sign in sign up
<<
Home , Glucagon, Insulin, SGK1

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 –Induced Insulinotropic Role by High- Altitude Hypoxia

Diabetes 2015;64:785–795 | DOI: 10.2337/db14-0500

We have shown that hypoxia reduces plasma , dysfunction and illness, particularly acute mountain sick- which correlates with corticotropin-releasing hormone ness (AMS) (1). During the construction of the Qinghai- (CRH) 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 corticosterone, and 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 supply on the train (3). Increas- a glucose- and CRH-level–dependent manner by activat- ing CRHR1 and thus the cAMP-dependent kinase ing evidence in both 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 , 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 -inducible kinase 1 (SGK1) altitude of 5,000 m for 5 days reduces plasma insulin in also played an inhibitory role. In 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 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 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 , 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 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 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 , 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 (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 and were immediately removed, frozen in liquid RESEARCH DESIGN AND METHODS , 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 were isolated by collagenase 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 immunosorbent glucose) supplemented with 10% FBS, 10 mmol/L HEPES, assay (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. The vol- HEPES buffer containing 2 mmol/L EGTA. Intact islets unteers’ oxygen saturation (SpO2) was determined with were immobilized with a wide-bore glass suction pipette a finger pulse oximeter. Fasting blood samples were col- under a Nikon TE2000 inverted microscope with a lected at 0700–0800 h before breakfast in Chengdu. Each Yokogawa spinning disk confocal system (PerkinElmer, diabetes.diabetesjournals.org Hao and Associates 787

Wellesley, MA). Calcium images were captured at 3-s ATP/ADP, and ATP/AMP ratios were assessed in both intervals and three different depths with 488-nm excita- the pancreas and the liver of rats under 5,000 m hypoxia. tion and 505–530-nm emission. At the end of each After 8 h of hypoxia, rat pancreas exhibited an elevated experiment, 0.5 mmol/L , a KATP channel in- lactate/pyruvate ratio, lowered ATP/ADP ratio, and elevated hibitor (Sigma, St. Louis, MO), was added to present the AMP/ATP ratio (Fig. 2A, C,andE). The lactate/pyruvate functional b-cells (25). As a rule, cells in islets were defined ratio was also elevated in the liver (Fig. 2B)butwithout as b-cells if fluorescence signals were markedly increased in detectable falls in the ATP/ADP or AMP/ATP ratio (Fig. 2D response to tolbutamide. The change of fluorescence inten- and F). However, after subacute hypoxia for 5 days, the sity (DF) was calculated as a percentage of the basal level lactate/pyruvate ratio was not changed (Fig. 2A and B), (F0, background subtracted), and frequency was calculated even though the ATP/ADP ratio decreased and AMP/ATP as events per minute. Methods for assays of plasma hor- ratio increased in both pancreas and liver (Fig. 2C–F). These mones and metabolism (glucose, lactate, pyruvate, ATP, changes indicate that acute or subacute hypoxia switches ADP, and AMP), drug treatment, cAMP assay, qPCR, im- glucose metabolism to anaerobic glycolysis and thus reduces munofluorescence staining, and Rhodiola rosea treatment ATP production in the pancreas. are described in the Supplementary Data. CRH Stimulates and Hypoxia Suppresses Insulin Statistical Analysis Release All data are presented as mean 6 SD and were analyzed To investigate the insulinotropic action of CRH and the using Student unpaired two-tailed t test and one-way effects of hypoxia on this process, isolated islets were ANOVA with Tukey post hoc test (GraphPad Prism 6 exposed to hypoxia and rotenone, an inhibitor of software). The paired t test was used in calcium imaging mitochondrial respiratory chain complex I. Treatment of and human data analyses. P , 0.05 was considered islets with CRH (10 pmol/L–10 nmol/L) for 1 h induced significant. dose-dependent increases in insulin secretion at 5.6 or 11.1 mmol/L glucose but not at 2.8 mmol/L glucose RESULTS (Fig. 3A). The CRH-induced maximum magnitude of in- Acute Hypobaric Hypoxia Affects Insulin and Glucose sulin at 11.1 mmol/L glucose (1.7-fold) was larger than at Levels in Rat Plasma Through CRHR1 5.6 mmol/L glucose (1.4-fold) (Fig. 3A). The insulino- To investigate the effect of hypoxic stress on plasma tropic action of CRH was persistently shown after 1, 12, glucose, insulin, CRH, and CORT, rats were exposed to and 24 h incubation at 5.6 mmol/L glucose, and this hypoxia of 2,000 or 5,000 m altitude for 8 h. Hypoxia of augmentation was completely abolished by pretreatment 5,000 m but not of 2,000 m decreased plasma insulin and with 1 mmol/L cp-154,526, a CRHR1 antagonist (Fig. 3B). increased plasma glucose, CRH, CORT, and HOMA insulin This CRH-insulinotropic action was blocked by cAMP- – sensitivity (HOMA-IS) (Fig. 1A E). All changes were reversed dependent protein kinase (PKA) inhibitors (H89 and by treatment with a CRHR1 antagonist (cp-154,526) (Fig. Rp-8-Br-cAMPs, 10 mmol/L) but not by inhibitors of pro- – 1A E). Therefore, after activation of the HPA axis, CRHR1 tein kinase C (Go 6983, 10 mmol/L) or C is likely involved in the regulation of insulin release and (U-73122, 10 mmol/L) (Fig. 3C). Moreover, this insulino- glucose metabolism under acute hypoxia. tropic effect of CRH depended on extracellular calcium Acute or Subacute Hypobaric Hypoxia Reduces ATP and was blocked by an L-type calcium channel inhibitor Level in Rat Pancreas (nifedipine, 10 mmol/L) (Fig. 3C). To determine how hypobaric hypoxia influences ATP Hypoxia reduced the CRH-insulinotropic action in homeostasis in the pancreas, the lactate/pyruvate, a manner that was inversely related to O2 supply. Under

Figure 1—CRHR1 is involved in the regulation of rat plasma insulin and glucose under hypoxia of 5,000 m altitude for 8 h. Hypoxia of 5,000 m significantly decreased plasma insulin (A) and increased plasma glucose (B)andHOMA-IS(C); these effects were blocked by pretreatment with a CRHR1 antagonist. CRHR1 antagonist administration reversed the hypoxia-induced high CRH (D) and high CORT (E) levels in plasma. n = 7 in each group. *P < 0.05, ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. cp group. cp, cp-154,526. 788 Hypoxia Inactivates Insulinotropic Role of CRH Diabetes Volume 64, March 2015

were assessed. CRH (0.1–1 nmol/L) significantly enhanced the accumulation of cAMP in normoxia, and 1 nmol/L CRH increased the cAMP levels at 0.1 or 1 nmol/L rotenone, whereas the basal cAMP levels were decreased and not stimulated by CRH at 10 nmol/L rotenone (Fig. 4A). Fur- thermore, 1 nmol/L CRH markedly elevated the ATP/ADP ratio under normoxia. This elevation was abolished by pre- treatment with 10 nmol/L rotenone (Fig. 4B), which in- creased the AMP/ATP ratio in a concentration-dependent manner (0.1–10 nmol/L) (Fig. 4D). Moreover, 1 and 10 nmol/L rotenone suppressed the basal ATP/ADP ratio (Fig. 4B), and the CRH-induced increases in ATP/ADP ratio were blocked by the CRHR1 antagonist with and without 1 nmol/L rotenone (Fig. 4C). CRH 1 nmol/L markedly increased the frequency of calcium oscillations in identified b-cells (Fig. 4E and F), and this increase was reversed by pretreatment with the CRHR1 antagonist (1 mmol/L) and the PKA inhibitor (10 mmol/L Rp-8-Br-cAMPs) (Fig. 4E). In calcium-free con- ditions, calcium oscillations were not induced by 1 nmol/L CRH or 1 mmol/L forskolin (Fig. 4I ). In the rotenone- induced ATP-deficient condition, the frequency of calcium Figure 2—Metabolic changes of rat pancreas and liver after 5,000 m oscillations was unaltered by CRH (Fig. 4E and G)but hypoxia for 8 h or 5 days. Hypoxia for 8 h increased the lactate/ pyruvate ratio in pancreas (A) and liver (B) and decreased the ATP/ was still increased by forskolin (Fig. 4E and H). These ADP ratio in pancreas (C), whereas 5 days of hypoxia reduced the findings strongly suggest that a deficiency of ATP and ATP/ADP ratio in both pancreas (C) and liver (D). The AMP/ATP reduced ATP/ADP ratio limit cAMP production and thus ratio in pancreas (E) was increased with 8 h or 5 days of hypoxia, lead to defective calcium oscillations in islets in a man- and AMP/ATP in liver (F) was elevated only with 5 days of hypoxia. n = 7 in each group. *P < 0.05, **P < 0.01 vs. each control group. C, ner that may contribute to the inactivation of the CRH- control; H, hypoxia. insulinotropic action.

Hypoxia Inhibits Insulin Release by Both Reducing ATP and Increasing CORT in Islets – 5% O2, CRH (0.01 1 nmol/L) failed to stimulate insulin To examine the effects of glucocorticoid and intracellular secretion at 5.6 mmol/L glucose (Fig. 3D), whereas 1% O2 ATP on insulin secretion under hypoxia, insulin secretion not only suppressed the insulinotropic action of CRH, but and GR target mRNA levels were assessed in isolated also reduced basal insulin secretion (Fig. 3D). To mimic rat islets. (DEX) concentration depen- the inhibition of oxidative and ATP pro- dently decreased insulin secretion under normoxia and duction in the absence of hypoxia, rotenone was used to 5% O2 and severely inhibited it under 1% O2 (Fig. 5A). inhibit complex I of the mitochondrial electron transport Reduction of insulin release correlated with increased DEX chain in islets. Rotenone treatment (0.1–10 nmol/L) grad- and rotenone (ATP reduction) concentration (Fig. 5B). DEX ually inhibited insulin secretion. Rotenone (0.1 nmol/L) elevated serum and glucocorticoid-inducible kinase 1 (Sgk1) suppressed the augmentation of insulin release by mRNA and inhibited Glut2 and Crhr1 mRNA expression 0.1 nmol/L CRH, whereas 1 or 10 nmol/L rotenone com- under normoxia (Fig. 5C). These changes were not affected fi pletely abolished 1 nmol/L CRH-stimulated insulin release by ATP de ciency (1 nmol/L rotenone) or hypoxia (5% O2) (Fig. 3E). despite that hypoxia increased the Crhr1 mRNA level (Fig. Of note, qPCR results indicated an increase in Crhr1 5C). These data suggest that both hypoxia-stimulated glu- mRNA in islets under 5% O2 hypoxia but not under cocorticoid release and a reduced ATP supply inhibit insulin 1 nmol/L rotenone-induced ATP deficiency (Fig. 3F). release from pancreatic islets. fi These ndings indicate that although hypoxia increases GR Mediates Inhibition of Insulin Release Under Crhr1 expression (Fig. 3F), the insulinotropic action of Hypobaric Hypoxia fi CRH was attenuated by hypoxia due to insuf cient sup- To test the role of glucocorticoid in insulin secretion and plies of O2 and ATP. glucose homeostasis under hypoxia in vivo, a GR antag- CRH-Insulinotropic Effect Is Inactivated by Gradually onist (RU486) was used in rats under hypoxia of 5,000 m Reducing cAMP, ATP, and Calcium Influx in Islets altitude for 8 h. RU486 pretreatment reversed the To determine the requirement for ATP as a substrate for hypoxia-induced and low plasma insulin the CRHR1-activated insulin release pathway, the cAMP (Fig. 6A). Immunofluorescence also showed a higher SGK1 level, ATP/ADP ratio, and calcium oscillations in islets signal in the nuclei of islet b-cells under hypoxia. The diabetes.diabetesjournals.org Hao and Associates 789

Figure 3—Hypoxia and ATP deficiency inactivates the insulinotropic action of CRH in isolated rat islets. CRH augmented insulin secretion at 5.6 and 11.1 mmol/L glucose but not at 2.8 mmol/L for 1 h. The CRH-induced maximum magnitude of insulin was at 1.7-fold at 11.1 mmol/L glucose and 1.4-fold at 5.6 mmol/L glucose (A). CRH persistently enhanced insulin secretion at 5.6 mmol/L glucose at 1, 12, and 24 h, and this was blocked by pretreatment with cp-154,526 (cp) (B). The insulinotropic action of CRH was blocked by PKA inhibitors (10 mmol/L H89 and Rp-8-Br-cAMPs [Rp-cAMP]), an L-type calcium channel inhibitor (10 mmol/L nifedipine), and in calcium-free solution (2 mmol/L EGTA) at 5.6 mmol/L glucose; this effect was not changed by inhibitors of protein kinase C (Go 6983) and phospholipase C (U-73122) (C). Hypoxia (5% or 1% O2) abolished the insulinotropic action of CRH on 24-h insulin secretion at 5.6 mmol/L glucose (D). CRH 1 nmol/L stimulated insulin secretion with 0.1 nmol/L rotenone, but this effect did not occur with 1 or 10 nmol/L rotenone at 5.6 mmol/L glucose. Linear regression analysis showed a correlation between rotenone concentration and insulin secretion at three doses of CRH (each slope is shown in the inset) (E). Crhr1 mRNA expression was high in islets under hypoxia (5% O2) but not under rotenone (1 nmol/L)-induced ATP-deficient conditions (F). n =3–4ineachgroup.*P < 0.05, **P < 0.01 vs. each control group; $P < 0.05, $$P < 0.01, $$$P < 0.001 vs. vehicle treatment group; #P < 0.05, ##P < 0.01, ###P < 0.001.

proportion of SGK1-positive b-cells increased under hyp- were analyzed. High-altitude hypoxia induced a dramatic oxia, and this was blocked by RU486 (Fig. 6B and C). decrease in SpO2 and increase in plasma CRH (Fig. 7A and These data suggest that hypoxia-evoked increases of D) but exerted no detectable effect on plasma insulin (Fig. SGK1 contribute to the inhibitory role of GR in insulin 7B). Despite unaffected plasma insulin levels, plasma glu- release in pancreatic islets. cose was significantly elevated relative to control (Fig. 7C). High-Altitude Hypoxia Affects Human Plasma Insulin Further analysis showed that plasma glucose increased in and Glucose Homeostasis volunteers with AMS (12 of 67) but not in those without To determine the regulatory effect of high-altitude AMS (55 of 67) (Fig. 7F). HOMA-IS declined only in AMS hypoxia on circulating hormones and plasma glucose in volunteers (Fig. 7G), who had a higher plasma CRH com- humans, 67 volunteers rapidly ascended to 3,860 m pared with those without AMS (Fig. 7D and H). Plasma altitude in Rikaze and stayed for 2 days. On the morning was decreased under hypoxia in all volunteers of day 3, plasma glucose, insulin, CRH, and cortisol levels (Fig. 7E). 790 Hypoxia Inactivates Insulinotropic Role of CRH Diabetes Volume 64, March 2015

Figure 4—Low cAMP and calcium oscillations suppress the insulinotropic action of CRH under the ATP-deficient condition. The CRH- induced high cAMP level (A) and ATP/ADP ratio (B) were attenuated or abolished by rotenone pretreatment. The CRH-induced increase of the ATP/ADP ratio was blocked by the CRHR1 antagonist with or without 1 nmol/L rotenone (C). The AMP/ATP ratio was elevated by rotenone pretreatment (D). CRH increased the calcium oscillatory frequency of b-cells in the normal condition, and this increase was abolished by cp-154,526 or Rp-8-Br-cAMPs (E). However, CRH did not increase the calcium oscillatory frequency in b-cells under the ATP- deficient condition (F–H). Calcium signaling patterns of single b-cells under the CRH challenge in normal (F), ATP-deficient (G and H), and calcium-free conditions (I). In A–D, n =3–4 per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. each control group; $P < 0.05 vs. vehicle treatment group; #P < 0.05, ##P < 0.01 vs. cp-154,526 group. In E, n =30–40 b-cells from more than five islets in five rats. &&&P < 0.001 vs. control group. TOL, tolbutamide.

DISCUSSION altitude acutely elevated plasma glucose without altering We addressed a different regulatory effect of CRH and plasma insulin. Furthermore, AMS volunteers exhibited on hypoxia-reduced insulin levels. Under reduced HOMA-IS and higher plasma CRH compared hypoxia, CRH-stimulated insulin release was abolished with non-AMS volunteers. due to hypoxia-reduced cellular ATP and cAMP levels, and CRH, a critical stress , plays a key role in a consequent inhibition of calcium influx in isolated rat regulating the HPA axis and adjustments in neural, islets, despite that hypoxia-activated CORT, still inhibited endocrine, metabolic, and glucose homeostasis to various insulin release (Fig. 8). In humans, rapid ascent to high stressors, including hypoxia (14,20,26,27). Consistent with diabetes.diabetesjournals.org Hao and Associates 791

Figure 5—DEX inhibits insulin secretion under normoxic, hypoxic, and ATP-deficient conditions in isolated rat islets. DEX inhibited 24-h insulin secretion under normoxia (21% O2) and hypoxia (5% and 1% O2)(A). Inhibition of insulin release by DEX was negatively correlated with ATP level (0.1–10 nmol/L rotenone) (B). DEX elevated Sgk1 mRNA and inhibited Glut2 and Crhr1 mRNA expression under normoxia, and these changes were not affected by ATP deficiency (1 nmol/L rotenone) or hypoxia (5% O2)(C). Hypoxia induced Crhr1 mRNA expression. n =3–4 in each group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. each control group; $P < 0.05, $$$P < 0.001 vs. vehicle treatment group.

our previous study (10), we show that acute hypobaric by CRH, increases calcium influx (17) and stimulates in- hypoxia induces marked increases in plasma CRH, which sulin secretion in a glucose-dependent manner (15,16,18). mediates reduced insulin and increased glucose in rat The current study demonstrates a 24-h persistent insulino- plasma (Fig. 1). Insulin, the hypoglycemic hormone, is tropic role of CRH under normoxia, depending on both only secreted from pancreatic islet b-cells and is mainly glucose and CRHR1 signaling pathways, namely PKA and regulated by glucose and neuroendocrine inputs (11,13). L-type calcium channel-mediated calcium influx (Fig. 3A– There have been reports that CRHR1 is expressed in hu- C). However, under hypoxia, this effect of CRH was inacti- man, mouse, and rat islets (15,16), which upon activation vated (Fig. 3D) despite Crhr1 mRNA being upregulated in

Figure 6—GR mediates hypoxia-induced plasma insulin reduction. GR antagonist RU486 administration reversed the increased glucose and decreased insulin in rat plasma induced by hypoxia (5,000 m, 8 h) (A). Immunofluorescence of SGK1 (red), insulin (green), and nuclei (DAPI blue) showed a high SGK1 signal (B) and an elevated percentage of SGK1-positive cells (C) in pancreatic islets under hypoxia; these increases were reversed by RU486. In A, n =7–8 rats in each group. In B and C, n =15–20 islets from five to seven rat pancreata. Scale bar = 100 mm; dotted line, islet region. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001 vs. hypoxia group. 792 Hypoxia Inactivates Insulinotropic Role of CRH Diabetes Volume 64, March 2015

Figure 7—High-altitude hypoxia results in changes of insulin and glucose homeostasis, and these are correlated with AMS in humans. Compared with low altitude (540 m), high altitude (3,860 m) decreased SpO2 (A) and caused no change in plasma insulin (B), increased plasma glucose (C) and CRH (D), and decreased plasma cortisol (E). Plasma glucose was elevated in volunteers with AMS (F), whereas their HOMA-IS was reduced (G). The increase in plasma CRH was significantly higher in volunteers with AMS than in those without (H). n = 67 (12 with AMS, 55 without). *P < 0.05, **P < 0.01, ***P < 0.001 vs. low altitude; $P < 0.05 AMS vs. no AMS. islets (Fig. 3F). These findings suggest that the insulino- reductions in substrate supply for AC-dependent cAMP tropic effect of CRH depends on sufficient O2 supply, and production. Hypoxia or ATP deficiency also induced an this view is supported by previous reports of attenuated increased AMP/ATP ratio (Figs. 2E and 4D), resulting in glucose-stimulated insulin secretion with stable basal insu- inhibited AC activity through binding to the intracellular lin levels under hypoxia of 6.7% (28) and 3% O2 (29). P site (33,34). Otherwise, a low ATP/ADP ratio under ATP It is well documented that decreased O2 supply switches cell respiration from aerobic metabolism toward anaerobic glycolysis. Consistent with this view, ATP pro- duction was deficient in rat pancreas under 5,000 m hyp- oxia (Fig. 2C). Islet b-cells express low levels of lactate dehydrogenase (30); thus, islets may have a lower capacity for anaerobic glycolysis and ATP production. The ATP/ ADP ratio of islet b-cells, therefore, may decrease more dramatically than that of pancreas under hypoxia. ATP is not only an energy molecule but also an important signal- ing molecule in islet b-cells, controlling insulin secretion. Under physiological conditions, insulin secretion is tightly correlated with the ATP/ADP ratio in islet b-cells, al- though it is still controversial whether increased ATP or decreased ADP contributes to the alteration of ATP/ADP ratio upon glucose stimulation (31,32). Other nutrients, including amino acids and free fatty acids, also have the capacity to modify the ATP/ADP ratio and insulin secre- tion (11). Therefore, islets may be more sensitive to ATP deficiency under hypoxia than other cells. Here, rotenone- induced ATP deficiency abolished the stimulatory role of CRH on insulin secretion (Fig. 3E), as did 5% O2 (Fig. 3D). Figure 8—Proposed inhibitory regulation of insulin release by hypoxia- Under normoxia, CRH increased cAMP production by activated CRH and CORT. Hypoxia switches from aerobic to an- aerobic glycolysis, resulting in low ATP/ADP and high AMP/ATP stimulating G-protein binding to CRHR1 and thus acti- ratios, which inhibit cAMP production and calcium oscillations in vating adenylate cyclase (AC), but cAMP production was rat islets. This consequently inactivates the insulinotropic role of lowered (not enhanced) when islets were deficient in ATP CRH, although CRHR1 is triggered by upregulated plasma CRH. (Fig. 4A). This reduction in cAMP likely contributes to the Meanwhile, hypoxia-stimulated plasma CORT inhibits insulin secre- tion through upregulated Sgk1 mRNA; Crhr1 mRNA is also inhibited suppression of CRHR1 signaling and might result from by CORT. a low ATP/ADP ratio under hypoxia and consequent diabetes.diabetesjournals.org Hao and Associates 793 deficiency (Fig. 4B and C) might limit the CRHR1 signal- ATP production and support hyperglycemia under acute ing pathway by gating of KATP channels, closure of L-type hypoxia (Fig. 2D). After 5 days of hypoxia, low insulin calcium channels, reduction of calcium oscillations (Fig. modulated by CRHR1 still occurs in rats. However, de- 4E), and consequent reductions in insulin secretion (Fig. creased blood glucose is not affected by CRHR1 (10). 3E). We conclude, therefore, that the insulinotropic role of Low insulin level and also occur in mice CRH is inactivated by a fall in ATP production under after 4 weeks of hypoxia (8) and likely result from adap- hypoxia, consequent reductions in the cAMP level and tations to hypoxia that consequently reduce the anaerobic ATP/ADP ratio, and, thus, inhibition of calcium oscillations glycolysis rate and ATP/ADP ratio in liver (Fig. 2D), de- in islets (Fig. 8). Consequently, high plasma CRH during spite that hepatic is sufficient and CORT hypoxia may not exert the expected insulinotropic effects. remains high (10). Therefore, CRHR1 regulation of blood Acute (Fig. 1) or subacute (10,26) hypoxia increases not glucose likely depends on high hepatic metabolism. In only plasma CRH but also CORT through activation of the man, 3,860 m hypoxia for 2 days only increased plasma HPA axis, raising the possibility that hypoxia-stimulated glucose and affected insulin sensitivity in the AMS group CORT is important in insulin regulation. Glucocorticoids (Fig. 7C and G) in association with low cortisol (Fig. 7E) coordinate various stress responses and glucose homeo- induced by R. rosea. Other studies have reported high stasis (35,36), and DEX is known to inhibit insulin secre- cortisol, glycemia, and insulin levels in man in the first tion in isolated islets (37,38). We found the same 1–2 days of exposure to 4,300 m hypoxia without any inhibitory effect of DEX on insulin secretion in cultured drugs (4,5). The sustained hyperglycemia with different islets (Fig. 5A and B) associated with upregulated Sgk1 insulin and cortisol levels in acute hypoxia may imply that and downregulated Glut2 and Crhr1 mRNA expression blood glucose is regulated by greater integration of sys- under ATP-deficient or hypoxic conditions (Fig. 5C). tems, including, for example, the autonomic nervous sys- These data suggest that CORT, activated by hypoxia, tem. In short, a greater increase of CRH in the AMS group may initiate a discrete inhibitory pathway from CRH may activate the sympathetic nervous system (42) and and correlate with the O2 and ATP supply. The current induce hyperglycemia (43). Furthermore, 5–10 days of in vivo study shows that acute hypoxia-induced high adaptation to hypoxia returns hyperglycemia to basal lev- CORT indeed contributes to the low plasma insulin and els in healthy people (5). Moreover chronic hypoxic expo- high plasma glucose mediated by GR (Fig. 6A) through sure reduces fasting insulin and improves insulin high SGK1 expression in islets (Fig. 6B and C). resistance in type 2 diabetic patients (44–46) and In humans, high-altitude hypoxia exposure for 2 days decreases the insulin dosageintype1diabeticpatients significantly reduced SpO2 (Fig. 7A) and increased plasma (47,48). Of note, lower insulin levels or reduced insulin CRH (Fig. 7D). To reduce the responsiveness to hypoxia, signaling is beneficial for health and longevity (49,50). It all volunteers took a Rhodiola capsule (extracted from is therefore tempting to speculate that fast travel to high R. rosea), a traditional Chinese herb to improve resistance altitude may benefit both healthy people and diabetic to AMS. Of note, R. rosea can reduce hypoxia-induced high patients in terms of glucose-insulin metabolic control. plasma CRH and CORT in rats (Supplementary Fig. 1). In conclusion, we propose a dynamic modulation of Consistent with the antistress effect in previous reports a CRH-insulinotropic role in pancreatic islets, which depends (39–41), R. rosea may thus contribute to low plasma cor- on CRH, glucose levels, and O2 availability. Under normoxia, tisol at high altitude (Fig. 7E). In the current study, cortisol the CRH-insulinotropic role increases with raised glucose lev- remained low, but high plasma CRH levels were still evoked els but is inactivated under hypoxia due to reductions in ATP, by hypoxia in human volunteers. Under these conditions, cAMP, and calcium influx into islets, even though plasma the raised plasma CRH level fails to stimulate insulin re- CRH and islet Crhr1 are upregulated. Additionally, hypoxia- lease during high-altitude hypoxia. These results greatly stimulated CORT inhibits insulin release through activated support the conclusion derived from animal experiments SGK1andinanO2- and ATP-dependent manner. Consistent that hypoxia increases CRH and inactivates its insulino- with these findings, rapid ascent to high altitude does not tropic role by inhibiting the CRHR1 signaling pathway. affect HOMA-IS in healthy volunteers but reduces HOMA-IS In the current study, acute hypoxia (5,000 m, 8 h) in AMS volunteers who usually exhibit high plasma CRH. induced low plasma insulin, high plasma glucose, and increased insulin sensitivity in rats (Fig. 1A–C). Other studies individually showed a gradual decrease in insulin Acknowledgments. The authors thank Iain C. Bruce (Department of level of rats during acute hypoxia for 1–4 days (6) or Physiology, School of Medicine, Zhejiang University) for editing the manuscript hyperglycemia in mice less than 1 day of hypoxia (7). and all the volunteers for participating in the study. Funding. This work was supported by grants from the Ministry of Science and We found that the CRH-induced high CORT mainly de- Technology of the People’s Republic of China, the National Basic Research Pro- creased insulin release and elevated blood glucose. High gram of China (973 Program no. 2012CB518200 and no. 2006CB504100), the CORT directly elevates blood glucose through decreased National Natural Science Foundation of China (no. 31171145 and no. 30871221), glycogen synthesis and glucose uptake in muscle (35). In and a Wellcome Trust Programme Grant (081195) to A.M.E. liver, the current study shows that a high anaerobic gly- Duality of Interest. No conflicts of interest relevant to this article were colysis rate and glycogen storage maintain stable liver reported. 794 Hypoxia Inactivates Insulinotropic Role of CRH Diabetes Volume 64, March 2015

Author Contributions. K.H. contributed to the experimental research, 19. Jeong KH, Sakihara S, Widmaier EP, Majzoub JA. Impaired expression data analysis, and writing and editing of the manuscript. F.-P.K. and J.-W.T. and abnormal response to fasting in corticotropin-releasing hormone-deficient contributed to the experimental research. Y.-Q.G. and J.C. contributed to the mice. Endocrinology 2004;145:3174–3181 collection of human plasma samples. A.M.E. contributed to the design of 20. Wang X, Meng FS, Liu ZY, et al. Gestational hypoxia induces sex-differential the nucleotide and the measurement of AMPK and the writing and editing of of Crhr1 linked to anxiety-like behavior. Mol Neurobiol 2013;48:544– the manuscript. S.L.L. contributed to the writing and editing of the manuscript. 555 X.-Q.C. and J.-Z.D. contributed to the study design, data analysis, and writing and 21. Xu JF, Chen XQ, Du JZ, Wang TY. CRF receptor type 1 mediates continual editing of the manuscript. X.-Q.C. and J.-Z.D. are the guarantors of this work hypoxia-induced CRF peptide and CRF mRNA expression increase in hypotha- and, as such, had full access to all the data in the study and take responsibility lamic PVN of rats. 2005;26:639–646 for the integrity of the data and the accuracy of the data analysis. 22. Sutton R, Peters M, McShane P, Gray DW, Morris PJ. Isolation of rat pancreatic islets by ductal of collagenase. Transplantation 1986;42: References 689–691 1. Basnyat B, Murdoch DR. High-altitude illness. Lancet 2003;361:1967– 23. Roach RC, Bärtsch P, Oelz O, Hackett PH; Lake Louise AMS Scoring Con- 1974 sensus Committee. The Lake Louise acute mountain sickness scoring system. In 2. Wu TY, Ding SQ, Liu JL, et al. Who should not go high: chronic disease and Hypoxia and Mountain Medicine. Sutton JR, Houston CS, Coates G, Eds. work at altitude during construction of the Qinghai-Tibet railroad. High Alt Med Burlington, VT, Charles S. Houston, 1993, p. 272–274 Biol 2007;8:88–107 24. Zhao Y, Ren JL, Wang MY, et al. Codon 104 variation of gene provides 3. Wu TY, Ding SQ, Zhang SL, et al. Altitude illness in Qinghai–Tibet railroad adaptive apoptotic responses to extreme environments in mammals of the Tibet passengers. High Alt Med Biol 2010;11:189–198 plateau. Proc Natl Acad Sci U S A 2013;110:20639–20644 4. Larsen JJ, Hansen JM, Olsen NV, Galbo H, Dela F. The effect of altitude 25. Quesada I, Nadal A, Soria B. Different effects of tolbutamide and hypoxia on glucose homeostasis in men. J Physiol 1997;504:241–249 in alpha, beta-, and delta-cells within intact islets of Langerhans. Diabetes 1999; 5. Barnholt KE, Hoffman AR, Rock PB, et al. Endocrine responses to acute and 48:2390–2397 chronic high-altitude exposure (4,300 meters): modulating effects of caloric re- 26. Chen XQ, Kong FP, Zhao Y, Du JZ. High-altitude hypoxia induces disorders striction. Am J Physiol Endocrinol Metab 2006;290:E1078–E1088 of the brain-endocrine-immune network through activation of corticotropin- 6. Simler N, Grosfeld A, Peinnequin A, Guerre-Millo M, Bigard AX. Leptin releasing factor and its type-1 receptors. Zhongguo Ying Yong Sheng Li Xue Za receptor-deficient obese Zucker rats reduce their food intake in response to Zhi 2012;28:481–487 hypobaric hypoxia. Am J Physiol Endocrinol Metab 2006;290:E591–E597 27. Carlin KM, Vale WW, Bale TL. Vital functions of corticotropin-releasing factor 7. Lee EJ, Alonso LC, Stefanovski D, et al. Time-dependent changes in glucose (CRF) pathways in maintenance and regulation of . Proc Natl – and insulin regulation during intermittent hypoxia and continuous hypoxia. Eur J Acad Sci U S A 2006;103:3462 3467 Appl Physiol 2013;113:467–478 28. Dionne KE, Colton CK, Yarmush ML. Effect of hypoxia on insulin secretion by – 8. Gamboa JL, Garcia-Cazarin ML, Andrade FH. Chronic hypoxia increases isolated rat and canine islets of Langerhans. Diabetes 1993;42:12 21 29. Hyder A, Laue C, Schrezenmeir J. Metabolic aspects of neonatal rat islet insulin-stimulated glucose uptake in mouse soleus muscle. Am J Physiol Regul hypoxia tolerance. Transpl Int 2010;23:80–89 Integr Comp Physiol 2011;300:R85–R91 30. Sekine N, Cirulli V, Regazzi R, et al. Low lactate dehydrogenase and high 9. Chintamaneni K, Bruder ED, Raff H. Effects of age on ACTH, corticosterone, mitochondrial phosphate dehydrogenase in pancreatic beta-cells. Po- glucose, insulin, and mRNA levels during intermittent hypoxia in the neonatal rat. tential role in nutrient sensing. J Biol Chem 1994;269:4895–4902 Am J Physiol Regul Integr Comp Physiol 2013;304:R782–R789 31. Doliba NM, Wehrli SL, Vatamaniuk MZ, et al. Metabolic and ionic coupling 10. Chen XQ, Dong J, Niu CY, Fan JM, Du JZ. Effects of hypoxia on glucose, factors in -stimulated insulin release in pancreatic beta-HC9 cells. Am insulin, , and modulation by corticotropin-releasing factor receptor type J Physiol Endocrinol Metab 2007;292:E1507–E1519 1 in the rat. Endocrinology 2007;148:3271–3278 32. Tanaka T, Nagashima K, Inagaki N, et al. Glucose-stimulated single pan- 11. Prentki M, Matschinsky FM, Madiraju SR. Metabolic signaling in fuel- creatic islets sustain increased cytosolic ATP levels during initial Ca2+ influx and induced insulin secretion. Cell Metab 2013;18:162–185 subsequent Ca2+ oscillations. J Biol Chem 2014;289:2205–2216 12. White MF. Insulin signaling in health and disease. Science 2003;302:1710– 33. Johnson RA, Shoshani I. Kinetics of “P”-site-mediated inhibition of adenylyl 1711 cyclase and the requirements for substrate. J Biol Chem 1990;265:11595– 13. Chandra R, Liddle RA. Neural and hormonal regulation of pancreatic se- 11600 – cretion. Curr Opin Gastroenterol 2009;25:441 446 34. Hardie DG. -acting through cyclic AMP as well as AMP? Cell 14. Bale TL, Vale WW. CRF and CRF receptors: role in stress responsivity and Metab 2013;17:313–314 – other behaviors. Annu Rev Pharmacol Toxicol 2004;44:525 557 35. Vegiopoulos A, Herzig S. Glucocorticoids, metabolism and metabolic dis- 15. Huising MO, van der Meulen T, Vaughan JM, et al. CRFR1 is expressed on eases. Mol Cell Endocrinol 2007;275:43–61 pancreatic beta cells, promotes proliferation, and potentiates insulin 36. Kainuma E, Watanabe M, Tomiyama-Miyaji C, et al. Association of gluco- secretion in a glucose-dependent manner. Proc Natl Acad Sci U S A 2010;107: corticoid with stress-induced modulation of body temperature, blood glucose and 912–917 innate immunity. Psychoneuroendocrinology 2009;34:1459–1468 16. Schmid J, Ludwig B, Schally AV, et al. Modulation of pancreatic islets-stress 37. Lambillotte C, Gilon P, Henquin JC. Direct glucocorticoid inhibition of insulin axis by hypothalamic releasing hormones and 11beta-hydroxysteroid de- secretion. An in vitro study of dexamethasone effects in mouse islets. J Clin hydrogenase. Proc Natl Acad Sci U S A 2011;108:13722–13727 Invest 1997;99:414–423 17. Kanno T, Suga S, Nakano K, Kamimura N, Wakui M. Corticotropin-releasing 38. Ullrich S, Berchtold S, Ranta F, et al. Serum- and glucocorticoid-inducible factor modulation of Ca2+ influx in rat pancreatic beta-cells. Diabetes 1999;48: kinase 1 (SGK1) mediates glucocorticoid-induced inhibition of insulin secretion. 1741–1746 Diabetes 2005;54:1090–1099 18. O’Carroll AM, Howell GM, Roberts EM, Lolait SJ. potentiates 39. Panossian A, Wikman G, Sarris J. Rosenroot (Rhodiola rosea): traditional corticotropin-releasing hormone-induced insulin release from mouse pancreatic use, chemical composition, pharmacology and clinical efficacy. Phytomedicine beta-cells. J Endocrinol 2008;197:231–239 2010;17:481–493 diabetes.diabetesjournals.org Hao and Associates 795

40. Panossian A, Hambardzumyan M, Hovhanissyan A, Wikman G. The adap- 45. Schobersberger W, Schmid P, Lechleitner M, et al. Austrian Moderate Al- togens rhodiola and schizandra modify the response to immobilization stress in titude Study 2000 (AMAS 2000). The effects of moderate altitude (1,700 m) on rabbits by suppressing the increase of phosphorylated stress-activated protein cardiovascular and metabolic variables in patients with . Eur kinase, and cortisol. Drug Target Insights 2007;2:39–54 J Appl Physiol 2003;88:506–514 41. Olsson EM, von Schéele B, Panossian AG. A randomised, double-blind, 46. de Mol P, Fokkert MJ, de Vries ST, et al. Metabolic effects of high al- -controlled, parallel-group study of the standardised extract shr-5 of the titude trekking in patients with . Diabetes Care 2012;35: roots of Rhodiola rosea in the treatment of subjects with stress-related fatigue. 2018–2020 Planta Med 2009;75:105–112 47. de Mol P, de Vries ST, de Koning EJ, Gans RO, Tack CJ, Bilo HJ. Increased 42. Nijsen MJ, Croiset G, Stam R, et al. The role of the CRH type 1 receptor in insulin requirements during at very high altitude in . autonomic responses to corticotropin- releasing hormone in the rat. Neuro- Diabetes Care 2011;34:591–595 psychopharmacology 2000;22:388–399 48. Moore K, Vizzard N, Coleman C, McMahon J, Hayes R, Thompson CJ. 43. Kalsbeek A, Bruinstroop E, Yi CX, Klieverik LP, La Fleur SE, Fliers E. Hy- Extreme altitude mountaineering and type 1 diabetes; the Diabetes Federation of pothalamic control of energy metabolism via the autonomic nervous system. Ann Ireland Kilimanjaro Expedition. Diabet Med 2001;18:749–755 N Y Acad Sci 2010;1212:114–129 49. Blüher M, Kahn BB, Kahn CR. Extended longevity in mice lacking the insulin 44. Schobersberger W, Leichtfried V, Mueck-Weymann M, Humpeler E. Austrian receptor in . Science 2003;299:572–574 Moderate Altitude Studies (AMAS): benefits of exposure to moderate altitudes 50. Taguchi A, Wartschow LM, White MF. Brain IRS2 signaling coordinates life (1,500-2,500 m). Sleep Breath 2010;14:201–207 span and nutrient homeostasis. Science 2007;317:369–372