425 Effects of pre-mortem stress on protein expression, steak color, and myofibrillar fragmentation index in the longissimus lumborum following harvest

Undesirable variation in beef tenderness and stability of flavor and color may be associated with the abundance of heat shock proteins (HSP). This study aimed to determine whether pre-mortem stress impacts HSP expression in the skeletal muscle following harvest. Forty Holstein steers were administered an i.v. bolus dose of adrenocorticotropic hormone (ACTH; 0.1 IU/Kg BW) to mimic an acute pre-mortem stress. Longissimus lumborum (LD) biopsy samples were taken prior to the ACTH challenge. Serum cortisol was measured every 0.5 h from -2 to 6 h relative to the ACTH challenge. Skeletal muscle and blood samples from 10 steers were collected at each harvest timepoint at (2, 12, 24 and 48 h post-challenge). Samples were collected from the LD immediately after harvest and after 14 d of aging. Protein expression of HSPβ1, P-HSPβ1, HSPβ5, and DJ-1 was analyzed in muscle samples taken prior to the ACTH challenge, at harvest, and after 14 d of post-mortem wet aging. In addition, steak color and myofibrillar fragmentation index (MFI) was analyzed in 14 d aged samples. Harvest time point following the ACTH challenge affected (P < 0.05) protein expression of HSPβ1 and P-HSPβ1. Protein expression of DJ-1 prior to the ACTH challenge was different (P < 0.05) among steers harvested at different timepoints. In addition, time of harvest had no effect on HSPβ5 expression (P > 0.05). Regarding steak color, time of harvest had an effect (P < 0.01) on a*, b*, hue, chroma, and ratio, but no effect (P > 0.05) on L*. Lastly, time of harvest had an effect (P < 0.05) on MFI. These data indicate that HSP expression, steak color, and MFI in the LD after harvest may be related to time of harvest following a stressful event pre-mortem.

Mediterranean Diet, Stress Resilience, and Aging in Nonhuman Primates

Persistent psychological stress increases the risk of many chronic diseases of aging. Little progress has been made to effectively reduce stress responses or mitigate stress effects suggesting a need for better understanding of factors that influence stress responses. Limited evidence suggests that diet may be a factor in modifying the effects of stress. However, long-term studies of diet effects on stress reactive systems are not available, and controlled randomized clinical trials are difficult and costly. Here we report the outcomes of a controlled, randomized preclinical trial of the effects of long-term consumption (31 months, ∼ equivalent to 9 human years) of Western versus Mediterranean - like diets on behavioral and physiological responses to acute (brief social separation) and chronic (social subordination) psychosocial stress in 38 adult, socially-housed, female cynomolgus macaques. Compared to animals fed a Western diet, those fed the Mediterranean diet exhibited enhanced stress resilience as indicated by lower sympathetic activity, brisker and more overt heart rate responses to acute stress, more rapid recovery, and lower cortisol responses to acute psychological stress and adrenocorticotropin (ACTH) challenge. Furthermore, age-related increases in sympathetic activity and cortisol responses to stress were delayed by the Mediterranean diet. Population level diet modification in humans has been shown to be feasible. Our findings suggest that population-wide adoption of a Mediterranean-like diet pattern may provide a cost-effective intervention on psychological stress and promote healthy aging with the potential for widespread efficacy.HighlightsThere is no population level treatment to reduce stress and associated disease.Mediterranean diet reduced sympathetic activity.Mediterranean diet reduced cortisol response to acute stress and to ACTH challenge.Mediterranean diet delayed age-related increases in sympathetic activity and cortisol responses to stress.These results suggest a dietary strategy to increase stress resilience.

Abstract P233: Inhibition Of Aldosterone Synthesis In Non-human Primates By PB6440, The Novel Highly Selective And Potent CYP11B2 Inhibitor

Aldosterone is an important mineralocorticoid responsible for fluid and electrolyte homeostasis produced by aldosterone synthase (CYP11B2). An aldosterone synthase inhibitor (ASI) may be a therapeutic option for primary aldosteronism-related conditions such as resistant hypertension. An ASI with sufficient selectivity for CYP11B2 versus the similar cortisol-producing enzyme CYP11B1 has remained elusive. PB6440 is a novel ASI that is potent and highly selective for CYP11B2. In vitro studies demonstrated 200-300-fold selectivity of PB6440 for human CYP11B2 compared to human CYP11B1. In single and multiple dose cynomolgus monkey studies of orally administered PB6440, dose-and concentration-dependent reduction of plasma aldosterone after ACTH challenge was observed with >90% reduction at higher doses. Consistent with its high selectivity, PB6440 had little effect on the CYP11B1 cortisol pathway. Plasma levels of cortisol, 11-deoxycortisol, and deoxycorticosterone, remained unchanged even at high doses of PB6440. Systolic and diastolic blood pressure was reduced in a dose-dependent manner. Circulating half-life of PB6440 was approximately 17 hours with high oral bioavailability. In summary, PB6440 is a highly selective ASI that demonstrated sustained aldosterone suppression for 14 days with no effect on the CYP11B1 pathway in non-human primates. In single and multiple dose studies, PB6440 appeared well tolerated, demonstrating good oral bioavailability, and a PK profile supportive of once daily dosing. These results suggest that PB6440 may be useful in humans as a novel therapeutic for treating hypertension or other conditions caused by excess aldosterone.

Cortisol and Dehydroepiandrosterone Response to Adrenocorticotropic Hormone and Frailty in Older Women

Background The response to adrenocorticotropic hormone (ACTH) is poorly characterized in old-old adults and may provide insight into the physiologic response to stress. Method We performed a standard 250 µg ACTH stimulation test in a home-based substudy of 51 women aged 85–96 years enrolled in the Women’s Health and Aging Study II who were not taking corticosteroids. We examined the cortisol and dehydroepiandrosterone (DHEA) responses at 0, 30, 60, and 120 minutes, overall and by frailty status. Results The peak cortisol response to ACTH could not be determined, with the highest levels at the 120-minute time point. Pre- and post-ACTH stimulated cortisol levels did not differ by frailty status over this time frame, with no difference in the characteristics of the dose-response curves. Pre- and post-ACTH stimulated DHEA levels also did not differ by frailty status, though the dose-response curves suggested divergence after stimulation, with a more rapid DHEA response with increasing frailty. Conclusions Our data demonstrate a robust cortisol response to ACTH challenge testing, but inadequate negative feedback in old-old women, resulting in prolonged exposure to cortisol. Future studies should examine dynamic cortisol and DHEA responses in this age group, using a less potent ACTH stimulus and longer collection period.

Validation of a noninvasive technique to quantify stress in northern bobwhite (Colinus virginianus)

Examination of the endocrine system through non-invasive fecal sampling may improve population management more than using demographic indicators alone. By addressing the physiological mechanisms that are influencing fitness, management actions can be proactively developed to alleviate stressors. Proactive determination of vulnerable populations is critical for species of concern, such as the Northern Bobwhite (Colinus virginianus), which have suffered decades of population decline. We validated an assay to noninvasively measure the adrenocortical response of captive reared bobwhite through fecal corticosterone metabolites (FCM). All individuals received three sequential 48-hour treatments in which samples were collected every 4 hours, including a reference period, an adrenocorticotropic hormone (ACTH) challenge and a biological stressor (exposure to a hunting dog). Reference FCM values had a mean concentration of 16.75 pg/mg (95% CrI: 13.68, 19.91) with adrenocortical activity increasing by 73% for the duration of the ACTH challenge (29.00 pg/mg; CrI: 25.01, 33.78). FCM concentrations remained similar to that of the reference levels during the biological stressor (16.56 pg/mg; CrI: 13.33, 19.92). Our study validates the use of feces to detect changes in FCM levels in our subject species but also demonstrates the complexity of FCM and the importance of both physiological and biological validation prior to field implementation.

Use of the ACTH challenge test to identify the predominant glucocorticoid in the southern sea otter (Enhydra lutris nereis)

After nearly being hunted to extinction during the fur trade of the late 20th Century, sea otter (Enhydra lutris) populations have recovered to varying degrees of their historical range. While overall population numbers and range have increased, there are regions in which expansion has occurred at a slower rate and/or animal numbers have decreased, which may be a result of chronic stress from a variety of sources. Some have employed glucocorticoid analysis in their attempts to validate these explanations. Our goal was to conduct a controlled study using sea otters managed under human care to validate the use of serum glucocorticoid analysis to monitor stress physiology in the sea otter. We used a standard ACTH challenge test to compare cortisol and corticosterone responses, thereby identifying the primary glucocorticoid in the sea otter. Fourteen sea otters of both sexes (five males, nine females), including juveniles, sub-adults and adults, participated in the study. The results of the testing supported cortisol as the primary glucocorticoid in the sea otter. Sex and age did not affect how the individual responded to the ACTH or saline injection. Interestingly, the saline injection not only confirmed the effects of the ACTH on glucocorticoid release from the adrenal glands but also provided information on how long it takes the sea otter’s glucocorticoid levels to return to baseline after capture and sedation. The insight gained from this study will aid in future efforts to better understand the role of stress in free-ranging sea otter populations. Recognition of the primary glucocorticoid will facilitate evaluation of more stable biological material, such as fur or whiskers, which tend to be less affected by the diurnal cycling of glucocorticoids.

Fecal stress, nutrition and reproductive hormones for monitoring environmental impacts on tigers (Panthera tigris)

Non-invasive stress and nutritional hormone analysis in relation to ecological and other biological indices have tremendous potential to address environmental disturbance impacts on wildlife health. To this end, we examined the relation between glucocorticoid (GC) and thyroid (T3) hormone indices of disturbance and nutritional stress in response to ACTH and TSH challenges in captive tigers, as well as how reproductive hormones vary by sex and reproductive condition. Glucocorticoid, thyroid, progesterone and androgen assays conducted on high-performance liquid chromatography separated fractions of biologically relevant fecal extracts revealed high cross-reactivity of these assays for their respective biologically relevant fecal hormone metabolites. Both adrenal and thyroid hormone metabolites were elevated in response to ACTH and TSH challenges. However, the adrenal and thyroid hormone responses to ACTH challenge were concurrent, whereas the adrenal response to TSH challenge was delayed relative to thyroid hormone elevation in both males and females. The concurrently elevated T3 in response to ACTH may serve to raise metabolic rate to maximize use of GC-mobilized glucose, whereas the relatively delayed GC rise following TSH challenge may be a response to glucose depletion due to increased metabolic rate associated with elevated T3. Progesterone, testosterone and androstenedione hormone metabolites were significantly elevated during gestation compared to lactation in a female monitored from conception through early lactation. Results suggest that the glucocorticoid, thyroid and reproductive hormone assays we tested can accurately measure the stress, nutrition and reproductive response from tiger feces, providing useful non-invasive tools to assess physiological responses to environmental stressors and their reproductive consequences in the wild.