Cachexia in MAC16 adenocarcinoma:Suppression of hunger despite normal regulation of leptin, insulin and hypothalamic neuropeptide Y

Weight loss normally stimulates hunger, through mechanisms that include falls in circulating leptin and insulin, leading to stimulation of hypothalamic neuropeptide Y (NPY). Here, we investigated the leptin, insulin and NPY to clarify why hunger is suppressed in mice with severe cachexia due to the MAC16 adenocarcinoma. MAC16-bearing mice progressively lost weight (19% below controls) and fat (-61%) over 16 days after tumour transplantation, while total food intake fell by 10%. Pair-fed mice showed less wasting, with final weight being 9% and fat mass 25% below controls. Plasma leptin fell by 85% in MAC16 and 51% in pair-fed mice, in proportion to loss of fat. Plasma insulin was also reduced by 49% in MAC16 and 53% in pair-fed groups. Hypothalamic leptin receptor (OB-Rb) mRNA was significantly increased in both MAC16 (+223%) and pair-fed (+192%) mice. Hypothalamic NPY mRNA was also significantly raised in MAC16 (+152%) and pair-fed (+99%) groups, showing negative correlations with plasma leptin and insulin, and a positive association with OB-Rb mRNA. In MAC16-induced cachexia, leptin production and hypothalamic OB-Rb and NPY expression are regulated appropriately in response to fat depletion. Therefore, suppression of hunger is probably due to tumour products that inhibit NPY transport or release, or that interfere with neuronal targets downstream of NPY.

Appetite regulatory mechanisms and food intake in mice are sensitive to mismatch in diets between pregnancy and postnatal periods

Human and animal studies suggest that obesity in adulthood may have its origins partly during prenatal development. One of the underlying causes of obesity is the perturbation of hypothalamic mechanisms controlling appetite. We determined mRNA levels of genes that regulate appetite, namely neuropeptide Y (NPY), pro-opiomelanocortin (POMC) and the leptin receptor isoform Ob-Rb, in the hypothalamus of adult mouse offspring from pregnant dams fed a protein-restricted diet, and examined whether mismatched post-weaning high-fat diet altered further expression of these gene transcripts. Pregnant MF1 mice were fed either normal protein (C, 18% casein) or protein-restricted (PR, 9% casein) diet throughout pregnancy. Weaned offspring were fed to adulthood a high-fat (HF; 45% kcal fat) or standard chow (21% kcal fat) diet to generate the C/HF, C/C, PR/HF and PR/C groups. Food intake and body weight were monitored during this period. Hypothalamic tissues were collected at 16 weeks of age for analysis of gene expression by real time RT-PCR. All HF-fed offspring were observed to be heavier vs. C groups regardless of the maternal diet during pregnancy. In the PR/HF males, but not in females, daily energy intake was reduced by 20% vs. the PR/C group (p <0.001). In PR/HF males, hypothalamic mRNA levels were lower vs. the PR/C group for NPY (p <0.001) and Ob-Rb (p <0.05). POMC levels were similar in all groups. In females, mRNA levels for these transcripts were similar in all groups. Our results suggest that adaptive changes during prenatal development in response to maternal dietary manipulation may have long-term sex-specific consequences on the regulation of appetite and metabolism following post-weaning exposure to an energy-rich nutritional environment. © 2008 Elsevier B.V. All rights reserved.

Role of the transgenic human thyrotropin receptor A-subunit in thyroiditis induced by A-subunit immunization and regulatory T cell depletion

Transgenic BALB/c mice that express intrathyroidal human thyroid stimulating hormone receptor (TSHR) A-subunit, unlike wild-type (WT) littermates, develop thyroid lymphocytic infiltration and spreading to other thyroid autoantigens after T regulatory cell (Treg) depletion and immunization with human thyrotropin receptor (hTSHR) adenovirus. To determine if this process involves intramolecular epitope spreading, we studied antibody and T cell recognition of TSHR ectodomain peptides (A–Z). In transgenic and WT mice, regardless of Treg depletion, TSHR antibodies bound predominantly to N-terminal peptide A and much less to a few downstream peptides. After Treg depletion, splenocytes from WT mice responded to peptides C, D and J (all in the A-subunit), but transgenic splenocytes recognized only peptide D. Because CD4+ T cells are critical for thyroid lymphocytic infiltration, amino acid sequences of these peptides were examined for in silico binding to BALB/c major histocompatibility complex class II (IA–d). High affinity subsequences (inhibitory concentration of 50% < 50 nm) are present in peptides C and D (not J) of the hTSHR and mouse TSHR equivalents. These data probably explain why transgenic splenocytes do not recognize peptide J. Mouse TSHR mRNA levels are comparable in transgenic and WT thyroids, but only transgenics have human A-subunit mRNA. Transgenic mice can present mouse TSHR and human A-subunit-derived peptides. However, WT mice can present only mouse TSHR, and two to four amino acid species differences may preclude recognition by CD4+ T cells activated by hTSHR-adenovirus. Overall, thyroid lymphocytic infiltration in the transgenic mice is unrelated to epitopic spreading but involves human A-subunit peptides for recognition by T cells activated using the hTSHR.

Investigation of the activation mechanism and structural characterization of the CGRP receptor

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor (GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode of activation of this receptor could be key in developing therapeutic agents for associated health conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this receptor (in complex with an antagonist) has been published, the details of receptor-agonist interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear. Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8) region, has not been well studied for its role in receptor signalling. This research project investigated these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2 and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural interaction study by surface plasmon resonance (SPR). Following expression and purification, these receptor proteins were found to individually retain their secondary structures when analysed by circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin family receptor paradigm. The research described in this thesis has produced novel data that contributes to a clearer understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a useful tool in determining novel interacting GPCR partners of RAMPs.

Structure-activity relationship of calcitonin gene-related peptide and its receptor

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Plasma vascular endothelial growth factor, angiopoietin-2, and soluble angiopoietin receptor tie-2 in diabetic retinopathy:effects of laser photocoagulation and angiotensin receptor blockade

Background: Proliferative diabetic retinopathy (PDR) may be a response to abnormal angiogenic growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), and the soluble angiopoietin receptor tie-2. The authors hypothesised the following: (a) there are differences in plasma levels of these growth factors in different grades of diabetic retinopathy; and (b) that the effects of intervention with panretinal laser photocoagulation (PRP) for PDR, and angiotensin receptor blockade (using eprosartan) for patients with other grades of diabetic retinopathy will be to reduce levels of the growth factors. Methods: Cross sectional and interventional study (using PRP and eprosartan) in diabetic patients. VEGF, Ang-2, and tie-2 were measured by ELISA. Results: VEGF (p<0.001) and Ang-2 levels (p<0.001) were significantly higher in 93 diabetic patients compared to 20 healthy controls, with the highest levels in grade 2 and grade 3 diabetic retinopathy (p<0.05). Tie-2 was lower in diabetics compared to controls (p = 0.008), with no significant differences between the diabetic subgroups. Overall, VEGF significantly correlated with Ang-2 (p<0.001) and tie-2 (p = 0.004) but the correlation between Ang-2 and tie-2 levels was not significant (p = 0.065). Among diabetic patients only, VEGF levels were significantly correlated with Ang-2 (p<0.001) and tie-2 (p<0.001); the correlation between Ang-2 and tie-2 levels was also significant (p<0.001). There were no statistically significant effects of laser photocoagulation on plasma VEGF, Ang-2, and tie-2 in the 19 patients with PDR, or any effects of eprosartan in the 28 patients with non-proliferative diabetic retinopathy. Conclusion: Increased plasma levels of VEGF and Ang-2, as well as lower soluble tie-2, were found in diabetic patients. The highest VEGF and Ang-2 levels were seen among patients with pre-proliferative and proliferative retinopathy, but there was no relation of tie-2 to the severity of retinopathy. As the majority of previous research into Ang-2 and tie-2 has been in relation to angiogenesis and malignancy, the present study would suggest that Ang-2 and tie-2 may be used as potential indices of angiogenesis in diabetes mellitus (in addition to VEGF) and may help elucidate the role of the angiopoietin/tie-2 system in this condition.

Identifying key residues important for CGRP binding to its receptor

The calcitonin gene related peptide (CGRP) is a 37 amino acid neuropeptide. Its receptor is a heterodimeric complex of calcitonin receptor-like receptor (CLR) – a family B G-protein coupled receptor – and a single-pass transmembrane protein, receptoractivity modifying protein 1 (RAMP1). Here, we identify residues, within the N-terminal extracellular domain (ECD) of CLR, potentially involved in ligand binding.Certain residues presumed to be possible sites of contact for the CGRP were picked from the CLR/RAMP1 ECD crystal structure (PDB 3N7S). Residues were mutated to alanine (A) bysite-directed mutagenesis (QuikChangeTM, Stratagene). Mutants were analysed for their ability to stimulate cAMP and cell surface expression as previously described [1]. All mutants showed reduced potency, though to varying degrees as indicated by their pEC50 values. W69A and D70Ashowed significant reduction in cell surface expression.These findings suggest that these residues are important for the interaction of CGRP with its receptor. W69A and D70A, part of the WDG motif of family B GPCRs, are thought to rather play a role in receptor stability [2]. The data is consistent with CGRP binding in agroove between CLR and RAMP1. This project was supported byAston School of Life and Health Sciences.References1. Barwell J, Conner A & Poyner D (2011) Extracellular loops 1and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function. Biochim Biophys Acta 1813, 1906–1916.2. Kumar S, Pioszak A, Zhang C et al. (2011) Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Cou-pled Receptors. PLoS One 6, e19682