Diabetes Volume 64, March 2015 785 Ke Hao,1 Fan-Ping Kong,1 Yu-Qi Gao,2 Jia-Wei Tang,1 Jian Chen,2 A. Mark Evans,3 Stafford L. Lightman,4 Xue-Qun Chen,1 and Ji-Zeng Du1 Inactivation of Corticotropin- Releasing Hormone–Induced Insulinotropic Role by High- Altitude Hypoxia Diabetes 2015;64:785–795 | DOI: 10.2337/db14-0500 We have shown that hypoxia reduces plasma insulin, dysfunction and illness, particularly acute mountain sick- which correlates with corticotropin-releasing hormone ness (AMS) (1). During the construction of the Qinghai- (CRH) receptor 1 (CRHR1) in rats, but the mechanism Tibet railway (at altitudes of 3,000–5,000 m) in China, remains unclear. Here, we report that hypobaric hypoxia at .100,000 construction workers were involved, and 51% an altitude of 5,000 m for 8 h enhances rat plasma CRH, of them developed AMS (2). Moreover, since the railway METABOLISM corticosterone, and glucose levels, whereas the plasma began service, .10 million travelers have visited the Tibet insulin and pancreatic ATP/ADP ratio is reduced. In islets region in 2012, of whom 31% developed AMS despite cultured under normoxia, CRH stimulated insulin release in traveling with an oxygen supply on the train (3). Increas- a glucose- and CRH-level–dependent manner by activat- ing CRHR1 and thus the cAMP-dependent protein kinase ing evidence in both humans and animals suggests that pathway and calcium influx through L-type channels. In exposure to either high-altitude or hypobaric hypoxia fl islets cultured under hypoxia, however, the insulinotropic in uences plasma insulin levels and glucose homeostasis, effect of CRH was inactivated due to reduced ATP and depending on the oxygen level and duration of exposure cAMP and coincident loss of intracellular calcium oscilla- (4–9). We previously showed that subacute hypoxia at an tions. Serum and glucocorticoid-inducible kinase 1 (SGK1) altitude of 5,000 m for 5 days reduces plasma insulin in also played an inhibitory role. In human volunteers rapidly rats, and this effect is blocked by a corticotropin-releasing ascended to 3,860 m, plasma CRH and glucose levels in- hormone (CRH) receptor 1 (CRHR1) antagonist in vivo creased without a detectable change in plasma insulin. By (10). However, the underlying mechanisms have not been contrast, volunteers with acute mountain sickness (AMS) clearly addressed. exhibited a marked decrease in HOMA insulin sensitivity Insulin, the unique hypoglycemic hormone, plays a (HOMA-IS) and enhanced plasma CRH. In conclusion, hyp- crucial role in maintaining glucose sensing in pancreatic oxia may attenuate the CRH-insulinotropic effect by re- b-cells and regulating glucose uptake in a variety of tis- ducing cellular ATP/ADP ratio, cAMP and calcium influx, sues and cells during health and disease (11,12). Apart and upregulated SGK1. Hypoxia may not affect HOMA-IS in healthy volunteers but reduces it in AMS volunteers. from glucose, many neural and endocrine hormones reg- ulate pancreatic insulin release (13). In particular, CRH is the key regulator of the hypothalamic-pituitary-adrenal To enjoy social activities, millions of people travel to high (HPA) axis; is activated by a variety of stressors, including altitudes every year. High-altitude hypoxia often induces hypoxia; and mediates a variety of neural and endocrine 1Division of Neurobiology and Physiology, Department of Basic Medical Sciences, Corresponding authors: Ji-Zeng Du, [email protected], and Xue-Qun Chen, School of Medicine, Key Laboratory of Medical Neurobiology of the Ministry of [email protected]. Health of China, Zhejiang University, Hangzhou, China Received 27 March 2014 and accepted 25 September 2014. 2Department of Pathophysiology and High Altitude Physiology, College of High This article contains Supplementary Data online at http://diabetes Altitude Military Medicine, Third Military Medical University, Chongqing, China .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0500/-/DC1. 3Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, U.K. © 2015 by the American Diabetes Association. Readers may use this article as 4Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, long as the work is properly cited, the use is educational and not for profit, and University of Bristol, Bristol, U.K. the work is not altered. 786 Hypoxia Inactivates Insulinotropic Role of CRH Diabetes Volume 64, March 2015 response to stress (14). Studies have shown that CRHR1 day, starting 2 days before the flight to Rikaze, China, at exists in human, mouse, and rat islets (15,16) and that 3,860 m altitude, volunteers were given a Rhodiola capsule CRH enhances calcium influx (17), increases insulin con- (Z10980020, Tibet Nuodikang Medicine, Lhasa, China) to tent, and elevates insulin secretion in a glucose-dependent improve endurance and resistance to hypoxia. On the manner in cultured islets (15,16,18). Furthermore, CRH third morning at Rikaze, SpO2 was measured, and fasting modulates development, proliferation, and antiapoptosis blood samples were collected again. The AMS score was in islets (15,16,19). These findings suggest that CRH and obtained using the Lake Louise Score $3 (23). Plasma was CRHR1 play significant roles in regulating insulin release obtained by centrifugation as soon as possible and stored under normal conditions. We previously showed that at 280°C until use. hypobaric hypoxia results in upregulated CRH in the para- ventricular nucleus and corticosterone (CORT) in the Hypoxia Exposure of Animals and Isolated Islets plasma of rats (20,21), which was associated with reduced The rats in the hypoxia group were placed in a hypobaric plasma insulin (reversed by a CRHR1 antagonist) and chamber (FLYDWC-50-IIC; AVIC Guizhou Fenglei Avia- glucose levels in vivo (10). tion Armament Co., Ltd., Guizhou, China) and exposed to In the current study, we address the mechanisms by hypoxia of 2,000 m altitude (79.97 kPa, equivalent to which CRHR1 mediates insulin secretion and glucose 16.0% O2 at sea level) or 5,000 m altitude (54.02 kPa, homeostasis during hypoxia. To achieve this goal, we 10.8% O2). The chamber was opened daily for 30 min to completed comparative studies on rats under hypobaric clean and replenish food and water during the 5 days of hypoxia and on humans following a rapid ascent to the exposure. The normoxia group was placed in an identical Tibetan plateau. Under hypoxia, the cell metabolic state chamber at sea level (100.08 kPa, 20.9% O2). Rats re- switches from aerobic metabolism toward anaerobic ceived intraperitoneal injections of the CRHR1 antagonist fi glycolysis, which may lead to reduced ATP production cp-154,526 (30 mg/kg) (donated by P zer, Groton, CT), the fi and plasma insulin levels (10). In the present article, the glucocorticoid receptor (GR)-speci c antagonist RU486 data suggest that a fall in the ATP/ADP ratio and loss of (50 mg/kg) (Tocris, Bristol, U.K.), or vehicle 30 min before cAMP signaling capacity during hypoxia attenuate voltage- exposure. After exposure, rats were killed by decapitation – gated calcium influx and, thus, inactivate insulinotropic at 1300 1400 h, and trunk blood was collected. Plasma was 2 action of CRH. obtained by centrifugation and stored at 80°C. The liver and pancreas were immediately removed, frozen in liquid RESEARCH DESIGN AND METHODS nitrogen, and stored at 280°C until use. Isolated islets in Animals the hypoxia group were incubated in 5% CO2 and various Male Sprague-Dawley rats weighing 200–220 g were pur- O2 conditions delivered by the hypoxia chamber (ProOx chased from the Laboratory Animal Center of Zhejiang model P110 and ProCO2 model P120 systems; BioSpherix, Province, China (certification no. SCXK2008-0033), and Lacona, NY) (24). maintained in a 12-h light/dark cycle (lights on at 0600 h) Insulin Secretion 6 at 20 2°C with food and water ad libitum. Rats were Size-matched islets were washed and preincubated for 1 h adapted for 1 week before experiments. All animal experi- in RPMI 1640 medium containing 2.8 mmol/L glucose, ments were approved by the Animal Care and Use Com- 10 mmol/L HEPES, and 0.1% BSA. Ten islets per well were mittee of the School of Medicine, Zhejiang University. then incubated in testing RPMI 1640 medium containing Islet Isolation 10 mmol/L HEPES and 0.1% BSA with the indicated glucose Pancreatic islets were isolated by collagenase digestion and drugs under hypoxia or normoxia. At the end of the from rats as previously described (22). Intact islets were experiments, the testing medium was collected for insulin cultured in RPMI 1640 medium (containing 8.3 mmol/L measurement using a rat insulin enzyme immunosorbent glucose) supplemented with 10% FBS, 10 mmol/L HEPES, assay kit (Mercodia, Uppsala, Sweden), whereas islets were and penicillin/streptomycin (Invitrogen, Carlsbad, CA) at placed in lysis buffer for quantitative PCR (qPCR) assay. 37°C under 5% CO and 21% O for overnight recovery 2 2 Calcium Imaging before experiments. Islets were washed and loaded with 5 mmol/L Fluo-4 AM Human Hypoxia Exposure (Molecular Probes, Eugene, OR) in Krebs-Ringer bicarbon- Sixty-seven healthy male volunteers (18–23 years old) ate HEPES buffer comprising (in mmol/L) 129 NaCl, 4.7 were recruited in Chengdu, China, at 540 m altitude, as KCl, 1.2 KH2PO4, 5.0 NaHCO3, 2.0 CaCl2, 1.2 MgSO4,10 basal lowland control subjects. They were informed about HEPES, and 0.1% BSA at pH 7.4 containing 5.6 mmol/L the objectives of the study and agreed to the experimental glucose for 1 h at 37°C. Calcium-free conditions were protocols. All studies were approved by the Ethics Com- achieved by use of calcium-free Krebs-Ringer bicarbonate mittee of the Third Military Medical University.