Sex differences in adult rat insulin and glucose responses to arginine: programming effects of neonatal separation, hypoxia, and hypothermia

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1 ORIGINAL RESEARCH Physiologicl Reports ISSN X Sex differences in dult rt insulin nd glucose responses to rginine: progrmming effects of neontl seprtion, hypoxi, nd hypothermi Ashley L. Gehrnd 1, Brin Hoeynck 1, Mck Jlonski 1, Cole Leonovicz 1, Risheng Ye 3, Philipp E. Scherer 3 & Hershel Rff 1,2 1 Endocrine Reserch Lortory, Auror St. Luke s Medicl Center, Auror Reserch Institute, Milwukee, Wisconsin 2 Deprtments of Medicine, Surgery, nd Physiology, Medicl College of Wisconsin, Milwukee, Wisconsin 3 Touchstone Dietes Center, Deprtment of Internl Medicine, University of Texs Southwestern Medicl Center, Dlls, Texs Keywords Body temperture, ody weight, glucgon, isothermi, neworn, sexul dimorphism. Correspondence Hershel Rff, Endocrinology, Auror St. Luke s Medicl Center, 2801 W KK River Pky Suite 245, Milwukee, WI Tel: (414) Fx: (414) E-mil: hrff@mcw.edu Funding Informtion Supported in prt y the Auror Reserch Institute, Juvenile Dietes Reserch Foundtion (JDRF 2-SRA Q-R), Ntionl Institutes of Helth (NIH) grnts R01-DK55758, R01-DK nd P01- DK (to P.E.S.) nd Nomi Berrie Reserch Fellowship from Nomi Berrie Dietes Center, Columi University Medicl Center (to R.Y.); Received: 19 August 2016; Accepted: 22 August 2016 doi: /phy Physiol Rep, 4 (18), 2016, e12972, doi: /phy Astrct Acute neontl hypoxi, common stressor, cuses spontneous decrese in ody temperture which my e protective. There is consensus tht hypothermi should e prevented during cute hypoxi in the humn neonte; however, this my e n dditionl stress with negtive consequences. We hypothesize tht mintining ody temperture during hypoxi in the first week of postntl life lters the susequent insulin, glucose, nd glucgon secretion in dult rts. Rt pups were seprted from their dm dily from postntl dys (PD) 2 6 for the following 90 min experimentl tretments: (1) normoxic seprtion (control), (2) hypoxi (8% O 2 ) llowing spontneous hypothermi, (3) normoxic hypothermi with externl cold, nd (4) exposure to 8% O 2 while mintining ody temperture using externl het. An dditionl normoxic non-seprted control group ws performed to determine if seprtion per se chnged the dult phenotype. Plsm insulin, glucose, nd glucgon responses to rginine stimultion were evluted from PD105 to PD133. Mternl seprtion (compred to non-seprted neontes) hd more pronounced effects on the dult response to rginine compred to the hypoxic, hypothermic, nd hypoxic-isothermic neontl tretments. Adult mles exposed to neontl mternl seprtion hd ugmented insulin nd glucose responses to rginine compred to unseprted controls. Additionlly, neontl tretment hd significnt effect on ody weight gin; dults exposed to neontl mternl seprtion were significntly hevier. Femle dults hd significntly smller insulin nd glucose responses to rginine regrdless of neontl tretment. Neontl mternl seprtion during the first week of life significntly ltered dult et-cell function in sexully dimorphic mnner. Introduction Acute hypoxi is common neontl stressor in premture infnts (Mrtin et l. 1986; Frnkel nd Stevenson 1987; Miller nd Mrtin 1992; Low et l. 1993); it cn led to spontneous hypothermi nd ltered metolism (Frppell et l. 1992; Clrk nd Fewell 1996; Wood nd Gonzles 1996; Mortol 2004). The generl consensus is tht hypothermi should e prevented in the humn neonte nd tht core ody temperture should e mintined (Lptook nd Jckson 2006; Wtkinson 2006; Lptook nd Wtkinson 2008; Clifford nd Hunt 2010). However, we nd others hve proposed tht prevention of the spontneous hypothermic response to hypoxi in ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society. This is n open ccess rticle under the terms of the Cretive Commons Attriution License, which permits use, distriution nd reproduction in ny medium, provided the originl work is properly cited. Pge 1

2 Sex Differences in Adult Insulin After Neontl Stress A. L. Gehrnd et l. the neonte my e detrimentl (Clrk nd Fewell 1996; Wood nd Gonzles 1996; Shnkrn et l. 2005; Bruder et l. 2011; Guenther et l. 2012). We hve developed rt model of neontl hypoxi with nd without prevention of spontneous hypothermi s model of humn premturity (Bruder et l. 2008; Guenther et l. 2012). As such, we hve chrcterized the spontneous decrese in core ody temperture in response to cute hypoxi (Bruder et l. 2011) nd the effects of ody temperture mintennce during cute hypoxi in postntl dy (PD) 2 nd PD8 rt pups (Guenther et l. 2012). We hve shown tht 1-h of continuous hypoxi while mintining isothermi cuses hyperinsulinemi in PD2 pups despite no chnge in plsm glucose. Bsed on these previous findings, it seems possile tht pplying externl het during hypoxic episodes to prevent hypothermi my e eliciting dditionl stress on the neonte nd my possily result in disruption of glucose homeostsis. In order to study hypoxi in the neonte without ltering mternl physiology due to exposure to hypoxi (Bruder et l. 2008), the normoxic control group for our model seprtes the neontl rt pups from their lctting dm (Bruder et l. 2008; Guenther et l. 2012). Since premture humn infnts re usully seprted from their prents for significnt periods of time for medicl cre, mternl seprtion dds to the strength of this model of premturity. Therefore, in this study, we evluted the long-term effects of neontl seprtion (normoxic seprtion time control), cute hypoxi llowing spontneous hypothermi, induced cute hypothermi per se, nd cute hypoxi while preventing spontneous hypothermi (isothermi) on the susequent control of insulin, glucose, nd glucgon in the dult mle nd femle rt. In the process, we were lso interested in the effects of cute, dily seprtion of the neontes from the nursing dms (needed s normoxic control) compred to neontes who were not seprted from their dms. We hypothesized tht hypoxi while mintining ody temperture t control levels will result in ltered insulin, glucose, nd glucgon secretion s n effect of long-term metolic progrmming. In the process, we lso evluted the hypothesis tht seprtion of the neontes from their lctting dms lters the dult control of pncretic islet cell hormone secretion. Mterils nd Methods Animl tretments nd experimentl protocols Federl guidelines ( for use nd cre of lortory nimls were followed nd the protocols were pproved y the Institutionl Animl Cre nd Use Committee of Auror Helth Cre. Timed pregnnt Sprgue Dwley rts (N = 10) t gesttionl dy E18 were otined nd housed in stndrdized environment (lights on h) nd provided stndrd diet nd wter d liitum t Auror St. Luke s Medicl Center. Dms were llowed to deliver normlly nd cre for their pups without interruption until experimenttion. A totl of 110 pups were studied s descried elow. Neontl tretments Rt pups of oth sexes on postntl dy 2 (PD2) were rndomly ssigned to the following cute, dily experimentl tretments: normoxi unseprted (Norm-Unsep s control for normoxic seprtion per se), normoxi seprted (Norm-Sep s normoxic control for the following neontl tretments), hypoxi (Hypoxi), hypothermi (Hypotherm), nd hypoxi while mintining isothermi (Hypoxi-Isotherm). Norm-Sep ws estlished priori s the seprtion control group for hypoxi, hypothermi, nd hypoxi-isothermi tretments. All neontl tretments were performed once dily for 90 min (etween 0830 nd 0 h) from PD2 to PD6. No edding chnges occurred during the week of experimenttion. Bedding chnges occurred once per week fter PD6. Normoxi unseprted pups (N = 23) were left undistured from PD2 to PD6. Normoxi seprted pups (N = 22) were removed from the dm nd plced into chmer with edding nd vrile setting heting pd set on the lowest setting required to prevent hypothermi in pups seprted from their lctting dms in normoxic environment (Guenther et l. 2012). Hypoxi pups (N = 25) were removed from the dm nd plced into chmer with edding nd vrile setting heting pd set on low het. Hypoxi ws induced y decresing the chmer inflow O 2 concentrtion to 8% (lnce nitrogen) s descried in detil previously (Bruder et l. 2008, 2011; Guenther et l. 2012). This leds to trnscutneous O 2 sturtion of pproximtely 80% (Bruder et l. 2008). Body temperture ws llowed to spontneously decrese during hypoxi nd ws mesured in sentinel pup s descried previously (Guenther et l. 2012). Body temperture hd spontneously decresed to C (n = 10) fter 90 min of hypoxi. Pups were wrmed to norml ody temperture rnge of C using vrile setting heting pd set on low efore returning the pups to the nest. Hypothermi pups (N = 15) were removed from the dm nd plced in chmer with edding on top of cold plte (Model #AHP-1200CPV; TECALAB, Chicgo, IL) set etween 24 Pge 2 ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society.

3 A. L. Gehrnd et l. Sex Differences in Adult Insulin After Neontl Stress nd 27 C, which could e djusted depending on ody temperture. Body temperture ws mesured in sentinel pup using RET-30-Iso rectl proes nd BAT-12 digitl thermometer connected to SBT-5 switchox (Physitemp Instruments, Clifton, NJ), nd ws decresed to 25 C over 30 min nd held t 25 C for the durtion of the cute exposure y djusting the temperture of the cold plte. After hypothermi ws completed, ody temperture ws llowed to increse to norml rnge (32 34 C) using vrile setting heting pd set on low efore returning the pups to the nest. Hypoxi-isothermi pups (N = 25) were treted identiclly s the hypoxi pups except tht ody temperture ws mintined t 32 C with het plte (Model #AHP-1200CPV; TECA- LAB) s descried previously (Guenther et l. 2012). Sentinel pups used for monitoring ody temperture were not used in susequent experiments. All rt pups were weighed on PD6 fter experimenttion efore eing returned to the nest. On PD22, ll nimls were wened nd housed y sex nd tretment group with two to three nimls per cge. Wened nimls were given stndrd diet nd wter d liitium nd only hndled during weekly edding chnges. Food nd wter intke ws not monitored. Animls were weighed every 2 weeks t PD22, PD37, PD51, PD65, PD79, nd PD93. Adult experiments: intrperitonel rginine stimultion tests Intrperitonel (ip) rginine stimultion tests were performed on dult rts ge PD105 to PD133 s previously descried (Ye et l. 2015). Arginine stimultion ws chosen rther thn glucose tolernce tests ecuse our previous dt suggested tht glucose tolernce tests re not suitly sutle (Gehrnd et l. 2015). Additionlly, we were interested in mesuring glucgon response to rginine stimultion (Ye et l. 2015). Rts were fsted for 16 h overnight efore ip rginine testing. Blood (pproximtely 200 ll) ws collected vi til clip to estlish seline smple (0 min) s descried previously (Wner nd Nysk 1994). L-rginine monohydrochloride (Sigm A4599, St. Louis, MO) ws diluted in isotonic sline nd dministered ip t 1 mg/kg fter the seline collection. Then, lood ws collected vi til clip 5, 15, nd 30 min post injection. Blood ws processed for plsm nd stored t 20 C until susequent nlysis. Insulin, glucose, nd glucgon ssys Plsm insulin (Crystl Chem, Downers Grove, IL) ws mesured y ELISA nd plsm glucose ws mesured spectrophotometriclly using the glucose-oxidse method (Pointe Scientific, Cnton, MI) s descried previously (Guenther et l. 2012). The plsm glucose ssy does not detect rginine t concentrtions up to 250 mg/ml. Plsm glucgon (Mercodi ctlog # ; Winston Slem, NC) ws mesured y ELISA. The intr-ssy vriility is %, the inter-ssy vriility is %, nd the sensitivity is 5.2 pg/ml. There is cross rectivity with rt glicentin (4.0%), nd humn/rt/mouse oxyntomodulin (2.0%). Body weight, plsm glucose, plsm insulin, nd plsm glucgon were nlyzed y two-wy ANOVA for repeted mesures (SigmPlot 12.5; Systt Softwre, Inc., Sn Jose, CA) nd post hoc multiple comprisons y Holm Sidk. Are under the curve (AUC) for plsm insulin, glucose, nd glucgon ws clculted for ech rt individully using trpezoidl rule (SigmPlot 12.5) prior to sttisticl nlysis. AUCs were nlyzed y two-wy ANOVA, t-test, nd post hoc multiple comprisons y Holm Sidk. All dt re expressed s men SE nd were normlly distriuted. Pncret immunohistochemistry Immeditely fter rginine stimultion testing, suset of pncret were removed nd fixed overnight t room temperture in 10% formlin. The pncret were wshed three times in 75% ethnol, nd stored in 75% ethnol t 4 C until nlysis. The pncret were then processed to prffin sections nd sujected to immunofluorescence of insulin nd glucgon s previously descried (Ye et l. 2014). The immunofluorescence signls from the whole sections were scnned into imges on Hmmtsu (Hmmtsu Photonics K.K., Bridgewter, NJ) Nnozoomer Digitl Slide Scnner (209 mode) y the University of Texs Southwestern Medicl Center Whole Brin Microscopy Fcility. On the imges from ech individul rt, the res of insulin nd glucgon signls were quntitted, respectively, with ImgeJ (Ntionl Institutes of Helth, Bethesd, MD), nd normlized ginst the totl pncres re. Two-tiled student s t-test ws pplied for pirwise comprisons. Results Effect of neontl tretments on ody weight All nimls were weighed t PD6 (t the conclusion of ll neontl experimentl tretments), t wening, nd then every 2 weeks until PD93 (Fig. 1). All nimls gined weight in similr pttern from PD6 to PD93. In mles, there ws no significnt difference in ody weight etween tretments until PD37. In femles, significnt differences in ody weight were oserved strting t PD21. In oth genders, the Norm-Unsep group hd the lowest ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society. Pge 3

4 Sex Differences in Adult Insulin After Neontl Stress A. L. Gehrnd et l Mles Normoxi Unseprted (N = 12) Normoxi Seprted (N = 13) Hypoxi (N = 14) Hypothermi (N = 8) Hypoxi Isothermi (N = 14) Femles Normoxi Unseprted (N = 11) Normoxi Seprted (N = 9) Hypoxi (N = 11) Hypothermi (N = 7) Hypoxi Isothermi (N = 11) Body Weight (g) Age (dys) Age (dys) Tretment Comprison PD6 PD21 PD37 PD51 PD65 PD79 PD93 Normoxi Seprted vs. Normoxi Unseprted <0.001 <0.001 <0.001 <0.001 <0.001 Hypoxi Isothermi vs. Normoxi Unseprted <0.001 <0.001 <0.001 <0.001 Hypothermi vs. Normoxi Unseprted <0.001 <0.001 < Hypoxi vs. Normoxi Unseprted <0.001 <0.001 <0.001 <0.001 Normoxi Seprted vs. Hypoxi <0.001 <0.001 Hypoxi Isothermi vs. Normoxi Seprted < Normoxi Seprted vs. Hypothermi <0.001 Hypoxi Isothermi vs. Hypoxi Hypoxi Isothermi vs. Hypothermi Hypothermi vs. Hypoxi Tretment Comprison PD6 PD21 PD37 PD51 PD65 PD79 PD93 Hypothermi vs. Normoxi Unseprted <0.001 <0.001 <0.001 <0.001 <0.001 Hypoxi Isothermi vs. Normoxi Unseprted <0.001 <0.001 <0.001 <0.001 Normoxi Seprted vs. Normoxi Unseprted <0.001 <0.001 <0.001 <0.001 Hypothermi vs. Hypoxi <0.001 <0.001 < Normoxi Seprted vs. Hypoxi Hypoxi Isothermi vs. Hypoxi Hypoxi vs. Normoxi Unseprted Hypoxi Isothermi vs. Hypothermi Normoxi Seprted vs. Hypothermi Hypoxi Isothermi vs. Normoxi Seprted Figure 1. (A) Mle nd femle ody weights over time from PD6 to PD93. Rts were weighed t the completion of ech tretment (PD6) nd every 2 weeks post-wening until PD93. (B) Significnt differences (P vlues) etween neontl tretments within gender re listed in the corresponding tles. Dt re presented s men SE. N vlues re shown in the figure lels. ody weight over time. Norm-Sep mles consistently hd the highest ody weight over time, nd y PD93, the Norm-Sep mle ody weight ( g, n = 12) ws significntly different from every other tretment (Norm- Unsep = g, n = 13; Hypoxi = g, n = 14; Hypotherm = g, n = 8; nd Hypoxi-Isotherm = g, n = 14). In femles, the Hypotherm group mintined the highest ody weight over time (PD21 to PD79). At PD93, Norm-Unsep femles weighed significntly less thn Norm-Sep, Hypotherm, nd Hypoxi-Isotherm neontl tretments ( g, n = 11; g, n = 9; g, n = 6.6; nd g, n = 11, respectively). Arginine stimultion testing in dults fter different neontl tretments Plsm insulin, glucose, nd glucgon levels during rginine stimultion testing re shown in Figures 2 4. Note tht the y-xis rnge is much smller for femle plsm insulin thn mle plsm insulin in Figures 2 nd 3. First, Norm-Sep nd Norm-Unsep mles nd femles were compred (Fig. 2 nd Tle 1). Within gender, femle Norm-Unsep plsm insulin ( ng/ml, n = 11) nd glucgon ( pg/ml, n = 11) were significntly lower thn Norm-Unsep mles ( ng/ml nd pg/ml, n = 12, respectively), while no differences were oserved in Norm-Sep tretment within gender (Tle 1). Additionlly, within mles, Norm-Sep plsm glucgon ws significntly lower thn Norm-Unsep ( pg/ml, n = 13, nd pg/ml, n = 12, respectively). Mle Norm-Sep plsm insulin levels t 5 nd 15 min fter rginine injection ( nd ng/ml, n = 13) were significntly higher thn Norm-Unsep mles ( nd ng/ml, n = 11, respectively). Femle Norm-Sep plsm insulin levels were lso significntly higher thn Norm-Unsep femles t 5 min fter rginine stimultion ( ng/ml, n = 9, nd ng/ml, n = 11, respectively). The mgnitude of the insulin response to rginine stimultion ws much less in femles thn mles. Mle Norm-Sep plsm glucose levels were significntly different thn Norm-Unsep t 15 min post rginine stimultion ( mg/dl, n = 13 nd mg/ dl, n = 12, respectively). No significnt chnges were oserved in femle plsm glucose levels over time in response to rginine stimultion. Plsm glucgon levels incresed in oth genders nd oth tretments (Norm- Pge 4 ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society.

5 A. L. Gehrnd et l. Sex Differences in Adult Insulin After Neontl Stress Plsm Insulin (ng/ml) Mles,, Normoxi Unseprted (N = 11) Normoxi Seprted (N = 13) Femles, Normoxi Unseprted (N = 11) Normoxi Seprted (N = 9) Plsm Glucose (mg/dl) , Normoxi Unseprted (N = 10-11) Normoxi Seprted (N = 12-13) Normoxi Unseprted (N = 9-11) Normoxi Seprted (N = 8-9) Plsm Glucgon (ng/ml) Normoxi Unseprted (N = 11) Normoxi Seprted (N = 13) , Normoxi Unseprted (N = 11) Normoxi Seprted (N = 9) Time (min) Time (min) Figure 2. Intrperitonel rginine stimultion test in dult rts previously exposed to neontl normoxi seprted nd normoxi unseprted tretments. Plsm glucose, insulin, nd glucgon were mesured t pre-rginine injection (0 min) nd t 5, 15, nd 30 min fter rginine injection (1 mg/kg ip). Significntly different from seline (0 min) within ech neontl tretment group. Significntly different from normoxi unseprted t sme time point. Dt re presented s men SE. N vlues re shown in the figure lels. Unsep nd Norm-Sep) fter rginine injection. There ws no significnt difference in plsm glucgon levels in Norm-Unsep nd Norm-Sep mles in response to rginine stimultion. In femles, Norm-Unsep rts hd pek plsm glucgon level significntly higher thn Norm-Sep femles t 5 min post rginine stimultion ( pg/ml, n = 11 nd pg/ml, n = 9, respectively). Figure 3 shows the plsm insulin, glucose, nd glucgon levels in Norm-Sep, Hypoxi, Hypotherm, nd Hypoxi-Isotherm mles nd femles in response to rginine stimultion. Plsm insulin ws significntly ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society. Pge 5

6 Sex Differences in Adult Insulin After Neontl Stress A. L. Gehrnd et l. Plsm Insulin (ng/ml) Mles,, Normoxi Seprted (N = 13) Hypoxi (N = 14) Hypothermi (N = 8) Hypoxi Isothermi (N = 14) Femles Normoxi Seprted (N = 9) Hypoxi (N = 11) Hypothermi (N = 7) Hypoxi Isothermi (N = 11) Plsm Glucose (mg/dl) , Normoxi Seprted (N = 13) Hypoxi (N = 11-14) Hypothermi (N = 8) Hypoxi Isothermi (N = 11-14) , Normoxi Seprted (N = 9) Hypoxi (N = 7-10) Hypothermi (N = 7) Hypoxi Isothermi (N = 8-10) Plsm Glucgon (ng/ml) , Normoxi Seprted (N = 13) Hypoxi (N = 14) Hypothermi (N = 8) Hypoxi Isothermi (N = 14) , Normoxi Seprted (N = 9) Hypoxi (N = 11) Hypothermi (N = 7) Hypoxi Isothermi (N = 11) Time (min) Time (min) Figure 3. Intrperitonel rginine stimultion test in dult rts previously exposed to neontl normoxi seprted, hypoxi, hypothermi, nd hypoxi isothermi mles nd femles. Plsm glucose, insulin, nd glucgon were mesured t pre-rginine injection (0 min) nd t 5, 15, nd 30 min fter rginine injection (1 mg/kg ip). Significntly different from seline (0 min) within ech neontl tretment group. Significntly different from normoxi seprted (control). Dt re presented s men SE. N vlues re shown in the figure lels. incresed in the Norm-Sep mles compred to the other tretment groups (Hypoxi, Hypotherm, nd Hypoxi- Isotherm) t 5 nd 15 min post rginine stimultion. Otherwise, there were no differences etween groups in mles. There were no significnt etween-group differences oserved in femle plsm insulin levels. Agin, note tht the mgnitude of the plsm insulin response to rginine ws much lower in the femles compred to the mles. Hypotherm nd Hypoxi-Isotherm mles hd significntly higher plsm glucose thn norm-sep mles t 5 min post rginine stimultion ( mg/dl, n = 8 mg/dl, mg/dl, n = 12, nd mg/dl, n = 13, respectively). The mgnitude of the plsm glucose response ws less in femles. At 5 min Pge 6 ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society.

7 A. L. Gehrnd et l. Sex Differences in Adult Insulin After Neontl Stress Mles Femles Mles Femles Insulin AUC (ng/ml/30min) d 0 5K Glucose AUC (mg/dl/30min) 4K 3K 2K 1K,,c, 0K 10K Glucgon AUC (ng/ml/30min) 8K 6K 4K 2K 0K Figure 4. Integrtion of individul niml plsm insulin, glucose, nd glucgon shown s res under the curve (AUCs) during intrperitonel rginine stimultion test in normoxi seprted, normoxi unseprted, hypoxi, hypothermi, nd hypoxi isothermi dult mles nd femles. Mles: normoxi unseprted (N = 12), normoxi seprted (N = 13), hypoxi (N = 11), hypothermi (N = 8), hypoxi isothermi (N = 11). Femles: normoxi unseprted (N = 11), normoxi seprted (N = 9), hypoxi (N = 7), hypothermi (N = 7), hypoxi isothermi (N = 8). Significntly different from unseprted (left pnel) nd seprted (right pnel) within sex. Significntly different from mle within tretment. c Significntly different from hypoxi within mles. d Significntly different within tretment y t-test. Dt re presented s men SE. post rginine stimultion, plsm glucose in Norm-Sep femles ( mg/dl, n = 9) ws significntly lower thn ll other tretment groups (Hypotherm = mg/dl, n = 7; Hypoxi = mg/ dl, n = 9; Hypoxi-Isotherm = mg/dl, n = 10). Only Hypoxi plsm glucose levels t 5 min post rginine ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society. Pge 7

8 Sex Differences in Adult Insulin After Neontl Stress A. L. Gehrnd et l. Tle 1. Bsl plsm insulin, glucose, nd glucgon in dult rts. Insulin (ng/ml) Glucose (mg/dl) Glucgon (pg/ml) Neontl tretment Mle Femle Mle Femle Mle Femle Normoxi unseprted Normoxi seprted Hypoxi Hypothermi Hypoxi isothermi Dt re men SEM. For mles: normoxi unseprted (N = 12), normoxi seprted (N = 13), hypoxi (N = 14), hypothermi (N = 8), hypoxi isothermi (N = 14). For femles: normoxi unseprted (N = 11), normoxi seprted (N = 9), hypoxi (N = 11), hypothermi (N = 7), hypoxi isothermi (N = 11). Dt re presented s men SE. 1 Femle different from mle within ech row. 2 Different from normoxi unseprted within ech column. stimultion were significntly different from seline in femles ( mg/dl, n = 9 nd mg/dl, n=10, respectively). Plsm glucgon levels were incresed in response to rginine stimultion for ll neontl tretments nd genders fter rginine stimultion. In mles, Norm-Sep plsm glucgon levels were significntly higher thn Hypoxi-Isotherm t 5 min post rginine stimultion ( pg/ml, n = 13 nd pg/ml, n = 14, respectively). In femles, Hypoxi plsm glucgon levels were significntly higher thn Norm-Sep nd Hypotherm t 5 min post rginine stimultion ( pg/ml, n = 11; pg/ml, n = 9; pg/ml, n = 7, respectively). In order to summrize the dt in Figures 2 nd 3, AUC nlyses for the response to rginine were clculted for plsm insulin, glucose, nd glucgon (Fig. 4). In most neontl tretment groups, dult mles hd higher AUCs for insulin, glucose, nd glucgon compred to dult femles. By post hoc nlysis fter ANOVA, there ws no significnt difference etween mle nd femle plsm insulin AUC within Norm-Unsep; however, the femle insulin AUC ws significntly lower y t-test (P < 0.001). Plsm insulin AUC for femle Norm-Sep ws significntly lower thn mle Norm-Sep. Within mles, Norm-Sep insulin AUC ws significntly higher thn Norm-Unsep. In mles, Hypoxi, Hypotherm, nd Hypoxi-Isotherm insulin AUCs were significntly lower thn the control (Norm-Sep). Plsm insulin AUCs of ll groups (Norm-Sep, Hypoxi, Hypotherm, nd Hypoxi- Isotherm) were significntly different etween genders. There ws no significnt difference in plsm insulin AUC etween tretments within femles. Mle Norm-Sep plsm glucose AUC ws significntly higher thn Norm-Unsep. Femle plsm glucose AUC ws significntly lower thn mle plsm glucose AUC in Norm-Unsep nd Norm-Sep tretments. Plsm glucose AUC in Hypotherm nd Hypoxi-Isotherm tretments were significntly higher thn control (Norm-Sep) in oth genders. Additionlly, Hypoxi-Isotherm plsm glucose AUC ws significntly higher thn Hypoxi in mles. Femle plsm glucose AUC ws significntly lower thn mles within ech tretment (Norm-Sep, Hypoxi, Hypotherm, nd Hypoxi-Isotherm). Femle plsm glucgon AUC ws significntly lower thn mle plsm glucgon AUC within Norm-Sep nd Hypotherm. No other significnt differences were oserved in plsm glucgon AUCs. Rndomly selected pncret from dult mle Norm- Unsep, Norm-Sep, nd Hypoxi rts were nlyzed for content of glucgon nd insulin in the islets of Lngerhns (Fig. 5). Ech row designtes three imges from one rt pncres. Overll, we did not oserve significnt differences in the glucgon (red) nd insulin (green) stining etween Norm-Unsep, Norm-Sep, nd Hypoxi mles. Totl immunofluorescent quntifiction of glucgon nd insulin nlysis ws performed on ech of these individul slides. No significnt differences in quntifiction of insulin or glucgon were oserved (numericl dt not shown). Discussion We evluted the effects of neontl-mternl seprtion, neontl hypoxi llowing hypothermi, neontl hypothermi per se, nd neontl hypoxi while mintining ody temperture (isothermi) on susequent insulin, glucose, nd glucgon responses to rginine in the dult mle nd femle rt. Our mjor findings were: (1) Adult mles seprted from their dms dily during the first week of neontl life hd ugmented insulin nd glucose responses to rginine compred to dult mles tht were not seprted s neontes. Additionlly, dult mles exposed to neontl Pge 8 ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society.

9 A. L. Gehrnd et l. Sex Differences in Adult Insulin After Neontl Stress 1 Normoxi seprted 2 Normoxi unseprted Hypoxi 6 Figure 5. Immunofluorescence of insulin nd glucgon in dult mle pncretic islets from the neontl normoxi seprted, normoxi unseprted, nd hypoxi exposures. Pncret were sectioned nd stined for insulin (green), glucgon (red), nd DAPI control (lue). Three individul islets from one pncres per rt [1 6] re shown. hypothermi nd hypoxi while mintining isothermi hd significntly higher levels of plsm glucose t 5 min post rginine stimultion compred to dult mles tht were seprted s neontes. (2) At 5 nd 15 min fter rginine stimultion, plsm insulin levels in dult mles tht hd een seprted from their dms s neontes ws significntly higher thn ll other neontl tretments (hypothermi, hypoxi, nd hypoxi while mintining isothermi). (3) The mgnitude of insulin nd glucose response to rginine ws significntly less in dult femles compred to dult mles regrdless of neontl tretment. (4) Overll, dult mles typiclly hd higher levels of insulin, glucose, nd glucgon compred to dult femles. (5) Normoxic, unseprted neontes of either sex mintining the lowest susequent ody weight gin over time. (6) Neontl hypoxi while mintining isothermi resulted in significntly higher plsm glucose levels in response to rginine stimultion in dult mles compred to dult mles exposed to neontl hypoxi; there were no pprecile differences in plsm insulin or glucgon. ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society. Pge 9

10 Sex Differences in Adult Insulin After Neontl Stress A. L. Gehrnd et l. Adult mle insulin, glucose, nd glucgon fter neontl seprtion We found tht the normoxic controls (pups seprted from their dms dily) demonstrted ugmented insulin nd glucose, ut not glucgon, responses to rginine in dults compred to dults not exposed to neontl seprtion. Furthermore, sl (fsting) plsm glucose tended to e higher. Postntl mternl seprtion hs een linked to ltered metolic sttes lter in life including decresed insulin secretion (Cook 1999; Mel et l. 2012), incresed insulin secretion (Hofmn et l. 1997), ltered glucose homeostsis (McPherson et l. 2009), incresed levels of glucocorticoids with susequent decresed levels of growth hormone (Fish et l. 2004), nd long-lsting decresed levels of plsm leptin (Mrco et l. 2015). It hs een suggested y Mel et l. (2012) tht there is n ltertion in centrl leptin sensitivity s result of n ltered metolic stte in dult rts tht hd een exposed to mternl seprtion, since plsm leptin levels remined low throughout dulthood with no pprecile differences oserved in hypothlmic mrna levels for the leptin receptor (Mel et l. 2012). Additionlly, Mel et l. found decreses in insulin levels in sex-dependent mnner; however, no chnges in insulin sensitivity were oserved. In this study, the ugmented insulin nd glucose responses to rginine in the dult mles exposed to neontl mternl seprtion suggests tht (1) the insulin-secreting et cells of the pncretic islets re hyperresponsive despite no pprent chnge in islet insulin content nd (2) tht these dults my e more insulin resistnt thn the neontlly unseprted controls. The ugmenttion of the insulin response to rginine is possily due to n increse in responsivity of the et cells, nd not due to n increse in the numer of et cells present in the pncres, ecuse we did not oserve ny differences in insulin content nd et cell numer in the islets. It is lso possile tht there is ugmented content of stored prepro- or proinsulin in the et cells of the normoxic, seprted mles. Of most interest in this study were the significnt differences oserved etween the non-seprted nd 90 min mternlly seprted groups. Mternl seprtion is well-known neontl stress model developed to study cute nd long-term physiologicl nd ehviorl effects. However, mternl seprtion protocols cn vry nywhere from 15 min to 5 h/dy nd spn nywhere from irth to wening t PD21 (McIntosh et l. 1999; Pryce et l. 2001; Klinichev et l. 2002; Rees et l. 2006; Hley et l. 2013). Some protocols move the dm to seprte room to minimize ultrsonic communiction from dm to pup (Foscolo et l. 2008), some seprte the pups individully from the dm (Pryce et l. 2001), nd some simply perform mternl seprtion for single 24-h period (Viveros et l. 2009; Mel et l. 2012; Clrke et l. 2013). Although rief periods of mternl seprtion of the dm from the pups in their nest is norml in the wild for rt species (Clhoun 1963), there does not seem to e generl consensus for how mternl seprtion should e performed in experimentl nimls. It is importnt to e wre tht the mternl seprtion protocols ring into ply other fctors side from just seprtion stress. Seprting the pup from the dm lso includes ltered mternl cre upon dm-pup reunion (Llorente et l. 2011), lck of proper nutrition (Viveros et l. 2009), nd decrese in ody temperture due to lck of huddling with the dm. These different protocols should e thought of s comintion of ll of these fctors (Mrco et l. 2015). We chose dily 90 min seprtion during PD2-6 s control for rief hypoxic exposures to model the hypoxi of premturity due to lung disese (Cummings et l. 1989) nd ecuse this is within the prmeters of norml rt ehvior (Clhoun 1963). Hypoxi with spontneous hypothermi, hypothermi, nd isothermic hypoxi The originl motivtion for this study ws to investigte the long-term effects of mintining isothermi during cute hypoxi during the first week of neontl life. We oserved significntly higher plsm glucose levels in response to rginine stimultion in dult mles exposed to neontl isothermic hypoxi compred to dult mles exposed to neontl hypoxi without significnt differences in plsm insulin or glucgon. Additionlly, plsm glucose AUCs for dult femles exposed to neontl hypothermi nd hypoxic isothermi were significntly higher thn the normoxic seprted control. We oserved significnt differences in plsm insulin, glucose, nd glucgon in response to rginine stimultion etween genders; however, there were no drmtic differences compring hypoxi with spontneous hypothermi, hypothermi, nd hypoxi while mintining control ody temperture. We were, therefore, surprised tht mintennce of ody temperture during neontl hypoxi did not hve mrked effects on susequent dult insulin, glucose, nd glucgon dynmics. Although there re not lrge numer of studies in dults exposed to postntl hypoxi in rts, there re numerous studies linking fetl hypoxi exposure to n incresed risk of insulin resistnce, chnges in glucose metolism, nd dietes development in the dult (Coughln et l. 2004; Cmm et l. 2011; Rued-Clusen et l. 2011; Co et l. 2012). We hve previously shown tht neontl cute hypoxi exposure with the mintennce of ody temperture, increses plsm insulin nd Pge 10 ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society.

11 A. L. Gehrnd et l. Sex Differences in Adult Insulin After Neontl Stress significntly lters the control of glucose in PD2 nd PD8 rts (Guenther et l. 2012). Additionlly, we hve previously shown tht neontl intermittent hypoxi exposure cutely elicits hyperglycemi nd hyperinsulinemi from PD2 to PD14 (Chintmneni et l. 2013) nd evokes chnges in hypothlmic pituitry drenl xis function nd n upregultion of inflmmtory-relted genes in the dult due to long-lsting progrmming effects (Chintmneni et l. 2014; Gehrnd et l. 2015). Even though hypoxi leds to spontneous hypothermi which is thought to e protective mechnism ginst severe rin dmge nd metolic stress in the neonte (Frppell et l. 1992; Clrk nd Fewell 1996; Steiner nd Brnco 2002; Ry et l. 2003), mintennce of isothermi in premture hypoxic humn neontes is encourged (Lptook nd Jckson 2006; Wtkinson 2006; Clifford nd Hunt 2010). However, the long-term effects of mintining isothermi during cute ut continuous hypoxic exposure re not fully studied. Our studies suggest tht these effects re not prticulrly drmtic suggesting tht cute neontl intermittent hypoxi hs significntly greter progrmming effects thn cute continuous neontl hypoxi (Chintmneni et l. 2014). Previously, we performed intrperitonel glucose tolernce tests in mle dult rts previously exposed to neontl intermittent hypoxi nd oserved no difference in insulin, glucose, or C-peptide from normoxic controls (Gehrnd et l. 2015). Therefore, in this study, we used rginine stimultion testing s more sensitive test to determine sutle differences in insulin dynmics (Ye et l. 2015) s rginine directly stimultes insulin relese from et cells in the pncretic islets (Cherrington nd Vrnic 1973; Fltt nd Biley 1981; Lrsson nd Ahren 1998; Thms nd Cpito 1999). Another dvntge of using rginine is its stimultion of glucgon secretion from the lph cell of the islet (Cherrington et l. 1974; Gerich et l. 1974; Plmer et l. 1976; Ye et l. 2015). Mles versus femles We oserved lrge (threefold) ugmenttion of the insulin response to rginine compred in dult mles compred to femles. It hs previously een reported tht neontl mternl seprtion during the first 2 weeks of life typiclly cuses hyperinsulinemi in the neontl mles, ut not femles (Viveros et l. 2009). Additionlly, plsm glucose levels were not significntly different etween sexes (Vitl et l. 2006; Viveros et l. 2009). Hley et l. (2013) did not find significnt differences in plsm glucose levels etween sexes or tretments of nonseprted control, pups seprted for 60 min/dy, or pups seprted for 60 min/dy from PD5 to PD9; however, plsm insulin levels in dult mles tht were mternlly seprted for 60 min/dy were significntly higher thn their dult non-seprted counterprts. These differences were not oserved in dult femles. Interestingly, in nother tretment group in Hley et l. (2013), mechnicl nd tcticl stimultion of the pups provided during seprtion from the dm ws le to prevent hyperinsulinemi in dult mles. These findings suggest tht consistent mternl cre is necessry for norml insulin response nd norml sensitivity to insulin. The lrge increse in glucose in response to rginine in mles compred to femles ws striking. Furthermore, the dult mles in the neontl seprtion group showed significntly higher plsm glucose responses compred to the unseprted neontes. The mechnism of the glucose response to rginine ppers to e n erly heptic response to incresed counterregultory hormones like glucgon (Cherrington et l. 1974). The glucose remined elevted with the sme increse in glucgon ut gret insulin response is further evidence of potentil for insulin resistnce in mle dult rts who were seprted dily from their dms during the first week of neontl life. Others hve demonstrted significntly lower insulin responses in dult femle compred to mle rts (Vitl et l. 2006). Previous studies hve shown tht the mmmlin hypothlmus is sexully dimorphic with mle hypothlmic neurons developing erlier thn femles (Crrer nd Cmisso 2002; Skum 2009). It hs een suggested tht differentil chnges in hypothlmic development cn e progrmmed through environmentl chnges, such s mternl seprtion, in sex-dependent mnner, nd thus induce differences in hypothlmic cell prolifertion nd mturtion tht ffect downstrem endocrine outcomes (Viveros et l. 2009). A possile meditor of this mechnism nd importnt fctor in norml development of specific hypothlmic circuits is leptin (Pinto et l. 2004), nd it hs een suggested tht ltered levels of leptin during importnt times of erly development cn result in n ltered metolic stte lter in life (McMillen et l. 2006). Viveros et l. (2009) demonstrted tht mternl seprtion performed during single 24-h period t PD9, led to leptin levels tht were nerly undetectle fter 12 h of seprtion, nd concluded tht femles were more sensitive to the effects of on the hypothlmus. Tht my e mechnism involved in the drmtic mle-femle differences we oserved in our study. Body weight gin The fetl insulin hypothesis ims to provide link etween low irth weight from mlnutrition nd et cell dysfunction nd type 2 dietes mellitus (Httersley nd Tooke 1999). It ws hypothesized tht n epigenetic ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society. Pge 11

12 Sex Differences in Adult Insulin After Neontl Stress A. L. Gehrnd et l. difference in et cell dysfunction progrmmed from mlnutrition cn led to decrese in insulin sensitivity nd ltered whole-ody glucose metolism in dulthood (Milner nd Hill 1984). Low irth weight with susequent rpid weight gin through dolescence in humns hs een linked with poor glucose tolernce nd insulin resistnce (Crowther et l. 1998; Yjnik 2000) nd this insulin resistnce is thought to e progrmmed through undernutrition (Hles et l. 1991; Phillips 1998). Birth weight ws not mnipulted in our study; however, s expected, difference in mternl cre, including rched-ck nursing nd excessive licking, ws oserved when the pups were returned to the dm fter seprtion (Frncis nd Meney 1999). This could cuse metolic chllenge y introducing different feeding ptterns, s we oserve significnt differences in ody weight etween tretments. All nimls, regrdless of neontl tretment, gined weight in similr pttern from PD6 to PD93. The neontl normoxic unseprted tretment typiclly mintined the lowest susequent ody weight over time in mles nd femles. The mles who were seprted from their dms s neontes mintined the gretest susequent ody weight gin from PD63 nd older, while the femles with neontl hypothermic exposure mintined the gretest susequent ody weight gin from PD21 to PD79. Some studies hve shown tht neontl-mternl seprtion does not lter susequent ody weight gin compred to non-seprted controls (Cook 1999; Fish et l. 2004; McPherson et l. 2009; Hley et l. 2013). On the other hnd, it hs een suggested tht mternl seprted neontes re hevier thn their control counterprts s dults (Slotten et l. 2006). Mny hve shown tht mternl seprtion resulted in reduction in ody weight up to wening t PD21 (Ogw et l. 1994; McIntosh et l. 1999; Pryce et l. 2001; Klinichev et l. 2002), nd t PD45 (Foscolo et l. 2008). In our study, dults exposed to neontl seprtion were significntly hevier compred to dults tht were not seprted, so it is possile tht some degree of metolic progrmming occurred during the first week of life resulting in incresed weight gin. Since food nd wter intke ws not mesured in our study, we cnnot comment on whether the differences in ody weights were due to chnge in ppetite or chnges in metolic progrmming from ltertions in mternl cre. In Clrke et l. (2013), pups in lrge litters (20 pups) showed reduced growth rte tht continued through dulthood compred to pups from smll litter (12 pups), nd the ody weight of pups from lrge litters were significntly lower thn pups from smll litter y PD70. In this study, litter size did not exceed 12 pups. Nursing rts in the wild will typiclly leve the nest for nywhere from 15 min to 3 h dy to gther food nd wter (Clhoun 1963). Additionlly, differences in ehvior in mternl cre re oserved when the pups re returned to the dm fter n extended seprtion; mong these re incresed licking nd grooming nd engging in rched-ck nursing (Frncis nd Meney 1999). Dms tht re never seprted from their pups exhiit more relxed ehvior (Frncis nd Meney 1999). We did not oserve ny differences in ody weight in mles t wening, ut did in femles. At wening (PD21), femles exposed to neontl hypothermi were significntly hevier thn hypoxic nd normoxic unseprted femles. This could e due to the dm supplying different mternl cre etween sexes. We conclude tht dily mternl seprtion for short periods of time during the first week of life lters the pncretic et cell response to rginine through metolic progrmming nd the effects of this re more pronounced in mles thn femles. The dt lso suggest sex-specific effect of mternl seprtion on insulin sensitivity, lthough this will require specific experimentl testing directed t this phenomenon. The cellulr mechnisms for these findings re unknown, ut we pln on investigting this further. Although we hd hypothesized tht dult rts exposed to neontl hypoxi while mintining isothermi would hve n ltered metolic stte compred to dults exposed to neontl hypoxi, we did not find mjor effects of this tretment. We relize tht this initil study did not evlute potentil mechnisms for the oserved chnges in insulin secretion nd the potentil for chnges in insulin sensitivity. This will require perfecting method to isolte pncretic islet cells (Kelly et l. 2003) to evlute potentil moleculr nd electrophysiologicl chnges in the dult due to neontl tretment. Furthermore, studies to evlute peripherl sensitivity to insulin re currently eing plnned. Although this study is descriptive nd, in fct, the mjor finding of drmtic effect of mternl seprtion ws n unexpected yproduct of our interest in hypoxi, we hope tht our findings stimulte others to study the phenomen descried. Mternl seprtion is now considered model of erly childhood stress in humns (Kuhn nd Schnerg 1998; Fturi et l. 2010; Mrco et l. 2015). Furthermore, ecuse of the ltricil nture of the rt (Romijn et l. 1991; Clncy et l. 2001, 2007; Cllghn et l. 2014), hypoxi nd hypothermi in the first dys of life in the rt cn e considered model of common stress of premturity (Sheldon et l. 1996; Hung et l. 2004; Wtkinson 2006; Bruder et l. 2008, 2011; Guenther et l. 2012). The mjor clinicl impliction of our study ws tht mternl cre during the first week of life seems to e mjor determinnt of pncretic islet cell function in dulthood nd my lso result in decrese in peripherl Pge 12 ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society.

13 A. L. Gehrnd et l. Sex Differences in Adult Insulin After Neontl Stress insulin sensitivity. Of interest is tht humn et cell popultions re estlished preterm, nd pek within the first 2 yers of life (Gregg et l. 2012). Since our rt experiments model humn premturity, it my e tht stress in the premture infnt lters the development of the pncretic islets, lthough we did not oserve significnt chnge in islet cell composition. Finlly, s lluded to ove, preterm humns hve propensity for insulin resistnce nd the metolic syndrome lter in life, so it is possile tht the erly-life stress of premture irth contriutes to this (Tinnion et l. 2014). Furthermore, our study dds to the growing concept of sex differences in the progrmming effects of neontl stress. Acknowledgments We thnk Pete Homr for his ssistnce with the nimls, Mhrj Singh for sttisticl dvice, nd John Corett nd Polly Hnsen for help with interprettion of the dt. Conflict of Interest The uthors hve no conflicts of interest to disclose. References Bruder, E. D., J. K. Tylor, K. J. Kmer, nd H. Rff Development of the ACTH nd corticosterone response to cute hypoxi in the neontl rt. Am. J. Physiol. Regul. Integr. Comp. Physiol. 295:R1195 R1203. Bruder, E. D., J. Vn Hoof, J. B. Young, nd H. Rff Epiderml growth fctor nd prthyroid hormone-relted peptide mrna in the mmmry glnd nd their concentrtions in milk: effects of postprtum hypoxi in lctting rts. Horm. Met. Res. 40: Bruder, E. D., K. J. Kmer, M. A. Guenther, nd H. Rff Adrenocorticotropic hormone nd corticosterone responses to cute hypoxi in the neontl rt: effects of ody temperture mintennce. Am. J. Physiol. Regul. Integr. Comp. Physiol. 300:R708 R715. Clhoun, J. B The ecology nd sociology of the Norwy rt. U.S. Deprtment of Helth, Eduction, nd Welfre, Pulic Helth Service, Bethesd, MD. Cllghn, B. L., R. M. Sullivn, B. Howell, nd N. Tottenhm The interntionl society for developmentl psychoiology Sckler symposium: erly dversity nd the mturtion of emotion circuits cross-species nlysis. Dev. Psychoiol. 56: Cmm, E. J., M. S. Mrtin-Gronert, N. L. Wright, J. A. Hnsell, S. E. Oznne, nd D. A. Giussni Prentl hypoxi independent of undernutrition promotes moleculr mrkers of insulin resistnce in dult offspring. FASEB J. 25: Co, L., C. Mo, S. Li, Y. Zhng, J. Lv, S. Jing, et l Heptic insulin signling chnges: possile mechnism in prentl hypoxi-incresed susceptiility of ftty liver in dulthood. Endocrinology 153: Crrer, H. F., nd M. J. Cmisso Sexul differentition of the rin: genes, estrogen, nd neurotrophic fctors. Cell. Mol. Neuroiol. 22: Cherrington, A. D., nd M. Vrnic Effect of rginine on glucose turnover nd plsm free ftty cids in norml dogs. Dietes 22: Cherrington, A. D., R. Kwmori, S. Pek, nd M. Vrnic Arginine infusion in dogs. Model for the roles of insulin nd glucgon in regulting glucose turnover nd free ftty cid levels. Dietes 23: Chintmneni, K., E. D. Bruder, nd H. Rff Effects of ge on ACTH, corticosterone, glucose, insulin, nd mrna levels during intermittent hypoxi in the neontl rt. Am. J. Physiol. Regul. Integr. Comp. Physiol. 304:R782 R789. Chintmneni, K., E. D. Bruder, nd H. Rff Progrmming of the hypothlmic-pituitry-drenl xis y neontl intermittent hypoxi: effects on dult mle ACTH nd corticosterone responses re stress specific. Endocrinology 155: Clncy, B., R. B. Drlington, nd B. L. Finly Trnslting developmentl time cross mmmlin species. Neuroscience 105:7 17. Clncy, B., B. L. Finly, R. B. Drlington, nd K. J. Annd Extrpolting rin development from experimentl species to humns. Neurotoxicology 28: Clrk, D. J., nd J. E. Fewell Decresed ody-core temperture during cute hypoxemi in guine pigs during postntl mturtion: regulted thermoregultory response. Cn. J. Physiol. Phrmcol. 74: Clrke, M., G. Ci, S. Sleh, K. M. Buller, nd S. J. Spencer Being suckled in lrge litter mitigtes the effects of erly-life stress on hypothlmic-pituitry-drenl xis function in the mle rt. J. Neuroendocrinol. 25: Clifford, M., nd R. W. Hunt Neontl resuscittion. Best Prct. Res. Clin. Anesthesiol. 24: Cook, C. J Ptterns of wening nd dult response to stress. Physiol. Behv. 67: Coughln, M. T., P. P. Vervrt, M. Permezel, H. M. Georgiou, nd G. E. Rice Altered plcentl oxidtive stress sttus in gesttionl dietes mellitus. Plcent 25: Crowther, N. J., N. Cmeron, J. Trusler, nd I. P. Gry Assocition etween poor glucose tolernce nd rpid post ntl weight gin in seven-yer-old children. Dietologi 41: Cummings, J. J., D. B. D Eugenio, nd S. J. Gross A controlled tril of dexmethsone in preterm infnts t high risk for ronchopulmonry dysplsi. N. Engl. J. Med. 320: ª 2016 The Authors. Physiologicl Reports pulished y Wiley Periodicls, Inc. on ehlf of the Americn Physiologicl Society nd The Physiologicl Society. Pge 13

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