Symptomatic response to testosterone treatment in dieting obese men with low testosterone levels in a randomized, placebo-controlled clinical trial.

BACKGROUND: Obese men commonly have reductions in circulating testosterone and report symptoms consistent with androgen deficiency. We hypothesized that testosterone treatment improves constitutional and sexual symptoms over and above the effects of weight loss alone. METHODS: We conducted a pre-specified analysis of a randomized double-blind, placebo-controlled trial at a tertiary referral center. About 100 obese men (body mass index (BMI)⩾30 kg m-2) with a repeated total testosterone level ⩽12 nmol l-1 and a median age of 53 years (interquartile range 47-60) receiving 10 weeks of a very-low-energy diet (VLED) followed by 46 weeks of weight maintenance were randomly assigned at baseline to 56 weeks of intramuscular testosterone undecanoate (n=49, cases) or matching placebo (n=51, controls). Pre-specified outcomes were the between-group differences in Aging Male Symptoms scale (AMS) and international index of erectile function (IIEF-5) questionnaires. RESULTS: Eighty-two men completed the study. At study end, cases showed significant symptomatic improvement in AMS score, compared with controls, and improvement was more marked in men with more severe baseline symptoms (mean adjusted difference (MAD) per unit of change in AMS score -0.34 (95% confidence interval (CI) -0.65, -0.02), P=0.04). This corresponds to improvements of 11% and 20% from baseline scores of 40 and 60, respectively, with higher scores denoting more severe symptoms. Men with erectile dysfunction (IIEF-5⩽20) had improved erectile function with testosterone treatment. Cases and controls lost the same weight after VLED (testosterone -12.0 kg; placebo -13.5 kg, P=0.40) and maintained this at study end (testosterone -11.4 kg; placebo -10.9 kg, P=0.80). The improvement in AMS following VLED was not different between the groups (-0.05 (95% CI -0.28, 0.17), P=0.65). CONCLUSIONS: In otherwise healthy obese men with mild to moderate symptoms and modest reductions in testosterone levels, testosterone treatment improved androgen deficiency symptoms over and above the improvement associated with weight loss alone, and more severely symptomatic men achieved a greater benefit.

Correlation of visceral adipose tissue measured by Lunar Prodigy dual X-ray absorptiometry with MRI and CT in older men.

Quantification of abdominal visceral adipose tissue (VAT) is important to understand obesity-related comorbidities. We hypothesized that dual X-ray absorptiometry (DXA) measurements of VAT would correlate with traditional gold standards of magnetic resonance imaging (MRI) and computed tomography (CT) in older men. Deming regression and Bland-Altman plots were used to assess the agreement between VAT measured simultaneously by DXA and MRI (n=95) in a cohort of older males participating in a randomized trial of testosterone replacement for diabetes. We also correlated DXA with single-slice CT (n=102) in a cohort of older males undergoing testosterone deprivation for prostate cancer. Lunar Prodigy DXA scanners using enCORE software was used to measure VAT. DXA VAT volume strongly correlated with MRI VAT volume (r=0.90, P<0.0001) and CT VAT area (r=0.83, P<0.0001). As DXA assesses VAT volume in a smaller compartment than MRI, Bland-Altman analysis demonstrated DXA systematically underestimated VAT by an approximately 30% proportional bias. DXA VAT volume measured by Lunar Prodigy DXA scanners correlate well with gold standard MRI and CT quantification methods, and provides a low radiation, efficient, cost-effective option. Future clinical studies examining the effects of interventions on body composition and regional fat distribution may find DXA an appropriate volumetric method to quantify VAT.

Testosterone levels increase in association with recovery from acute fracture in men.

UNLABELLED: In this longitudinal case-control study, acute fracture was associated with low serum testosterone, which was transient in 43% of men. While assessment of gonadal status is part of the assessment of bone fragility, measurement of testosterone in the early period after fracture may overestimate the prevalence of androgen deficiency. INTRODUCTION: Measurement of circulating testosterone is recommended in the evaluation of bone fragility in men. Since acute illness can transiently decrease circulating testosterone, we quantified the association of acute fracture and serum testosterone levels. METHODS: A case-control study was conducted involving 240 men with a radiologically confirmed minimal trauma fracture presenting to a tertiary referral hospital and 89 age-matched men without a history of minimal trauma fracture serving as controls. Follow-up testosterone levels 6 months after baseline were available for 98 cases and 27 controls. Results were expressed as the median and interquartile (IQR) range. RESULTS: Compared to controls, cases had lower total testosterone [TT, 7.2 (3.5, 10.8) vs 13.6 (10.9, 17.1) nmol/L, p < 0.001]. The 143 cases treated as inpatients had lower testosterone levels than the 97 cases treated as outpatients [TT 4.7 (2.3, 8.1) vs 10.3 (7.5, 12.7) nmol/L, p < 0.001]. Group differences in calculated free testosterone (cFT) were comparable to the group differences in TT. At follow-up, in 98 cases, median TT increased from 6.5 nmol/L (3.2, 8.5) to 9.6 nmol/L (6.9, 12.0) p < 0.0001, and SHBG remained unchanged. Of cases with low testosterone, 43% with TT <10 nmol/L and/or cFT <230 pmol/L at presentation were reclassified as androgen sufficient at follow-up. TT was unchanged in the controls. CONCLUSIONS: Low testosterone levels in men presenting with an acute fracture may, at least in part, be due to an acute, fracture-associated, stress response. To avoid over diagnosis, evaluation for testosterone deficiency should be deferred until recovery from the acute event.

A comparison of precipitants and mortality when acute decompensated heart failure occurs in the community and hospital settings.

AIM: We aimed to compare the precipitants of acute decompensated heart failure (ADHF) among patients admitted with diagnoses inclusive of ADHF (community patients) and patients admitted without ADHF but who developed it during their stay (hospital patients). METHODS: This was a prospective, analytical, observational study undertaken in the Austin Hospital, a major metropolitan teaching hospital (September 2008-February 2010). Consecutive patients admitted to a general medicine unit, and diagnosed and treated for ADHF were enrolled. The unit medical staff completed a specifically designed data collection document. RESULTS: Three hundred and fifty-nine patients were enrolled (42.9% male, mean age 81.9 years). The community (n=312) and hospital (n=47) patient groups did not differ in age, gender, risk variables (living alone, cognitive impairment, multiple medications, compliance), cardiac failure medication use or cause of known heart failure (ischaemia, hypertension, valve dysfunction, 'other') (p>0.05). The ADHF precipitants comprised infection (39.8% patients), myocardial ischaemia (17.3%), tachyarrhythmia (16.2%), non-compliance with fluid and salt restriction (9.2%), non-compliance with medication (6.7%), renal failure (5.8%), medication reduction (5.0%), intravenous fluid complication (3.9%) and 'other' causes (13.9%). Significantly more hospital patients had their ADHF precipitated by intravenous fluid complications (25.5% versus 0.6%, p<0.001). Hospital patients also had a significantly greater death rate (25.5% versus 9.3%, p<0.01). CONCLUSION: Acute decompensated heart failure precipitated in hospital is a dangerous condition with a high mortality. While infection and myocardial ischaemia are the common precipitants, complications of intravenous fluid use, an iatrogenic condition, may be considerable and are potentially avoidable.

Increase in visceral and subcutaneous abdominal fat in men with prostate cancer treated with androgen deprivation therapy.

OBJECTIVE: Androgen deprivation therapy (ADT) for prostate cancer is associated with increases in fat mass and risk of type 2 diabetes; however, the relationship between sex steroid deficiency and abdominal fat distribution remains controversial. DESIGN: We conducted a 12-month prospective observational study at a tertiary referral centre. PATIENTS AND MEASUREMENTS: We investigated changes in abdominal fat distribution and insulin resistance in 26 men (70.6±6.8 years) with nonmetastatic prostate cancer during the first year of ADT. RESULTS: Twelve months of ADT increased visceral abdominal fat area by 22% (from 160.8±61.7 to 195.9±69.7 cm(2) ; P<0.01) and subcutaneous abdominal fat area by 13% (from 240.7±107.5 to 271.3±92.8 cm(2) ; P<0.01). Fat mass increased by 14% (+3.4 kg; P<0.001) and lean tissue mass decreased by 3.6% (-1·9 kg; P<0.001). Insulin resistance (HOMA-IR) increased by 12% (2.50±1.12 to 2.79±1.31, P<0.05). There was no change in fasting glucose or glycated haemoglobin levels. Total testosterone (TT) was inversely associated with visceral fat area independent of oestradiol (E2), but E2 was not associated with visceral fat area independent of TT. Visceral fat area, not TT or E2, was independently associated with insulin resistance. CONCLUSIONS: ADT for prostate cancer results in accumulation of both visceral and subcutaneous abdominal fat. Increased visceral fat area appears more closely linked to testosterone than oestradiol deficiency. Increased insulin resistance may arise secondary to visceral fat accumulation, rather than as a direct result of sex steroid deficiency.

Generation and analysis of an androgen-responsive myoblast cell line indicates that androgens regulate myotube protein accretion.

Androgens have anabolic actions in skeletal muscle and could potentially act to: (a) increase proliferation of myoblasts; (b) delay differentiation to myotubes; and (c) induce protein accretion in post-proliferative myofibers. To identify the site of androgens action, we investigated the proliferative response of the C2C12 mouse myoblast cell line to testosterone and dihydrotestosterone (DHT) treatment. Neither androgens affected cell proliferation after up to 7 days treatment, nor was there a synergistic effect of androgens on the proliferative response of C2C12 cells to IGF-I treatment. However, proliferating C2C12 cells expressed 0.1% of the level of androgen receptor (AR) mRNA found in adult mouse gastrocnemius muscle (p<0.01). Therefore, we generated mouse C2C12 myoblast cell lines stably transfected with the mouse AR cDNA driven by the SV40 promoter (C2C12-AR). C2C12-AR cell proliferation, differentiation, and protein content were analyzed in response to androgen treatment. Our data demonstrated that androgen treatment does not alter either proliferation rate or differentiation rate of C2C12-AR cells. However, treatment of differentiated C2C12-AR myotubes with 100 nM DHT for 3 days caused a 20% increase in total protein content vs vehicle treatment (p<0.05). This effect was not observed in control C2C12 cells transfected with empty vector. These data suggest that androgens act via the AR to upregulate myotube protein content. This model cell line will be useful to further investigate the molecular mechanisms via which androgens regulate protein accretion.

Routine use of HbA1c amongst inpatients hospitalised with decompensated heart failure and the association of dysglycaemia with outcomes.

Diabetes is an independent risk factor for development of heart failure and has been associated with poor outcomes in these patients. The prevalence of diabetes continues to rise. Using routine HbA1c measurements on inpatients at a tertiary hospital, we aimed to investigate the prevalence of diabetes amongst patients hospitalised with decompensated heart failure and the association of dysglycaemia with hospital outcomes and mortality. 1191 heart failure admissions were identified and of these, 49% had diabetes (HbA1c ≥ 6.5%) and 34% had pre-diabetes (HbA1c 5.7-6.4%). Using a multivariable analysis adjusting for age, Charlson comorbidity score (excluding diabetes and age) and estimated glomerular filtration rate, diabetes was not associated with length of stay (LOS), Intensive Care Unit (ICU) admission or 28-day readmission. However, diabetes was associated with a lower risk of 6-month mortality. This finding was also supported using HbA1c as a continuous variable. The diabetes group were more likely to have diastolic dysfunction and to be on evidence-based cardiac medications. These observational data are hypothesis generating and possible explanations include that more diabetic patients were on medications that have proven mortality benefit or prevent cardiac remodelling, such as renin-angiotensin system antagonists, which may modulate the severity of heart failure and its consequences.

Female mice haploinsufficient for an inactivated androgen receptor (AR) exhibit age-dependent defects that resemble the AR null phenotype of dysfunctional late follicle development, ovulation, and fertility.

The role of classical genomic androgen receptor (AR) mediated actions in female reproductive physiology remains unclear. Female mice homozygous for an in-frame deletion of exon 3 of the Ar (AR(-/-)) were subfertile, exhibiting delayed production of their first litter (AR(+/+) = 22 d vs. AR(-/-) = 61 d, P < 0.05) and producing 60% fewer pups/litter (AR(+/+): 8.1 +/- 0.4 vs. AR(-/-): 3.2 +/- 0.9, P < 0.01). Heterozygous females (AR(+/-)) exhibited an age-dependent 55% reduction (P < 0.01) in pups per litter, evident from 6 months of age (P < 0.05), compared with AR(+/+), indicating a significant gene dosage effect on female fertility. Ovulation was defective with a significant reduction in corpora lutea numbers (48-79%, P < 0.01) in 10- to 12- and 26-wk-old AR(+/-) and AR(-/-) females and a 57% reduction in oocytes recovered from naturally mated AR(-/-) females (AR(+/+): 9.8 +/- 1.0 vs. AR(-/-): 4.2 +/- 1.2, P < 0.01); however, early embryo development to the two-cell stage was unaltered. The delay in first litter, reduction in natural ovulation rate, and aromatase expression in AR(+/-) and AR(-/-) ovaries, coupled with the restored ovulation rate by gonadotropin hyperstimulation in AR(-/-) females, suggest aberrant gonadotropin regulation. A 2.7-fold increase (AR(+/+): 35.4 +/- 13.4 vs. AR(-/-): 93.9 +/- 6.1, P < 0.01) in morphologically unhealthy antral follicles demonstrated deficiencies in late follicular development, although growing follicle populations and growth rates were unaltered. This novel model reveals that classical genomic AR action is critical for normal ovarian function, although not for follicle depletion and that haploinsufficiency for an inactivated AR may contribute to a premature reduction in female fecundity.

Related individuals with different androgen receptor gene deletions.

We have identified different members of one family affected by androgen insensitivity syndrome who have deletions of different exons of the X-linked androgen receptor (AR) gene. Two affected (XY) siblings have a deletion of exon E of the AR gene and their affected (XY) aunt has a normal exon E, but a deletion of exons F and G of the same gene. The mother and maternal grandmother of the children both carry the exon E deletion, but not the exon F, G deletion. Both deletions are 5 kb in length and have one breakpoint within a 200-bp region in intron 5; however, they extend in opposite directions. The probability that these two different deletions have arisen at random is extremely low, but the cause of this intriguing phenomenon remains to be found.

Impaired regulation of hepatic fructose-1,6-bisphosphatase in the New Zealand obese mouse model of NIDDM.

The New Zealand obese mouse, a model of NIDDM, is characterized by hyperglycemia, hyperinsulinemia, and hepatic and peripheral insulin resistance. The aim of this study was to investigate the biochemical basis of hepatic insulin resistance in NZO mice. Glycolytic and gluconeogenic enzyme activities were measured in fed and overnight fasted 19- to 20-wk-old NZO and control New Zealand chocolate mice. The NZO mice were twice as heavy as the NZC mice. The activity of the glycolytic enzymes glucokinase and pyruvate kinase was higher, whereas that of the gluconeogenic enzymes PEPCK and glucose-6-phosphatase was lower in fed and fasted NZO mice. These enzyme changes are consistent with a normal response to the hyperinsulinemia in NZO mice. In contrast, the activity of the third regulated gluconeogenic enzyme, fructose-1,6-bisphosphatase, was similar in fed and fasted NZO and NZC mice despite the higher insulin and glucose levels in the NZO mouse. This enzyme is primarily regulated by the powerful inhibitor fructose-2,6-bisphosphate. The levels of this metabolite were measured and found to be increased in both the fed and fasted states in the NZO mouse, suggesting that the activity of the bifunctional enzyme that regulates the level of inhibitor (6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase) is normally regulated in the NZO mouse. We conclude that most insulin-responsive gluconeogenic and glycolytic enzymes are normally regulated in the NZO mouse, but an abnormality in the regulation of fructose-1,6-bisphosphatase may contribute to the increase hepatic glucose production in these mice.

Genomic actions of the androgen receptor are required for normal male sexual differentiation in a mouse model.

Androgens mediate their effects in target cells via the androgen receptor (AR), which acts predominantly as a ligand-dependent transcription factor. In addition, androgens induce rapid activation of second messenger signal transduction cascades, and this is thought to occur via non-genomic mechanisms. We have used the Cre/loxP system to generate an AR knockout (ARKO) mouse targeting exon 3, which encodes the second zinc finger of the DNA-binding domain. To generate universal ARKO mice, floxed AR mice were mated with CMV-Cre mice, which express Cre recombinase ubiquitously. Deletion of the floxed allele in our mice does not disrupt the reading frame, and has been designed so that the mutant AR can bind ligand but not target genes. ARKO males displayed a complete androgen insensitivity phenotype, with female external genitalia and a reduction in body weight compared with wild-type males (P < 0.001). Testes of ARKO males were smaller than control males (P < 0.0001) and were located intra-abdominally. We have demonstrated that genotypically XY mice lacking the second zinc finger of the AR have a female phenotype, and we conclude that the genomic actions of the AR (mediated by DNA binding) are indispensable for normal male sexual differentiation.

Inhibition of parathyroid hormone responsiveness in clonal osteoblastic cells expressing a mutant form of 3',5'-cyclic adenosine monophosphate-dependent protein kinase.

PTH activates multiple acute intracellular signals within responsive target cells, but the importance of cAMP vs. other second messenger signals in mediating different biological responses to PTH is not known. To address these questions, we developed a genetic approach to block activation of the cAMP-dependent protein kinase (PK-A) in PTH-responsive cell lines. Clonal rat osteosarcoma cells (UMR 106-01) were stably transfected with REV-I, a plasmid that directs synthesis of a mutant cAMP-resistant form of the type I regulatory subunit of PK-A. In the transfected bone cells, most of the catalytic subunits of PK-A were associated with the mutant regulatory subunit, and activation of PK-A by cAMP was correspondingly inhibited. We have characterized one such mutant (UMR 4-7) that expressed large amounts of mutant mRNA and exhibited inducible blockade of PK-A via the REV-1 metallothionein promoter. In the absence of metallothionein induction, these cells exhibited nearly normal PTH responsiveness, but after REV-1 induction by Zn2+, they were resistant to PTH-induced activation of PK-A and regulation of membrane phospholipid synthesis by both PTH and cAMP analogs. The mutant UMR 4-7 cell provides a model system in which the consequences of cAMP production by PTH or other agonists that activate adenylate cyclase in osteoblasts may be specifically inhibited by brief exposure to Zn2+. Such mutant cell lines will facilitate further investigation of the linkage between early signalling events and subsequent biological responses in the action of PTH and other agonists on target cells in bone.

Impaired suppression of gluconeogenesis induced by overexpression of a noninsulin-responsive phosphoenolpyruvate carboxykinase gene.

Despite detailed knowledge of the regulation of individual steps in the gluconeogenic pathway, the relative importance of each step to the overall control of gluconeogenesis by insulin is not known. The aim of this study was to determine the role of phosphoenolpyruvate carboxykinase (PEPCK) in the regulation of gluconeogenesis by insulin. Clones of the rat hepatoma cell line H4IIE-C3 were produced, overexpressing a PEPCK gene, driven by a promoter not responsive to insulin. In these cells basal gluconeogenesis from 2-[14C]pyruvate was increased 2.1-fold compared to controls (4.63 +/- 0.49 nmol/10(5) cells vs. 2.21 +/- 0.24 nmol/10(5) cells after 3 h, P < 0.05, n = 5). Increased gluconeogenesis was associated with an increase in basal PEPCK mRNA levels (1.9-fold) and enzyme activity (2.8-fold). Insulin (10(-7) M) suppressed basal gluconeogenesis, PEPCK mRNA levels, and enzyme activity in control cells, but no detectable decrease was observed in PEPCK-transfected cells. These experiments provide direct evidence in intact cells that PEPCK is the rate-limiting enzyme in gluconeogenesis from pyruvate and show that insulin's action to inhibit gluconeogenesis is predominantly on the inhibition of PEPCK transcription.

Impaired glucose tolerance and increased weight gain in transgenic rats overexpressing a non-insulin-responsive phosphoenolpyruvate carboxykinase gene.

The effects of an overexpressed, non-insulin-responsive gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (PEPCK; EC 4.1.1.32), on glucose homeostasis were investigated. Transgenic rats harboring a metallothionein-driven PEPCK gene (lacking the entire PEPCK upstream-regulatory region) expressed transgene PEPCK mRNA in the key gluconeogenic tissues, liver and kidney. Female transgenic rats, studied at 10 weeks of age, showed mild fasting hyperglycemia (6.9 +/- 0.2 vs. 5.9 +/- 0.1 mM P = 0.002 n = 6), hyperinsulinemia (92.2 +/- 4.0 vs. 54.0 +/- 6.6 pM, P = 0.001, n = 6), impaired glucose tolerance and increased weight gain (178.3 +/- 3.2 vs. 153.4 +/- 2.5 g, P = 0.001, n = 16 and n = 13 transgenic and control rats, respectively). Despite hyperinsulinemia at this age, kidneys of transgenic rats maintained a significant 20% elevation of total PEPCK enzyme activity, while total liver PEPCK activity was not reduced. This study suggests that an insulin-resistant step in the gluconeogenic pathway can lead to glucose intolerance and an increase in weight. These rats offer the unique opportunity to study the metabolic consequences of chronic, mild excess glucose supply, as seen in non-insulin-dependent diabetes.

Biosynthesis of calcitonin by human lung cancer cells.

The ectopic secretion of calcitonin (CT) by a wide variety of nonthyroidal human tumors has been studied by CT RIA, but little information is available concerning the biosynthesis of CT in these tumors. In the present study, a human lung cancer cell line (BEN), secreting high mol wt forms of CT was investigated to characterize the CT gene products synthesized. When conditioned medium from BEN cells was chromatographed through a Bio-Gel P-30 column, larger species of immunoreactive CT were detected with mol wt of approximately 8,000 and 18,000. Little, if any, CT of 3,500 mol wt was detected. To examine CT gene products produced in BEN cells, poly A+ RNA was isolated from BEN cells and subjected to cell-free translation assays and DNA/RNA hybridization assays. In the wheat germ cell-free translation assay, a single BEN cell product which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent mol wt of 17,000 could be specifically immunoprecipitated with CT antisera. A similar sized CT-related translation product is produced in wheat germ assays programmed by mRNA prepared from human medullary thyroid carcinomas. In DNA/RNA hybridization assays, a single BEN cell mRNA species of 1,000 base pairs, identical in size to human thyroidal CT mRNA, hybridized to a radiolabeled CT cDNA probe. Hybridization of the CT cDNA probe with BEN cell mRNA was confirmed by RNA dot blot hybridization and cytoplasmic RNA blotting procedures. These results indicate that larger mol wt forms of CT secreted by BEN cells are derived from a translation product and a mRNA which are of similar, if not identical, size as CT gene products produced in human thyroidal tissues. The inability of lung tumor cells to process the CT precursor to calcitonin of 3,500 mol wt may reflect a lack of specific prohormone processing enzymes in these tumor cells or may be due to structural polymorphism in the CT precursor expressed in the lung cells.

Glucocorticoid treatment facilitates cyclic adenosine 3',5'-monophosphate-dependent protein kinase response in parathyroid hormone-responsive osteogenic sarcoma cells.

Late passage cultures of a clonal osteogenic sarcoma line (ROS 17/2.8) failed to respond to PTH with activation of cAMP-dependent protein kinase isoenzymes despite showing a sensitive and dose-dependent increase in cAMP after treatment with the hormone. When cells were treated with hydrocortisone or dexamethasone, protein kinase responsiveness to PTH was readily demonstrated; such treatment also resulted in enhanced cAMP production. Forskolin preincubation resulted in a cAMP response to PTH of similar magnitude to that seen with hydrocortisone but no activation of cAMP-dependent protein kinase occurred. Thus, the effect of glucocorticoid cannot be explained merely by the increased amplitude and sensitivity of the cAMP response which developed with glucocorticoid treatment in these cells. The data indicate that cellular activation of cAMP-dependent protein kinase does not automatically follow cAMP generation and that information transfer can be restored by pharmacological means.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent and cAMP-independent regulation of parathyroid hormone receptors on UMR 106-01 osteoblastic osteosarcoma cells.

The osteoblast-like cells, UMR 106-01, express PTH receptors that are coupled to adenylate cyclase. Recently, we reported the isolation of a UMR 106-01 subclone, UMR 4-7, that is stably transfected with a Zn(++)-inducible mutant of the regulatory subunit of protein kinase A. Incubation of UMR 4-7 cells with Zn++ renders the cells unresponsive to cAMP agonists. This subclone, therefore, seemed particularly suitable for studies of PTH receptor regulation. In UMR 106-01 cells, PTH receptors are strikingly down-regulated by pretreatment with 8-Br-cAMP or 3-isobutyl-1-methylxanthine for 2 days. In UMR 4-7 cells, this effect is totally prevented by prior and concurrent treatment with Zn++. Zn++ addition to UMR 106 cells does not modify these responses. Treatment with the PTH agonist [Nle8,18,Tyr34]bovine PTH(1-34)NH2 [(NlePTH(1-34)] also markedly down-regulates PTH receptors in UMR 106 cells, but this effect is only partially inhibited in Zn(++)-induced UMR 4-7 cells. At high doses, the PTH antagonist, [Nle8,18,Tyr34]bovine PTH(3-34)NH2 [NlePTH(3-34)] also (partially) reduces PTH receptor availability. Receptor regulation by NlePTH(3-34) is not blocked in the cAMP-resistant cells, however. Coincubation of submaximal doses of NlePTH(1-34) (1 nM) with NlePTH(3-34) (1 microM) reduces receptor availability more than when the cells are exposed to either ligand alone. This decrease is only partially inhibited in Zn(++)-induced UMR 4-7 cells. In contrast to its additive effect on receptor regulation, NlePTH(3-34) efficiently competes for binding to the PTH receptor in UMR 106-01 cells and antagonizes the stimulatory effects of NlePTH(1-34) on both intracellular cAMP accumulation and gene expression driven by a transiently transfected synthetic cAMP-responsive enhancer. In conclusion, homologous down-regulation of PTH receptors is mediated by activation of both cAMP-dependent (via protein kinase A) and cAMP-independent pathways. PTH activates both pathways, whereas the effect of NlePTH(3-34) appears to be exclusively cAMP-independent. These results give new insights into mechanisms of PTH receptor regulation.

Identification of calcitonin and calcitonin gene-related peptide messenger ribonucleic acid in medullary thyroid carcinomas by hybridization histochemistry.

Synthesis and secretion of calcitonin and calcitonin gene-related peptide (CGRP) were studied in medullary thyroid carcinomas (MTC) by hybridization histochemistry on tissue sections and by Northern gel analysis of mRNA. Five patients with MTC and elevated serum levels of calcitonin and CGRP were studied. Surgically obtained tumor samples (four primary and three lymph node metastases) were extracted after freezing, and the RNA was fractionated on Northern gels. Hybridization was carried out with 32P-labeled synthetic oligodeoxyribonucleotides coding specifically for calcitonin and CGRP. Calcitonin- and CGRP-specific mRNAs approximately 1000 nucleotides in length were demonstrated in all 7 tumor samples. However, neither calcitonin nor CGRP mRNA was detected in a pheochromocytoma from 1 of the patients who had multiple endocrine neoplasia type II. A series of unselected lung carcinomas yielded the same result. Hybridization histochemistry was carried out on sections from the same tumors using the same probes. The mRNAs for calcitonin and CGRP were located in all cells of neoplastic MTC appearance, with CGRP mRNA at significantly lower levels. This demonstrated that both calcitonin and CGRP mRNA were present within the same tumor cells. The lung tumors and pheochromocytoma were negative with both probes. Hybridization histochemistry is likely to be of use in diagnosis of medullary thyroid cancer and in studying the calcitonin-CGRP mRNA processing mechanism in whole cells.

Abnormal androgen receptor binding affinity in subjects with Kennedy's disease (spinal and bulbar muscular atrophy).

We have investigated androgen-binding properties of the androgen receptor (AR) in cultured suprapubic skin fibroblasts from six subjects with Kennedy's disease (X-linked spinal and bulbar muscular atrophy). Binding of the synthetic androgen methyltrienolone (R1881) was measured in a monolayer assay, and Scatchard analysis was performed to determine the total number of binding sites and the apparent binding affinity (Kd) of the AR for androgen. Five of the six subjects investigated had an abnormal apparent binding affinity, with Kd values ranging from 0.34-11.7 nmol/L, more than 2 SD from the mean of the normal range (0.19 +/- 0.06 nmol/L). In this group of six patients, there was a significant correlation between the AR Kd and the severity of testicular atrophy and gynecomastia. The number of CAG repeats in the expanded region of exon A of the AR gene was determined in all subjects from whom suprapubic skin fibroblasts were cultured and an additional 12 subjects with Kennedy's disease. In the total group of 18 subjects investigated, there was a trend for an increasing number of CAG repeats associated with decreasing age at onset of different symptoms; however, this correlation was not statistically significant. Thus, we report for the first time a quantitative abnormality of the AR apparent binding affinity in subjects with Kennedy's disease, which appears to be related to the severity of the symptoms of androgen insensitivity.

Germline dinucleotide mutation in codon 883 of the RET proto-oncogene in multiple endocrine neoplasia type 2B without codon 918 mutation.

The autosomal dominant multiple endocrine neoplasia type 2 syndromes (MEN 2) comprise three clinically distinct entities, MEN 2A, familial medullary thyroid carcinoma and MEN 2B, which share a common clinical feature: medullary thyroid carcinoma (MTC). MEN 2B is considered to have the most aggressive form of MTC. Therefore, early detection of MEN 2B in order to prevent potentially lethal MTC is important. More than 95% of all MEN 2B cases are caused by germline mutation at codon 918 (M918T) in exon 16 of the RET proto-oncogene. In this study, we demonstrate the presence of germline codon 883 mutation (A883F) in 2 of 3 unrelated MEN 2B cases without codon 918 mutation. Our data demonstrate a novel etiologic event which may have roles in predisposition to MEN 2B when present in the germline and in the pathogenesis of sporadic MTC when somatic.