The objective is to evaluate the association of arterial stiffness [brachial-ankle pulse wave velocity (ba-PWV)] with glycemic control and duration of type 2 diabetes in diabetic subjects with or without hypertension. One thousand Chinese diabetic patients (562 with hypertension, 438 without hypertension) were included in this study. All patients underwent ba-PWV and biochemical measurements. Ba-PWV was significantly higher in diabetic subjects with hypertension than those without hypertension (1779 +/- 341 vs. 1691 +/- 342 cm/s, P < 0.0001), and these subjects were under similar glycemic control (6.9 +/- 1.5 vs. 6.9 +/- 1.3, P = 0.86). In diabetic and hypertensive subjects, ba-PWV was positively associated with HbA1c (1715 +/- 314 vs. 1851 +/- 335 cm/s, in subjects with HbA1c < 6.5% and with HbA1c > 7.0%, respectively, P < 0.0001), as well as duration of diabetes (1671 +/- 342 vs. 1791 +/- 313 vs. 1861 +/- 338 cm/s, in subjects with duration of diabetes <5 years, 5-10 years, and >10 years, respectively, P all < 0.05). In diabetic subjects without hypertension, ba-PWV was not associated with HbA1c (P = 0.47), but associated with duration of diabetes (1503 +/- 272 vs. 1692 +/- 354 cm/s, in subjects with duration of diabetes <5 years and >10 years, respectively, P = 0.0013). Ba-PWV was higher in diabetic subjects with hypertension than in diabetic subjects without hypertension, suggesting the independent effect of hypertension on arterial stiffness in diabetic subjects. Ba-PWV positively correlated with HbA1c and duration of diabetes in subjects with diabetes and hypertension, suggesting the importance of early glycemic control in the prevention of arterial stiffness and vascular complications.
Estrogen receptors (ERα and ERβ) mediate the neuroprotection of estrogens against MPTP-induced striatal dopamine (DA) depletion. Pain is an important and distressing symptom in Parkinson's disease (PD). Voltage-gated sodium channels in sensory neurons are involved in the development of neuropathic pain. In this study, MPTP caused changes in nociception and alterations of gene expression of voltage-gated sodium channels in dorsal root ganglion (DRG) neurons in ER knockout (ERKO) mice were investigated. We found that administration of MPTP (11 mg/kg) to WT mice led to an extensive depletion of DA and its two metabolites, αERKO mice were observed to be more susceptible to MPTP toxicity than βERKO or WT mice. In addition, we found that the mRNA levels of TTX-S and TTX-R sodium channel subtypes were differentially affected in MPTP-treated WT animals. The MPTP-induced up-regulation of Nav1.1 and Nav1.9, down-regulation of Nav1.6 in DRG neurons may be through ERβ, up-regulation of Nav1.7 and down-regulation of Nav1.8 are dependent on both ERα and ERβ. Therefore, the MPTP-induced alterations of gene expression of sodium channels in DRG neurons could be an important mechanism to affect excitability and nociceptive thresholds, and the ERs appear to play a role in nociception in PD.
Six monkeys were rendered hemiparkinsonian with a unilateral injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. These monkeys displayed ipsilateral circling under basal conditions, and after dopaminergic stimulation with levodopa they decreased their ipsilateral circling and started turning to the contralateral side of their lesion. The effect of 17beta-estradiol and dehydroepiandrosterone (DHEA) was investigated in these animals. 17beta-Estradiol (0.1 mg/kg) added to a threshold dose of levodopa significantly potentiated contralateral circling (mean/30 min) compared to saline or threshold levodopa treatment whereas the duration of circling remained unchanged. DHEA (1-15 mg/kg) alone induced contralateral circling, compared to saline treatment, for 90 min. In addition, DHEA (1-15 mg/kg) potentiated the contralateral circling (mean/30 min) induced by a threshold dose of levodopa and did not change the duration of levodopa circling. A maximal response was observed with 1 or 5 mg/kg of DHEA combined with levodopa depending on the monkey. No correlation was found between the dose for the maximal DHEA response and baseline circling or threshold dose of levodopa. These results suggest that 17beta-estradiol or DHEA is able to potentiate locomotor activity of hemiparkinsonian monkeys. The DHEA doses investigated are similar to those presently used in humans. DHEA may be an alternative to 17beta-estradiol to modulate dopaminergic activity.
Hyperandrogenemia modifies phenotypic characteristics of women with polycystic ovary syndrome (PCOS). The aim of the present study is to evaluate (a) the prevalence of hyperandrogenemia in PCOS women (Rotterdam criteria) and (b) the impact of either the degree or the type of hyperandrogenemia on phenotype. Anthropometric, clinical, hormonal, metabolic and ultrasound characteristics of 1,218 women with PCOS were analyzed in this cross-sectional study. The prevalence of hyperandrogenemia was 58.8 %. Women with hyperandrogenemia had higher luteinizing hormone (LH), follicle-stimulating hormone (FSH), free androgen index, lower sex-hormone-binding globulin (SHBG) and fasting glucose levels compared to women with normal androgens (p < 0.001 for all comparisons; p = 0.001 for fasting glucose). Regarding the presence of isolated hyperandrogenemia, the group with only elevated testosterone levels was termed GT and an analogous categorization was made for dehydroepiandrosterone sulfate (GD) and androstenedione (Δ4) (GΔ4), respectively. GT, GD and GΔ4 comprised the 17.2, 7.6 and 4.1 % of total cohort, respectively. These groups differed significantly between them in LH, LH/FSH ratio, and SHBG (p < 0.001). Hyperandrogenemia is found in almost 60 % of women with PCOS (Rotterdam criteria), and it affects hormonal characteristics of these women such as LH and SHBG values. Regarding the impact of isolated hyperandrogenemia on PCOS characteristics, it appears that Δ4 and testosterone elevations are associated with increased LH levels.
YoungHyp mice malabsorb phosphate from the jejunum at 4 weeks of age. This has been attributed to both low plasma levels of 1,25-dihydroxyvitamin D and to intestinal resistance to stimulation by 1,25-dihydroxyvitamin D. To differentiate between these two hypotheses, 4 week old normal andHyp mice were treated with 0, 17, 50, or 150 ng/kg/day of 1,25-dihydroxyvitamin D(3) by Alzet osmotic mini pumps (n=10-12/group). After 4 days, the jejunum was isolated by sutures and 0.5 ml 2 mM Na(2)HPO(4) in 150MM: NaCl with 1.0 µCi(32)PO(4) was injected into the lumen. After 8 min, plasma, jejunal tissue and lumenai contents were measured for(32)P content. Absorption was measured as counts removed from the lumen. Both normal andHyp mice responded to the 1,25-dihydroxyvitamin D(3) with increased absorption, increased tissue(32)P and increased plasma(32)P.Hyp mice responded less than normal mice to the 50 ng/kg/day dose in plasma(32)P levels (significant dose by genotype interaction,P<0.05). Plasma was pooled by genotype and dose for the measurement of plasma 1,25-dihydroxyvitamin D. This yielded 13 samples (7 normal and 6Hyp). Absorption of(32)P (r=0.75, p=0.002) and jejunal tissue content of(32)P (r=0.66, p=0.02) were correlated to plasma 1,25-dihydroxyvitamin D. Analysis of covariance revealed a significant difference in phosphate absorption between normal andHyp mice (p=0.02). In conclusion, there is a partial resistance of intestinal phosphate absorption to 1,25-dihydroxyvitamin D stimulation.
The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10(-7) M 1,25(OH)2D3 for 24h resulted in a 20-30% decrease in PKI mRNA. 1,25(OH)2D3 treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)2D3 directly and tissue-specifically downregulates PKI mRNA in the chick kidney.
The osteopetrotic (op/op) rat mutation is a lethal mutation in which decreased osteoclast function (bone resorption) coexists with markedly elevated serum levels of 1 ,25-dihydroxyvitamin D3[1,25(OH)2D3]. Increased circulating levels of 1,25(OH)2D3 have been reported in other osteopetrotic animal mutations and in some osteopetrotic children. This study examined the effects of 1,25(OH)2D3 infusions on serum and skeletal parameters in normal and mutant rats of op stock. We also examined vitamin D receptor expression and binding in bone cells from op normal and mutant animals. Four-week-old normal and mutant rats were infused either with propylene glycol (used as controls) or with 12.5-125 ng of 1,25(OH)2D3/d using osmotic minipumps implanted subcutaneously for 1 wk. Sera were analyzed for calcium, phosphorus, and 1,25(OH)2D3 levels. Histomorphometric analyses of proximal tibiae from treated normal (50 ng/d) and op mutant (125 ng/d) rats and their vehicle-infused controls were performed. Normal animals infused with 1,25(OH)2D3 exhibited a dose-dependent increase in serum calcium levels. Histomorphometric analyses of metaphyseal bone within the primary spongiosae region showed that 1,25(OH)2D3 increased osteoclast number with a reduction in osteoblast surface associated with a decrease in growth plate cartilage thickness. However, similar analyses on secondary spongiosae showed a decrease in osteoclast number and surface associated with an anabolic response. Op mutants infused with 1,25(OH)2D3 did not exhibit any change in serum calcium levels or histomorphometric parameters related to growth plate cartilage and metaphyseal bone compared with mutant controls. Vitamin D mRNA and protein levels were increased twoto threefold in op mutants compared to age-matched normal rats. However, binding affinity of 1,25(OH)2D3 to its receptor was similar between op mutant and normal animals. High dose calcitriol therapy, under the conditions and period of treatment used in this study, failed to stimulate bone turnover in op rats, suggesting that they are resistant to the skeletal effects of 1,25(OH)2D3. The failure of osteoclast activation in response to 1,25(OH)2D3 treatment may be associated with osteoblast incompetence in this mutation.
The aim of the present study was to examine the hypothesis that primary cultures of osteoblasts obtained from bones of young animals respond to hormones better than cell cultures obtained from old animals. We studied in cultured osteoblastic cells the effects of 1,25(OH)2D3 and sex steroid hormones on several mouse osteoblastic phenotypic expressions including transforming growth factor-beta (TGF-beta) and interleukin-1beta (IL-1beta) mRNAs. Second passages of long bone-derived osteoblastic cells from young donors (5-12 wk) and old donors (10-12 mo old) were used for this study. The cells obtained from old animals had decreased ALP activity and cAMP compared with cells obtained from young animals with no change in collagen production and mineralization. The addition of 17beta-estradiol and testosterone increased ALP activity and mineralization in the cultured cells from both age groups and collagen production in cells obtained from old mice. Using in situ hybridization IL-1beta and TGF-beta mRNA expression was observed to be higher in the osteoblasts from young than from old donors. 1,25(OH)2D3 increased IL-1beta mRNA expression in the cells derived from young mice. Testosterone and 17beta-estradiol inhibited IL-1beta mRNA expression only in cells derived from young mice. Sex steroid hormones did not change TGF-beta mRNA expression in any of the cell lines, but 1,25(OH)2D3 increased its expression in cells derived from old donors. The results of the present study indicate that cells obtained from old mice are generally less active than those obtained from young animals.
The goal of the present work was to use IEC-6 cells to investigate the possible mechanisms underlying the regulation of alkaline phosphatase (ALP) activity (ALPA) by 1α, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and retinoids. Here we demonstrate that the vitamin D analogs, 25(OH)(2)-16-ene-23-yne-D(3) and 1α, 24S-(OH)(2)-22-en-26, 27-dehydro-vitamin D(3), which have been shown by others to bind to the intracellular vitamin D receptor (VDR), have similar effects to 1, 25(OH)(2)D(3) in increasing ALPA of IEC-6 cells. A third vitamin D analog, 25-(OH)-16-ene-23-yne-D(3) (AT), which activates membrane 1,25(OH)(2)D(3) effects, but binds poorly to the intracellular VDR, did not stimulate ALPA of IEC-6 cells. These data suggest that the effects of 1,25(OH)(2)D(3) to increase ALPA are mediated by intracellular VDR rather than by membrane actions of the hormone. The all-trans and 9-cis retinoic acids alone each caused increased ALPA of IEC-6 cells without altering steady-state levels of ALP mRNA, suggesting that retinoic acids may regulate ALPA of IEC-6 cells at a posttranscriptional level. Vitamin D analogs which bind intracellular receptors showed synergistic effects with either retinoid to increase ALPA, but there was no interaction with AT. Although the retinoids alone did not alter ALP mRNA levels, addition of 1,25(OH)(2)D(3) in combination with either retinoid increased ALP mRNA more than did 1,25(OH)(2)D(3) alone. These data suggest that the synergistic effects of 1,25(OH)(2)D(3) and retinoids on IEC-6 cell ALPA are mediated by intracellular VDR. The results of these experiments indicate that 1,25(OH)(2)D(3) alters IEC-6 cell ALPA via increased mRNA levels, while retinoids appear to both have post-transcriptional effects and the capacity to interact with 1,25(OH)(2)D(3) in altering ALP mRNA levels.
The measure of glycated hemoglobin (HbA1c) concentration is the gold standard of glycemic control index in diabetes management and is well known as a marker for diabetes complications. However, HbA1c level neither accurately reflect glucose fluctuations, nor does it provide a clear indication of glycemic control in recent days or weeks. HbA1c concentration measurement can be confounded in patients with anemia, hemoglobinopathy, liver disease, or renal impairment. 1,5-Anhydroglucitol (1,5-AG) structurally resembles glucose. It can be influenced by diet or medication, gender and race, especially severe renal disease and various pathological conditions. Most notably, 1,5-AG level is reflective of short-term glucose status, postprandial hyperglycemia, and glycemic variability which are not captured by HbA1c assay. 1,5-AG may suggest an alternative index of subtypes of diabetes and a warning sign of diabetes complications. This review provides an overview of our current understanding of the role of 1,5-AG marker in diabetes. However, further investigations on the associations between this glycemic marker and diabetes complications are needed.
The aim of this study was to estimate the associations between tumor necrosis factor (TNF) gene polymorphisms and type 1 diabetes (T1D) using meta-analysis. Relevant studies were searched using PubMed and Embase up to August 2013. A total of 32 comparisons from 21 studies examining the associations between TNF polymorphisms and T1D were included in the present meta-analysis. Our meta-analysis identified a significant association between TNF -308 A/G polymorphism A allele and T1D in all subjects [odds ratio (OR) 2.001, 95 % confidence interval (CI) 1.732-2.312). Significant associations of AA and AA+AG genotype of TNF -308 A/G polymorphism with genetic susceptibility to T1D were also found (OR 3.203, 95 % CI 2.373-4.324; OR 2.232, 95 % CI 1.881-2.649). After stratification by ethnicity, significant associations of T1D with TNF -308 A/G polymorphism under all genetic models (A allele and AA, AA+AG genotype) were still detected in European (OR 1.952, 95 % CI 1.675-2.274; OR 3.108, 95 % CI 2.169-4.455; OR 2.249, 95 % CI 1.870-2.706, respectively) and non-European populations (OR 2.152, 95 % CI 1.488-3.112; OR 3.439, 95 % CI 2.000-5.914; OR 2.207, 95 % CI 1.496-3.257, respectively). Our meta-analysis also revealed an association of TNF -857 T/C polymorphism T allele with T1D risk (OR 1.647, 95 % CI 1.431-1.896). Furthermore, analysis of TT and TT+TC genotype indicated the same result patterns as shown by the TNF -857 T/C polymorphism T allele (OR 2.206, 95 % CI 1.467-3.317; OR 1.762, 95 % CI 1.490-2.083). In conclusion, our meta-analysis results indicate that TNF -308 A/G and -857 T/C polymorphisms are involved in the genetic background of T1D.
Chronic glucocorticoid therapy causes rapid bone loss and clinical osteoporosis. We previously found that dexamethasone, a potent glucocorticoid, increased renal expression of vitamin D-24-hydroxylase, which degrades such vitamin D metabolites as 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 (1,25[OH]2D3). We therefore investigated the mechanisms of this increase in UMR-106 osteoblast-like cells and LLC-PK1 kidney cells. To induce 24-hydroxylase expression, 1,25(OH)2D3 (10(-7)M) and dexamethasone were added simultaneously to the medium of LLC-PK1 cells, and 24 h before dexamethasone treatment, 1,25(OH)2D3 was added to the medium of UMR-106 cells. Dexamethasone dose dependently increased 24-hydroxylase mRNA and enzymatic activity in 1,25(OH)2D3-treated LLC-PK1 and UMR-106 cells. Maximal stimulation was observed with 10(-6) M dexamethasone in both cell lines. The addition of 10(-6) M dexamethasone significantly increased the abundance of 24-hydroxylase mRNA by 24 and 8 h in 1,25(OH)2D3-treated LLC-PK1 and UMR-106 cells, respectively. Stimulation for dexamethasone in UMR-106 cells persisted for up to 48 h. Dexamethasone stimulation of 24-hydroxylase mRNA expression in UMR-106 cells was abolished by pretreatment with cycloheximide, an inhibitor of protein synthesis. Northern and Western analyses indicated that 10(-6) M dexamethasone markedly increased the abundance of c-fos mRNA at 20 min and c-fos protein concentration at 60 min in 1,25(OH)2D3-treated UMR-106 cells but only slightly induced the abundance of c-jun mRNA. The addition of phorbol 12-myristate 13-acetate increased mRNA expression for both c-fos and 24-hydroxylase in 1,25(OH)2D3-treated UMR-106 cells. The effect of dexamethasone on 24-hydroxylase mRNA expression was blocked by RO31-8220, a specific inhibitor of protein kinase C. Thus, dexamethasone in the presence of 1,25(OH)2D3 enhances expression of 24-hydroxylase in UMR-106 osteoblastic cells via new protein synthesis. The mechanism of this effect appears to involve activation of the AP-1 site by increased c-fos protein.
Parathyroid hormone (PTH) elicits many of its physiological effects by activating distinct G-proteincoupled signaling cascades that lead to synthesis of cyclic AMP and hydrolysis of phosphatidylinositol 4,5-bisphosphate. Using the nonhydrolyzable photoreactive GTP analog [α-32P]GTP-γ-azidoanilide (GTP-AA) and peptide antisera raised against G-protein α-subunits, we studied coupling of the PTH receptor to G-proteins in rat osteoblast-like cells (ROS 17/2.8), and in human embryonal kidney cells expressing the cloned human PTH/parathyroid hormone-related peptide (PTHrP) receptor at 40,000 receptors/cell (C20) or 400,000 receptors/cell (C21). Incubation of C21 membranes (but not C20 membranes) with [Nle8,18, Tyr34]-bovine PTH(1-34) amide (bPTH[1-34]) led to concentration-dependent incorporation of GTP-AA into the two isoforms of Gα
s, into Gα
q/11, and to a much lesser extent into Gα
i(1). In ROS 17/2.8 cells, bPTH(1-34) increased the incorporation of GTP-AA into Gα
s, but not into Gα
q/11 or Gα
i. The ability of bPTH(1-34) to increase labeling of Gα
s and Gα
q/11 was correlated with the receptor-dependent sensitivity of the adenylyl cyclase and phospholipase C signaling pathways to the hormone.
Endothelial nitric oxide synthase (eNOS)-derived nitric oxide is a major vasorelaxing factor and a mediator of vasopermeability and angiogenesis. Vasoinhibins, a family of antiangiogenic prolactin fragments that include 16 K prolactin, block most eNOS-mediated vascular effects. Vasoinhibins activate protein phosphatase 2A, causing eNOS inactivation through dephosphorylation of eNOS at serine residue 1179 in bovine endothelial cells and thereby blocking vascular permeability. In this study, we examined whether human eNOS phosphorylation at S1177 (analogous to bovine S1179) influences other actions of vasoinhibins. Bovine umbilical vein endothelial cells were stably transfected with human wild-type eNOS (WT) or with phospho-mimetic (S1177D) or non-phosphorylatable (S1177A) eNOS mutants. Vasoinhibins inhibited the increases in eNOS activity, migration, and proliferation following the overexpression of WT eNOS but did not affect these responses in cells expressing S1177D and S1177A eNOS mutants. We conclude that eNOS inhibition by dephosphorylation of S1177 is fundamental for the inhibition of endothelial cell migration and proliferation by vasoinhibins.
The majority of the known transthyretin (TTR) variants are associated with amyloidosis, but there are also variants associated with euthyroid hyperthyroxinemia and others are apparently nonpathogenic. TTR Met 119 is a nonpathogenic variant found to be frequent in the Portuguese population. Previous studies on thyroxine (T4) binding to semi-purified TTR from heterozygotic carriers of TTR Met 119, reported by us and other groups, revealed different results. Therefore, to further characterize T4 binding to TTR Met 119 we performed T4-TTR binding studies in homotetrameric-recombinant TTR Met 119 variant and normal TTR. We also studied T4 binding to TTR purified from serum of different heterozygotic carriers of TTR Met 119 including compound heterozygotic individuals carriers of a TTR mutation in the other allele. We observed an increased T4 binding affinity to TTR Met 119 from heterozygotic individuals and compound heterozygotes and this effect of increasing T4 binding affinity was consistent and independent from the mutation present in the other allele. Recombinant homotetrameric TTR Met 119 and heterotetrameric protein from heterozygotic carriers of TTR Met 119 presented similar T4 binding affinity demonstrating the increased T4 binding affinity of TTR Met 119. X-ray crystallography studies performed on the recombinant TTR Met 119 variant revealed structural alterations mainly at the level of residue Leu 110 allowing a closer contact between the hormone and the mutant protein. These results are consistent with the observed T4 binding results.
The diagnostic performance of positron emission tomography using (11)C-methionine (MET-PET) in detecting parathyroid adenoma has been investigated by several studies with conflicting results. Aim of our study is to meta-analyze published data about this topic. A comprehensive computer literature search of studies published in PubMed/MEDLINE, Scopus and Embase databases through May 2012 and regarding the diagnostic performance of MET-PET in patients with parathyroid adenoma was carried out. No language restriction was used. Only articles in which at least five patients with parathyroid adenoma underwent MET-PET were included in the meta-analysis. Pooled sensitivity and detection rate (DR) on a per patient-based analysis were calculated to assess the diagnostic performance of MET-PET. Nine studies comprising 258 patients with suspected parathyroid adenoma were included in this meta-analysis. Pooled sensitivity and DR values of MET-PET in patients with suspected parathyroid adenoma were 81 % (95 % confidence interval [95 %CI] 74-86 %) and 70 % (95 %CI 62-77 %), respectively, on a per patient-based analysis. The included studies were heterogeneous in their estimate of sensitivity and DR. Our meta-analysis demonstrates that MET-PET is a sensitive and reliable tool in patients with suspected parathyroid adenoma. Thus, this imaging method could be helpful in patients with diagnosis of primary hyperparathyroidism when conventional imaging techniques are negative or inconclusive in localizing parathyroid adenoma.
Lactogenic hormone-dependent Nb2-11C cells proliferate in response to prolactin (PRL) or human growth hormone (hGH). We have investigated the activation of p21ras
and mitogen-activated protein kinase (MAP-kinase) by hGH in lactogen-dependent Nb2-11C and in autonomous hormone-independent Nb2-SP rat lymphoma cells. Exposure of Nb2-11C cells to hGH resulted in a dose-dependent activation of p21ras
and of MAP-kinase. Activation occurs at physiological hGH concentration and with a rapid onset (∼1 min) reaching maximal level at 10–20 min. In contrast, in Nb2-SP autonomous lactogen-independent cells, p21ras
and MAP-kinase are constitutively activated and a challenge with lactogenic hormone had a modest additional activating effect. TPA, an activator of protein kinase C, enhanced p21ras
and MAP-kinase activity in Nb2-11C cells but failed to induce proliferation. The mechanism of activation of p21ras
in Nb2-11C cells by lactogenic hormones involves both an increased binding of guanine nucleotides to p21ras
as well as an increase in GTP/GDP+GTP ratio. In summary, we have demonstrated here that activation of the p21ras
/MAP-kinase pathway follows PRL receptor activation but is not sufficient for the lactogenic hormone-dependent mitogenesis.
Recently, the succinate dehydrogenase subunit D (SDHD) gene has been reported as one of the major susceptibility genes for pheochromocytoma (PCC) and paraganglioma (PGL). In addition, loss of heterozygosity (LOH) on chromosome 11, mainly in 11q23 and 11q13, is observed frequently in PGL. Based on the fact that mutation frequency of the SDHD gene is less than that of allelic loss at chromosome11q, where the SDHD gene is located, this region may contain other candidate tumor-suppressor genes involved in pathogenesis of PCC/PGL. The tumor-suppressor gene Men1 located in 11q13 is responsible for multiple endocrine neoplasia type 1 (Men1). However, the involvement of the Men1 gene in tumorigenesis of sporadic PCC/PGL is yet to be determined. To understand the roles of the two tumor-suppressor genes and LOH on chromosome 11q in Chinese patients with sporadic PCC or PGL, we performed mutation detection of the SDHD and Men1 genes in tumors from 35 Chinese patients with PCC/PGL; we also did LOH analysis at chromosome 11q for 25 patients out of the 35. No mutation was found in all of 35 patients. However, LOH was detected at one or more loci in 11 of the 25 (44%) tumor samples. The highest frequency of LOH occurred at D11S2006 (41%). Our results suggested that mutation in SDHD or Men1 gene was not found in Chinese patients with sporadic PCC/PGL. However the loss of chromosome 11q might be critical in development of PCC or PGL.
In some patients with multiple endocrine neoplasia type 1 (MEN1) it is not possible to identify a germline mutation in the MEN1 gene. We sought to document the loss of expression and function of the MEN1 gene product, menin, in the tumors of such a patient. The proband is an elderly female patient with primary hyperparathyroidism, pancreatic islet tumor, and breast cancer. Her son has primary hyperparathyroidism. No germline MEN1 mutation was identified in the proband or her son. However, loss of heterozygosity at the MEN1 locus and complete lack of menin expression were demonstrated in the proband's tumor tissue. The proband's cultured parathyroid cells lacked the normal reduction in proliferation and parathyroid hormone secretion in response to transforming growth factor- beta. This assessment provided insight into the molecular pathogenesis of the patient and provides evidence for a critical requirement for menin in the antiproliferative action of transforming growth factor-beta.
Patients with ectopic ACTH syndrome often develop hypertension and hypokalemic alkalosis with an abnormal increase in the ratio of plasma cortisol to cortisone, indicating that 11β-hydroxysteroid dehydrogenase (11βHSD) activity is inhibited. Inhibition of 11βHSD allows access of cortisol or corticosterone to the mineralocorticoid receptor where it act as a mineralocorticoid. Two isozymes, 11βHSD-1 and 11βHSD-2, have been cloned and characterized. The rat adrenal expresses the mRNAs for 11βHSD-2 and, in lesser amounts, 11βHSD-1. We investigated the effect of ACTH on the 11 11βHSD-2 activity in the rat adrenal. Rat adrenal cells zone fasciculata (ZF) were dispersed and incubated separately with increasing concentrations of ACTH for 90 min, and secretion of corticosterone (B) and 11-dehydrocorticosterone (A) in the media was measured by enzyme-linked immunoabsorbent assays (ELISA). The conversion of [3H]B to [3H]A in the presence of 0.5 mM NAD+ was evaluated in microsomes prepared from dispersed cells preincubated for 30 min with cyanoketone and metyrapone followed by incubation for 30 min with the same inhibitors, with and without 10 nM ACTH. The dispersed cells of the ZF produced significant amounts of A which increased with ACTH. The basal B/A ratio was 0.97±0.05. ACTH caused a concentration-dependent increase in the ratio of B/A with a maximum ratio of 9.58±0.20. ACTH also inhibited the conversion of [3H]B to [3H]A in microsomes in which endogenous B production was inhibited by cyanoketone and metyrapone. ACTH did not change theK
m for B conversion, but theV
max was reduced significantly (1.73±0.43 pmol/min. mg protein), indicating that ACTH suppressed the 11βHSD-2 in a noncompetitive fashion. Dibutyryl cyclic AMP (dcAMP) also produced a concentration-dependent increase in the B/A ratio, but various concentrations of calcium did not affect the enzyme activity. In summary, adrenal cells treated with ACTH results in a significant increase in the ratio of B/A in the ZF owing a noncompetitive inhibition of the 11βHSD-2 via the ACTH receptor.
Endogenous glucocorticoids (GC) rapidly increase after acute exercise, and the phosphodiesterase's type 5 inhibitor (PDE5i) tadalafil influences this physiological adaptation. No data exist on acute effects of both acute exercise and PDE5i administration on 11β-hydroxysteroid dehydrogenases (11β-HSDs)-related GC metabolites. We aimed to investigate the rapid effects of exercise on serum GC metabolites, with and without tadalafil administration. A double blind crossover study was performed in eleven healthy male volunteers. After the volunteers randomly received a short-term administration of placebo or tadalafil (20 mg/die for 2 days), a maximal exercise test to exhaustion on cycle ergometer was performed. Then, after a 2-week washout period, the volunteers were crossed over. Blood samples were collected before starting exercise and at 5 and 30 min of recovery (+5-Rec, +30-Rec). Serum ACTH, corticosterone (Cn), cortisol (F), cortisone (E), tetrahydrocortisol (THF), tetrahydrocortisone (THE), cortols, cortolones and respective ratios were evaluated. Pre-Ex THF was higher after tadalafil. Exercise increased ACTH, Cn, F, E, THE, cortols and cortolones after both placebo and tadalafil, and THF after placebo. The F/E ratio increased at +5-Rec and decreased at +30-Rec after placebo. Compared to placebo, after tadalafil lower ACTH, F and Cn, higher THF/F and THE/E, and not E (at +5-Rec) and F/E modifications were observed. Acute exercise rapidly influences serum GC metabolites concentrations. Tadalafil influences both GC adaptation and 11β-HSDs activity during acute exercise. Additional researches on the effects of both exercise and PDE5i on tissue-specific 11β-HSDs activity at rest and during physiological adaptation are warranted.
Excessive glucocorticoid exposure (Cushing's syndrome) results in increased adiposity associated with dysmetabolic features (including insulin resistance, hyperlipidaemia, and hypertension). Circulating cortisol levels are not elevated in idiopathic obesity, although cortisol production and clearance are increased. However, tissue glucocorticoid exposure may be altered independently of circulating levels by 11beta-hydroxysteroid dehydrogenase type 1 (11HSD1), an enzyme which generates active glucocorticoid within tissues, including in adipose tissue. Transgenic overexpression of 11HSD1 in mice causes obesity. In human obesity, 11HSD1 is altered in a tissue-specific manner with reduced levels in liver but elevated levels in adipose, which may lead to glucocorticoid receptor activation and contribute to the metabolic phenotype. The reasons for altered 11HSD1 in obesity are not fully understood. Although some polymorphisms have been demonstrated in intronic and upstream regions of the HSD11B1 gene, the functional significance of these is not clear. In addition, there is mounting evidence that 11HSD1 may be dysregulated secondarily to factors that are altered in obesity, including substrates for metabolism, hormones, and inflammatory mediators. 11HSD1 is a potential therapeutic target for the treatment of the metabolic syndrome. 11HSD1 knockout mice are protected from diet-induced obesity and associated metabolic dysfunction. Although many specific inhibitors of 11HSD1 have now been developed, and published data support their efficacy in the liver to reduce glucose production, their efficacy in enhancing insulin sensitivity in adipose tissue remains uncertain. The therapeutic potential of 11HSD1 in human obesity therefore remains highly promising but as yet unproven.
Proinflammatory cytokines, just like glucocorticoids (GCs), have been reported to upregulate 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) expression in many cell types. This concerted regulation of 11β-HSD1 by interleukin-1β (IL-1β) and GCs is in marked contrast to their antagonistic effects on inflammation. Further, the molecular mechanisms underlying the induction of 11β-HSD1 by IL-1β are not very well understood. In this study, we demonstrated that IL-1β dramatically stimulated 11β-HSD1 expression and enzyme activity as well as promoter activity including the -64 bp fragment upstream to the transcription start site in human fetal lung fibroblasts (HFL-1). Nucleotide mutations of the proximal CCAAT box within this region abolished the induction of 11β-HSD1 promoter activity by IL-1β. Western blotting analysis demonstrated that IL-1β induced the expression of C/EBPβ dramatically while C/EBPα was barely detectable in HFL-1 cells. Global inhibition of CCAAT/enhancer-binding proteins (C/EBPs) with transfection of C/EBP-specific dominant-negative expression plasmid (CMV500-A-C/EBP) significantly attenuated the induction of 11β-HSD1 by IL-1β, whereas over-expression of C/EBPβ enhanced the expression of 11β-HSD1. Chromatin immunoprecipitation assay revealed the recruitment of C/EBPβ to the promoter region containing the C/EBP binding site. In conclusion, IL-1β induces the expression of 11β-HSD1 mRNA in the fetal lung tissue through mechanisms that involve C/EBPβ binding to the promoter. This impact of IL-1β on the expression of 11β-HSD1 in human fetal lung cells may explain the alternate mechanism for the lung maturation that appears to occur when there is a risk of premature delivery of the fetus due to the presence of infection.