Presence of CD3+ and CD79a+ Lymphocytes in the Pituitary Gland of Dogs at Post-mortem Examination.

Hypophysitis has been reported occasionally in dogs, with most cases resembling primary lymphocytic hypophysitis in man. Although it is generally assumed that lymphocytes are not present normally in the canine pituitary gland, few studies have investigated this hypothesis. However, lymphocytes are recognized in the pituitary gland of people and horses without signs of pituitary disease. It is unknown to what degree lymphocyte infiltration of the pituitary gland might occur as an incidental finding in dogs. The aim of the present study was to investigate the presence and distribution of lymphocytes in the pituitary gland of dogs without clinical suspicion of pituitary disease. Twenty dogs were subjected to routine necropsy examination. Formalin-fixed and paraffin wax-embedded sections of pituitary were stained with haematoxylin and eosin (HE) or subjected to immunohistochemistry (IHC) using primary antibodies specific for the T-cell marker CD3 and the B-cell marker CD79a. The number of CD3+ and CD79a+ cells per area unit (CPA) was determined for different pituitary regions. Two dogs had extensive neoplastic lesions in the pituitary gland and were excluded from analysis. In the remaining 18 dogs, occasional scattered CD3+ cells were found in the pituitary gland. There was a significant difference in CD3+ CPA between pituitary regions (P = 0.001). The highest CD3+ CPA was found in the pars tuberalis (median 41.3 cells/mm2, interquartile range 20.9-50.5 cells/mm2). In six of the 18 dogs (33%), CD79a+ cells were detected in small number (median total cell number 0 cells/section, interquartile range 0-1.0 cells/section). This study shows that T cell, and fewer B cells, may be found in the pituitary gland of dogs without clinical suspicion of pituitary disease. Regional difference in T-cell density, with the highest CD3+ CPA in the pars tuberalis, may imply regional immunoregulatory functions in the canine pituitary gland.

Weight loss after bariatric surgery normalizes brain opioid receptors in morbid obesity.

Positron emission tomography (PET) studies suggest opioidergic system dysfunction in morbid obesity, while evidence for the role of the dopaminergic system is less consistent. Whether opioid dysfunction represents a state or trait in obesity remains unresolved, but could be assessed in obese subjects undergoing weight loss. Here we measured brain µ-opioid receptor (MOR) and dopamine D2 receptor (D2R) availability in 16 morbidly obese women twice-before and 6 months after bariatric surgery-using PET with [(11)C]carfentanil and [(11)C]raclopride. Data were compared with those from 14 lean control subjects. Receptor-binding potentials (BPND) were compared between the groups and between the pre- and postoperative scans among the obese subjects. Brain MOR availability was initially lower among obese subjects, but weight loss (mean=26.1 kg, s.d.=7.6 kg) reversed this and resulted in ~23% higher MOR availability in the postoperative versus preoperative scan. Changes were observed in areas implicated in reward processing, including ventral striatum, insula, amygdala and thalamus (P's<0.005). Weight loss did not influence D2R availability in any brain region. Taken together, the endogenous opioid system plays an important role in the pathophysiology of human obesity. Because bariatric surgery and concomitant weight loss recover downregulated MOR availability, lowered MOR availability is associated with an obese phenotype and may mediate excessive energy uptake. Our results highlight that understanding the opioidergic contribution to overeating is critical for developing new treatments for obesity.

Nitric oxide increases cyclic GMP levels, AMP-activated protein kinase (AMPK)alpha1-specific activity and glucose transport in human skeletal muscle.

AIMS/HYPOTHESIS: We investigated the direct effect of a nitric oxide donor (spermine NONOate) on glucose transport in isolated human skeletal muscle and L6 skeletal muscle cells. We hypothesised that pharmacological treatment of human skeletal muscle with N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) would increase intracellular cyclic GMP (cGMP) levels and promote glucose transport. METHODS: Skeletal muscle strips were prepared from vastus lateralis muscle biopsies obtained from seven healthy men. Muscle strips were incubated in the absence or presence of 5 mmol/l spermine NONOate or 120 nmol/l insulin. The L6 muscle cells were treated with spermine NONOate (20 micromol/l) and incubated in the absence or presence of insulin (120 nmol/l). The direct effect of spermine NONOate and insulin on glucose transport, cGMP levels and signal transduction was determined. RESULTS: In human skeletal muscle, spermine NONOate increased glucose transport 2.4-fold (p < 0.05), concomitant with increased cGMP levels (80-fold, p < 0.001). Phosphorylation of components of the canonical insulin signalling cascade was unaltered by spermine NONOate exposure, implicating an insulin-independent signalling mechanism. Consistent with this, spermine NONOate increased AMP-activated protein kinase (AMPK)-alpha1-associated activity (1.7-fold, p < 0.05). In L6 muscle cells, spermine NONOate increased glucose uptake (p < 0.01) and glycogen synthesis (p < 0.001), an effect that was in addition to that of insulin. Spermine NONOate also elicited a concomitant increase in AMPK and acetyl-CoA carboxylase phosphorylation. In the presence of the guanylate cyclase inhibitor LY-83583 (10 micromol/l), spermine NONOate had no effect on glycogen synthesis and AMPK-alpha1 phosphorylation. CONCLUSIONS/INTERPRETATION: Pharmacological treatment of skeletal muscle with spermine NONOate increases glucose transport via insulin-independent signalling pathways involving increased intracellular cGMP levels and AMPK-alpha1-associated activity.

The second extracellular loop of alpha2A-adrenoceptors contributes to the binding of yohimbine analogues.

BACKGROUND AND PURPOSE: Rodent alpha(2A)-adrenoceptors bind the classical alpha(2)-antagonists yohimbine and rauwolscine with lower affinity than the human alpha(2A)-adrenoceptor. A serine-cysteine difference in the fifth transmembrane helix (TM; position 5.43) partially explains this, but all determinants of the interspecies binding selectivity are not known. Molecular models of alpha(2A)-adrenoceptors suggest that the second extracellular loop (XL2) folds above the binding cavity and may participate in antagonist binding. EXPERIMENTAL APPROACH: Amino acids facing the binding cavity were identified using molecular models: side chains of residues 5.43 in TM5 and xl2.49 and xl2.51 in XL2 differ between the mouse and human receptors. Reciprocal mutations were made in mouse and human alpha(2A)-adrenoceptors at positions 5.43, xl2.49 and xl2.51, and tested with a set of thirteen chemically diverse ligands in competition binding assays. KEY RESULTS: Reciprocal effects on the binding of yohimbine and rauwolscine in human and mouse alpha(2A)-adrenoceptors were observed for mutations at 5.43, xl2.49 and xl2.51. The binding profile of RS-79948-197 was reversed only by the XL2 substitutions. CONCLUSIONS AND IMPLICATIONS: Positions 5.43, xl2.49 and xl2.51 are major determinants of the species preference for yohimbine and rauwolscine of the human versus mouse alpha(2A)-adrenoceptors. Residues at positions xl2.49 and xl2.51 determine the binding preference of RS-79948-197 for the human alpha(2A)-adrenoceptor. Thus, XL2 is involved in determining the species preferences of alpha(2A)-adrenoceptors of human and mouse for some antagonists.

Exercise training increases insulin-stimulated glucose disposal and GLUT4 (SLC2A4) protein content in patients with type 2 diabetes.

AIMS/HYPOTHESIS: Exercise enhances insulin-stimulated glucose transport in skeletal muscle through changes in signal transduction and gene expression. The aim of this study was to assess the impact of acute and short-term exercise training on whole-body insulin-mediated glucose disposal and signal transduction along the canonical insulin signalling cascade. METHODS: A euglycaemic-hyperinsulinaemic clamp, with vastus lateralis skeletal muscle biopsies, was performed at baseline and 16 h after an acute bout of exercise and short-term exercise training (7 days) in obese non-diabetic (n=7) and obese type 2 diabetic (n=8) subjects. RESULTS: Insulin-mediated glucose disposal was unchanged following acute exercise in both groups. Short-term exercise training increased insulin-mediated glucose disposal in obese type 2 diabetic (p<0.05), but not in obese non-diabetic subjects. Insulin activation of (1) IRS1, (2) IRS2, (3) phosphotyrosine-associated phosphatidylinositol-3 kinase activity and (4) the substrate of phosphorylated Akt, AS160, a functional Rab GTPase activating protein important for GLUT4 (now known as solute carrier family 2 [facilitated glucose transporter], member 4 [SLC2A4]) translocation, was unchanged after acute or chronic exercise in either group. GLUT4 protein content was increased in obese type 2 diabetic subjects (p<0.05), but not in obese non-diabetic subjects following chronic exercise. CONCLUSIONS/INTERPRETATION: Exercise training increased whole-body insulin-mediated glucose disposal in obese type 2 diabetic patients. These changes were independent of functional alterations in the insulin-signalling cascade and related to increased GLUT4 protein content.