Denise D. Belsham

The 56/58 kDa androgen-binding protein in male genital skin fibroblasts with a deleted androgen receptor gene

Human genital skin fibroblasts (GSF) make a relatively abundant 56/58 kDa protein that binds androgens. The protein shares many properties with the approximately 100 kDa androgen receptor that is encoded by a locus in the q12 region of the X chromosome. It does not appear to be androgen-induced, yet is absent in GSF of most patients with complete androgen insensitivity (CAI). A precursor-product relation with the androgen receptor (AR) protein has been largely excluded; that it may be an unorthodox product of the AR gene has not. The 56/58 kDa protein is made by the GSF of a mentally retarded subject who has CAI because of a complete deletion of the coding portion of the AR gene. Hence, the strong constitutional and statistical correlations that have been demonstrated between the two proteins cannot arise because they share the same gene. The subjects genomic DNA hybridizes normally with 11 single-copy probes from Xq11-Xq13. Therefore, we cannot attribute her mental retardation to a contiguous gene syndrome.

Analysis of the CAG repeat region of the androgen receptor gene in a kindred with X-linked spinal and bulbar muscular atrophy

Herein we describe a family with X-linked spinal and bulbar muscular atrophy (SBMA or Kennedys disease), an adult onset neuromuscular disease characterized by slow progression, predominant proximal and bulbar muscle weakness. One frequent association is the appearance of gynecomastia. This disorder was previously shown to be linked to the locus DXYS1 on the proximal long arm of the X chromosome. Recently, a report implicated a mutation at the N-terminus of the androgen receptor gene involving amplification of CAG repeats as the cause of X-linked SBMA. We studied this region of the androgen receptor in a kindred clinically suspected but not confirmed of having X-linked SBMA by the polymerase chain reaction (PCR) followed by Southern analysis and DNA sequencing. The mutated allele was found to have an increased number of 51 CAG repeats confirming the clinical diagnosis of SBMA. Normal individuals revealed 23 repeat numbers within the normal range, while another unrelated X-linked SBMA patient had an enlarged CAG repeat region. The carrier or disease status could be established or confirmed in 12 individuals of this family on the basis of detecting normal and disease alleles reflected by the number of CAG repeats.

Glucose sensing mechanisms in hypothalamic cell models: Glucose inhibition of AgRP synthesis and secretion

Glucose-sensing neurons play a role in energy homeostasis, yet how orexigenic neurons sense glucose remains unclear. As models of glucose-inhibited (GI) neurons, mHypoE-29/1 and mHypoA-NPY/GFP cells express the essential orexigenic neuropeptide AgRP and glucose sensing machinery. Exposure to increasing concentrations of glucose or the glucose analog 2-deoxyglucose (2-DG) results in a decrease in AgRP mRNA levels. Taste receptor, Tas1R2 mRNA expression was reduced by glucose, whereas 2-DG reduced Tas1R3 mRNA levels. Increasing glucose concentrations elicited a rise in Akt and neuronal nitric oxide synthase (nNOS) phosphorylation, CaMKKβ levels, and a reduction of AMP-kinase alpha phosphorylation. Inhibitors of NOS and the cystic fibrosis transmembrane conductance regulator (CFTR) prevented a decrease in AgRP secretion with glucose, suggesting a pivotal role for nNOS and the CFTR in glucose-sensing. These models possess the hallmark characteristics of GI neurons, and can be used to disentangle the mechanisms by which orexigenic neurons sense glucose.

The 56 kDa androgen-binding protein in human genital skin fibroblasts: its relation to the human androgen receptor

We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein with androgen-binding activity. This protein is missing in GSF of most patients with complete androgen insensitivity syndrome (CAI). The protein has many characteristics compatible with the androgen receptor; it has in fact been tentatively considered as a precursor or degradation form of the prototypic (approximately 100 kDa) human androgen receptor. We have prepared an antiserum to this protein, which allowed us to detect it as a direct product by in vitro translation of mRNA from GSF. It is thus very unlikely to be a degradation product of a larger precursor. Furthermore, covalent photolytic labeling of this protein with the androgen analogue [3H]mibolerone revealed a much lower affinity for this protein than is known for the androgen receptor. Finally, the GSF of two exceptional patients with complete androgen insensitivity syndrome due to negligible androgen receptor-binding activity express this protein normally, as determined on two-dimensional gels by Western blot analysis with the antiserum and by photolytic covalent labeling with androgen analogues. These data indicate that the protein is not a precursor or a degradation product of the receptor; nor is it androgen-induced. They are more compatible with the idea that the protein is another member of the steroid/thyroid/retinoic acid receptor supergene family, perhaps as an unorthodox product of the human androgen receptor gene.

Gene array analysis of embryonic- versus adult-derived hypothalamic NPY-expressing cell lines

Few studies have utilized microarray analysis to understand the genome wide changes involved in the development of the hypothalamus despite its overall importance to basic physiology. Gene expression profiling of immortalized, clonal hypothalamic neurons, embryonic-derived mHypoE-46 and adult-derived mHypoA-2/12, reveals that the expression of 1225 probes was significantly changed between the two neuronal models. Further comparison of the gene expression profiles identified two categories of genes that were confirmed with qRT-PCR: (i) genes implicated in the Wnt signaling pathway; and (ii) transcription factors previously implicated in the development of the central nervous system. Yet, functional analysis of the two cell lines, including hormonal responses and secretion, indicate that they are comparable despite their developmental origin. This study provides a comprehensive analysis of embryonic- and adult-derived hypothalamic neuronal cell models that both express neuropeptide Y, and identifies novel genes as candidates for mediating the development of specific hypothalamic neurons.

The 56 kDa protein of human genital skin fibroblasts is identical to that radiolabelled by [3H]dihydrotestosterone 17β-bromoacetate

Analysis of soluble proteins from human genital skin fibroblasts by two-dimensional polyacrylamide gel electrophoresis reveals an abundant protein doublet of mol. wt 56,000 with isoelectric points (pI) of 6.7 and 6.5. This protein is absent in non-genital skin fibroblasts as well as in genital skin fibroblasts of most patients with complete forms of androgen insensitivity. The protein specifically binds androgen. A protein of similar estimated molecular weight (58,000) from human genital skin fibroblasts has recently been found to be covalently radiolabelled by the affinity ligand dihydrotestosterone 17 beta-bromoacetate (DHT-BA). In the present study these proteins have been found to be indistinguishable on one- and two-dimensional gel electrophoresis. Antibodies raised against the 56 kDa pI 6.7/6.5 protein also recognized the protein covalently radiolabelled by DHT-BA. A third protein of estimated mol. wt 59,000 has been found to be associated with several steroid hormone receptor complexes but has no known ligand binding activity. This protein was found to be clearly separable from the 56/58 kDa protein on two-dimensional gel electrophoresis as it has a more acidic pI of approximately 5.4. Furthermore, antibodies against the 59 kDa protein do not recognize the 56 kDa species, and vice versa.

Partial androgen insensitivity—are all tissues equal? *

Nonaromatizable androgens, administered in high doses to an adult patient with partial androgen insensitivity, failed to result in a change in phallic size despite a clear decline in SHBG and gonadotropin levels. These findings raise the question of differential tissue sensitivity to androgens. Because ancillary laboratory testing does not predict reliably the genital response, a therapeutic trial should be advocated in such cases.

Phoenixin: uncovering its receptor, signaling and functions

Phoenixin (PNX) is a newly discovered peptide that has been linked to reproductive function, both in the hypothalamus and pituitary. This review will focus on the most recent discoveries related to this novel neuropeptide. Initially, it was found that PNX increased gonadotropin releasing hormone (GnRH)-stimulated luteinizing hormone (LH) release from pituitary cells. Importantly, knockdown of PNX in female rats extended the estrous cycle by 2.3 days. Using novel hypothalamic cell lines, we found that PNX has a stimulatory role on kisspeptin (Kiss) and GnRH gene expression and secretion. The PNX receptor was uncovered using siRNA knockdown of GPR173, an orphan receptor postulated to bind PNX. We have found that the PNX-R signaling through protein kinase A (PKA) in hypothalamic neurons. Althuogh a number of studies demonstrate that PNX plays an important role in reproductive function, there is also evidence that it may have other functions, regulating the heart, feeding, memory, and anxiety, both in the brain and the periphery.

Palmitate induces an anti-inflammatory response in immortalized microglial BV-2 and IMG cell lines that decreases TNFα levels in mHypoE-46 hypothalamic neurons in co-culture.

Background and Objectives: Elevated levels of saturated fatty acids (SFA) induce a state of neuroinflammation in the hypothalamus. It has been suggested that microglia sense palmitate, a prevalent circulating SFA, and act as mediators of this inflammatory process by communicating with neurons, particularly those involved in appetite regulation. In this study, we examined the inflammatory response to palmitate in immortalized microglial cell lines, BV-2 and IMG, and the subsequent effects on inflammatory gene expression in a model of NPY/AgRP neurons, mHypoE-46. Methods: The BV-2 cells were treated with 50 µM palmitate for 4 and 24 h, and the transcriptional regulation of markers for inflammation and cellular stress was assessed using an RT2 Profiler PCR Array. Select genes were verified with qRT-PCR. The BV-2 and IMG cells were then co-cultured using 1.0-µm cell culture inserts with an immortalized hypothalamic cell line, mHypoE-46, to investigate potential intercellular communication between microglia and neurons. Results: We found that palmitate increased the mRNA levels of specific inflammatory genes, and a general anti-inflammatory profile was revealed in the microglia cells. The mRNA changes in TNFα at 4 and 24 h in BV-2 cells were abrogated with the toll-like receptor 4 (TLR4) inhibitor, TAK-242, indicating the involvement of TLR4. Co-culture of mHypoE-46 neurons with microglia pre-treated with palmitate resulted in repression of TNFα expression in the hypothalamic neurons. As palmitate significantly increased IL-13 expression in microglia, the effect of this cytokine was tested in mHypoE-46 neurons. The addition of IL-13 to neuronal cultures normalized the palmitate-mediated increase in IL-6 and AgRP expression, suggesting that microglia may protect surrounding neurons, at least in part, through the release of IL-13. Conclusions: These results suggest a potential anti-inflammatory role of microglia towards the palmitate-induced neuroinflammation, and potentially energy homeostasis, in hypothalamic neurons.