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Pleiotrophin (PTN) is a heparin-binding cytokine that is widely expressed during early development and increases in maternal circulation during pregnancy.Aged PTN-deficient mice exhibit insulin resistance, suggesting a role in metabolic control. The objectives of this study were to determine if PTN is expressed in mouse pancreatic β-cells in young vs. adult animals, and its effects on DNA synthesis, β-cell gene expression and glucose-stimulated insulin secretion (GSIS). The Ptn gene was expressed in isolated fractions of young mouse β-cells, especially within immature β-cells with low glucose transporter 2 expression. Expression was retained in the adult pancreas but did not significantly change during pregnancy. PTN and its receptor, phosphotyrosine phosphatase-β/ζ, were also expressed in the proliferative INS1E β-cell line. Fluorescence immunohistochemistry showed that PTN peptide was present in islets of Langerhans in adult mice, associated predominantly with β-cells. The percentage of β-cells staining for PTN did not alter during mouse pregnancy, but intense staining was seen during β-cell regeneration in young mice following depletion of β-cells with streptozotocin. Incubation of INS1E cells with PTN resulted in an increased DNA synthesis as measured by Ki67 localization and increased expression of Pdx1 and insulin. However, both DNA synthesis and GSIS were not altered by PTN in isolated adult mouse islets. The findings show that Ptn is expressed in mouse β-cells in young and adult life and could potentially contribute to adaptive increases in β-cell mass during early life or pregnancy., Competing Interests: The authors declare that they have no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2022 Sevillano, Liang, Strutt, Hill, Szlapinski, Ramos-Álvarez and Hill.)

Gestational diabetes mellitus results, in part, from a sub-optimal β-cell mass (BCM) during pregnancy. Artemisinins were reported to increase BCM in models of diabetes by α- to β-cell conversion leading to enhanced glucose tolerance. We used a mouse model of gestational glucose intolerance to compare the effects of an artemisinin (artesunate) on glycemia of pregnant mice with vehicle treatment (acetone) or no treatment. Animals were treated daily from gestational days (GD) 0.5 to 6.5. An intraperitoneal glucose tolerance test was performed prior to euthanasia at GD18.5 or post-partum. Glucose tolerance was significantly improved in both pregnant and non-pregnant mice with both artesunate and vehicle-alone treatment, suggesting the outcome was primarily due to the acetone vehicle. In non-pregnant, acetone-treated animals, improved glucose tolerance was associated with a higher BCM and a significant increase in bihormonal insulin and glucagon-containing pancreatic islet cells, suggesting α- to β-cell conversion. BCM did not differ with treatment during pregnancy or post-partum. However, placental weight was higher in acetone-treated animals and was associated with an upregulation of apelinergic genes. Acetone-treated animals had reduced weight gain during treatment despite comparable food consumption to non-treated mice, suggesting transient effects on nutrient uptake. The mean duodenal and ileum villus height was reduced following exposure to acetone. We conclude that acetone treatment may mimic transient fasting, resulting in a subsequent improvement in glucose tolerance during pregnancy.

The apelin receptor (Aplnr) and its ligands, Apelin and Apela, contribute to metabolic control. The insulin resistance associated with pregnancy is accommodated by an expansion of pancreatic β-cell mass (BCM) and increased insulin secretion, involving the proliferation of insulin-expressing, glucose transporter 2-low (Ins + Glut2 LO ) progenitor cells. We examined changes in the apelinergic system during normal mouse pregnancy and in pregnancies complicated by glucose intolerance with reduced BCM. Expression of Aplnr, Apelin and Apela was quantified in Ins + Glut2 LO cells isolated from mouse pancreata and found to be significantly higher than in mature β-cells by DNA microarray and qPCR. Apelin was localized to most β-cells by immunohistochemistry although Aplnr was predominantly associated with Ins + Glut2 LO cells. Aplnr-staining cells increased three- to four-fold during pregnancy being maximal at gestational days (GD) 9-12 but were significantly reduced in glucose intolerant mice. Apelin-13 increased β-cell proliferation in isolated mouse islets and INS1E cells, but not glucose-stimulated insulin secretion. Glucose intolerant pregnant mice had significantly elevated serum Apelin levels at GD 9 associated with an increased presence of placental IL-6. Placental expression of the apelinergic axis remained unaltered, however. Results show that the apelinergic system is highly expressed in pancreatic β-cell progenitors and may contribute to β-cell proliferation in pregnancy., (© 2021. The Author(s).)

ANITA's fourth long-duration balloon flight in 2016 detected 29 cosmic-ray (CR)-like events on a background of 0.37_{-0.17}^{+0.27} anthropogenic events. CRs are mainly seen in reflection off the Antarctic ice sheets, creating a phase-inverted waveform polarity. However, four of the below-horizon CR-like events show anomalous noninverted polarity, a p=5.3×10^{-4} chance if due to background. All anomalous events are from locations near the horizon; ANITA-IV observed no steeply upcoming anomalous events similar to the two such events seen in prior flights.

We report on an upward traveling, radio-detected cosmic-ray-like impulsive event with characteristics closely matching an extensive air shower. This event, observed in the third flight of the Antarctic Impulsive Transient Antenna (ANITA), a NASA-sponsored long-duration balloon payload, is consistent with a similar event reported in a previous flight. These events could be produced by the atmospheric decay of an upward-propagating τ lepton produced by a ν_{τ} interaction, although their relatively steep arrival angles create tension with the standard model neutrino cross section. Each of the two events have a posteriori background estimates of ≲10^{-2} events. If these are generated by τ-lepton decay, then either the charged-current ν_{τ} cross section is suppressed at EeV energies, or the events arise at moments when the peak flux of a transient neutrino source was much larger than the typical expected cosmogenic background neutrinos.

We report on four radio-detected cosmic-ray (CR) or CR-like events observed with the Antarctic Impulsive Transient Antenna (ANITA), a NASA-sponsored long-duration balloon payload. Two of the four were previously identified as stratospheric CR air showers during the ANITA-I flight. A third stratospheric CR was detected during the ANITA-II flight. Here, we report on characteristics of these three unusual CR events, which develop nearly horizontally, 20-30 km above the surface of Earth. In addition, we report on a fourth steeply upward-pointing ANITA-I CR-like radio event which has characteristics consistent with a primary that emerged from the surface of the ice. This suggests a possible τ-lepton decay as the origin of this event, but such an interpretation would require significant suppression of the standard model τ-neutrino cross section.

For 50 years, cosmic-ray air showers have been detected by their radio emission. We present the first laboratory measurements that validate electrodynamics simulations used in air shower modeling. An experiment at SLAC provides a beam test of radio-frequency (rf) radiation from charged particle cascades in the presence of a magnetic field, a model system of a cosmic-ray air shower. This experiment provides a suite of controlled laboratory measurements to compare to particle-level simulations of rf emission, which are relied upon in ultrahigh-energy cosmic-ray air shower detection. We compare simulations to data for intensity, linearity with magnetic field, angular distribution, polarization, and spectral content. In particular, we confirm modern predictions that the magnetically induced emission in a dielectric forms a cone that peaks at the Cherenkov angle and show that the simulations reproduce the data within systematic uncertainties.

The ability of pancreatic β-cells to undertake glucose-stimulated insulin secretion (GSIS) depends on the generation of adenosine triphosphate (ATP) within the mitochondria from pyruvate, a major rate-limiting enzyme being pyruvate dehydrogenase (PDH) complex (PDC). However, glucose metabolism also controls β-cell mass. To examine the role of PDC in the regulation of pancreatic β-cell development and maturation, we generated β-cell-targeted PDHα subunit knock-out male mice (β-PDHKO) and compared these with control males (β-PDHCT) from birth until 6-8 weeks age. Pancreas morphology, transcription factor expression, pancreatic insulin content, and circulating glucose and insulin values were compared. Compared to β-PDHCT male mice, β-PDHKO animals had significantly reduced pancreatic insulin content from birth, a lower serum insulin content from day 15, and relative hyperglycemia from day 30. Isolated islets from β-PDHKO mice demonstrated a reduced GSIS. The number of islets per pancreatic area, mean islet area, and the proportion of islet cells that were β-cells were all reduced in β-PDHKO animals. Similarly the number of insulin-immunopositive, extra-islet small endocrine cell clusters, a possible source of β-cell progenitors, was lower in β-PDHKO mice. Analysis of pancreatic expression of transcription factors responsible for β-cell lineage commitment, proliferation, and maturation, Pdx1, Neurogenin3, and NeuroD1 showed that mRNA abundance was reduced in the β-PDHKO. This demonstrates that PDC is not only required for insulin expression and glucose-stimulated secretion, but also directly influences β-cell growth and maturity, and positions glucose metabolism as a direct regulator of β-cell mass and plasticity., (© 2014 by the Society for Experimental Biology and Medicine.)

Objective: The role of NOX2-containing NADPH oxidase in the development of diabetes is not fully understood. We hypothesized that NOX2 deficiency decreases reactive oxygen species (ROS) production and immune response and protects against streptozotocin (STZ)-induced β-cell destruction and development of diabetes in mice., Research Design and Methods: Five groups of mice--wild-type (WT), NOX2(-/-), WT treated with apocynin, and WT adoptively transferred with NOX2(-/-) or WT splenocytes--were treated with multiple-low-dose STZ. Blood glucose and insulin levels were monitored, and an intraperitoneal glucose tolerance test was performed. Isolated WT and NOX2(-/-) pancreatic islets were treated with cytokines for 48 h., Results: Significantly lower blood glucose levels, higher insulin levels, and better glucose tolerance was observed in NOX2(-/-) mice and in WT mice adoptively transferred with NOX2(-/-) splenocytes compared with the respective control groups after STZ treatment. Compared with WT, β-cell apoptosis, as determined by TUNEL staining, and insulitis were significantly decreased, whereas β-cell mass was significantly increased in NOX2(-/-) mice. In response to cytokine stimulation, ROS production was significantly decreased, and insulin secretion was preserved in NOX2(-/-) compared with WT islets. Furthermore, proinflammatory cytokine release induced by concanavalin A was significantly decreased in NOX2(-/-) compared with WT splenocytes., Conclusions: NOX2 deficiency decreases β-cell destruction and preserves islet function in STZ-induced diabetes by reducing ROS production, immune response, and β-cell apoptosis.

We have previously shown that artificial rearing of newborn female rat pups on a high-carbohydrate (HC) milk formula resulted in chronic hyperinsulinemia and adult-onset obesity (HC phenotype) and that the maternal HC phenotype was transmitted to their progeny (2-HC rats) because of fetal development in the HC female rat. The aims of this study were to investigate 1) the fetal adaptations that predisposed the progeny for the expression of the HC phenotype in adulthood and 2) whether the transfer of the HC phenotype to the progeny could be reversed by maternal food restriction. Fetal parameters such as plasma insulin and glucose levels, mRNA level of preproinsulin gene, pancreatic insulin content, and islet insulin secretory response in vitro were determined. On gestational day 21, 2-HC fetuses were hyperinsulinemic, had increased insulin content and mRNA level of the preproinsulin gene in their pancreata and demonstrated an altered glucose-stimulated insulin secretory response by isolated islets. Modification of the intrauterine environment in HC female rats was achieved by pair feeding them to the amount of diet consumed by age-matched control rats from the time of their weaning. This mild dietary restriction reversed their HC phenotype and also prevented the development of the HC phenotype in their progeny. These findings show that mal-programming of the progeny of the hyperinsulinemic-obese HC female for the expression of the HC phenotype is initiated in utero and that normalization of the maternal environment in HC female rats by mild food restriction resulted in the normal phenotype in their progeny.

Aims/hypothesis: We hypothesised that nutritional taurine, which is important for the development of the endocrine pancreas and reduces cytokine-induced apoptosis in pancreatic beta cells, would prevent or delay the onset of autoimmune diabetes, if given early in life to the non-obese diabetic (NOD) mouse., Methods: Pregnant NOD mice received a diet supplemented with taurine throughout gestation or until weaning, and the pancreas of the offspring was examined using immunohistochemistry. This was done at postnatal day 14 and after 8 weeks (assessment of insulitis). The animals were also monitored until they became diabetic., Results: At 14 days, pancreatic islet mass was significantly greater in animals treated with taurine than in controls. This finding was associated with a greater incidence of islet cell proliferation and a lower incidence of apoptosis. At age 8 weeks the number of islets manifesting insulitis was reduced by more than half, and the area of insulitis was reduced by 90%. Taurine treatment delayed the mean onset time of diabetes from 18 to 30 weeks in females, and from 30 to 38 weeks in males, while 20% of treated females remained free of diabetes after one year., Conclusions/interpretation: Taurine supplementation in early life altered islet development, reduced insulitis and delayed the onset of diabetes in NOD mice.

We show that transplantation of adult bone marrow-derived cells expressing c-kit reduces hyperglycemia in mice with streptozotocin-induced pancreatic damage. Although quantitative analysis of the pancreas revealed a low frequency of donor insulin-positive cells, these cells were not present at the onset of blood glucose reduction. Instead, the majority of transplanted cells were localized to ductal and islet structures, and their presence was accompanied by a proliferation of recipient pancreatic cells that resulted in insulin production. The capacity of transplanted bone marrow-derived stem cells to initiate endogenous pancreatic tissue regeneration represents a previously unrecognized means by which these cells can contribute to the restoration of organ function.

Studies in vitro and in vivo have shown that glucocorticoids and sex steroids play an important role in bone physiology and pathophysiology. In this study we investigated glucocorticoid and sex steroid conversion in osteoblasts derived from lumbar vertebrae of adult male and female rats. Progesterone was converted to inactive 20alpha-OH-progesterone and the conversion at day 5 was 16-fold greater than that at day 13 in both sexes (male/ female, 2.7/1.7 and 0.16/0.10 nM/10(5)cells/24 h, respectively). The conversion of inactive androstenedione to active androgen testosterone in males and females was 1.2- and 2.4-fold greater at day 5 than at day 13, respectively (male/female, 0.40/0.70 and 0.34/0.30 nM/ 10 cells/24 h, respectively). These results suggest that osteoblasts possess 20alpha-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD and that their activities are dependent on the stage of cell differentiation. At day 5, dehydroepiandrosterone was converted to androstenedione (male/female, 0.25/0.098 nM/10(5)cells/24 h), to 7alpha-OH-dehydroepiandrosterone (male/female, 0.49/0.39 nM/10(5)cells/24 h) and to 5-androstene-3beta,17beta-diol (male/female, 0.18/0.37 nM/10(5)cells/24 h), indicating the presence of 3beta-HSD, 7alpha-hydroxylase and 17beta-HSD, respectively. Both 3beta-HSD and 7alpha-hydroxylase activities declined with cell differentiation. Hormonally inactive cortisone was converted to active cortisol (male/female, 0.34/0.29 microM/10(6)cells/6 h) while conversion of cortisol to cortisone was not detectable, suggesting the presence of oxoreductase activity of 11beta-HSD-1. These results show, for the first time, the presence of 7alpha-hydroxylase and 20alpha-HSD in osteoblasts, and provide further evidence that osteoblasts metabolize a variety of steroid hormones and can thus regulate tissue responsiveness to them.

In rats, a proportion of pancreatic beta-cells are deleted by apoptosis in the second week of postnatal life and replaced by endocrine cell neogenesis from pancreatic ductal epithelium. This coincides with a reduction in pancreatic insulin-like growth factor II (IGF-II) expression, and IGF-II has been shown to act as a beta-cell survival factor in vitro. To examine whether IGF-II regulates beta-cell apoptosis in vivo, an IGF-II transgenic mouse model was used in which mouse IGF-II is overexpressed in skin, gut, and uterus driven by a keratin promoter, so that circulating IGF-II is retained postnatally. Mice were killed between postnatal days 7 and 26, and the pancreas was examined histologically. Apoptotic cells were visualized by the terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling method, and proliferating cells were examined by immunohistochemistry for proliferating cell nuclear antigen. In nontransgenic mice, serum IGF-II was absent by 26 days, but mean (+/-SEM) values were 45+/-9 ng/ml (n = 5) in transgenic animals. A 2- to 3-fold rise in islet cell apoptosis was seen in normal animals between days 11 and 16, but this was substantially decreased in IGF-II transgenic mice (day 11; control, 12+/-1%; transgenic, 6+/-1%; P < 0.01; n = 5). Consequently, islets from IGF-II transgenic mice had a significantly greater mean area from days 11-16, but the proportions of beta- and alpha-cells and circulating insulin levels were not changed. Islet cell DNA synthesis was increased in transgenic mice on days 13 and 16. The total islet number per section did not alter. The results show that a persistent presence of circulating IGF-II postnatally alters endocrine pancreatic ontogeny in the mouse and largely prevents the wave of developmental apoptosis that precipitates beta-cell turnover in neonatal life.

Obesity is frequently associated with insulin-resistance and abnormal glucose homeostasis. Recent evidence indicates that TNFalpha may play a role in mediating the insulin-resistance of obesity through its overexpression in adipose tissue. Previously, we have shown that human adipose stromal cells contain 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) mRNA and activity. The present study was designed to examine the effects of insulin on 11beta-HSD1 expression in human adipose stromal cells under basal and TNFalpha-stimulated conditions. The cells were obtained from breast adipose tissue by collagenase digestion, and grown to confluence under replicating conditions in 10% fetal bovine serum. The cells were transferred to serum-free medium for 24 h prior to treatment with either TNFalpha, insulin or both for a further 24 h. The level of 11beta-HSD1 reductase activity was determined by measuring the conversion of [(3)H]-cortisone to [(3)H]-cortisol at a substrate concentration of 10 nM. Treatment with TNFalpha at concentrations of 0.1-10 ng/ml resulted in a dose dependent increase in 11beta-HSD1 reductase activity from 1.5 to 10-fold. Insulin (0.1-100 nM) had no effect under basal conditions, but inhibited the stimulatory effects of TNFalpha (5 ng/ml) on 11beta-HSD1 reductase activity in a dose dependent fashion (8-66%) inhibition). Northern blot analysis revealed corresponding changes in the level of 11beta-HSD1 mRNA, suggesting that the effects of TNFalpha and insulin on 11beta-HSD1 activity are mediated at the level of gene transcription. The interaction between insulin and TNFalpha suggests that local and systemic factors may act in a concerted fashion to modulate glucocorticoid activity in adipose and other peripheral tissues.

The biological activity of glucocorticoids in target tissues can be influenced by locally produced 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), the enzyme responsible for the interconversion of cortisol and its inactive metabolite cortisone. In human adipose stromal cells, glucocorticoids are potent stimulators of the conversion of androgens to estrogens (aromatase activity). The present study was designed to determine whether 11 beta-HSD activity was present in human adipose stromal cells, and if changes in the activity of this enzyme could influence aromatase activity. 11 beta-HSD activity was determined by a radiometric conversion assay in breast adipose tissue from six patients. It was found that both dehydrogenase (cortisol to cortisone) and reductase (cortisone to cortisol) activities were present in all six subjects, and the reductase activity was always predominant. Carbenoxolone (CBX), a potent inhibitor of 11 beta-HSD, added to the culture medium at 50 and 200 microM, resulted in 39 +/- 4% and 85 +/- 1% inhibition, respectively, of both reductase and dehydrogenase activity of 11 beta-HSD. To determine whether alterations in 11 beta-HSD could influence aromatase activity, the effect of CBX (200 microM) on cortisol- and cortisone-induced changes in the conversion of androstenedione to estrone was examined. CBX prevented the stimulatory effect of cortisone and minimally potentiated the stimulatory effect of cortisol on aromatase activity, reflecting an inhibition of the local activation of cortisone and the local metabolism of cortisol, respectively. In order to determine whether the product of the 11 beta-HSD 1 gene was responsible for the observed 11 beta-HSD activity, total RNA extracts from these cells were subjected to Northern blot analysis using human 11 beta-HSD 1 cDNA as the probe. A single 1.8 11 beta-HSD 1 transcript was detected, and its abundance was reduced by CBX. No 11 beta-HSD 2 mRNA was detected. The present results demonstrate that the 11 beta-HSD 1 gene is expressed and functional in human breast adipose stromal cells and that changes in 11 beta-HSD 1 activity result in alterations in aromatase activity.

Human adipose stromal cells provide an excellent model for studying a variety of metabolic processes in an in vitro system These are normal cells derived from subcutaneous or omental adipose tissue. Under specific culture condition, they will differentiate without replication into cells resembling mature adipocytes or will replicate, become confluent, and grow in subculture. The initial observation that the stromal vascular fraction of human omental adipose tissue contained a fibroblast-like cell that was a possible adipocyte precursor was made by Poznanski et al (1). Subsequent studies demonstrated enzymological and morphological properties that developed during differentiation (2-4). The primary interest has focused on the conditions required to stimulate these cells to differentiate into adipocytes, which can accumulate lipid and possess the enzymes involved in lipogenesis, including glycerol-3-phosphate dehydrogenase (GPDH) and lipoprotein lipase (LPL).

The metabolism of dehydroepiandrosterone (DHA) and androstenedione (A-dione) was studied in cultured human adipose stromal cells obtained from breast tissue of six premenopausal patients undergoing reduction mammoplasty. Cells were maintained in culture in the presence of 10% fetal bovine serum. Studies were carried out during the proliferative and confluent phases of culture with radiolabelled substrates (2 microCi, 10 nM). During the early phases of replication 7 alpha-hydroxydehydroepiandrosterone (7 alpha-OHDHA) was formed from DHA. As the cells reached confluence, the major metabolite of DHA in cells from all patients was A-dione indicating the presence of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD). The conversion of DHA to A-dione was variable among patients when cells were confluent with 30-80% of substrate being metabolized to this product. Adipose stromal cells synthesized estrone (E1) from DHA once A-dione formation was established. Under basal conditions E1 was obtained in cells from three of the six patients examined with up to 36% substrate converted to this product. Dexamethasone (Dex 10(-7) M) stimulated E1 formation in cells from all subjects with up to 50% of substrate being converted. Parallel studies comparing the conversion of DHA with A-dione to E1 revealed that as the cells became confluent, E1 formation from both substrates was similar. The pattern of steroid metabolism was also examined in primary culture and in subculture. Passage 1 cells continued to form A-dione as a major metabolite of DHA, and did not revert to the pattern of metabolism found in primary cells during the early stages of replication, when 7 alpha-hydroxylation predominated. Human adipose stromal cells actively metabolize DHA, producing 7 alpha-OHDHA, A-dione and E1 as principal metabolites. Changes in the circulating levels of DHA may directly influence the formation of E1 in peripheral tissues. This source of E1 will be modulated by factors controlling 3 beta-HSD and aromatase activities.

7 alpha-Hydroxydehydroepiandrosterone (7 alpha-OHDHA) is a major metabolite of dehydroepiandrosterone (DHA) using adipose stromal cells. To gain a better understanding of the factors regulating DHA metabolism, we examined the effect of dexamethasone and cytochrome P450 inhibitors on the formation of 7 alpha-OHDHA. Dexamethasone (10(-9) to 10(-7) M) stimulated 7 alpha-OHDHA formation in a dose-dependent manner with a 2- to 5-fold stimulation at 10(-7) M. The dexamethasone stimulated 7 alpha-OHDHA formation was inhibited by RU486 in a dose-dependent manner with suppression to basal levels at 10(-6) M. Progesterone (10(-7) M) had no effect on 7 alpha-OHDHA formation suggesting that the dexamethasone stimulation was acting through the glucocorticoid receptor. Conversion of DHA to 7 alpha-OHDHA was inhibited by ketoconazole and metyrapone. An inhibition of 70-80% was obtained with ketoconazole and 25-60% with metyrapone at concentrations of 10(-5) M. Aminoglutethimide phosphate was less effective than either ketoconazole or metyrapone in inhibiting 7 alpha-OHDHA formation with < 30% inhibition at 10(-5) M. These studies indicate that 7-hydroxylation provides an alternative pathway for the metabolism of DHA in peripheral tissues. This pathway, which is regulated by glucocorticoids, may influence the amount of DHA available for conversion to androstenedione and its subsequent aromatization to estrone. The biological role of the 7-oxygenated metabolites and their effects on other steroidogenic pathways have not been established.

Studies of the metabolism of dehydroepiandrosterone (DHA) by cultured human adipose stromal cells revealed that the most abundant metabolite detected by HPLC was a polar compound accounting for up to 45% of total radioactivity. This metabolite was isolated by chromatography on Lipidex 5000 from the culture medium of breast adipose stromal cells cultured with unlabelled DHA (5 microM) and identified by combined capillary gas chromatography and mass spectrometry as 7 alpha-hydroxydehydroepiandrosterone (7 alpha-OHDHA). In breast adipose stromal cells, the conversion of DHA to 7 alpha-OHDHA was linear from a substrate concentration of 10 nM to 1 microM. At 1 microM substrate concentration, the formation of 7 alpha-OHDHA in four patients ranged from 6.1 to 22.5 ng/10(5) cells/24 h. Incubations carried out in primary culture and up to the fifth subculture revealed continued formation of 7 alpha-OHDHA. Adipose stromal cells from abdomen, flank and perinephric fat also produced 7 alpha-OHDHA from DHA. These studies have shown that 7 alpha-OHDHA is a major metabolite of DHA in human adipose stromal cells. The variability from patient to patient and the magnitude of this conversion suggests that this pathway may play an important role in the peripheral metabolism of DHA.

We have induced teratocarcinomas from female embryos heterozygous for electrophoretic variants of the X-linked gene coding for phosphoglycerate kinase (PGK). An embryonal carcinoma cell line, P10, has been isolated from such a teratocarcinoma. It has a normal female karyotype and cultures contain both PGK isoenzymic forms. Clonal populations derived from P10 also contain both PGK electrophoretic variants. In addition, both X chromosomes in these cells replicate in synchrony with the autosomes during early S phase of the cell cycle. These data indicate that the undifferentiated P10 embryonal carcinoma cells contain two active X chromosomes. When cultured under the appropriate conditions, the P10 cells differentiate to form a variety of tissue types. At least some of these differentiated cells contain an inactive X chromosome as determined by cytogenetic analysis. Apparently X chromosome inactivation accompanies the differentiation of these female embryonal carcinoma cells.