorptive state (P < 0.0001). In conclusion, 1) the fasting lipolytic rate is associated with insulin levels; 2) OW and DW have more adipose tissue insulin resistance than LW; 3) OW and DW have a brisker lipolysis in the femoral area; and 4) in DW, higher visceral mass is associated with elevated free testosterone and FFA concentrations. Obesity in the black population is therefore characterized by a marked degree of adipose tissue lipolysis. This degree of resistance together with increasing body fat mass may predispose the obese women to developing type 2 diabetes. Once this disease is established, the onset of adipose tissue vascular insulin resistance will sustain ongoing insulin resistance, even in the presence of relative insulinopenia.

There is a higher prevalence of ischemic heart disease (IHD) in South African white than black women. The objective of this study was to determine biochemical explanations for this prevalence. The study group contained 15 obese black women (OBW) and 14 obese white women (OWW), all premenopausal, who were examined after an overnight fast. Anthropometric measurements and blood concentrations of glucose, non-esterified fatty acids (NEFAs), catecholamines, plasminogen activator inhibitor-1, C-peptide, proinsulin, lipograms, cortisol, growth hormone, and post-heparin lipoprotein lipase activity were measured during an oral glucose tolerance test (OGTT). Body composition was measured using bioelectrical impedance analysis, and subcutaneous and visceral fat mass were assessed with CT-scans. Visceral fat area was higher in OWW (139.7 +/- 10.7 cm(2)) than in OBW (72.3 +/- 3.9 cm(2)) (P < 0.01), as were fasting and 3 h triglyceride concentrations (P < 0.05 for all). OWW also had higher NEFA levels than OBW at 3 and 4 h compared with OBW (P < 0.05 for both). Fasting cortisol (266 +/- 24 vs. 197 +/- 19 nmol/l; P < 0.05) was higher in OWW than in OBW. These data demonstrate that OWW have higher visceral fat mass than OBW, which may lead to a more atherogenic fasting and postprandial lipid profile. The higher cortisol levels of the OWW may promote visceral fat deposition.

Aims/hypothesis: This study aimed to assess the effects of fetal and childhood growth on beta-cell activity and insulin sensitivity in 7-year-old children., Methods: Insulin, des-31,32 proinsulin, proinsulin, non-esterified fatty acids and glucose concentrations were measured in oral glucose tolerance tests in 152 South African children for whom longitudinal weight data was available., Results: Children with low weights at birth and 7 years (low-low) had relatively low beta-cell activity whereas children with low birth weight and high weight at 7 years (low-high) had relatively high beta-cell activity. The low-low group had higher 30-min glucose concentrations than children with high birth weights. When each insulin-related peptide was expressed as a percentage of all these peptides the low-low children had the highest percentage of insulin but the lowest of the prohormones. The low-high children had the lowest percentage of insulin but the highest of the prohormones. Non-esterified fatty acid concentrations were lowest and their suppression post-glucose load highest in the low-high group., Conclusion/interpretation: Poor fetal and neonatal growth give rise to low beta-cell numbers compensated for by increased efficiency of proinsulin processing to insulin. Poor fetal followed by higher postnatal growth results in low beta-cell numbers and reduced whole-body glucose uptake which leads to reduced efficiency in the processing of proinsulin. Growth in utero and postnatally therefore have profound effects on beta-cell activity and insulin sensitivity with poor fetal coupled with high postnatal growth being detrimental to these processes but not detrimental to the suppression of lipolysis.

Objective: The goal of this study was to quantify differences in lipid metabolism and insulin sensitivity in black and white subjects to explain ethnic clinicopathological differences in type 2 diabetes., Research Methods and Procedures: The in vitro lipolytic activity of adipocytes isolated from obese black and white women was measured in the presence of insulin and isoproterenol. Insulin resistance was assessed in vivo using the euglycemic hyperinsulinemic clamp technique., Results: Fasting plasma levels of insulin and nonesterified fatty acid (NEFA) in black and white women were 67 +/- 5 pM vs. 152 +/- 20 pM (p < 0.01) and 863 +/- 93 microM vs. 412 +/- 34 microM (p < 0.01), respectively. Euglycemic hyperinsulinemic clamp studies showed that obese black subjects were more insulin-resistant than their white counterparts (glucose infusion rates: 1.3 +/- 0.2 vs. 2.2 +/- 0.3 mg/kg per min; p < 0.05). Isolated adipocytes from white women were more responsive to insulin than those from black women with 0.7 nM insulin causing a 55 +/- 4% inhibition of isoproterenol-stimulated lipolysis compared with 27 +/- 10% in black women (p < 0.05)., Discussion: The low responsiveness of adipocyte lipolytic activity to insulin in black women in the presence of a relative insulinopenia may account for the high plasma NEFA levels seen in these women, which may, in turn, account for their higher in vivo insulin resistance. High NEFA levels may also contribute to the low insulin secretory activity observed in the obese black females. These data suggest that the pathogenesis of insulin resistance and type 2 diabetes within the black obese community is strongly influenced by their adipocyte metabolism.

Objective: To investigate the relationship between leptin concentrations, various metabolic indices and body composition in six different groups., Design and Measurements: Anthropometric measurements, fasting plasma glucose, serum insulin, C-peptide, FFA and leptin levels were performed. In the obese and diabetic subjects, body composition was analysed with bio-impedance equipment and as a 5 level CT scan., Subjects: Five lipoatrophic diabetes mellitus (LDM) patients, five normal subjects (N), nine white and nine black obese women (WW, BW), and nine white and nine black diabetic women (DWW, DBW) were investigated after an overnight fast., Results: In both ethnic groups there was a positive correlation between leptin and BMI (black group: r=0.8; P<0.0001, white group: r=0.7, P<0.002) and leptin and SC fat mass (black group: r=0.6; P<0.005, white group: r=0.6; P<0.004)., Conclusions: Across the groups, there were positive linear correlations between leptin concentrations, BMI, SC fat mass and FFA levels. Leptin and FFA concentrations are higher and insulin levels lower in both groups of black women compared to the two groups of white women, despite a similar BMI and body fat mass. In the DBW the large increase in visceral fat mass may be indicative of a more complex relationship between compensatory insulin resistance, elevated FFA levels and leptin secretion.

To measure interstitial glycerol and lactate production from the sc adipose tissue of two regions in nine black and nine white lean men, sc microdialysis was performed in combination with adipose tissue blood flow rates measured with 133Xe clearance. In the postabsorptive state, the plasma glucose and insulin levels of the black men and white men were similar. The black men had higher plasma free fatty acids (825+/-97 vs. 439+/-58 micromol/L; P < 0.005), glycerol (99.5+/-5.1 vs. 54.1+/-3.3 micromol/L; P < 0.0001), and lactate (1056+/-95 vs. 729+/-45 micromol/L; P < 0.01). Interstitial glycerol concentrations in the black and white men were 227 vs. 163 micromol/L (P < 0.01) and 230 vs. 162 micromol/L (P < 0.05) in the abdominal and femoral regions. The adipose tissue blood flow rate was higher in the black men in the abdominal (7.9+/-0.9 vs. 3.1+/-0.5 mL/100 g x min; P < 0.01) and femoral area (5.2+/-0.6 vs. 2.8+/-0.3; P < 0.01). Interstitial lactate concentrations in black and white men were 1976 vs. 1364 micromol/L (P < 0.004) and 1953 vs. 1321 micromol/L (P < 0.004) in the abdominal and femoral regions, respectively. Glycerol release was higher in black men vs. white men for abdominal (0.21+/-0.02 vs. 0.14+/-0.02 micromol/100 g x min; P < 0.02) and femoral (0.22+/-0.02 vs. 0.15+/-0.01; P < 0.05) areas. Postprandially, black men had higher plasma glucose levels [1 h, 9.6+/-0.4 vs. 8.2+/-0.5 mmol/L (P < 0.05); 2 h, 8.9+/-0.4 vs. 7.2+/-0.4 mmol/L (P < 0.01)], but lower plasma insulin levels [1 h, 173+/-13 vs. 264+/-48 pmol/L (P < 0.05); 2 h, 136+/-20 vs. 209+/-34 pmol/L (P < 0.05)]. Plasma free fatty acid, lactate, and glycerol levels remained higher in the black men. After 1 h, lactate release was higher in the black men vs. that in the white men for abdominal (20.5+/-1.6 vs. 14.7+/-2.5 micromol/100 g x min;P < 0.05) and femoral (15.6+/-1.1 vs. 12.1+/-1.8; P < 0.03) areas. We conclude that the black men, who are relatively insulinopenic postprandially, have a brisker lipolysis and also release more lactate from sc fat tissue than white men. These differences in adipose tissue metabolism may be related to differences in the lipid profiles and glucose metabolism previously documented in these ethnic groups.

Interstitial glycerol and lactate production was measured in the s.c. adipose tissue of two anatomical regions in 10 obese urban black women (BW) and 10 obese urban white women (WW) matched for age, body mass index, waist-hip ratio, diet, and physical activity. This was done with the s.c. microdialysis technique and combined with adipose tissue blood flow (ATBF) rates calculated from 133Xe clearance. Biochemical measurements were done in the postabsorptive and postprandial state. Bioimpedance and computed tomography scans were used for analyses of body composition. BW responded with lower plasma insulin levels, but higher glucose levels, during the oral glucose tolerance test. BW have higher lactate release from the s.c. adipose tissue, compared with WW, in the postabsorptive state (abdominal: 7.8 +/- 0.9 vs. 2.4 +/- 0.3 micromol/kg x min, P < 0.0001; femoral: 9.1 +/- 0.9 vs. 2.1 +/- 0.3 micromol/kg x min, P < 0.0001) and during the postprandial period (at 1 h, abdominal = 7.3 +/- 0.8 vs. 3.0 +/- 0.4 micromol/kg x min, P < 0.0001, femoral area = 8.1 +/- 1.0 vs. 2.7 +/- 0.4 micromol/kg x min, P < 0.0001; at 2 h, abdominal = 5.7 +/- 0.4 vs. 3.1 +/- 0.3 micromol/kg x min, P < 0.001). The BW also released more glycerol from the sc adipose tissue in the postabsorptive state (abdominal = 1.15 +/- 0.17 vs. 0.65 +/- 0.03 micromol/ kg x min, P < 0.009; femoral = 1.55 +/- 0.19 vs. 0.72 +/- 0.05 micromol/kg x min, P < 0.001) and during the postprandial period (at 1 h, abdominal = 1.05 +/- 0.15 vs. 0.11 +/- 0.02 micromol/kg x min, P < 0.001, femoral = 1.05 +/- 0.12 vs. 0.21 +/- 0.03 micromol/kg x min, P < 0.001; at 2 h, abdominal = 0.31 +/- 0.06 vs. 0.04 +/- 0.01 micromol/kg x min, P < 0.001, femoral = 0.28 +/- 0.07 vs. 0.05 +/- 0.01 micromol/kg x min, P < 0.003). Postprandially, the BW had higher ATBF rates in the abdominal and femoral areas. WW have more visceral fat (150 +/- 2.0 vs. 110 +/- 5.0 cm2, P < 0.05). In conclusion, the insulinopenic BW have a brisker lipolysis and ATBF and release more glycerol and lactate from their sc adipose tissue, both in the postabsorptive state and after an oral glucose tolerance test. These variations in adipose tissue metabolism may contribute to differences observed in the disease profiles of these two groups of women.

A number of studies have shown that glucose tolerance falls with decreasing birth weight and that people with low birth weight and high body mass index (BMI) as adults are those at greatest risk of developing Type II (non-insulin-dependent) diabetes mellitus. No such studies have been carried out in African populations. Therefore we investigated the relation between glucose tolerance and birth weight in a group of 7-year-old black South Africans for whom longitudinal anthropometric data were available. Oral glucose tolerance tests (OGTTs) were carried out on 152 subjects and inverse correlations were found between birth weight and the total amount of insulin secreted during the first 30 min (r = -0.19, p = 0.04) and last 90 min (r = -0.19, p = 0.04) of the oral glucose tolerance test and also between birth weight and the 30 min glucose concentrations (r = -0.20, p = 0.02). Children born with low birth weights but who had high weights at 7 years had higher insulin concentrations and indices of obesity compared with those with low birth weights and low weights at 7 years. There were also positive correlations between weight velocity and BMI (r = 0.24, p = 0.02) and weight velocity and insulin resistance (r = 0.18, p = 0.04) as measured using homeostasis model assessment (HOMA). Thus, low birth weight in conjunction with rapid childhood gains in weight especially as subcutaneous fat, produces poor glucose tolerance in 7-year-old children and can make them susceptible to the development of Type II diabetes later in life.

It is becoming well established that poor fetal and early postnatal growth can have long-term effects on adult health, including susceptibility to non-insulin-dependent diabetes mellitus, cardiovascular disease and hypertension. It is suggested that this results from poor nutrition during early life having permanent effects on the structure and metabolism of certain organs and tissues. In the present study we investigated the effect of a low-protein diet during pregnancy and lactation on adipocyte properties and glucose tolerance. Rat dams were fed on a diet containing either 200 (control) or 80 (low protein) g protein/kg during pregnancy and lactation. In addition cross-fostering techniques were employed to enable a separate evaluation of the prenatal and postnatal periods. All offspring were weaned onto a 200 g protein/kg diet at 21 d of age and then studied at 6 weeks of age. The mothers' protein supply during lactation appeared to be the most critical time window for long-term growth. In contrast, the offspring of mothers fed on a low-protein diet during pregnancy or lactation were significantly more glucose tolerant than controls, suggesting that both time windows can have long-term effects on glucose tolerance. In addition offspring of mothers fed on a low-protein diet during pregnancy or lactation had significantly smaller adipocytes than controls. However the largest reduction in adipocyte size was observed when there was a low-protein diet during both pregnancy and lactation. The amount of insulin receptor present in adipocyte membranes was increased in the three animal groups that had been exposed to the low-protein diets while levels of the insulin responsive glucose transporter (GLUT 4) were similar in adipocyte membranes from all groups.

Recent epidemiological studies in people whose birth weights were recorded many years ago suggest links between impaired growth during early life and the development of diseases, including diabetes, much later in life. The long-term effects of retarded early growth are proposed to result from malnutrition at critical periods of fetal or infant development leading to reduction in the growth of organs and permanent changes in their metabolism or structure, or both. In order to investigate this, a rat model was established which involved feeding either a diet containing 200 g protein/kg or an isoenergetic diet containing 80 g protein/kg to pregnant and lactating rats. In addition, cross-fostering techniques were employed which allowed a separate evaluation of the prenatal or the postnatal periods. The offspring were studied at 21 d of age or were weaned onto a normal laboratory chow and studied at 11 months of age. The 80 g protein/kg diet during pregnancy did not affect the overall reproductive although more subtle differences were evident. Permanent growth retardation was evident in offspring subjected to maternal protein restriction during the postnatal period. At 21 d of age the offspring of protein-restricted mothers exhibited selective changes in organ growth: compared with the body weight, the lung and brain experienced a smaller decrease in weight: the heart, kidney and thymus decreased proportionately: whereas, the pancreas, spleen, muscle and liver showed a greater reduction in weight. In older animals the muscle weight was lower in the male rats and the relative weight of pancreas was increased in the female rats.

Residues belonging to epitopes on human insulin that were recognized by a panel of three monoclonal antibodies were located using mutated insulins and insulins from a number of different animal species. Epitopes on human proinsulin recognized by two monoclonal antibodies were also identified using partially processed proinsulin species. Epitopes were located on the C-A and B-C junctions of proinsulin and on the N-termini of the A- and B-chains and the central region of the B-chain of human insulin. Antibodies that bound proinsulin were found to induce conformational changes in the prohormone. The presence of a well-defined interaction between the C-peptide portion and the N-terminus of the A-chain of the insulin moiety of intact proinsulin has also been demonstrated. The relevance of these studies to the development of two-site assays for the measurement of partially processed proinsulin species in human sera is also discussed.

Since porcine islets are considered a likely tissue source for islet transplantation we have studied the insulin secretory responses to stimuli and some of the cell surface antigen characteristics of porcine islet cells. In a static incubation system, the threshold level of glucose required for the stimulation of insulin secretion from freshly isolated porcine islets was found to be between 2.8 and 4.2 mmol glucose/l. Arginine (5 mmol/l) and 3-isobutyl-l-methylxanthine (1 mmol/l) potentiated insulin release induced by 8.3 mmol glucose/l. Leucine (5 mmol/l) initiated release in the presence of 2 mmol glucose/l. Neither beta-hydroxybutyrate (10 mmol/l) nor octanoate (5 mmol/l) potentiated insulin release induced by 8.3 mmol glucose/l, but beta-hydroxybutyrate initiated release in the presence of 2 mmol glucose/l while octanoate did not. A 125I-labelled protein A binding assay and an enzyme-linked immunosorbent assay system were used to detect antibody binding to islet and non-islet cells. Monoclonal antibodies raised against intact rat islets were shown to bind to both porcine and rat islet cells but not to rat hepatoma tissue culture cells or rat insulinoma cells. The serum from recently diagnosed type I diabetics was shown to bind to rat islet cells in a 125I-labelled protein A binding assay, while serum from control subjects showed little, if any, binding. Porcine islet cells were unable to distinguish between the sera of recently diagnosed type I diabetics and controls in a similar assay. In conclusion, porcine islets respond to many of the major insulin secretagogues to which human islets are sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Porcine islets were isolated by infusion of a warm collagenase solution into whole pancreata followed by static incubation at 37 degrees C for 15 minutes. The pancreata were then chopped into small pieces and the free islets purified by filtration and centrifugation over a ficoll gradient. The insulin:amylase ratio of the islets compared to that in the intact pancreas was determined in 19 pancreata and indicates that the isolated islets were of a high degree of purity. The distribution of insulin, glucagon, somatostatin and pancreatic polypeptide containing cells in pig pancreas sections was compared with that in rat. Porcine islets were much smaller and less well defined than rat islets with infiltration of acinar material even into the islet core. The levels of insulin, glucagon and somatostatin in porcine pancreas and isolated porcine islets were measured using conventional radioimmunoassay techniques. The ratio of these hormones in the pancreas was 105.1:5.8:1 respectively, and in the islets 105.1:0.68:0.087 respectively. Fragmentation of the islets during the isolation may have led to the loss of glucagon and somatostatin-containing cells. Islets cultured overnight and tested with a range of glucose concentrations for one hour did not show a significant stimulation of insulin secretion in the presence of 8.3 mM or 16.7 mM glucose compared to that in 2.8 mM glucose. However freshly isolated islets challenged with 8.3 mM, 13.9 mM and 22.2 mM glucose showed a 1.8 fold, 2.0 fold and 2.3 fold response respectively, over that in 2.8 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)