Articles in PresS. Am J Physiol Regul Integr Comp Physiol (September 6, 2017). doi:10.1152/ajpregu.00271.2017
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Insulin Sensitivity, Leptin, Adiponectin, Resistin, and Testosterone in Adult Male
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and Female Rats After Maternal-Neonatal Separation and Environmental Stress.
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Hershel Raffa,b, Brian Hoeyncka, Mack Jablonskia, Cole Leonovicza, Jonathan M.
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Phillipsa, Ashley L. Gehranda,
6 Endocrine Research Laboratory, Aurora St. Luke’s Medical Center, Aurora Research Institute, Milwaukee, WI 53215
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a
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Keywords: HOMA-IR, adipokines, leptin, androgens, neonatal stress, sex difference
b
Departments of Medicine, Surgery, and Physiology, Medical College of Wisconsin, Milwaukee, WI 53226
Running Head: Adult Insulin Resistance after Neonatal Stress
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Correspondence: Hershel Raff, PhD Endocrinology Aurora St. Luke’s Medical Center 2801 W KK River Pky Suite 245 Milwaukee WI 53215 USA Phone (414) 649-6411 Fax (414) 649-5747 Email:
[email protected]
Copyright © 2017 by the American Physiological Society.
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ABSTRACT:
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Care of premature infants often requires parental and caregiver separation
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particularly during hypoxic and hypothermic episodes. We have established a neonatal
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rat model of human prematurity involving maternal-neonatal separation and hypoxia with
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spontaneous hypothermia prevented with external heat. Adults previously exposed to
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these neonatal stressors show a sex difference in the insulin and glucose response to
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arginine stimulation suggesting a state of insulin resistance. The current study used this
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cohort of adult rats to evaluate insulin resistance (Homeostatic Model Assessment of
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Insulin Resistance [HOMA-IR]), plasma adipokines (reflecting insulin resistance states),
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and testosterone. The major findings were that daily maternal-neonatal separation led to
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an increase in body weight and HOMA-IR in adult male and female rats and increased
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plasma leptin in adult male rats only; neither prior neonatal hypoxia (without or with
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body temperature control) nor neonatal hypothermia altered subsequent adult HOMA-IR
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or plasma adiponectin. Adult male-female differences in plasma leptin were lost with
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prior exposure to neonatal hypoxia or hypothermia; male-female differences in resistin
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were lost in the adults with prior neonatal hypoxia allowing spontaneous hypothermia
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Exposure of neonates to daily hypoxia while preventing spontaneous hypothermia led to
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a decrease in plasma testosterone in adult male rats. We conclude that neonatal stressors
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result in subsequent adult sex-dependent increases in insulin resistance and adipokines,
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and that our rat model of prematurity with hypoxia with the prevention of hypothermia
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alters adult testosterone dynamics.
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INTRODUCTION:
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Neonatal stress has dramatic acute effects in the premature infant as well as long-
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lasting, metabolic effects that are evident in adulthood (4, 30-34, 56, 66, 79). Premature
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birth often requires separation from direct, tactile parental or caregiver care (i.e. incubator
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therapy) that can be mimicked in rodents by maternal-neonatal separation (1, 2, 7, 14, 38,
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41, 45, 46, 57, 64, 85). Furthermore, premature infants often have periods of hypoxia due
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to apneas or immature lung function that may worsen during “kangaroo care” used to
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minimize the negative effects of neonatal separation (1-3, 7, 35, 45, 64, 71). In addition,
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premature infants can experience significant hypothermia even when normoxic (27, 40,
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41, 55, 76). Finally, hypoxia in the neonate results in profound, endogenous hypothermia
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(19, 59). The consensus seems to be that isothermia should be maintained during periods
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of acute hypoxia in the neonate even though therapeutic hypothermia is commonly used
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to prevent the sequelae of hypoxia at birth, particularly in the central nervous system (73,
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74).
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Exploiting the altricial nature of the newborn rat, we have developed a rodent
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model of prematurity and its treatment. This is accomplished by exposing neonatal rat
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pups periodically separated from their dams to hypoxia to mimic the apnea of the
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prematurity, while allowing or preventing the spontaneous hypothermia during hypoxic
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exposure (9, 10, 23). We have previously shown a significant additional neonatal stress
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response due to the prevention of hypoxia-induced spontaneous hypothermia in the
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neonate (9, 10, 23). Furthermore, we have shown long-term alterations in adult
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physiology subsequent to exposure to maternal-neonatal separation without and with
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concomitant hypoxia (13, 20, 21).
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Of relevance to the current study is our previous finding of sex differences in
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acute adult rat insulin and glucose responses to arginine after neonatal separation and the
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environmental stresses described above (20). In particular, the adult rats exposed to prior
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neonatal stress demonstrated hyperinsulinemia without hypoglycemia and greater weight
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gain reminiscent of the metabolic syndrome and early type 2 diabetes mellitus in humans
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(48, 62). We now follow up these prior studies by correlating the calculated Homeostatic
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Model Assessment of Insulin Resistance (HOMA-IR), a validated index of insulin
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resistance in adult rats (11, 53, 54, 61, 68) with measurement of the adipokines leptin,
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adiponectin, and resistin which are known to reflect the existence of, and be involved in
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the adaptation to insulin resistance (5, 16, 39, 42, 50, 51, 69, 72, 77, 87). Furthermore, in
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order to explore the significantly larger insulin response to arginine in males but not
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females, we measured adult plasma testosterone levels by LC-MS/MS. We hypothesize
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that maternal-neonatal separation without (normoxic) or with application of neonatal
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environment stressors will result in related increases in adult insulin resistance and
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associated increases in plasma leptin and resistin, and decreases in.adiponectin.
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METHODS: This is the second in a series of studies in this cohort of rats exposed to neonatal
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stress (20). Federal guidelines (http://grants1/nih/gov/grants/olaw/references/phspol.htm)
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for use and care of laboratory animals were followed and the protocols were approved by
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the Institutional Animal Care and Use Committee of Aurora Health Care. Timed pregnant
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Sprague–Dawley rats (N = 10) were obtained at 18 days of gestation and housed in a
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standardized environment (lights on 0600–1800 h). Rats were provided ad libitum
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standard diet and water. Dams were delivered normally and cared for their pups until
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experimentation. Each litter was assigned to a unique treatment group to avoid cross-
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fostering (47). A total of 110 pups were studied as described below.
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Neonatal stressors: Male and female rat pups were randomly assigned to different (90
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min) morning neonatal treatments from postnatal day (PD) 2 to PD6 as described below:
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1) Normoxia-Unseparated – the control group for normoxic separation: These pups (N = 23) were left undisturbed except for weekly cage changes.
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2) Normoxia-Separated – the normoxic control for the environmental neonatal
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stressors listed below: These pups (N = 22) were separated from their dams and placed
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into an environmental chamber with bedding and a variable setting heating pad on the
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lowest setting required to prevent a decrease in basal body temperature during separation
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from the dams (23). Pups were allowed to huddle normally.
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3) Hypoxia allowing spontaneous, endogenous hypothermia: These pups (N = 25)
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were separated from the dam and placed into a chamber with bedding and a variable
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setting heating pad on low heat. Hypoxia was induced by decreasing the chamber O2
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concentration to 8% as described in detail previously (9, 10, 23). This results in a plateau
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nadir transcutaneous O2 saturation of approximately 80% (10). Body temperature was
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allowed to spontaneously decrease during hypoxia and was measured in a sentinel pup in
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each chamber as described previously (23). Sentinel pups were not used for the adult
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experiments. Body temperature had spontaneously decreased to 23.9±0.5° C (n = 10)
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after 90 min of hypoxia. After 90 min, the chamber was opened to room air and the pups
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were warmed to a normal body temperature range of 32–34 °C using a variable setting
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heating pad set on low before returning the pups to the nest.
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4) Induced Hypothermia: These pups (N = 15) were separated from the dam and
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placed in a normoxic chamber with bedding on top of a cold plate (Model
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#AHP-1200CPV; TECALAB, Chicago, IL) set between 24 and 27 °C and adjusted
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depending on measured body temperature. Body temperature was measured in a sentinel
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pup using RET-30-Iso rectal probes and a BAT-12 digital thermometer connected to a
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SBT-5 switchbox (Physitemp Instruments, Clifton, NJ). Body temperature was
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decreased to 25 °C over 30 min and held at 25° C by adjusting the temperature of the
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cold plate. After hypothermia was completed, body temperature was allowed to increase
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as described for hypoxia above.
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5) Hypoxia while maintaining isothermia: These pups (N = 25) were treated the
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same as the hypoxia pups except that body temperature was maintained at 32 °C with a
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heat plate (Model #AHP-1200CPV; TECALAB) as described previously (23).
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At PD22, pups were weaned and housed by sex and treatment group. Weaned
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animals were given a standard diet and water ad libitum and handled only during weekly
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bedding changes.
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Adult Measurements: These measurements were obtained after an overnight fast. Rats
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between PD105 and PD133 had been accustomed to daily handling for 5-10 min for
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several days before experimentation in order to obtain basal stress hormone levels on the
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day of experimentation (65). For plasma insulin and glucose measurements, blood was
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obtained by tail nick as described previously (84). These data have been reported
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previously and are provided here to support the HOMA-IR calculations (shown below).
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Rats were exposed to inhaled isoflurane for 5-10 seconds and then killed by rapid
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decapitation to obtain trunk blood in EDTA-treated tubes to generate plasma for the
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measurement of hormones described below:
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Plasma immunoassays: Plasma leptin and adiponectin were measured by ELISA
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(Crystal Chem #90040 and #80570, respectively, Downer’s Grove, IL)(8). Plasma
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resistin was measured by ELISA (BioVendor #RD391016200R, Karasek, Czech
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Republic). The limit of detection is 0.05 ng/ml and intraassay and interassay coefficients
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of variation are 4.9-5.2% and 4.9-9.3%, respectively.
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Plasma testosterone: Plasma testosterone was measured by LC-MS/MS. Plasma
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samples, quality controls or standards (100 µl) were each combined with 100 ng/dl
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deuterium labeled d3-testosterone internal standard (100 µl) and extracted with 0.4 ml of
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acetonitrile; the organic phase was evaporated to dryness under nitrogen in a 50 ºC water
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bath and reconstituted in 100 µl of water/methanol (50:50, v:v). Testosterone
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measurements were performed using a 1290 Infinity HPLC (Agilent Technologies, Palo
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Alto, CA, USA) and a Triple Quad LC-MS (Agilent Technologies, Palo Alto, CA, USA)
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with an ESI ion source in positive mode. The two dimensional LC separation technique
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was performed with a Zorbax SB-C8 (2.1 x 15 mm, 3.5 µm) loading column (Agilent
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Technologies, Palo Alto, CA, USA) and a Poroshell 120, EC-C18 (2.1 x 50 mm, 2.7 µm)
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analytical column (Agilent Technologies, Palo Alto, CA, USA) both maintained at 50º C
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combined with a 0.3 µm inline filter. The injection volume and flow rate range while
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injections were on the columns was 5 µl and from 0.3-0.6 ml/min, respectively.
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Introduction of sample onto the loading column was performed by a gradient elution of
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mobile phase consisting of 5 mM ammonium formate in water (solvent A) and 5 mM
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ammonium formate in methanol (solvent B) at 0.5 ml/min A:B 50:50-41:59 (0-1.2). The
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analytical pump transferred the sample to the analytical column and then introduced it to
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the mass spectrometer with a gradient elution of A:B 45:55-2:98 (1.1-4.4 min). The flow
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rate of the analytical pump increased from 0.3 ml/min to 0.6 ml/min (4-4.1 min).
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Additional, gradients used for eluting waste components and equilibration of columns to
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initial conditions. MassHunter software (Agilent Technologies, Palo Alto, CA, USA)
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was used to control the instruments and for data analysis. The mass spectrometer scan
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type utilized was multiple reaction monitoring with total testosterone quantified and
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qualified by the ion transition m/z 289.2/97.1 and m/z 289.2/109.1, respectively. d3-
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testosterone internal standard was analyzed by the ion transition m/z 292.2/97.1. The
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following source conditions were used: gas temperature, 300 °C; gas flow, 5 l/min;
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nebulizer pressure, 60 psi; sheath gas temperature, 400 °C; sheath gas flow, 11 l/min;
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capillary voltage, 3,500 V; nozzle voltage, 0 V; and an electron multiplier voltage of 400
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V. Fragmentor voltage was 123 V for all compounds. Collision energy was 20 V for
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internal standard and testosterone quantifier ions and 24 V for testosterone qualifier ions.
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The functional sensitivity is 0.7 ng/dl, and intraassay and interassay coefficients of
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variation are 1.5-3.9% and 2.6-7.8%.
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Calculations and Statistical Analyses: HOMA-IR, a validated index of insulin
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resistance in rats, was calculated as the product of fasting plasma glucose and fasting
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plasma insulin divided by a constant (11, 61). Data were analyzed by two factor analysis
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of variance (ANOVA) followed by the Holm-Sidak multiple comparisons method
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(Sigmaplot 12.5). Since pups had to be separated from their dams to expose them to the
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environment stressors, two different null hypotheses were evaluated. For the comparison
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of separated (normoxia) vs unseparated (control for normoxic separated), the two
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ANOVA factors were sex and separation. For the comparison of environmental stressors
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(that required separation) to their control (normoxic separated), the two factors were sex
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and environmental stressor. Data are presented as median (25%-75% percentile) or mean
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± standard error of the mean with P
