Distinct tumor-infiltrating lymphocyte landscapes are associated with clinical outcomes in localized non-small-cell lung cancer.

BACKGROUND: Despite the importance of tumor-infiltrating T lymphocytes (TILs) in cancer biology, the relationship between TIL phenotypes and their prognostic relevance for localized non-small-cell lung cancer (NSCLC) has not been well established. PATIENTS AND METHODS: Fresh tumor and normal adjacent tissue was prospectively collected from 150 patients with localized NSCLC. Tissue was comprehensively characterized by high-dimensional flow cytometry of TILs integrated with immunogenomic data from multiplex immunofluorescence, T-cell receptor sequencing, exome sequencing, RNA sequencing, targeted proteomics, and clinicopathologic features. RESULTS: While neither the magnitude of TIL infiltration nor specific TIL subsets were significantly prognostic alone, the integration of high-dimensional flow cytometry data identified two major immunotypes (IM1 and IM2) that were predictive of recurrence-free survival independent of clinical characteristics. IM2 was associated with poor prognosis and characterized by the presence of proliferating TILs expressing cluster of differentiation 103, programmed cell death protein 1, T-cell immunoglobulin and mucin-domain containing protein 3, and inducible T-cell costimulator. Conversely, IM1 was associated with good prognosis and differentiated by an abundance of CD8+ T cells expressing cytolytic enzymes, CD4+ T cells lacking the expression of inhibitory receptors, and increased levels of B-cell infiltrates and tertiary lymphoid structures. While increased B-cell infiltration was associated with good prognosis, the best prognosis was observed in patients with tumors exhibiting high levels of both B cells and T cells. These findings were validated in patient tumors from The Cancer Genome Atlas. CONCLUSIONS: Our study suggests that although the number of infiltrating T cells is not associated with patient survival, the nature of the infiltrating T cells, resolved in distinct TIL immunotypes, is prognostically relevant in NSCLC and may inform therapeutic approaches to clinical care.

Effects of slow- v. fast-digested protein supplementation combined with mixed power training on muscle function and functional capacities in older men.

Ageing leads to a progressive loss of muscle function (MF) and quality (MQ: muscle strength (MS)/lean muscle mass (LM)). Power training and protein (PROT) supplementation have been proposed as efficient interventions to improve MF and MQ. Discrepancies between results appear to be mainly related to the type and/or dose of proteins used. The present study aimed at determining whether or not mixed power training (MPT) combined with fast-digested PROT (F-PROT) leads to greater improvements in MF and MQ in elderly men than MPT combined with slow-digested PROT (S-PROT) or MPT alone. Sixty elderly men (age 69 (sd 7) years; BMI 18-30 kg/m2) were randomised into three groups: (1) placebo + MPT (PLA; n 19); (2) F-PROT + MPT (n 21) and (3) S-PROT + MPT (n 20) completed the intervention. LM, handgrip and knee extensor MS and MQ, functional capacity, serum metabolic markers, skeletal muscle characteristics, dietary intake and total energy expenditure were measured. The interventions consisted in 12 weeks of MPT (3 times/week; 1 h/session) combined with a supplement (30 g:10 g per meal) of F-PROT (whey) or S-PROT (casein) or a placebo. No difference was observed among groups for age, BMI, number of steps and dietary intake pre- and post-intervention. All groups improved significantly their LM, lower limb MS/MQ, functional capacity, muscle characteristics and serum parameters following the MPT. Importantly, no difference between groups was observed following the MPT. Altogether, adding 30 g PROT/d to MPT, regardless of the type, does not provide additional benefits to MPT alone in older men ingesting an adequate (i.e. above RDA) amount of protein per d.

Muscle adaptation in response to a high-intensity interval training in obese older adults: effect of daily protein intake distribution.

BACKGROUND: Aging is associated with declines in muscle mass, strength and quality, leading to physical impairments. An even protein distribution in daily meals has recently been proposed along with adequate total protein intake as important modulators of muscle mass. In addition, due to its short duration, high-intensity interval training (HIIT) has been highlighted as a promising intervention to prevent physical deterioration. However, the interaction between daily protein intake distribution and HIIT intervention in elderlies remain unknown. OBJECTIVE: To investigate muscle adaptation following HIIT in older adults according to daily protein intake distribution. METHODS: Thirty sedentary obese subjects who completed a 12-week elliptical HIIT program were matched [criteria: age (± 2 years), sex, BMI (± 2 kg/m2)] and divided a posteriori into 2 groups according to the amount of protein ingested at each meal: < 20 g in at least one meal (P20-, n = 15, 66.8 ± 3.7 years) and ≥ 20 g in each meal (P20+, n = 15, 68.1 ± 4.1 years). Body composition, functional capacity, muscle strength, muscle power, physical activity level, and nutritional intakes were measured pre- and post-intervention. A two way repeated ANOVA was used to determine the effect of the intervention (HIIT) and protein distribution (P20- vs P20+, p < 0.05). RESULTS: No difference was observed at baseline between groups. Following the HIIT intervention, we observed a significant decrease in waist and hip circumferences and improvements in functional capacities in both P20- and P20 + group (p < 0.05). However, no protein distribution effect was observed. CONCLUSION: A 12-week HIIT program is achievable and efficient to improve functional capacities as well as body composition in obese older adults. However, consuming at least 20 g of proteins in every meal does not further enhance muscle performance in response to a 12-week HIIT intervention.

Differences in muscle adaptation to a 12-week mixed power training in elderly men, depending on usual protein intake.

INTRODUCTION: Normal aging is often associated with a decline of muscle mass (MM), strength (MS) and quality (MQ: MS/MM), leading to functional incapacities. This aging-related deterioration of muscles may involve a decreased protein intake. Mixed power training has been recently shown to induce positive effects on MM, MS and MQ. However, to our knowledge, no study has examined if muscle adaptations following mixed power training could be influenced by the daily amount of protein ingested in elderly men. METHODS: Twenty-one men completed the intervention and were divided into 2 groups based on their usual protein intake: PROT 1.1- (<1.1 g·kg-1·d-1 [n = 10; 73 ±â€¯3 years]) and PROT 1.2+ (>1.2 g·kg-1·d-1 [n = 11; 73 ±â€¯3 years]). Body composition (DXA: lean and fat masses), MS (1-maximal repetition on leg-press and handgrip strength), MQ (MS/body mass and MS/lower limb lean mass), functional capacities (Short Physical Performance Battery/Senior Fitness Test), dietary intake (3-day food record) and energy expenditure (accelerometer; 7 days) were measured. Mixed power training intervention consisted in power and functional exercises (12 weeks; 3 times/week; 1 h/session). RESULTS: Lower limb MS increase in the PROT 1.2+ group was greater from that of the PROT 1.1- group when normalized to lower limbs lean mass (p = 0.036). In addition, a trend for greater gain in lower limb MS normalized to body mass (p = 0.053) was observed in the PROT 1.2+. CONCLUSION: To optimize mixed power training effects on muscle function, healthy older men should ingest daily at least 1.2 g·kg-1·d-1 of protein. These beneficial effects of a higher usual protein intake were observed especially for MQ, which is one of the best predictors of functional capacities in older adults.

Muscle Mass Index and Animal Source of Dietary Protein Are Positively Associated with Insulin Resistance in Participants of the NuAge Study.

OBJECTIVES: Contribute evidence towards the complex interrelationships of body composition, insulin sensitivity and protein intake independently from adiposity in an older population. DESIGN: This is a cross-sectional analysis of an existing dataset in which a literature-supported model linking together the variables of interest is tested using path analysis. SETTING: The loss of muscle mass has been implicated in the development of insulin resistance. We propose to test associations of muscle mass with insulin sensitivity and their respective associations with animal and vegetable sources of protein intake, independently from adiposity. PARTICIPANTS: Non-diabetic participants aged 68-82 years from the NuAge study with all available measures (n=441) were included. MEASUREMENTS: A model considering age, sex, chronic diseases, physical activity; smoking and sources of protein intake influencing body composition components and insulin sensitivity was created and tested with Path Analysis for their independent associations. Muscle mass index (MMI; kg/height in m2) and % body fat were derived from DXA and BIA. Insulin resistance was estimated by the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) score and physical activity by the Physical Activity Scale for the Elderly (PASE) questionnaire. Protein intakes were obtained from three non-consecutive 24h-diet recalls. RESULTS: In the final model, direct positive associations were observed between HOMA-IR score and MMI (ß=0.42; 95%CI: 0.24; 0.6) and % body fat (ß=0.094; 95%CI: 0.07; 0.11). There were no direct associations between animal protein intake and MMI or with HOMA-IR. There was a significant direct negative association between plant protein intake and MMI (ß= -0.068; 95%CI: -0.13; -0.003) and significant indirect associations mediated through MMI and % body fat between HOMA-IR and animal protein intake (ß=0.0321; 95%CI: 0.01; 0.05), as well as plant protein intake (ß= -0.07; 95%CI: -0.1; 0.0). CONCLUSIONS: Our final model indicated that MMI and HOMA score were significantly positively associated. Protein intake sources were related to HOMA-IR score differently through MMI and % body fat, respectively.

Intakes of folate, vitamin B6 and B12 and risk of depression in community-dwelling older adults: the Quebec Longitudinal Study on Nutrition and Aging.

BACKGROUND/OBJECTIVES: Depression can decrease quality of life and affect health outcomes in older population. We investigated whether different intake levels of folate, vitamin B6 and B12 were associated with a 3-year depression incidence among generally healthy, community-dwelling older men and women. SUBJECTS/METHODS: Participants in the Québec Longitudinal Study on Nutrition and Aging (NuAge), free of depression (that is, 30-item Geriatric Depression Scale (GDS) <11) at baseline (N=1368; 74 ± 4 years old; 50.5% women), were screened annually for incident depression (GDS ⩾ 11) or antidepressant medication. Tertiles of intakes (food only and food+supplements) were obtained from the mean of three non-consecutive 24-h recalls at baseline. Sex-stratified multiple logistic regression models were adjusted for age, physical activity, physical functioning, stressful life events and total energy intake. RESULTS: Over 3 years, 170 participants were identified as depressed. Women in the highest tertile of B6 intake from food were 43% less likely to become depressed when adjusting for demographic and health factors (multivariate odds ratio (OR) 0.57, 95% confidence interval (CI) 0.39-0.96), but adjustment for energy intake attenuated the effect. Men in the highest tertile of dietary B12 intake had decreased risk of depression (energy-adjusted multivariate OR 0.42, 95% CI 0.20-0.90). No other association was observed. CONCLUSIONS: This study provides some evidence of decreased depression risk among women with higher intakes of vitamin B6 from food, which was dependent on total energy intake, and among men with higher intakes of B12 from food, independently of energy intake.

Dietary patterns and incidence of depression in a cohort of community-dwelling older Canadians.

OBJECTIVES: To investigate the association of dietary patterns with a 3-year incidence of depression among healthy older adults. DESIGN: Multiple logistic regression models adjusted for age, sex, marital status, smoking, education, total energy intake, physical activity, body mass index, hypertension, functional autonomy, cognitive functioning, social activities, and stressful life events. Energy and macronutrient intakes were also analyzed as potential predictors of depression. SETTING: Cities of Montréal, Laval, and Sherbrooke in Quebec, CA. PARTICIPANTS: Community-dwelling older adults, free of depression at baseline (N=1,358, 67-84 y), followed for 3y in the Québec Longitudinal Study on Nutrition and Aging (NuAge). MEASUREMENTS: Dietary patterns derived from principal components analysis of three 24 h-recalls at baseline, and depression incidence as measured by the 30-item Geriatric Depression Scale (≥11) and/or use of antidepressants at follow-up years. RESULTS: 170 people (63% women) became depressed over the 3 years. People in the highest tertile of adherence to the "varied diet" had lower risk of depression before adjustment (OR 0.58, 98% C.I. 0.38-0.86) but not significant once age and sex were controlled. No other dietary pattern was associated with the incidence of depression. The highest tertile of energy intake was associated with lower depression incidence after controlling for all confounders (OR 0.55, 95%CI 0.34-0.87). CONCLUSION: Among healthy older adults, dietary patterns do not appear to be related to depression. Those who eat less, however, possibly reflecting declining health, are at higher risk of becoming depressed.

Baseline determinants of global diet quality in older men and women from the NuAge cohort.

UNLABELLED: Judicious food choices are of prime importance during aging. OBJECTIVES: This study was conducted to identify individual and collective attributes determining global diet quality (DQ). METHODOLOGY: Participants were 1,793 adults (52% women) from the NuAge study on nutrition and successful aging. Subjects aged 67 to 84 years in relatively good health were recruited from the Québec Medicare Database. Sociodemographic, affective, and cognitive data, health conditions, perceived physical health and functional status, dietary habits and dietary attributes and community resources were obtained using questionnaires. Body weight and height were measured and body mass index (BMI) was calculated. Three non-consecutive 24-hour diet recalls were collected at recruitment. DQ, assessed using the Canadian Healthy Eating Index (C-HEI, /100), was computed on the mean intakes from the diet recalls. Analyses were stratified by gender. Variables significantly related to DQ in bivariate analyses (p<.05) were entered into backward stepwise multiple regression analyses. RESULTS: Among men, the final model showed higher education (ß=0.23, p=.01), diet knowledge (ß=0.96, p<.0001), number of daily meals (ß=1.91, p=.02) and perceived physical health (ß=0.06, p=.01) to be positive determinants of DQ, whereas alcohol consumption (ß=-2.25, p=.05), wearing dentures (ß=-2.31, p=.01) and eating regularly in restaurants (ß=-1.65, p=.03) were negative determinants of DQ (adjusted R2 = 13.7%). Among women, higher education (ß=0.29, p=.002), diet knowledge (ß=0.54, p=.002), number of daily meals (ß=3.61, p<.0001), and hunger (ß=0.61, p<.0001) were positive determinants of global DQ; greater BMI (ß=-0.16, p=.03) and chewing problems (ß=-0.48, p=.03) were negative determinants of DQ (adjusted R2 = 7.8%). DISCUSSION: These results point to several key factors influencing global DQ in older adults and also show gender-based differences. More research must be done to better understand how these factors change with aging and exert their impact on diet, particularly since variance in DQ was largely unexplained. As diet knowledge was an independent predictor for both genders, targeted, sustainable interventions are needed to ensure good diet quality as people age.

Biophysical model estimation of neurovascular parameters in a rat model of healthy aging.

Neuronal, vascular and metabolic factors result in a deterioration of the cerebral hemodynamic response with age. The interpretation of neuroimaging studies in the context of aging is rendered difficult due to the challenge in untangling the composite effect of these modifications. In this work we integrate multimodal optical imaging in biophysical models to investigate vascular and metabolic changes occurring in aging. Multispectral intrinsic optical imaging of an animal model of healthy aging, the LOU/c rat, is used in combination with somatosensory stimulation to study the modifications of the hemodynamic response with increasing age. Results are fitted with three macroscopic biophysical models to extract parameters, providing a phenomenological description of vascular and metabolic changes. Our results show that 1) biophysical parameters are estimable from multimodal data and 2) parameter estimates in this population change with aging.

Cerebrovascular hemodynamic correlates of aging in the Lou/c rat: a model of healthy aging.

The LOU/c rat is an inbred strain considered a model of healthy aging. It exhibits a longer free disease lifespan and a low adiposity throughout life. While this animal model has been shown to maintain eating behavior and neuroendocrine, metabolic and cognitive functions with age, no study has yet investigated vascular correlates in this model of healthy aging. In the present work, multispectral optical imaging was used to investigate the hemodynamic response in the somatosensory cortex of LOU/c rats following forepaw stimulation in three age groups, 4, 24 and 40months. Results indicate reduced hemodynamic responses in the contralateral somatosensory cortex between young (4months) and older groups following stimulation. This decrease was associated with an increase in the spatial extent of activation. The ipsilateral response did not change with aging leading to decreased laterality. Estimations of the relative change in the local cerebral metabolic rate of oxygen during stimulation based on multimodal data showed no significant change with age. The exponent describing the relation between blood volume and blood flow changes, Grubb's parameter, did display a significant change with age which may suggest vessel compliance modifications. This work finds its relevance in recent findings underlying the importance of vascular changes with aging and its impact on neurodegenerative disease.

Regulation of the pituitary growth hormone-releasing hormone receptor in ageing male and female LOU rats: new insights into healthy ageing.

Ageing is characterised by a decrease of somatotroph functionality, involving growth hormone-releasing hormone receptor (GHRH-R). The present study was conducted in LOU/C/jall (LOU) rats, a strain described as a model of healthy ageing, which is characterised by a low adiposity and long life expectancy without developing severe pathologies. Effects of age and diet (chow versus self-selection), on levels of anterior pituitary GHRH-R mRNA transcripts, were assessed in male and female LOU rats. The effect of age on pituitary GHRH-R functionality was examined in the anterior pituitary of both males and females fed chow diet. Moreover, serum insulin-like growth factor-I (IGF-I), T4 and leptin were measured because changes in their concentration could affect GHRH-R expression. In the pituitary of 18-month-old male and female LOU/C/jall rats fed standard chow, the level of 2.5-kb GHRH-R mRNA transcript, coding for functional GHRH-R, was significantly decreased. In 24- to 34-month-old males and females, it progressively returned to the level of younger animals, suggesting an enrichment of the group with survivors maintaining functional GHRH-R. In males and females repeatedly submitted to self-selection, this phenomenon was not observed. Studies with the GHRH-R agonist, Fluo-GHRH, revealed that 73% of 16-18-month-old male and female rats studied did not show an increase of fluorescence density characteristic of receptor-mediated internalisation upon incubation at 37 degrees C. In the other 27%, the increase of fluorescence was identical to that observed in pituitaries of young rats, suggesting the presence of an optimal level of functional GHRH-R. Serum levels of leptin, free T4 and total IGF-I decreased more drastically in ageing males and in rats fed a self-selection diet. A positive correlation was demonstrated between leptin and IGF-I levels in ageing males and females fed standard chow and ageing females submitted to a self-selection regimen. In conclusion, healthy ageing in LOU rats fed chow diet appears to be associated with a maintenance of functional pituitary GHRH-R levels found in younger rats but not necessarily with those of serum leptin, T4 and IGF-I.

Cellular distribution of growth hormone-releasing hormone receptor in human reproductive system and breast and prostate cancers.

Growth hormone releasing hormone receptor (GHRH-R) mRNA and protein was first localized to the anterior pituitary gland, consequent with the action of its ligand on GH synthesis and release. Subsequent studies found GHRH-R also expressed in the hypothalamus and in systemic tissues including those of the reproductive system. In the present work, we studied the distribution of GHRH-R in human reproductive system of males and females by immunohistochemical method. GHRH-R immunostaining was localized in male reproductive system: Leydig cells, Sertoli and basal germ cells of the seminiferous tubules and prostate secretory cells. GHRH-R immunostaining was also demonstrated in the ovary: oocytes, follicular cells, granulosa, thecal and corpus luteum cells. Endometrial glands, placenta and normal mammary glands also showed GHRH-R immunostaining. Our results demonstrate the localization of GHRH-R in the reproductive system, which may mediate the direct action of GHRH in these tissues. Moreover, GHRH-R was demonstrated in prostate and breast carcinomas, opening a variety of possibilities for the use of GHRH antagonists in the treatment of prostatic and mammary tumors.

Similar ultrastructural distribution of the 5-HT(2A) serotonin receptor and microtubule-associated protein MAP1A in cortical dendrites of adult rat.

As visualized by light and electron microscopic immunocytochemistry, the distribution of the neuronal serotonin-2A (5-HT(2A)) receptor is mainly intracellular throughout adult rat brain. This localization is particularly striking in the pyramidal cells of cerebral cortex, the dendrites of which are intensely immunoreactive, but without any labeling of their spines. In view of recent yeast two-hybrid and biochemical results suggesting an association of 5-HT(2A) receptors with the cytoskeletal microtubule-associated protein MAP1A, the respective subcellular distributions of the receptors and of MAP1A were compared by quantitative electron microscopic immunocytochemistry in dendrites of adult rat frontoparietal cortex. Counts of silver-intensified immunogold particles revealed a higher density of 5-HT(2A) receptors in smaller rather than larger dendrites, and an apportionment between pre-defined compartments representing the plasma membrane and the cytoplasm that was proportional to the relative surface area of these compartments. MAP1A immunoreactivity also predominated in smaller versus larger dendrites, but with a slightly lower proportion of labeling in the plasma membrane versus cytoplasmic compartment. The co-localization of 5-HT(2A) receptors and MAP1A protein in the same dendrites could be demonstrated in double immunolabeling experiments. These results confirmed the predominantly somato-dendritic, intracellular localization of 5-HT(2A) receptors in cerebral cortex, showed their higher concentration in distal as opposed to proximal dendrites, and suggested their potential association to the cytoskeleton in cortical neurons in vivo. Such a distribution of 5-HT(2A) receptors reinforces our earlier hypothesis that 5-HT(2A) receptors participate in intraneuronal signaling processes involving the cytoskeleton, and raises the possibility that their activation could be dependent upon that of another co-localized, plasma membrane-bound, 5-HT receptor.

Effects of peripheral and central administration of GHRH on feeding in aging LOU rats.

In aging LOU rats, a decreased protein intake is restored by GH administration. To study the contribution of GHRH to macronutrient selection, hGHRH NH(2) was administered sc. (1 mg/kg B.W./day/14 days) or icv. (4 and 40 pmol/rat) to 11-, 19-, 24- and 28-month-old rats. Sc. administration induced a decreased food and lipid intakes from 24 months of age and a transient stimulation of protein intake in 19-month-old and older low protein eaters (<10% protein/total intake). Icv. administration induced decreased food and lipid intakes in all age groups. These results suggest that GHRH may regulate feeding through pituitary and/or hypothalamic GHRH receptor mechanisms.

Dopamine D-5 receptor modulates hippocampal acetylcholine release.

Dopamine is intimately involved in cognitive processes in the brain. Of the several subtypes of dopamine receptors, the possible role of dopamine D1-like receptors in brain functions, especially in learning and memory, has recently generated much interest. However, molecularly the D1-like receptors are comprised of at least two subtypes, namely D-1 and D-5, and it has not been possible to ascertain which of these two receptor classes is responsible for these functions due to the lack of selective ligands. In the present study, utilizing a combined antisense-in vivo dialysis approach, we show that the D-5 subtype is the dopamine D1-like receptor involved in modulating hippocampal acetylcholine (ACh) release, a transmitter implicated in a variety of cognitive processes. This is one of the first evidence for a functional role for the D-5 receptor.

Antiangiogenic effects of the oral administration of liquid cartilage extract in humans.

BACKGROUND: The antiangiogenic properties of shark cartilage extracts have been demonstrated in animal models but there are no data in human subjects. MATERIALS AND METHODS: A placebo or one of two doses of a liquid shark cartilage extract was orally administered daily, from Day 1 to Day 23 of the study protocol, to 29 healthy male volunteers randomized into three groups. On Day 12, a polyvinyl alcohol sponge threaded in a perforated silicone tubing was inserted subcutaneously on the anterior side of the arm and removed on Day 23. Evaluation of endothelial cell density, with factor VIII immunostaining, an indirect measurement of angiogenesis, was performed on histological sections of the implant using a semiquantitative numerical scale ranging from 1 (low density) to 5 (high density). The hydroxyproline content of the sponges was measured by HPLC. RESULTS: The mean endothelial cell density was significantly lower in groups that had received the liquid cartilage extract: grades 2.24 +/- 0.10, 2.47 +/- 0.10, and 3.15 +/- 0.11 for 7 and 21 ml liquid cartilage extract and placebo, respectively (P < 0.01 for both comparisons). No grade 1 was observed in the placebo group, whereas 9 treated subjects received a grade 1. Hydroxyproline content of the sponges did not differ between groups and there was no significant correlation between hydroxyproline content and endothelial cell density in the sponges. CONCLUSIONS: These results demonstrate that the liquid cartilage extract contains an antiangiogenic component bioavailable in humans by oral administration. This is the first report of an inhibition of wound angiogenesis in healthy men.

Development of a site-directed polyclonal antibody against the pituitary growth hormone-releasing hormone receptor and its use to estimate GHRH receptor concentration in normal and hypothyroid rats.

A site-directed polyclonal antipeptide antibody was generated in rabbits against segment 392-404 of the rat pituitary growth hormone-releasing hormone receptor (GHRH-R), using a multiple antigenic peptide system strategy of immunization. This C-terminal intracellular region of the rat GHRH-R exhibits 85% sequence identity with the human GHRH-R. The purified anti-GHRH-R(392-404) IgGs were characterized in cell lines expressing the human GHRH-R and in rat and human anterior pituitary, using immunoblotting. The polyclonal antibody recognized a 45-kD protein in human GHRH-R-transfected BHK 570 cell membrane preparations but not in wild-type cells. A 45-kD N(alpha)-tagged human GHRH-R was immunodetected with both antitag and anti-GHRH-R antibodies in human GHRH-R-transfected HEK 293 cells. Cross-linking of [(125)I-Tyr(10)]hGHRH(1-44)NH(2) to GHRH-R-transfected BHK cells led to the detection of a major and specific 45-kD radioactive complex. Its probing with the anti-GHRH-R(392-404) IgGs led also to the detection of a 45-kD entity. In rat anterior pituitary homogenates or membrane preparations, immunoblotting led to the detection of 44-, 47- and 65-kD proteins. In human anterior pituitary membrane preparations, immunoblotting led to the detection of 52- and 55-kD proteins. No immunoreactive signal was observed in the rat liver. Cross-linking of [(125)I-Tyr(10)]hGHRH(1-44)NH(2) to rat anterior pituitary homogenates revealed the presence of specific 28-, 47- and 65-kD radioactive complexes. Probing of these radioactive complexes with the anti-GHRH-R(392-404) IgGs resulted in the visualization of 28-, 47- and 65-kD entities and of an additional immunoreactive 44-kD protein. To assess the usefulness of this GHRH-R antibody, estimation of changes in the concentration of rat anterior pituitary GHRH-R was performed by immunoblotting and compared to binding data after a 3-week antithyroid treatment. The treatment known to depress the 2.5- and 4-kb GHRH-R mRNA transcripts by at least 1.7-fold decreased the apparent maximal concentration of high (B(max1)) and low (B(max2)) affinity binding sites by 4.6- and 15.2-fold, respectively, and the 47- and 65-kD GHRH-R proteins by 3.5- and 1. 25-fold, respectively. Altogether, the characteristics of the anti-GHRH-R(392-404) polyclonal antibody indicate that it specifically recognizes the human and rat GHRH-R. It also represents an additional valuable tool to estimate variations of GHRH-Rs in physiopathological conditions known to affect GHRH-R mRNA and/or GHRH binding site concentrations.

Restricted presence of the growth hormone-releasing hormone receptor to somatotropes in rat and human pituitaries.

Specific binding of growth hormone-releasing hormone (GHRH) to its plasma membrane receptor represents the first step of cellular signals leading to exocytotic GH secretion in the anterior pituitary. The GHRH receptor (GHRH-R) has been cloned and belongs to the secretin/glucagon/vasoactive intestinal peptide subfamilly of G-protein-coupled receptors. To study its characteristics in rat and human pituitaries and examine its cellular and subcellular localization, a site-directed polyclonal antibody recognizing the C-terminal portion 392-404 of the rat and human GHRH-R was used. Immunohistochemistry was performed on paraffin-embedded pituitary sections while ultrastructural immunocytology was done on frozen and Lowicryl-resin-embedded ultrathin sections. GHRH-R-like immunoreactivity was restricted to somatotropes and colocalized with GH in both rat and human tissues. No signal was detected in gonadotropes, lactotropes, corticotropes and thyrotropes. At the subcellular level, gold particles were associated with the plasma membrane (observed on ultrathin frozen sections), secretory granule membrane, cytoplasmic matrix, nuclear membrane and nuclear matrix. In the nucleus, gold particles were mainly observed at the junction between eu- and heterochromatin. The highest density of labeling was observed in the cytoplasm (55 vs. 45% in the nucleus), mainly in secretory granules (59% of cytoplasmic labeling) and the plasma membrane. These results support the hypothesis that GHRH-mediated actions in the pituitary are specific to somatotropes and that GHRH-R isoforms and/or ligand-receptor complexes are involved in intracellular trafficking, recycling processes and nuclear functions.

Peripheral injection of growth hormone stimulates protein intake in aged male and female Lou rats.

It is well established that growth hormone (GH) induces growth rate and food efficiency and stimulates protein accretion in young mammals. Senescence is characterized by metabolic and hormonal disorders, particularly a decrease in protein turnover, which could be correlated to a decrease in GH and insulin-like growth factor I (IGF-I) secretion. We have shown that body weight, protein intake, and IGF-I plasma levels are greatly decreased with aging in Lou/C rats, particularly in males. In order to specify the GH effect on protein intake during aging, males and females (6, 19, and 24 mo) placed on a self-selection regimen were injected daily with a physiological dose of human GH (0.023 mg/rat sc). No GH effect on caloric intake was observed. Nevertheless, GH treatment stimulated body weight in older rats. It also increased protein intake in females and older males (19-24 mo). This stimulating effect was positively correlated with the degree of weight loss in senescent rats, suggesting that the decrease in protein intake observed with aging could be a marker of senescence.

Differential in vivo regulation of the pituitary growth hormone-releasing hormone (GHRH) receptor by GHRH in young and aged rats.

In aging, alterations of pituitary GH-releasing hormone (GHRH) receptor (GHRH-R)-binding sites have been proposed as one of the initiating factors contributing to the loss of somatotroph responsiveness to GHRH. Changes in the characteristics and/or concentration of the functional GHRH-R could take place in the course of aging and reduce the sensitivity of the somatotroph axis to GHRH. Because chronic exposure to GHRH has been proposed to resensitize aged somatotroph cells, better knowledge of its effects on the regulation of the somatotroph axis is required, particularly at the level of GHRH-R. Two- and 18-month-old male Sprague Dawley rats were treated for 14 days with a daily s.c. injection of 0.5 or 1.0 mg/kg BW human GHRH-(1-29)NH2 or saline. In 2-month-old rats, treatment with 0.5 mg/kg GHRH increased the number of high affinity pituitary GHRH-R-binding sites by 2-fold (P < 0.05) and hypothalamic somatostatin (SRIF) content by 45% (P < 0.05). It did not affect hypothalamic GHRH content, serum total insulin-like growth factor I (IGF-I), or body weight gain. Treatment with 1.0 mg/kg GHRH decreased the number of high affinity pituitary GHRH-R-binding sites by 2.4-fold compared with that in rats treated with 0.5 mg/kg BW (P < 0.05) and increased hypothalamic SRIF content by 45% (P < 0.05), but did not affect GHRH content. It also decreased circulating levels of IGF-I by 13% (P < 0.05) and slowed the growth rate by 17% (P < 0.05). In 18-month-old rats, treatment with 0.5 mg/kg GHRH for 14 days was not sufficient to rejuvenate pituitary GHRH binding parameters. However, treatment with 1.0 mg/kg GHRH restored the affinities of high and low affinity classes of GHRH-binding sites to values similar to those found in 2-month-old rats. Binding capacities of the high and low affinity classes of sites were increased by 1.8- and 3-fold, respectively, although significance was only reached for the low affinity site (P < 0.05). These changes were associated with a normalization of the level of 2.5-kb GHRH-R messenger RNA transcript, which was decreased by 31% in aging rats (P < 0.05), and by a trend for an increase in the 4-kb GHRH-R messenger RNA transcript, which was already increased by 49% in 18-month-old rats (P < 0.05). A normalization of serum IGF-I levels, which were decreased by 11% in 18-month-old control rats (P < 0.01), was also observed. No treatment effect was detected on body weight or hypothalamic SRIF and GHRH contents. We conclude that a 14-day administration of GHRH induces a differential GHRH-R-mediated regulation at the level of the pituitary and probably the hypothalamus as a function of age.