Exercise and insulin resistance in PCOS: muscle insulin signalling and fibrosis.

OBJECTIVE: Mechanisms of insulin resistance in polycystic ovary syndrome (PCOS) remain ill defined, contributing to sub-optimal therapies. Recognising skeletal muscle plays a key role in glucose homeostasis we investigated early insulin signalling, its association with aberrant transforming growth factor ß (TGFß)-regulated tissue fibrosis. We also explored the impact of aerobic exercise on these molecular pathways. METHODS: A secondary analysis from a cross-sectional study was undertaken in women with (n = 30) or without (n = 29) PCOS across lean and overweight BMIs. A subset of participants with (n = 8) or without (n = 8) PCOS who were overweight completed 12 weeks of aerobic exercise training. Muscle was sampled before and 30 min into a euglycaemic-hyperinsulinaemic clamp pre and post training. RESULTS: We found reduced signalling in PCOS of mechanistic target of rapamycin (mTOR). Exercise training augmented but did not completely rescue this signalling defect in women with PCOS. Genes in the TGFß signalling network were upregulated in skeletal muscle in the overweight women with PCOS but were unresponsive to exercise training except for genes encoding LOX, collagen 1 and 3. CONCLUSIONS: We provide new insights into defects in early insulin signalling, tissue fibrosis, and hyperandrogenism in PCOS-specific insulin resistance in lean and overweight women. PCOS-specific insulin signalling defects were isolated to mTOR, while gene expression implicated TGFß ligand regulating a fibrosis in the PCOS-obesity synergy in insulin resistance and altered responses to exercise. Interestingly, there was little evidence for hyperandrogenism as a mechanism for insulin resistance.

Informing Translation: The Accuracy of Information on Websites for Lifestyle Management of Polycystic Ovary Syndrome.

Lifestyle (diet, physical activity [PA], and/or behavioral) interventions are recommended for all women with polycystic ovary syndrome (PCOS) in international guidelines. The internet is a widely used health information resource. However, the accuracy of lifestyle information on PCOS websites is unknown and is reviewed here to inform translation of the international guideline on PCOS. An internet search was conducted with three search engines across different web browsers and countries. Accuracy was assessed through a checklist of 29 questions based on international guidelines for diet, PA, or weight management for the general population and for PCOS with higher scores indicating greater accuracy. Fifteen websites were eligible out of 72 (20%). The total accuracy score was 56 ± 13 (mean ± standard deviation; potential range: -29 to 87) comprising 23 ± 6 for diet (-11 to 33), 15 ± 5 for PA (-9 to 27), and 14 ± 3 for weight management (-8 to 24). A moderate proportion of websites provided general information on appropriate diet (40-80%) or weight management strategies (47-60%), but only 10 to 40% of websites provided information on core foods, discretionary foods, exercise quantity/intensity, energy deficits, or behavioral strategies. Limited websites on PCOS contain information on lifestyle management. The majority provided information on general diet, PA, and weight recommendations but less information on practical implementation of lifestyle change as an identified translation gap for the international guideline on PCOS.

Use of an inverse agonist radioligand [3H]A-317920 reveals distinct pharmacological profiles of the rat histamine H3 receptor.

Selective radioligands for histamine H(3) receptors have been used to characterize H(3) receptor pharmacology by radioligand binding assays and to determine H(3) receptor distribution by tissue autoradiography. Here we report the synthesis and receptor binding characterization of [(3)H]A-317920 (furan-2-carboxylic acid(2-[4-[3-([3,5-(3)H]4-cyclopropanecarbonyl-phenoxy)-propyl]-piperazin-1-yl]-1-methyl-2-oxo-ethyl)-amide), a high affinity inverse agonist radioligand for the rat H(3) receptor. The binding of [(3)H]A-317920 to rat cortical and cloned H(3) receptors revealed fast on- and slower off-rate kinetics with calculated K(d) values in agreement with those determined in saturation binding assays (0.2 nM for both receptors). Further, we compared [(3)H]A-317920 with the agonist [(3)H](N)-alpha-methylhistamine ([(3)H]NalphaMH) as radioligand tools to study receptor pharmacology. Agonists and antagonists displaced [(3)H]NalphaMH with one-site binding characteristics and Hill slopes approached unity. In contrast, although antagonists exhibited one-site binding, [(3)H]A-317920 displacement by agonists was best fit by two-site binding models, and the potencies of the high affinity, GDP-sensitive sites correlated with the potencies defined in [(3)H]NalphaMH binding. Unlike [(125)I]iodoproxyfan, [(3)H]A-317920 exhibits potent and selective binding to rat H(3) receptors with low binding to non-H(3) sites, including cytochrome P450. These findings show that [(3)H]A-317920 is a potent rat H(3) receptor antagonist radioligand and has utility for studying H(3) receptor pharmacology.

Reduced expression of regeneration associated genes in chronically axotomized facial motoneurons.

Chronically axotomized motoneurons progressively fail to regenerate their axons. Since axonal regeneration is associated with the increased expression of tubulin, actin and GAP-43, we examined whether the regenerative failure is due to failure of chronically axotomized motoneurons to express and sustain the expression of these regeneration associated genes (RAGs). Chronically axotomized facial motoneurons were subjected to a second axotomy to mimic the clinical surgical procedure of refreshing the proximal nerve stump prior to nerve repair. Expression of α1-tubulin, actin and GAP-43 was analyzed in axotomized motoneurons using in situ hybridization followed by autoradiography and silver grain quantification. The expression of these RAGs by acutely axotomized motoneurons declined over several months. The chronically injured motoneurons responded to a refreshment axotomy with a re-increase in RAG expression. However, this response to a refreshment axotomy of chronically injured facial motoneurons was less than that seen in acutely axotomized facial motoneurons. These data demonstrate that the neuronal RAG expression can be induced by injury-related signals and does not require acute deprivation of target derived factors. The transient expression is consistent with a transient inflammatory response to the injury. We conclude that transient RAG expression in chronically axotomized motoneurons and the weak response of the chronically axotomized motoneurons to a refreshment axotomy provides a plausible explanation for the progressive decline in regenerative capacity of chronically axotomized motoneurons.

Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: differential regulation of GAP-43, tubulins, and neurofilament-M.

Axotomized motoneurons regenerate their axons regardless of whether axotomy occurs proximally or distally from their cell bodies. In contrast, regeneration of rubrospinal axons into peripheral nerve grafts has been detected after cervical but not after thoracic injury of the rubrospinal tract. By using in situ hybridization (ISH) combined with reliable retrograde tracing methods, we compared regeneration-associated gene expression after proximal and distal axotomy in spinal motoneurons versus rubrospinal neurons. Regardless of whether they were axotomized at the iliac crest (proximal) or popliteal fossa (distal), sciatic motoneurons underwent highly pronounced changes in ISH signals for Growth Associated Protein 43 (GAP-43) (10-20x increase) and neurofilament M (60-85% decrease). In contrast, tubulin ISH signals substantially increased only after proximal axotomy (3-5x increase). To compare these changes in gene expression with those of axotomized rubrospinal neurons, the rubrospinal tract was transected at the cervical (proximal) or thoracic (distal) levels of the spinal cord. Cervically axotomized rubrospinal neurons showed three- to fivefold increases in ISH signals for GAP-43 and tubulins (only transient) and a 75% decrease for neurofilament-M. In sharp contrast, thoracic axotomy had only marginal effects. After implantation of peripheral nerve transplants into the spinal cord injury sites, retrograde labeling with the sensitive retrograde tracer Fluoro-Gold identified regenerating rubrospinal neurons only after cervical axotomy. Furthermore, rubrospinal neurons specifically regenerating into the transplants were hypertrophied and expressed high levels of GAP-43 and tubulins. Taken together, these data support the concept that, even if central nervous system (CNS) axons are presented with a permissive/supportive environment, appropriate cell body responses to injury are a prerequisite for CNS axonal regeneration.

Molecular modeling and pharmacological analysis of species-related histamine H(3) receptor heterogeneity.

Presynaptic histamine H(3) receptors (H(3)R) regulate neurotransmitter release in the central nervous system, suggesting an important role for H(3) ligands in human diseases such as cognitive disorders, sleep disturbances, epilepsy, or obesity. Drug development for many of these human diseases relies upon rodent-based models. Although there is significant sequence homology between the human and rat H(3)Rs, some compounds show distinct affinity profiles. To identify the amino acids responsible for these species disparities, various mutant receptors were generated and their pharmacology studied. The N-terminal portion was shown to determine the species differences in ligand binding since a chimeric H(3)R containing N-terminal human and C-terminal rat receptor sequences exhibited similar pharmacology to the human receptor. Sequence analysis and molecular modeling studies suggested key amino acids at positions 119 and 122 in transmembrane region 3 play important roles in ligand recognition. Mutant receptors changing amino acids 119 or 122 of the human receptor to those in the rat improved ligand binding affinities and functional potencies of antagonist ligands, confirming the significant role that these amino acids play in species-related pharmacological differences. A model has been developed to elucidate the ligand receptor interactions for H(3)Rs, and pharmacological aspects of this model are described.

Influence of level and depth on recurrence rate in thin melanomas.

A population-based study of the biology of the thin-level melanoma according to site, Breslow's thickness, and Clark's level was undertaken. Two hundred fifteen patients were studied with a mean follow-up of 41 months. Overall, 23 patients (10.7%) had recurrences, 8 locally, 9 regionally, and 6 systemically, despite an adequate local excision. A multivariate analysis was done. In the patients with thin lesions (less than 1 mm), increasing level (p < 0.002) and head and neck site (p < 0.04) increased the risk of recurrence. Increasing thickness of melanoma up to 1 mm did not influence the risk. This study identifies a group of high-risk melanoma patients for whom adjuvant therapy to decrease recurrences should be studied.

Identification and expression of two baculovirus gp37 genes.

The gp37 genes of the Mamestra brassicae and Lymantria dispar multicapsid nucleopolyhedroviruses (MbMNPV and LdMNPV) have been identified and characterized. Both genes were similar to other baculovirus gp37 genes and to entomopoxvirus fusolin genes. Phylogenetic analysis showed that baculovirus gp37 genes and entomopoxvirus fusolin genes form two distinct and well-separated clades. There was no evidence of recent gene transfer between the two groups. The gp37 genes also showed a distant similarity to bacterial cellulose- and chitin-binding protein genes, but the significance of this is unclear. MbMNPV and LdMNPV gp37 were both transcribed from consensus baculovirus late transcription start sites. MbMNPV gp37 was additionally transcribed from a putative early transcription start site. Tunicamycin treatment of MbMNPV-infected cells confirmed that MbMNPV GP37 is N-glycosylated. Confocal immunofluorescence microscopy revealed that the protein is located exclusively in the cytoplasm, probably in the endoplasmic reticulum.