Weight loss in men in late life and bone strength and microarchitecture: a prospective study.

Weight loss in men in late life was associated with lower bone strength. In contrast, weight gain was not associated with a commensurate increase in bone strength. Future studies should measure concurrent changes in weight and parameters of bone strength and microarchitecture and evaluate potential causal pathways underlying these associations. INTRODUCTION: Our aim was to determine associations of weight loss with bone strength and microarchitecture. METHODS: We used data from 1723 community-dwelling men (mean age 84.5 years) who attended the MrOS study Year (Y) 14 exam and had high-resolution peripheral quantitative computed tomography (HR-pQCT) scans at ≥ 1 skeletal sites (distal tibia, distal radius, or diaphyseal tibia). Weight change from Y7 to Y14 exams (mean 7.3 years between exams) was classified as moderate weight loss (loss ≥ 10%), mild weight loss (loss 5 to < 10%), stable weight (< 5% change), or weight gain (gain ≥ 5%). Mean HR-pQCT parameters (95%CI) were calculated by weight change category using linear regression models adjusted for age, race, site, health status, body mass index, limb length, and physical activity. The primary outcome measure was estimated failure load. RESULTS: There was a nonlinear association of weight change with failure load at each skeletal site with different associations for weight loss vs. weight gain (p < 0.03). Failure load and total bone mineral density (BMD) at distal sites were lower with greater weight loss with 7.0-7.6% lower failure loads and 4.3-5.8% lower BMDs among men with moderate weight loss compared to those with stable weight (p < 0.01, both comparisons). Cortical, but not trabecular, BMDs at distal sites were lower with greater weight loss. Greater weight loss was associated with lower cortical thickness at all three skeletal sites. CONCLUSION: Weight loss in men in late life is associated with lower peripheral bone strength and total BMD with global measures reflecting cortical but not trabecular parameters.

Genetic analysis of vertebral trabecular bone density and cross-sectional area in older men.

We investigated 383 bone candidate genes for associations between single nucleotide polymorphisms and vertebral trabecular volumetric bone mineral density (vBMD) and cross-sectional area (CSA) in 2,018 Caucasian men aged ≥ 65 years. SNPs in TGFBR3, SOST, KL, CALCR, LEP, CSF1R, PTN, GNRH2, FGFR2, and MEPE were associated with vBMD and SNPs in CYP11B1, DVL2, DLX5, WNT4, and PAX7 were associated with CSA in independent study samples (p < 0.005). INRODUCTION: Vertebral bone mineral density and cross-sectional area are important determinants of vertebral bone strength. Little is known about the specific genetic variants that influence these phenotypes in humans. METHODS: We investigated the potential genetic variants associated with vertebral trabecular volumetric BMD and CSA measured by quantitative computed tomography. We initially tested for association between these phenotypes and 4608 tagging and potentially functional single nucleotide polymorphisms (SNPs) in 383 candidate genes in 862 community-dwelling Caucasian men aged ≥ 65 years in the Osteoporotic Fractures in Men Study. RESULTS: SNP associations were then validated by genotyping an additional 1,156 randomly sampled men from the same cohort. We identified 11 SNPs in 10 genes (TGFBR3, SOST, KL, CALCR, LEP, CSF1R, PTN, GNRH2, FGFR2, and MEPE) that were consistently associated with trabecular vBMD and five SNPs in five genes (CYP11B1, DVL2, DLX5, WNT4, and PAX7) that were consistently associated with CSA in both samples (p < 0.005). CONCLUSION: None of the SNPs associated with trabecular vBMD were associated with CSA. Our findings raise the possibility that at least some of the loci for vertebral trabecular BMD and bone size may be distinct.

Long-term Variation in Agricultural Edge-of-Field Phosphorus Transport during Snowmelt, Rain, and Mixed Runoff Events.

Runoff of nutrients and erosion of soil from agricultural lands affect soil fertility and are important nonpoint contributors of P and N to surface and ground waters, yet studies of edge-of-field nutrient transport from snowmelt or rainfall runoff on frozen ground are limited. The objective of this study was to quantify the temporal and spatial variation in edge-of-field snowmelt, rain, and mixed (rain on snow) runoff events for sediment and P loadings in five agricultural subwatersheds over a 12-yr period. Edge-of-field runoff events from five subwatersheds at Pioneer Farm near Platteville, WI, ranging in size from approximately 4 to 30 ha were sampled using automated samplers from 2002 through 2014 to determine sediment and P yields (mass loads). Mean dissolved reactive P (DRP) runoff concentrations for each event type (rain = 1.24 mg L, snow = 1.90 mg L, mix = 2.23 mg L) were above total P (TP) water quality guidelines for surface waters. The percentages of TP that was DRP for snow, mixed, and rain events were 74, 84, and 39%, respectively. Although variation in total annual P yield in edge-of-field runoff was noted between years and among sites within a given year, when aggregated over the study period, the subwatersheds showed similar transport characteristics with respect to DRP and TP yield. This study highlights the importance of examining long-term datasets in quantifying annual yields and understanding the timing of DRP and TP transport for developing best management practices and improving model accuracy in cold weather agricultural systems.

The Association between Objectively Measured Physical Activity and the Gut Microbiome among Older Community Dwelling Men.

OBJECTIVES: To determine the relationship between objectively measured physical activity (PA) and the gut microbiome among community-dwelling older men. DESIGN: Cross-sectional study. SETTING: Osteoporotic Fractures in Men (MrOS) cohort participants at Visit 4 (2014-16). PARTICIPANTS: Eligible men (n=373, mean age 84 y) included participants with 5-day activity assessment with at least 90% wear time and analyzed stool samples. MEASUREMENTS: PA was measured with the SenseWear Pro3 Armband and stool samples analyzed for 16S v4 rRNA marker genes using Illumina MiSeq technology. Armband data together with sex, height, and weight were used to estimate total steps, total energy expenditure, and level of activity. 16S data was analyzed using standard UPARSE workflow. Shannon and Inverse Simpson indices were measures of (within-participant) α-diversity. Weighted and unweighted Unifrac were measures of (between-participant) ß-diversity. We used linear regression analysis, principal coordinate analysis, zero-inflated Gaussian models to assess association between PA and α-diversity, ß-diversity, and specific taxa, respectively, with adjustments for age, race, BMI, clinical center, library size, and number of chronic conditions. RESULTS: PA was not associated with α-diversity. There was a slight association between PA and ß-diversity (in particular the second principal coordinate). Compared to those who were less active, those who had higher step counts had higher relative abundance of Cetobacterium and lower relative abundance of taxa from the genera Coprobacillus, Adlercreutzia, Erysipelotrichaceae CC-115 after multivariable adjustment including age, BMI, and chronic conditions. There was no consistent pattern by phylum. CONCLUSION: There was a modest association between levels of PA and specific gut microbes among community-dwelling older men. The observed associations are consistent with the hypothesis that underlying health status and composition of the host microbiome are related.

Developmental and hormonal regulation of glucocorticoid receptor messenger RNA in the rat.

The biological action of glucocorticoids is dependent upon tissue-specific levels of the glucocorticoid receptor (GR). During stress, the hypothalamic-pituitary-adrenal axis is stimulated, and high levels of glucocorticoids circulate. This axis is modulated by negative feedback by glucocorticoids, which inhibit hypothalamic and pituitary hormone secretion and downregulate GR gene expression. To study the developmental tissue-specific regulation of the GR, we measured the relative concentration of GR mRNA in fetal, neonatal, adult, and aged rats and examined the effects of dexamethasone on GR gene expression. Three different tissue-specific developmental patterns of GR mRNA accumulation were found. In addition, there was an age-dependent tissue-specific pattern in the feedback regulation of GR mRNA by glucocorticoids. In the fetus and neonate, GR mRNA abundance was not regulated by circulating glucocorticoids. The adult pattern of glucocorticoid feedback inhibition of GR mRNA expression appeared between 2 and 7 d of life in liver, and after 7 but before 14 d of age in brain. The GR was biologically active in the 2-d-old neonate, however, since dexamethasone enhanced gene expression of angiotensinogen, which is another glucocorticoid responsive gene. These data demonstrate that the GR gene is regulated by both developmental and tissue-specific factors, and provide another molecular basis for ontogenic variations in the hypothalamic-pituitary-adrenal response to stress.

Regulation of lipoprotein lipase in the diabetic rat.

Diabetes mellitus is associated with a reduction of lipoprotein lipase (LPL) activity and development of hypertriglyceridemia. In the current experiments the mechanisms involved in the regulation of LPL have been examined in control rats, streptozocin-induced diabetic rats, and diabetic rats treated chronically or with a single injection of insulin. Diabetes decreased adipose tissue LPL activity partially by decreasing immunoreactive LPL protein and the steady-state levels of LPL mRNA, but primarily by reducing the catalytic activity of LPL. Both chronic and acute insulin increased adipose tissue LPL activity by correcting the defect in the catalytic activity of LPL and increasing immunoreactive LPL protein; however, only chronic insulin restored LPL mRNA levels to normal. In the heart, LPL activity tended to be elevated with diabetes in parallel to an increase in immunoreactive LPL protein even though levels of LPL mRNA declined. Both chronic and acute insulin normalized LPL activity and immunoreactive LPL protein, while only chronic insulin corrected the levels of LPL mRNA. No changes in the catalytic activity of LPL in heart were detected among the groups. Thus, diabetes and insulin treatment regulate LPL expression pretranslationally, translationally, and post-translationally, with tissue-specific differences apparent in the mechanisms involved.

Cross-species clues of an epigenetic imprinting regulatory code for the IGF2R gene.

The epigenetic marks on the IGF2R gene that encodes a receptor responsible for IGF-II degradation consist of differentially methylated DNA in association with multiple modifications on the associated histones. We review these epigenetic marks across various species during the evolution of IGF2R imprinting. Both IGF2 and IGF2R genesare imprinted in the mammal lineage that diverged from Monotremata approximately 150 million years ago. While IGF2 is consistently imprinted in all mammals following its divergence, IGF2R imprinting disappears in the Euarchonta lineage, including human species, approximately 75 million years ago. Differential DNA methylation marks on the two parental alleles correlate with imprinting in all imprinted genes including IGF2R. While the DNA methylation marks in the IGF2R promoter region 1 (DMR1) correlate with IGF2R allelic expression, the DNA methylation marks in the intron region 2 (DMR2) fail to correlate with IGF2R imprinting status in a number of species. Human IGF2R and mouse neuronal Igf2r are not imprinted despite the presence of DMR2. We have noted that human IGF2R is not imprinted in more than 100 informative samples including various tumor tissues. Furthermore, opossum (Marsupialia) IGF2R is consistently imprinted despite the absence of DMR2. These lines of evidence indicate that DNA methylation marks in DMR2 are neither necessary nor sufficient for consistent imprinting of IGF2R across species. Histone modification marks, however, correlate more consistently with the tissue-specific and species-specific imprinting status of IGF2R in human and mouse. Acetylated histone H3 and H4 and methylated lysine 4 of H3 (H3-K4Me) associate with transcriptionally active alleles while tri-methylated lysine 9 of H3 (H3-K9Me3) marks the silenced alleles. In the mouse, an antisense non-coding transcript called Air is transcribed from DMR2 on the paternal allele, and this imprinted transcript plays a central role in Igf2r imprinting. Mouse Igf2r imprinting depends on an Air RNA while the existence of AIR in other species is unknown. Overall, DNA methylation, histone acetylation, and histone methylation play a vital role in coordinating IGF2R allelic expression across all species. Rare monoallelic or skewed allelic expression of human IGF2R and their biological importance warrants further rigorous study.

Catecholestrogen synthesis and metabolism by human breast tumors in vitro.

The activities of enzymes which synthesize and metabolize catecholestrogens were studied in biopsy samples of human breast neoplasms. Estrogen 2-hydroxylase, a cytochrome P-450-dependent enzyme, was present in both benign and malignant neoplasms but not in normal breast tissue. Catechol O-methyltransferase activity was present in all samples examined and was significantly higher in malignant tumors [549 +/- 31 (S.E.) pmol/20 min/mg protein] than in benign neoplasms (226 +/- 41 pmol/20 min/mg protein) or in normal breast tissue (133 +/- 28 pmol/20 min/mg protein). There was no correlation, however, between estrogen 2-hydroxylase and catechol O-methyltransferase activities. The enzymes responsible for the synthesis and metabolism of catecholestrogens are present in some breast tumor specimens, suggesting that in such tissues these metabolites may be formed in vivo.

Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts.

The correlation between telomerase activity, telomere lengths, and cellular replicative capacity has led to the theory that maintenance of telomere lengths by telomerase acts as a molecular clock to control replicative capacity and senescence. Regulation of this molecular clock may have applications in the treatment of cell aging and tumorigenesis, although little is presently known about the regulation of telomerase activity. To investigate possible mechanisms of regulation, we examined telomerase activity and the expression of the human telomerase RNA subunit and the human telomerase reverse transcriptase protein (hTERT) during human fetal heart, liver, and kidney development. The human telomerase RNA subunit is expressed in all three tissues at all gestational ages examined. hTERT expression correlates with telomerase activity in the liver, where both are expressed at all ages surveyed, and in the heart, where both are present until the 11th gestational week but not thereafter. However, although telomerase activity in the kidney is suppressed after the 15th gestational week, the hTERT transcript can be detected until at least the 21st week. Reverse transcription-PCR using primers within the reverse transcriptase domain of hTERT show the presence of multiple alternately spliced transcripts in these tissues, corresponding to full-length message as well as spliced messages with critical reverse transcriptase motifs deleted. Of note, telomerase activity in the kidney is only present at those gestational ages when full-length hTERT message is expressed (until approximately week 15), with spliced transcripts continuing to be expressed at later stages of development. The tissue-specific and gestational-age dependent expression of hTERT mRNA seen in human development suggests the presence of at least two regulatory mechanisms controlling the activity of telomerase: transcriptional control of the hTERT gene and alternate splicing of hTERT transcripts.

GH safety workshop position paper: a critical appraisal of recombinant human GH therapy in children and adults.

Recombinant human GH (rhGH) has been in use for 30 years, and over that time its safety and efficacy in children and adults has been subject to considerable scrutiny. In 2001, a statement from the GH Research Society (GRS) concluded that 'for approved indications, GH is safe'; however, the statement highlighted a number of areas for on-going surveillance of long-term safety, including cancer risk, impact on glucose homeostasis, and use of high dose pharmacological rhGH treatment. Over the intervening years, there have been a number of publications addressing the safety of rhGH with regard to mortality, cancer and cardiovascular risk, and the need for long-term surveillance of the increasing number of adults who were treated with rhGH in childhood. Against this backdrop of interest in safety, the European Society of Paediatric Endocrinology (ESPE), the GRS, and the Pediatric Endocrine Society (PES) convened a meeting to reappraise the safety of rhGH. The ouput of the meeting is a concise position statement.

Repression of hepatitis B virus (HBV) transgene and HBV-induced liver injury by low protein diet.

Persistent infection with hepatitis B virus (HBV) is one of the primary risk factors for human hepatocellular carcinoma (HCC). In a human ecological study, we have shown that, in addition to HBV, animal food consumption also significantly contributes to the variance of HCC. To test the interacting effect of HBV and animal food consumption on the development of HCC, we investigated HBV expression in HBV transgenic mice fed three levels of casein diet. HBV expression in transgenic animals was substantially inhibited when dietary casein was reduced from the traditional level of 22% to the level of 6%. Northern analysis revealed that suppression of HBV was derived from both the upstream albumin promoter and the internal HBV promoter. Immunochemical staining of liver sections indicated that only a few hepatocytes around the central vein expressed viral surface antigen (HBsAg) in the 6% casein animals, whereas virtually all hepatocytes stained positively for HBsAg in the 22% dietary casein animals. Serum HBsAg concentrations at 4 months were increased by 1.6-, 2.1-, and 5.1- fold over baseline for animals fed the 6%, 14%, and 22% casein diets, respectively. Correspondingly, liver injury was much less severe in animals fed 6% casein diet than in those fed 14% and 22% casein diets. These results demonstrate that a low casein diet is a potent suppresser of HBV transgene and HBV-induced liver injury, suggesting that diet management may be a practical means to aid in the control HBV infection.

Allelic expression of the putative tumor suppressor gene p73 in human fetal tissues and tumor specimens.

p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic 'hit' or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms' tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms' tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms' tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.

Differential biallelic activation of three insulin-like growth factor II promoters in the mouse central nervous system.

Imprinting of the insulin-like growth factor II gene (IGF-II) is conserved in human, rat, and mouse. In human liver and chondrocytes, IGF-II transcripts from promoter hP1 are always derived from both parental alleles, while transcripts from promoters hP2-hP4 are from one parental allele. To examine the promoter-specific imprinting pattern of mouse IGF-II, we examined IGF-II expression in F1 generation mice derived from crossing M. spretus with M. musculus using a novel BsaA1 polymorphism in mouse IGF-II exon 6. There was maintenance of maternal IGF-II imprinting in all non-central nervous system (CNS) tissues in the F1 generation animals. However, there was biallelic expression of the IGF-II gene in CNS. Allelic expression of each IGF-II promoter transcript was examined by full-length cDNA amplification with promoter-specific primers. In every tissue in which IGF-II was imprinted, IGF-II transcripts were derived from paternal promoters mP1-mP3, while the maternal allele was suppressed. In the CNS, however, promoters mP1-mP3 of the imprinted maternal allele became activated, leading to the biallelic expression of IGF-II. Moreover, the expression of IGF-II from each parental allele differed in various CNS regions. In leptomeninges, mP1-mP3 drive IGF-II expression predominantly from the paternal allele, while in some CNS regions, the promoter transcripts were primarily from the maternal allele. The coordinate regulation of mouse IGF-II promoters suggests the presence of an upstream imprinting complex controlling IGF-II imprinting.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-specific imprinting of the mouse insulin-like growth factor II receptor gene correlates with differential allele-specific DNA methylation.

Imprinted genes may be expressed uniparentally in a tissue- and development-specific manner. The insulin-like growth factor II receptor gene (Igf2r), one of the first imprinted genes to be identified, is an attractive candidate for studying the molecular mechanism of genomic imprinting because it is transcribed monoallelically in the mouse but biallelically in humans. To identify the factors that control genomic imprinting, we examined allelic expression of Igf2r at different ages in interspecific mice. We found that Igf2r is not always monoallelically expressed. Paternal imprinting of Igf2r is maintained in peripheral tissues, including liver, kidney, heart, spleen, intestine, bladder, skin, bone, and skeletal muscle. However, in central nervous system (CNS), Igf2r is expressed from both parental alleles. Southern analysis of the Igf2r promoter (region 1) revealed that, outside of the CNS where Igf2r is monoallelically expressed, the suppressed paternal allele is fully methylated while the expressed maternal allele is completely unmethylated. In CNS, however, both parental alleles are unmethylated in region 1. The importance of DNA methylation in the maintenance of the genomic imprint was also confirmed by the finding that Igf2r imprinting was relaxed by 5-azacytidine treatment. The correlation between genomic imprinting and allelic Igf2r methylation in CNS and other tissues thus suggests that the epigenetic modification in the promoter region may function as one of the major factors in maintaining the monoallelic expression of Igf2r.

Modulation of Igf2 genomic imprinting in mice induced by 5-azacytidine, an inhibitor of DNA methylation.

The adjacent genes, insulin-like growth factor 2 (Igf2) and H19, are imprinted in both mouse and human. While Igf2 is expressed from the paternal allele, H19 is transcribed exclusively from the maternal allele. To explore the underlying mechanism of Igf2 and H19 imprinting, we studied the effect of DNA demethylation on allelic expression by injecting mice with the demethylating agent 5-azacytidine (5-aza-C). We observed a > or = 2-fold increase in the abundance of Igf2 mRNA in liver from treated mice compared with that of control mice. There was no significant change in Igf2 or H19 expression in brain. In the 5-aza-C-treated mice, there was dramatic modulation of Igf2 imprinting. In some tissues, Igf2 was expressed biallelically, while in other tissues, the paternal allele was silenced and the normally imprinted maternal allele was expressed, an example of allelic switching. There was no change in the normal biallelic pattern of Igf2 expression in brain. H19, on the other hand, remained imprinted in all tissues in mice treated with 5-aza-C. These results provide the first example of a pharmacological manipulation of genomic imprinting of an endogenous gene in vivo and further implicate DNA methylation as an important factor in maintaining the differential allelic expression of the Igf2 gene.

Milk-alkali syndrome in patients treated with calcium carbonate after cardiac transplantation.

Heart and heart-lung transplant recipients at Stanford (Calif) University Medical Center were routinely prescribed long-term calcium carbonate antacid therapy to aid in the prevention of peptic ulcer disease and osteoporosis associated with glucocorticoid immunosuppressive therapy. Patients consumed 4 to more than 10 g/d of elemental calcium. Since calcium carbonate also provides the essential ingredients for the development of the milk-alkali syndrome, the laboratory flow sheets of 297 heart and heart-lung transplant recipients were reviewed to examine the incidence of hypercalcemia. Sixty-five patients developed significant hypercalcemia after transplantation. Thirty-one patients were alkalotic at the time of hypercalcemia; 37 had impairment in renal function. It is likely that most of these patients had the milk-alkali syndrome. While most patients became eucalcemic by discontinuing calcium carbonate therapy, intravenous hydration and forced diuresis were used to treat severe cases. It is possible that the incidence of the milk-alkali syndrome will increase with the current popularity of prescribing calcium carbonate for the prevention and treatment of osteoporosis.

Interactions of growth hormone and parathyroid hormone in renal phosphate, calcium, and calcitriol metabolism and bone remodeling in postmenopausal women.

The mechanisms underlying the effects of recombinant human growth hormone (rhGH) on vitamin D, mineral, and bone metabolism are not known. We examined whether these effects are mediated by parathyroid hormone (PTH) by measuring renal phosphorus (P) and calcium (Ca) handling, serum calcitriol, and markers of bone turnover for 24 h before and 72 h after an infusion of hPTH(1-34) in eight healthy postmenopausal women at baseline and following short-term (1 week) and sustained (5 weeks) rhGH treatment. On short-term rhGH, serum phosphorus and basal TmP/GFR were unaffected, but the fall in TmP/GFR after hPTH infusion was exaggerated (integrated response: -99.2 +/- 22.3 versus -144.1 +/- 15.0 minute-mg/dl, P = 0.0021). Basal calcitriol levels rose from 115 +/- 17 to 163 +/- 16 pM (P = 0.0002), but the increase in calcitriol following hPTH infusion was unaffected by short-term rhGH. The basal Ca excretion index (CEI) rose from 0.054 +/- 0.005 to 0.073 +/- 0.007 mM (P = 0.0095), but markers of bone turnover were unaffected. With sustained rhGH treatment, serum P (1.47 +/- 0.05 mM), basal TmP/GFR (4.29 +/- 0.24 mg/dl), and basal CEI (0.067 +/- 0.005 mM) were elevated compared with control values, and the PTH-induced lowering of TmP/GFR was again enhanced (-158.7 +/- 22.8 minute-mg/dl, P = 0.0021). Basal calcitriol concentrations returned to control levels (108 +/- 10 pM), but the calcitriol response to hPTH remained unchanged. Markers of bone remodeling were elevated with sustained rhGH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of recombinant insulin-like growth factor-I and growth hormone on bone turnover in elderly women.

We evaluated the effects of recombinant insulin-like growth factor-I (IGF-I) and growth hormone (GH) on calciotropic hormones and bone turnover markers in 16 healthy elderly women 71.9 +/- 1.3 years of age (mean +/- SEM). Subjects consumed a fixed diet providing 1000 mg of calcium and 0.9 g/kg of protein for 10 days before starting baseline 24-h urine and blood collections. Specimens were collected for 6 consecutive days before initiating subcutaneous injections of GH (25 micrograms/kg/day, n = 5) and IGF-I at 60 micrograms/kg b.i.d. (high-dose, n = 5) or at 15 micrograms/kg b.i.d. (low-dose, n = 6) for 28 days. Resorption markers included urine hydroxyproline (OHP), total pyridinolines (PYD), and N-telopeptide; formation markers include osteocalcin, skeletal alkaline phosphatase (sALP), and type I procollagen carboxy-terminal extension peptide (CICP). For each subject, baseline daily turnover markers varied substantially (DV = 16-22%). With GH and high-dose IGF-I, resorption and formation markers increased progressively to maximum levels at day 21. For GH, the increase in day 21 PYD, N-telopeptide, osteocalcin, and CICP was 143, 111, 53, and 81%, respectively (p < 0.96-0.02). For high-dose IGF-I, these increases were 108, 81, 77, and 111% (p < 0.02-0.002). However, with low-dose IGF-I no change was observed in resorption markers while osteocalcin and CICP increased progressively (day 21, % increases = 88 +/- 51, 36 +/- 14). Twenty-four hour urine collections during the last days of baseline and of study drug were taken as six 4 h aliquots. When deoxyPYD was measured on these samples in the low-dose IGF-I group, a significant increase was observed only on the 0800-1200 h aliquot. Serum phosphorus concentrations increased with GH (21.2 +/- 3.3%) and high-dose IGF-I (8.8 +/- 3.6%) by day 21 but actually decreased by day 28 (-9.7 +/- 2.7, p < 0.02) with low-dose IGF-I. Urinary phosphorus excretion decreased with high-dose IGF-I only. Twenty-four hour calcium excretion increased with all treatments. These results indicate that both GH and high-dose IGF-I activate remodeling osteons. By contrast, low-dose IGF-I may directly increase osteoblastic function with only a minimal increase in bone resorption and may therefore provide a useful means to increase bone mass. The results also suggest some of the GH action on renal phosphorus handling represents a direct action of GH on the nephron which does not involve the intermediacy of IGF-I. Finally, even under controlled conditions bone turnover markers exhibit substantial daily variation so that a very large treatment effect will be required for these markers to have clinical utility.

Glucocorticoid modulation of corticotropin-releasing factor desensitization in cultured rat anterior pituitary cells.

The ability of ovine corticotropin-releasing factor (CRF) to stimulate both ACTH release and intracellular cAMP accumulation is rapidly and reversibly desensitized when rat anterior pituitary cells are cultured in the absence of added glucocorticoids. Since this desensitization has not been readily apparent in vivo, where initial CRF exposure results in high levels of ambient glucocorticoids, we examined the effect of glucocorticoids on the desensitization process in vitro. Rat anterior pituitary cells were cultured for 3-5 days in the absence of added steroid hormones. Dexamethasone was then added to some culture wells 24 h before the desensitization experiment began. Desensitization of CRF was achieved by preincubating the cells for 4 h with varying concentrations (10(-11)-10(-7) M) of ovine CRF, washing the cells with medium alone, and then reexposing the cells to CRF. In the absence of glucocorticoid, the ED50 for CRF desensitization (the preincubation dose causing 50% desensitization of subsequent ACTH release) was 3 X 10(-10) M, but cells that had been preexposed to dexamethasone desensitized less readily. With concentrations of dexamethasone of 10(-8) M or greater, no desensitization occurred. When cells were incubated in the absence of added glucocorticoids, CRF-stimulated intracellular cAMP accumulation was diminished by prior exposure to CRF. No decrease in intracellular cAMP accumulation was seen in those cells that had been preincubated with dexamethasone, however. Similar changes in CRF desensitization of ACTH release were observed when cells were incubated with corticosterone, but not with 10(-8) M testosterone, progesterone, aldosterone, or estradiol. These data demonstrate that glucocorticoids profoundly alter the development of CRF desensitization in vitro and suggest that high ambient glucocorticoid concentrations prevent the development of substantial CRF desensitization in vivo.