Human dietary exposure to heavy metals via the consumption of greenshell mussels (Perna canaliculus Gmelin 1791) from the Bay of Islands, northern New Zealand.

Cadmium (Cd), mercury (Hg), arsenic (As), lead (Pb) and tin (Sn) concentrations were determined using ICP-MS in soft tissues (wet wt.) from whole greenshell mussels (Perna canaliculus) collected from Urapukapuka-Rawhiti Island, Opua Marina, Waitangi Bridge and Opua Wharf from the Bay of Islands, northern New Zealand (NZ). All samples had metal concentrations well below the Food Standards Australia and New Zealand (FSANZ) maximum limits and were comparable to, or less than, concentrations observed in previous NZ studies. Based on the average values detected in the current study, the concentrations of heavy metals ingested in a 'typical diet' containing greenshell mussels are below the provisional tolerable weekly intake (PTWI). However, Maori (indigenous people of New Zealand), Pacific Islanders and Asians consume a far greater quantity of seafood (and therefore heavy metals) than the general public of New Zealand and could potentially consume enough shellfish to exceed the PTWI for Cd (but not for Hg, As, Pb or Sn). Although our results, based on the current PTWIs, indicate no significant health risk to greenshell mussel consumers in this region, PTWIs change over time; concentrations which were thought to be safe are later found to be harmful. Additionally, differences in individual human susceptibilities to various toxins could increase the risk of harm for consumers with low tolerance to heavy metals. We suggest that a survey of the frequency, amount and species consumed by groups whose diet may be largely shellfish-based is required to enable a more comprehensive risk assessment to be made.

Developing the public health practitioner workforce in England: lessons from theory and practice.

FOCUS: The paper focuses on public health practitioners who collectively represent one of three key workforce groups identified by England's Chief Medical Officer as critical to the successful delivery of national public health policy priorities. QUESTION: We report on two areas of work which attempt to address the following two-part question: in developing the public health practitioner workforce in England, what is needed, and how do we do it? APPROACH: First, we describe a five-component conceptual framework for developing the public health workforce which is grounded in data derived from a national Open Space event hosted by the University of the West of England in March 2005. The five components are (i) strategic support and oversight; (ii) national technical and professional support; (iii) national career building; (iv) local organisational development, and (v) sub-regional skills development. Key elements of each component are described in the paper. Second, we describe in some detail a new multidisciplinary skills development programme which illustrates one of the framework components (sub-regional skills development). The programme, established in January 2005, is aimed at three key groups of public health practitioners: health visitors (specialist community public health nurse), school nurses and environmental health officers. Its main features and some initial evaluation findings are presented. CONCLUSIONS: To be effective, activities aimed at supporting the development of the public health practitioner workforce should, where possible, aim to be congruent with core public health principles of self-determination and collective responsibility. We also conclude that leadership and vision at a national level, combined with local implementation of evidence-based training programme such as the one described could help to achieve much greater and more rapid progress in skilling up the existing public health practitioner workforce than has been possible up to now. But we note that this requires sustained investment, robust sector-wide delivery frameworks, and a group of committed local public health champions.

Towards smaller and faster gas chromatography-mass spectrometry systems for field chemical detection.

Gas chromatography-mass spectrometry (GC-MS) is already an important laboratory method, but new sampling techniques and column heating approaches will expand and improve its usefulness for detection and identification of unknown chemicals in field settings. In order to demonstrate commercially-available technical advances for both sampling and column heating, we used solid phase microextraction (SPME) sampling of both water and air systems, followed by immediate analysis with a resistively heated analytical column and mass spectrometric detection. High-concern compounds ranging from 140 to 466 amu were analyzed to show the applicability of these techniques to emergency situations impacting public health. A field portable (about 35 kg) GC-MS system was used for analysis of water samples with a resistively heated analytical column externally mounted as a retrofit using the air bath oven of the original instrument design to heat transfer lines. The system used to analyze air samples included a laboratory mass spectrometer with a dedicated resistive column heating arrangement (no legacy air bath column oven). The combined sampling and analysis time was less than 10 min for both air and water sample types. By combining dedicated resistive column heating with smaller mass spectrometry systems designed specificallyfor use in the field, substantially smaller high performance field-portable instrumentation will be possible.

Measurements of alkali-labile DNA damage and protein-DNA crosslinks after 2450 MHz microwave and low-dose gamma irradiation in vitro.

In vitro experiments were performed to determine whether 2450 MHz microwave radiation induces alkali-labile DNA damage and/or DNA-protein or DNA-DNA crosslinks in C3H 10T(1/2) cells. After a 2-h exposure to either 2450 MHz continuous-wave (CW) microwaves at an SAR of 1.9 W/kg or 1 mM cisplatinum (CDDP, a positive control for DNA crosslinks), C3H 10T(1/2) cells were irradiated with 4 Gy of gamma rays ((137)Cs). Immediately after gamma irradiation, the single-cell gel electrophoresis assay was performed to detect DNA damage. For each exposure condition, one set of samples was treated with proteinase K (1 mg/ml) to remove any possible DNA-protein crosslinks. To measure DNA-protein crosslinks independent of DNA-DNA crosslinks, we quantified the proteins that were recovered with DNA after microwave exposure, using CDDP and gamma irradiation, positive controls for DNA-protein crosslinks. Ionizing radiation (4 Gy) induced significant DNA damage. However, no DNA damage could be detected after exposure to 2450 MHz CW microwaves alone. The crosslinking agent CDDP significantly reduced both the comet length and the normalized comet moment in C3H 10T(1/2) cells irradiated with 4 Gy gamma rays. In contrast, 2450 MHz microwaves did not impede the DNA migration induced by gamma rays. When control cells were treated with proteinase K, both parameters increased in the absence of any DNA damage. However, no additional effect of proteinase K was seen in samples exposed to 2450 MHz microwaves or in samples treated with the combination of microwaves and radiation. On the other hand, proteinase K treatment was ineffective in restoring any migration of the DNA in cells pretreated with CDDP and irradiated with gamma rays. When DNA-protein crosslinks were specifically measured, we found no evidence for the induction of DNA-protein crosslinks or changes in amount of the protein associated with DNA by 2450 MHz CW microwave exposure. Thus 2-h exposures to 1.9 W/ kg of 2450 MHz CW microwaves did not induce measurable alkali-labile DNA damage or DNA-DNA or DNA-protein crosslinks.

Repeatability characteristics of simple indices of insulin resistance: implications for research applications.

The objectives of this study were to evaluate test characteristics, such as normality of distribution, variation, and repeatability, of simple fasting measures of insulin sensitivity and to use the results to choose among these measures. Duplicate fasting samples of insulin and glucose were collected before 4 h of euglycemic hyperinsulinemic clamping using insulin infusion rates ranging from 40-600 mU/m2 x min. Currently recommended estimates of insulin sensitivity, including the fasting insulin, 40/insulin, the homeostasis model assessment, the logarithmic transformation of the homeostasis model assessment, and the Quantitative Insulin Sensitivity Check Index, were evaluated. The normality of distribution and the variability of the tests (coefficient of variation and discriminant ratio) were compared between the measures and against the "gold standard" hyperinsulinemic clamp. Data from 253 clamp studies in 152 subjects were examined, including 79 repeated studies for repeatability analysis. In subjects ranging from lean to diabetic, the log transformed fasting measures combining insulin and glucose had normal distributions and test characteristics superior to the other simple indices (logarithmic transformation of the homeostasis model assessment coefficient of variation, 0.55; discriminant ratio, 13; Quantitative Insulin Sensitivity Check Index coefficient of variation, 0.05; discriminant ratio, 10) and statistically comparable to euglycemic hyperinsulinemic clamps (coefficient of variation, 0.10; discriminant ratio, 6.4). These favorable characteristics helped explain the superior correlations of these measures with the hyperinsulinemic clamps among insulin-resistant subjects. Furthermore, therapeutic changes in insulin sensitivity were as readily demonstrated with these simple measures as with the hyperinsulinemic clamp. The test characteristics of the logarithmic transformation of the homeostasis model assessment and the Quantitative Insulin Sensitivity Check Index are superior to other simple indices of insulin sensitivity. This helps explain their excellent correlations with formal measures both at baseline and with changes in insulin sensitivity and supports their broader application in clinical research.

Polycystic ovary syndrome is associated with endothelial dysfunction.

BACKGROUND: We recently reported endothelial dysfunction as a novel cardiovascular risk factor associated with insulin resistance/obesity. Here, we tested whether hyperandrogenic insulin-resistant women with polycystic ovary syndrome (PCOS) who are at increased risk of macrovascular disease display impaired endothelium-dependent vasodilation and whether endothelial function in PCOS is associated with particular metabolic and/or hormonal characteristics. METHODS AND RESULTS: We studied leg blood flow (LBF) responses to graded intrafemoral artery infusions of the endothelium-dependent vasodilator methacholine chloride (MCh) and to euglycemic hyperinsulinemia in 12 obese women with PCOS and in 13 healthy age- and weight-matched control subjects (OBW). LBF increments in response to MCh were 50% lower in the PCOS group than in the OBW group (P:<0.01). Euglycemic hyperinsulinemia increased LBF above baseline by 30% in the PCOS and 60% in OBW group (P:<0.05 between groups). Across all subjects, the maximal LBF response to MCh exhibited a strong inverse correlation with free testosterone levels (r=-0.52, P:<0.007). This relationship was stronger than with any other parameter, including insulin sensitivity. CONCLUSIONS: PCOS is characterized by (1) endothelial dysfunction and (2) resistance to the vasodilating action of insulin. This endothelial dysfunction appears to be associated with both elevated androgen levels and insulin resistance. Given the central vasoprotective role of endothelium, these findings could explain, at least in part, the increased risk for macrovascular disease in women with PCOS.

Evidence for physiological coupling of insulin-mediated glucose metabolism and limb blood flow.

We hypothesized that the vasodilation observed during insulin stimulation is closely coupled to the rate of glucose metabolism. Lean (L, n = 13), obese nondiabetic (OB, n = 13), and obese type 2 diabetic subjects (Type 2 DM, n = 16) were studied. Leg blood flow (LBF) was examined under conditions of euglycemic hyperinsulinemia (EH) and hyperglycemic hyperinsulinemia (HH), which produced a steady-state whole body glucose disposal rate (GDR) of approximately 2,000 micromol. m(-2). min(-1). At this GDR, under both conditions, subjects across the range of insulin sensitivity exhibited equivalent LBF (l/min EH: L, 0.42 +/- 0.03; OB, 0.43 +/- 0. 03; Type 2 DM, 0.38 +/- 0.07; P = 0.72 by ANOVA. HH: L, 0.44 +/- 0. 04; OB, 0.39 +/- 0.05; Type 2 DM, 0.41 +/- 0.04; P = 0.71). The continuous relationship between LBF and GDR did not differ across subject groups [slope x 10(-5) l/(micromol. m(-2). min(-1)) by ANOVA. EH: L, 8.6; OB, 9.2; Type 2 DM, 7.9; P = 0.91. HH: L, 4.2; OB, 2.5; Type 2 DM, 4.1; P = 0.77], although this relationship did differ between the EH and HH conditions (P = 0.001). These findings support a physiological coupling of LBF and insulin-mediated glucose metabolism. The mechanism(s) linking substrate delivery and metabolism appears to be intact in insulin-resistant states.

Micronucleated erythrocyte frequency in peripheral blood of B6C3F(1) mice from short-term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program.

The mouse peripheral blood micronucleus (MN) test was performed on samples collected from 20 short-term, 67 subchronic, and 5 chronic toxicity and carcinogenicity studies conducted by the National Toxicology Program (NTP). Data are presented for studies not previously published. Aspects of protocol that distinguish this test from conventional short-term bone marrow MN tests are duration of exposure, and absence of repeat tests and concurrent positive controls. Furthermore, in contrast to short-term bone marrow MN tests where scoring is limited to polychromatic erythrocytes (PCE), longer term studies using peripheral blood may evaluate MN in both, or either, the normochromatic (NCE) or PCE populations. The incidence of MN-PCE provides an index of damage induced within 72 hr of sampling, whereas the incidence of MN in the NCE population at steady state provides an index of average damage during the 30-day period preceding sampling. The mouse peripheral blood MN test has been proposed as a useful adjunct to rodent toxicity tests and has been effectively incorporated as a routine part of overall toxicity testing by the NTP. Data derived from peripheral blood MN analyses of dosed animals provide a useful indication of the in vivo potential for induced genetic damage and supply an important piece of evidence to be considered in the overall assessment of toxicity and health risk of a particular chemical. Although results indicate that the test has low sensitivity for prediction of carcinogenicity, a convincingly positive result in this assay appears to be highly predictive of rodent carcinogenicity.

Interaction between insulin sensitivity and muscle perfusion on glucose uptake in human skeletal muscle: evidence for capillary recruitment.

Insulin and glucose delivery (muscle perfusion) can modulate insulin-mediated glucose uptake. This study was undertaken to determine 1) to what extent insulin sensitivity modulates the effect of perfusion on glucose uptake and 2) whether this effect is achieved via capillary recruitment. We measured glucose disposal rates (GDRs) and leg muscle glucose uptake (LGU) in subjects exhibiting a wide range of insulin sensitivity, after 4 h of steady-state (SS) euglycemic hyperinsulinemia (>6,000 pmol/l) and subsequently after raising the rate of leg blood flow (LBF) 2-fold with a superimposed intrafemoral artery infusion of methacholine chloride (Mch), an endothelium-dependent vasodilator. LBF was determined by thermodilution: LGU = arteriovenous glucose difference (AVGdelta) x LBF. As a result of the 114+/-12% increase in LBF induced by Mch, the AVGdelta decreased 32+/-4%, and overall rates of LGU increased 40+/-5% (P < 0.05). We found a positive relationship between the Mch-modulated increase in LGU and insulin sensitivity (GDR) (r = 0.60, P < 0.02), suggesting that the most insulin-sensitive subjects had the greatest enhancement of LGU in response to augmentation of muscle perfusion. In separate groups of subjects, we also examined the relationship between muscle perfusion rate and glucose extraction (AVGdelta). Perfusion was either pharmacologically enhanced with Mch or reduced by intra-arterial infusion of the nitric oxide inhibitor N(G)-monomethyl-L-arginine during SS euglycemic hyperinsulinemia. Over the range of LBF, changes in AVGdelta were smaller than expected based on the noncapillary recruitment model of Renkin. Together, the data indicate that 1) muscle perfusion becomes more rate limiting to glucose uptake as insulin sensitivity increases and 2) insulin-mediated increments in muscle perfusion are accompanied by capillary recruitment. Thus, insulin-stimulated glucose uptake displays both permeability- and perfusion-limited glucose exchange properties.

Free fatty acid elevation impairs insulin-mediated vasodilation and nitric oxide production.

The effect and time course of free fatty acid (FFA) elevation on insulin-mediated vasodilation (IMV) and the relationship of FFA elevation to changes in insulin-mediated glucose uptake was studied. Two groups of lean insulin-sensitive subjects underwent euglycemic-hyperinsulinemic (40 mU x m(-2) x min(-1)) clamp studies with and without superimposed FFA elevation on 2 occasions approximately 4 weeks apart. Groups differed only by duration of FFA elevation, either short (2-4 h, n = 12) or long (8 h, n = 7). On both occasions, rates of whole-body glucose uptake were measured, and changes in leg blood flow (LBF) and femoral vein nitric oxide nitrite plus nitrate (NOx) flux in response to the clamps were determined. Short FFA infusion did not have any significant effect on the parameters of interest. In contrast, long FFA infusion decreased rates of whole-body glucose uptake from 47.7 +/-2.8 to 32.2 +/- 0.6 micromol x kg(-1) x min(-1) (P < 0.01), insulin-mediated increases in LBF from 66 +/- 8 to 37 +/- 7% (P < 0.05), and insulin-induced increases in NOx flux from 25 +/- 9 to 5 +/- 9% (P < 0.05). Importantly, throughout all groups, FFA-induced changes in whole-body glucose uptake correlated significantly with FFA-induced changes in insulin-mediated increases in LBF (r = 0.706, P < 0.001), which indicates coupling of metabolic and vascular effects. In a different protocol, short FFA elevation blunted the LBF response to NG-monomethyl-L-arginine (L-NMMA), which is an inhibitor of NO synthase. LBF in response to L-NMMA decreased by 17.3 +/- 2.4 and 9.0 +/- 1.4% in the groups without and with FFA elevation, respectively (P < 0.05), which indicates that FFA elevation interferes with shear stress-induced NO production. Thus, impairment of shear stress-induced vasodilation and IMV by FFA elevation occurs with different time courses, and impairment of IMV occurs only if glucose metabolism is concomitantly reduced. These findings suggest that NO production in response to the different stimuli may be mediated via different signaling pathways. FFA-induced reduction in NO production may contribute to the higher incidence of hypertension and macrovascular disease in insulin-resistant patients.

Type II diabetes abrogates sex differences in endothelial function in premenopausal women.

BACKGROUND: Obesity is a more potent cardiovascular risk factor (CVRF) in men than in women. Because traditional CVRFs cannot fully account for this sex difference, we tested the hypothesis that compared with men, women exhibit more robust endothelial function independent of obesity and that this sex difference is abrogated by diabetes. METHODS AND RESULTS: We studied leg blood flow (LBF) responses to graded intrafemoral artery infusions of the endothelium-dependent vasodilator methacholine chloride (Mch) and the endothelium-independent vasodilator sodium nitroprusside (SNP) in groups of lean, obese (OB), and type II diabetic (DM) premenopausal women and age- and body mass index-matched men. LBF response to intrafemoral administration of L-NMMA, an inhibitor of nitric oxide synthase, was also assessed in normal men and women. Maximum LBF increments in response to Mch were 347+/-57% versus 231+/-22% in lean women versus men (P<0.05) and 203+/-25% versus 111+/-17% in OB women versus men (P<0.01), respectively. In DM, maximum LBF increments in response to Mch were 104+/-24% and 138+/-33% in women and men, respectively, (P=NS). LBF decrements in response to L-NMMA were 34.9+/-4.1% and 17.1+/-4.2% in women and men, respectively (P<0.01). The response to SNP was not different between sexes and groups. CONCLUSIONS: Premenopausal nondiabetic women exhibit more robust endothelium-dependent vasodilation owing to higher rates of nitric oxide release than men. Given the protective vascular action of nitric oxide, this difference may partially explain the lower incidence of macrovascular disease in women. In premenopausal women, DM causes impairment of endothelial function beyond that observed with obesity alone and leads to endothelial dysfunction similar to that observed in DM men. These findings may help explain the similar rates of coronary artery disease and mortality in diabetic men and women.

Treatment of breast abscesses with US-guided percutaneous needle drainage without indwelling catheter placement.

PURPOSE: To assess ultrasonographically (US) guided needle aspiration of breast abscesses as an alternative to surgical incision and drainage or indwelling catheter placement. MATERIALS AND METHODS: The authors reviewed hospital records from 1995 to 1997 for patients undergoing US-guided aspiration of breast abscesses. Two radiologists reviewed the US, mammographic, and US-guided aspiration studies for the size, appearance, and drainage of abscesses. The medical records were reviewed for follow-up data. RESULTS: Thirteen patients aged 15-69 years underwent US-guided percutaneous aspiration of 13 breast abscesses. All patients presented with a palpable mass, nine of which were retroareolar. At US, four abscesses were oval, nine (including three with septa) were irregularly shaped, and five had a thick rind. Of seven abscesses 2.4 cm or smaller, two were almost completely drained and five were completely aspirated. All seven abscesses resolved without surgery. Of six women with incompletely aspirated abscesses larger than 2.4 cm (one 3 cm, four 4 cm, one 7 cm), five required surgical referral; one of these cases was referred after repeat aspiration had been performed. CONCLUSION: Percutaneous aspiration of breast abscesses can enable diagnosis of abscesses and be used to treat small abscesses if they are completely drained. Partial drainage of abscesses larger than 3 cm may be palliative, but incision and drainage still may be necessary for definitive treatment.

Dual energy X-ray absorptiometry assessment of fat mass distribution and its association with the insulin resistance syndrome.

OBJECTIVE: To determine which dual energy X-ray absorptiometry (DXA)-derived indices of fat mass distribution are the most informative to predict the various parameters of the metabolic syndrome. RESEARCH DESIGN AND METHODS: A total of 87 healthy men, 63 lean (% fat < or =26) and 24 obese (% fat >26), underwent DXA scanning to evaluate body composition with respect to the whole body and the trunk, leg, and abdominal regions from L1 to L4 and from L3 to L4. These regions were correlated with insulin sensitivity determined by the euglycemic-hyperinsulinemic clamp, insulin area under the curve after oral glucose tolerance test (AUC I); triglyceride; total, HDL, and LDL cholesterol; free fatty acids; and blood pressure. The analyses were performed in all subjects, as well as in lean and obese groups separately. RESULTS: Among the various indices of body fat, DXA-determined adiposity in the abdominal cut at L1-4 level was the most predictive of the metabolic variables, showing significant relationships with glucose infusion rate ([GIR], mg kg(-1) lean body mass x min(-1)), triglyceride, and cholesterol, independent of total-body mass (r = -0.267, P<0.05; r = 0.316, P<0.005; and r = 0.319, P<0.005, respectively). Upon subanalysis, these correlations remained significant in lean men, whereas in obese men, only BMI and the amount of leg fat (negative relationship) showed significant correlations with triglyceride and cholesterol (r = 0.438, P<0.05; r = 0.458, P<0.05; r = -0.439, P<0.05; and r = -0.414, P<0.05, respectively). The results of a multiple regression analysis revealed that 47% of the variance in GIR among all study subjects was predicted by AUC I, fat L1-4, diastolic blood pressure (dBP), HDL, and triglyceride as independent variables. In the lean group, fat L1-4 alone accounted for 33% of the variance of GIR, whereas in obese men, AUC I and dBP explained 68% of the variance in GIR. CONCLUSIONS: The DXA technique applied for the evaluation of fat distribution can provide useful information regarding various aspects of the insulin resistance syndrome in healthy subjects. DXA can be a valid, accurate, relatively inexpensive, and safer alternative compared with other methods to investigate the role of abdominal body fat distribution on cardiovascular risk factors.

Recommendations for statistical designs of in vivo mutagenicity tests with regard to subsequent statistical analysis.

A workshop was held on September 13 and 14, 1993, at the GSF, Neuherberg, Germany, to start a discussion of experimental design and statistical analysis issues for three in vivo mutagenicity test systems, the micronucleus test in mouse bone marrow/peripheral blood, the chromosomal aberration tests in mouse bone marrow/differentiating spermatogonia, and the mouse dominant lethal test. The discussion has now come to conclusions which we would like to make generally known. Rather than dwell upon specific statistical tests which could be used for data analysis, serious consideration was given to test design. However, the test design, its power of detecting a given increase of adverse effects and the test statistics are interrelated. Detailed analyses of historical negative control data led to important recommendations for each test system. Concerning the statistical sensitivity parameters, a type I error of 0.05 (one tailed), a type II error of 0.20 and a dose related increase of twice the background (negative control) frequencies were generally adopted. It was recommended that sufficient observations (cells, implants) be planned for each analysis unit (animal) so that at least one adverse outcome (micronucleus, aberrant cell, dead implant) would likely be observed. The treated animal was the smallest unit of analysis allowed. On the basis of these general consideration the sample size was determined for each of the three assays. A minimum of 2000 immature erythrocytes/animal should be scored for micronuclei from each of at least 4 animals in each comparison group in the micronucleus assays. A minimum of 200 cells should be scored for chromosomal aberrations from each of at least 5 animals in each comparison group in the aberration assays. In the dominant lethal test, a minimum of 400 implants (40-50 pregnant females) are required per dose group for each mating period. The analysis unit for the dominant lethal test would be the treated male unless the background frequency of dead implants (DI) is so low that multiple males would need to be integrated to meet the minimum observation of one adverse outcome (DI) per analysis unit. A three-step strategy of data analysis was proposed for the cytogenetic assays. Use of negative historical controls was allowed in certain circumstances for interpretation of results from micronucleus tests and chromosomal aberration tests.

Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance.

Insulin resistance is instrumental in the pathogenesis of type 2 diabetes mellitus and the Insulin Resistance Syndrome. While insulin resistance involves decreased glucose transport activity in skeletal muscle, its molecular basis is unknown. Since muscle GLUT4 glucose transporter levels are normal in type 2 diabetes, we have tested the hypothesis that insulin resistance is due to impaired translocation of intracellular GLUT4 to sarcolemma. Both insulin-sensitive and insulin-resistant nondiabetic subgroups were studied, in addition to type 2 diabetic patients. Biopsies were obtained from basal and insulin-stimulated muscle, and membranes were subfractionated on discontinuous sucrose density gradients to equilibrium or under nonequilibrium conditions after a shortened centrifugation time. In equilibrium fractions from basal muscle, GLUT4 was decreased by 25-29% in both 25 and 28% sucrose density fractions and increased twofold in both the 32% sucrose fraction and bottom pellet in diabetics compared with insulin-sensitive controls, without any differences in membrane markers (phospholemman, phosphalamban, dihydropyridine-binding complex alpha-1 subunit). Thus, insulin resistance was associated with redistribution of GLUT4 to denser membrane vesicles. No effects of insulin stimulation on GLUT4 localization were observed. In non-equilibrium fractions, insulin led to small GLUT4 decrements in the 25 and 28% sucrose fractions and increased GLUT4 in the 32% sucrose fraction by 2.8-fold over basal in insulin-sensitive but only by 1.5-fold in both insulin-resistant and diabetic subgroups. The GLUT4 increments in the 32% sucrose fraction were correlated with maximal in vivo glucose disposal rates (r = +0.51, P = 0.026), and, therefore, represented GLUT4 recruitment to sarcolemma or a quantitative marker for this process. Similar to GLUT4, the insulin-regulated aminopeptidase (vp165) was redistributed to a dense membrane compartment and did not translocate in response to insulin in insulin-resistant subgroups. In conclusion, insulin alters the subcellular localization of GLUT4 vesicles in human muscle, and this effect is impaired equally in insulin-resistant subjects with and without diabetes. This translocation defect is associated with abnormal accumulation of GLUT4 in a dense membrane compartment demonstrable in basal muscle. We have previously observed a similar pattern of defects causing insulin resistance in human adipocytes. Based on these data, we propose that human insulin resistance involves a defect in GLUT4 traffic and targeting leading to accumulation in a dense membrane compartment from which insulin is unable to recruit GLUT4 to the cell surface.

Endothelial dysfunction is associated with cholesterol levels in the high normal range in humans.

BACKGROUND: The purpose of this study was to test the hypothesis that cholesterol levels in the high normal range are associated with impaired endothelium-dependent vasodilation. METHODS AND RESULTS: We studied leg blood flow (LBF) responses to graded intrafemoral artery infusions of the endothelium-dependent vasodilator methacholine chloride (MCh) or the endothelium-independent vasodilator sodium nitroprusside (SNP) in normal volunteers exhibiting a wide range of total cholesterol levels within the normal range (<75th percentile). LBF increased in a dose-dependent fashion in response to the femoral artery infusions of MCh and SNP (P<.001). LBF responses to MCh were significantly blunted (P<.001) in subjects with high normal cholesterol (195+/-6 mg/dL, n=13) compared with subjects with low normal cholesterol (146+/-5 mg/dL, n=20). Maximal endothelium-dependent vasodilation in the high normal group was decreased by nearly 50% compared with the low normal group (146+/-13% versus 268+/-34%, P<.01). There was a negative correlation between total cholesterol levels and maximal endothelium-dependent vasodilation (total cholesterol, r=-.41, P<.02; LDL cholesterol, r=-.42, P<.02). On the other hand, LBF responses to the endothelium-independent vasodilator SNP did not differ between groups. CONCLUSIONS: These data suggest that an inverse and continuous relationship exists between the prevailing cholesterol level and endothelium-dependent vasodilation. Moreover, cholesterol levels even in the normal range may be associated with endothelial dysfunction, thus potentially contributing to the increased risk of macrovascular disease conferred by cholesterol elevations.