Impaired activation of protein kinase C-zeta by insulin and phosphatidylinositol-3,4,5-(PO4)3 in cultured preadipocyte-derived adipocytes and myotubes of obese subjects.

Insulin resistance in obesity is partly due to diminished glucose transport in myocytes and adipocytes, but underlying mechanisms are uncertain. Insulin-stimulated glucose transport requires activation of phosphatidylinositol (PI) 3-kinase (3K), operating downstream of insulin receptor substrate-1. PI3K stimulates glucose transport through increases in PI-3,4,5-(PO(4))(3) (PIP(3)), which activates atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). However, previous studies suggest that activation of aPKC, but not PKB, is impaired in intact muscles and cultured myocytes of obese subjects. Presently, we examined insulin activation of glucose transport and signaling factors in cultured adipocytes derived from preadipocytes harvested during elective liposuction in lean and obese women. Relative to adipocytes of lean women, insulin-stimulated [(3)H]2-deoxyglucose uptake and activation of insulin receptor substrate-1/PI3K and aPKCs, but not PKB, were diminished in adipocytes of obese women. Additionally, the direct activation of aPKCs by PIP(3) in vitro was diminished in aPKCs isolated from adipocytes of obese women. Similar impairment in aPKC activation by PIP(3) was observed in cultured myocytes of obese glucose-intolerant subjects. These findings suggest the presence of defects in PI3K and aPKC activation that persist in cultured cells and limit insulin-stimulated glucose transport in adipocytes and myocytes of obese subjects.

Surface protein characterization of human adipose tissue-derived stromal cells.

Human bone marrow stromal cells are a multipotent population of cells capable of differentiating into a number of mesodermal lineages as well as supporting hematopoeisis. Their distinct protein and gene expression phenotype is well characterized in the literature. Human adipose tissue presents an alternative source of multipotent stromal cells. In this study, we have defined the phenotype of the human adipose tissue-derived stromal cells in both the differentiated and undifferentiated states. Flow cytometry and immunohistochemistry show that human adipose tissue-derived stromal cells have a protein expression phenotype that is similar to that of human bone marrow stromal cells. Expressed proteins include CD9, CD10, CD13, CD29, CD34, CD44, CD 49(d), CD 49(e), CD54, CD55, CD59, CD105, CD106, CD146, and CD166. Expression of some of these proteins was further confirmed by PCR and immunoblot detection. Unlike human bone marrow-derived stromal cells, we did not detect the STRO-1 antigen on human adipose tissue-derived stromal cells. Cells cultured under adipogenic conditions uniquely expressed C/EBPalpha and PPARdelta, two transcriptional regulators of adipogenesis. Cells cultured under osteogenic conditions were more likely to be in the proliferative phases of the cell cycle based on flow cytometric analysis of PCNA and Ki67. The similarities between the phenotypes of human adipose tissue-derived and human bone marrow-derived stromal cells could have broad implications for human tissue engineering.

Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: biochemical, cellular, and molecular analysis.

While adipocyte differentiation has been studied extensively in murine cultures, the lack of a readily available preadipocyte model has hindered equivalent studies in man. We describe methods for the isolation and culture of primary human stromal cells from surgical adipose tissue specimens. In vitro, the stromal cells rapidly differentiate in response to a combination of adipogenic agents. Among these, glucocorticoids and thiazolidinediones act together to induce the formation of lipid vacuoles within the cells. These morphologic changes accompany the increased expression of 2 characteristic adipocyte proteins, the cytoplasmic enzyme glycerol phosphate dehydrogenase (GPDH) and the secreted cytokine leptin. Likewise, stromal cell differentiation results in elevated mRNA levels for the fatty acid binding protein aP2 and the adipogenic regulatory transcription factors CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) in addition to leptin. The in vitro differentiated stromal cells exhibit a lipolytic response to beta-adrenergic agonists, comparable to that reported with primary human adipocytes. These studies demonstrate the validity of human adipose tissue-derived stromal cells as a reliable in vitro model for investigations of adipocyte metabolism in humans.

Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous.

The current study was done to assess if heterogeneity existed in the degree of adipogenesis in stromal cells (preadipocytes) from multiple donors. In addition to conventional lipid-based methods, we have employed a novel signal amplification technology, known as branched DNA, to monitor expression of an adipocyte specific gene product aP2. The fatty acid binding protein aP2 increases during adipocyte differentiation and is induced by thiazolidinediones and other peroxisome proliferator activated receptor gamma ligands. The current work examined the adipogenic induction of aP2 mRNA levels in human adipose tissue stromal cells derived from 12 patients (mean age +/- SEM, 38.9 +/- 3.1) with mild to moderate obesity (mean body mass index +/- SEM, 27.8 +/- 2.4). Based on branched DNA technology, a rapid and sensitive measure of specific RNAs, the relative aP2 level in adipocytes increased by 679 +/- 93-fold (mean +/- SEM, n=12) compared to preadipocytes. Normalization of the aP2 mRNA levels to the housekeeping gene, glyceraldehyde phosphate dehydrogenase, did not significantly alter the fold induction in a subset of 4 patients (803.6 +/- 197.5 vs 1118.5 +/- 308.1). Independent adipocyte differentiation markers were compared between adipocytes and preadipocytes in parallel studies. Leptin secretion increased by up to three-orders of magnitude while measurements of neutral lipid accumulation by Oil Red O and Nile Red staining increased by 8.5-fold and 8.3-fold, respectively. These results indicate that preadipocytes isolated from multiple donors displayed varying degrees of differentiation in response to an optimal adipogenic stimulus in vitro. This work also demonstrates that branched DNA measurement of aP2 is a rapid and sensitive measure of adipogenesis in human stromal cells. The linear range of this assay extends up to three-orders of magnitude and correlates directly with independent measures of cellular differentiation.

Delivery of comprehensive children's dental services using portable dental clinics in NYC public schools. A six-year analysis.

In 1990, the Bureau of Dental Health Services of the New York City Department of Health launched a major initiative to modernize a network of school-based dental clinics located throughout the city. Since 1913, the bureau has provided dental care to public school children; however, the clinics were not properly maintained or upgraded, and were in a state of disrepair and obsolescence. Anticipating that the survival of the program was in question, the school program was converted to a fleet of state-of-the-art portable dental clinics permitting targeting of underserved, high-risk poor and immigrant populations. Demographics had changed dramatically over the years; the program could now situate services where they were needed most, and provide a broader array of care where access was a problem. This paper presents a six-year analysis of the program and builds a strong case to show that a portable delivery system can equal or in many ways surpass the effectiveness and capabilities of a fixed-state approach.

The use of multiple parameters to characterize cadmium-induced renal dysfunction resulting from occupational exposure.

Renal function has been examined in a group of 77 subjects occupationally exposed to cadmium fume and dust, together with a referent group of 103 age- and socioeconomically matched subjects. Fourteen biochemical parameters were measured on each subject. Three different ways of combining the information from all 14 tests were used to identify those subjects with renal dysfunction. These were first to count the number of parameters in which a subject recorded an abnormal test result. Second, the z value was computed for each parameter for each person by comparison with the mean and standard deviation of a derived normal population; these z scores were then summed. Lastly a multivariate distance measure, Mahalanobis D2, was determined for each subject from the distribution of normal subjects. The three approaches showed a considerable degree of agreement in identifying subjects with renal dysfunction, but they also displayed complementary strengths and weaknesses. The consensus of the three techniques was then taken to define truly dysfunctional subjects and each of the 14 parameters, and some combinations of pairs of parameters were tested as to their sensitivity and specificity. For this group of subjects, it was not possible to improve greatly on the use of retinol binding protein on its own. Were a second parameter to be chosen, it would be desirable to choose one reflecting the glomerular filtration rate, but the absence of a suitable sensitive biological monitoring parameter precludes a firm recommendation.

The prevalence and incidence of Toxoplasma antibodies in pregnant women.

A group of 213 pregnant Jewish women of Israeli and North-African/Asian origin in the Upper Gallilee in Israel were tested for Toxoplasma antibody, first at 4-12 weeks gestation and again 5-6 months later. Immunofluorescent antibody, Sabin-Feldman tests, and specific IgM estimation were used. The prevalence rates for seropositive women were lower in both groups (total 21%) than the rate found in a 1973 study in Israel. The incidence rate for infection acquired in pregnancy was 1.4%. There were no cases of congenital toxoplasmosis, as far as is known up to 3 years of age. More information on the prevalence and incidence of seropositivity, and on congenital toxoplasmosis, is required before a policy decision can be taken as to whether an antenatal screening program for toxoplasmosis should be instituted in Israel.

An improved in vivo neutron activation system for measuring kidney cadmium.

An in vivo neutron activation system for measuring kidney cadmium has been redesigned, firstly to reduce ambient dose levels and, secondly, to improve the cadmium signal to neutron dose ratio by modifying the neutron spectrum from 238Pu/Be, by interposing a beryllium premoderator. The ambient dose was reduced by a factor of seven. The overall system performance (lower limit of detection for a given dose) was improved by 40-50%. This 238Pu/Be based system now performs as well as or better than analogous 252Cf systems, without the drawback of the relatively short half life of 252Cf.

In vivo neutron activation analysis of organ cadmium burdens. Referent levels in liver and kidney and the impact of smoking.

In vivo neutron activation measurements of liver and kidney cadmium have been made in 77 exposed workers and 101 referents. Cadmium levels were higher in exposed workers than in referents; both in liver, 25.7 cf. 0.6 micrograms/g, and kidney, 17.9 cf. 2.7 mg. The 19 referents who never smoked had lower mean organ cadmium burdens than the other referents, the difference achieving statistical significance in the kidney, p less than .01. Cigarette smoking was estimated to increase cadmium body burden by 370 +/- 140 micrograms/pack year. These referent cadmium levels are similar to, although slightly below, previous in vivo and autopsy data.

Relations between liver cadmium, cumulative exposure, and renal function in cadmium alloy workers.

Detailed biochemical investigations of renal function were made on 75 male workers exposed to cadmium and an equal number of referents matched for age, sex, and employment status. The exposed group consisted of current and retired workers who had been employed in the manufacture of copper-cadmium alloy at a single factory in the United Kingdom for periods of up to 39 years and for whom cumulative cadmium exposure indices could be calculated. In vivo measurements of liver and kidney cadmium burden were made on exposed and referent workers using a transportable neutron activation analysis facility. Significant increases in the urinary excretion of albumin, retinol binding protein, beta 2 microglobulin, N-acetylglucosaminidase (NAG), alkaline phosphatase, gamma-glutamyl transferase and significant decreases in the renal reabsorption of calcium, urate, and phosphate were found in the exposed group compared with the referent group. Measures of glomerular filtration rate (GFR) (creatinine clearance, serum creatinine, and beta 2 microglobulin) indicated a reduction in GFR in the exposed population. Many of these tubular and glomerular function indicators were significantly correlated with both cumulative exposure index and liver cadmium burden. Using cumulative exposure index and liver cadmium as estimates of dose, a two phase linear regression model was applied to identify an inflection point signifying a threshold level above which changes in renal function occur. Many biochemical variables fitted this model; urinary total protein, retinol binding protein, albumin, and beta 2 microglobulin gave similar inflection points at cumulative exposure levels of about 1100 y.micrograms/m3 whereas changes in the tubular reabsorption of urate and phosphate occurred at higher cumulative exposure indices. Measures of GFR, although fitting the threshold model did not give well defined inflection points. Fewer variables fitted the two phase model using liver cadmium; those that did gave threshold levels in the range 20.3-55.1 ppm. When cadmium workers with cumulative exposure indices of less than 1100 y.micrograms/m3 were compared with their respective referents only serum beta 2 microglobulin and urinary NAG were significantly increased in the exposed group and these differences were not related to the degree of cadmium exposure.(ABSTRACT TRUNCATED AT 400 WORDS)

In-vivo and in-vitro measurements of lead and cadmium.

Tibia lead is measured in vivo using X-ray fluorescence. A(109)Cd source is used to excite Pb K X-rays, and this signals is normalized to that from Rayleigh scattering to remove geometrical variations. The lower limit of detection is 10 µg/g for a mean absorbed dose, to the exposed section of the leg, of 100 µGy. Tibia lead correlated positively with age in normal volunteers (r=0.615,p=0.004) and with duration of exposure in occupationally exposed subjects (r=0.847,p=0.0001). When the X-ray fluorescence technique was applied to autopsy specimens previously analyzed by atomic absorption spectrometry there was excellent agreement between measurement techniques.Cadmium is measured in vivo by neutron activation analysis. The detection limit in liver is 6.5 µg/g for a local skin dose equivalent of 0.5 mSv and in kidney is 6.4 mg for a dose equivalent of 0.9 mSv to the skin. Detailed analysis of the γ-ray spectrum will produce only slight improvements in detection limit. Uncertainties in organ position during measurement, even after ultrasonic localization, are likely to produce uncertainties of 20-25% in cadmium measurement. Autopsy samples were measured, using a fast neutron activation method, from people previously measured in vivo. The results are broadly consistent, but show differences greater than those accounted for by counting statistics.