To test whether paternal obesity impacted maternal metabolic adaptations to pregnancy, separate cohorts of pregnant females were subjected to a glucose tolerance test (GTT) at mid-gestation (E14.5, n = 10 per group), and term gestation (E18.5, n = 10–13 per group). Dams mated with CON and PHF males were fasted for 6 h prior to a GTT. At E14.5, glucose was administered via intraperitoneal (i.p.) injection of glucose (G5767; Sigma-Aldrich; 1.5 g/kg). Due to the difficulty of i.p. injection with a full gravid uterus at term, glucose was orally administered to E18.5 pregnant dams by gavage (G5767; Sigma-Aldrich; 2 g/kg). At both timepoints, maternal blood glucose was repeatedly measured through tail vein sampling using a commercial blood glucometer (an Accu-Check Aviva Roche Diagnostics) prior to glucose administration (0) and 15, 20, 30, 40, 60, 90, and 120 min after glucose administration.
G5767
Manufactured by Merck Group
G5767 is a multi-channel pipette designed for precise and efficient liquid handling in laboratory settings. It features adjustable volume ranges and digital display for accurate volume dispensing. The product is intended for general laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Humulin r, by Eli Lilly (5 mentions)
Accu check aviva, by Roche (2 mentions)
Glucometer, by Bayer (1 mentions)
Bacto yeast extract, by BD (1 mentions)
Difco mueller hinton broth, by BD (1 mentions)
S318 500, by Thermo Fisher Scientific (1 mentions)
A144 500, by Thermo Fisher Scientific (1 mentions)
Comprehensive lab animal monitoring system clams, by Columbus Instruments (1 mentions)
Endothelial cell medium (ecm), by ScienCell (1 mentions)
Endothelial growth supplement, by ScienCell (1 mentions)
Fcs500, by ExCell Bio (1 mentions)
Fns 18 2, by Kent Scientific (1 mentions)
11 protocols using g5767
1
Maternal Glucose Tolerance in Obesity
2
Glucose and Insulin Tolerance Tests
3
Optimized Media for Bacillus Cultures
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All samples were cultured on a complex MYPGP medium adapted from previously published protocols [11 , 44 (link)] with added agonists of P. larvae spore germination (i.e., L-tyrosine and uric acid [45 (link)]). For 1 L of media, 10 g of Difco™ Mueller Hinton Broth (BD, 275730), 15 g of Bacto™ Yeast Extract (BD, 212750), 3 g of potassium phosphate dibasic (Fisher BioReagents, BP363-1), 1 g of sodium pyruvate (Fisher BioReagents, BP356-100), and 15 g of agar (Fisher BioReagents, BP1423-2) were autoclaved in 880 mL of distilled water and combined with 20 mL of separately autoclaved, 10% glucose (Sigma, G-5767). To attain a final volume of 1 L, 50 mL of 60 mM L-tyrosine (Alfa Aesar, A11141) dissolved in 1 M hydrochloric acid (Fisher Chemical, A144-500), 50 mL of 60 mM uric acid (Alfa Aesar, A13346) dissolved in 1 M sodium hydroxide (Fisher Chemical, S318-500), and 1 mL of 20 mg/mL Nalidixic acid (Alfa Aesar, J63550), each sterilized through separate, 0.22 micron filters, were immediately added to the molten media. The resulting MYPGP medium enhanced by germination agonists is hereafter referred to as enhanced MYPGP medium. Plates were refrigerated at 4°C until use.
4
Metabolic Profiling of CON and PHF Offspring
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Metabolic profiling was performed on male and female CON (n = 14/sex) and PHF (n = 8/sex) offspring using a Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments) at P53 [25 (link)], which monitors the mice and records a number of outcome measures. Offspring were placed into individual CLAMS cages at 15:00 h and acclimatized for 24 h prior to acquiring measurements. These included: food consumption, total horizontal motor activity, heat production, oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory exchange ratio (RER), carbohydrate oxidation (carbOx), and lipid oxidation (lipOx). Measurements were acquired every 20 min for 48 h.
At P60, a standard i.p. GTT was performed on fasted male and female CON (n = 6/sex) and PHF (n = 7–8/sex) offspring. Offspring were injected with glucose (G5767; Sigma-Aldrich; 2 g/kg, i.p.) and blood glucose was repeatedly measured through tail vein sampling using a commercial blood glucometer (an Accu-Check Aviva Roche Diagnostics) prior to glucose injection (0) and 20, 30, 40, 60, 90, and 120 min after glucose injection.
At P60, a standard i.p. GTT was performed on fasted male and female CON (n = 6/sex) and PHF (n = 7–8/sex) offspring. Offspring were injected with glucose (G5767; Sigma-Aldrich; 2 g/kg, i.p.) and blood glucose was repeatedly measured through tail vein sampling using a commercial blood glucometer (an Accu-Check Aviva Roche Diagnostics) prior to glucose injection (0) and 20, 30, 40, 60, 90, and 120 min after glucose injection.
5
Retinal Cell Culture Under Glucose Conditions
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Primary human retinal endothelial cells (HREC, Angio-Proteomie) were cultured in endothelial cell media (ECM, ScienCell) containing 5% fetal bovine serum, 1% penicillin/streptromycin (30-002-CI, Corning) with endothelial growth supplement (ScienCell). Human retinal pigment epithelial cells (ARPE-19, ATCC CRL-2302) were cultured in DMEM (Corning) containing 10% fetal bovine serum (FCS500, Excell Bio) and 1% penicillin/streptromycin. Normal medium (low glucose) contained 5 mM D-glucose (G5767, Sigma). High-glucose medium contained 40 mM D-glucose. The HRECs or RPE cells were cultured in normal or high-glucose medium for 6, 24 and 48 hours respectively, and then subjected to experiments.
6
Glucose and Insulin Tolerance Tests in Mice
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For glucose tolerance test (GTT), mice were fasted for 12 h, and then injected intraperitoneally with glucose (Sigma-Aldrich, G5767) saline solution (1.5 g/kg body weight). For insulin tolerance test (ITT), mice were fasted for 6 h and injected intraperitoneally with insulin (Novolin R, HH20170016) saline solution (1 U/kg of body weight). Blood glucose levels were measured by tail-snip blood sampling pre-injection and 15-, 30-, 60-, and 120-min after injection.
7
Glucose and Insulin Tolerance Assays
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Oral glucose tolerance and intraperitoneal insulin tolerance tests were performed as described previously [30] (link). A fixed dose of glucose (G5767; Sigma, 60 mg/mouse in 300 μl water) was administered rather than a dose adjusted by body weight to eliminate the confounding effects of obesity. For insulin tolerance tests, insulin (Humulin R; Eli Lilly, 0.5 units/kg lean mass) i.p. was administered. The total area under the curve (AUC) was calculated using the trapezoidal rule.
A glucose-stimulated insulin secretion assay was performed according to our published protocol [48] (link).
A glucose-stimulated insulin secretion assay was performed according to our published protocol [48] (link).
8
Glucose and Insulin Tolerance in Mice
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Fifteen-week-old mice were fasted for 18 h (6 PM–12 PM) previous to a glucose tolerance test or for 4 h (8 AM–12 PM) for the insulin tolerance test. For basal fasting levels, blood samples were taken from the tail tip and glucose concentration measured with a One Touch R glucometer (LifeScan, Johnson & Johnson). Following a d -glucose (1 g/kg; Sigma, G5767) intraperitoneal (i.p.) injection, blood samples were taken at 15, 30, 60 and 120 min. For the insulin tolerance test, insulin (1 IU/kg, Humulin R; Lilly; i.p.) was administered and blood samples taken at 15, 30, 60 and 120 min. The total area under the curve (AUC) was calculated using the trapezoidal rule.
9
Glucose and Insulin Tolerance Assessments in Mice
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Mice were fasted on separate occasions for 6 h (8:00 a.m. –2:00 p.m. ) before being subjected to oral glucose tolerance and insulin tolerance tests. For oral glucose tolerance tests (OGTTs), glucose (fixed dose of 60 mg per mouse [G5767; Sigma]) was delivered into the stomach by an 18-gauge gavage needle (FNS-18-2; Kent Scientific Corporation), and blood was sampled at 0, 15, 30, 60, and 120 min for glucose measurements by an AlphaTRAK 2 glucometer. A fixed dose of glucose was given rather than a dose adjusted by body weight to eliminate the confounding effects of obesity. This method is recommended and has been validated for evaluating glucose tolerance in mice (25 (link),26 (link)). Moreover, glucose tolerance in humans is assessed based on a fixed dose (75 g) of glucose administration. For experiments involving epinephrine (Fig. 7D ), the OGTT was carried out 60 min after epinephrine 0.3 mg/kg lean mass i.p. or saline injection.
For insulin tolerance tests (ITTs), insulin 0.5 units/kg lean mass i.p. (Humulin R; Lilly) was administered and blood sampled at 0, 15, 30, 60, and 120 min for glucose measurements as described for OGTT. The total area under the curve (AUC) was calculated using the trapezoidal rule.
For insulin tolerance tests (ITTs), insulin 0.5 units/kg lean mass i.p. (Humulin R; Lilly) was administered and blood sampled at 0, 15, 30, 60, and 120 min for glucose measurements as described for OGTT. The total area under the curve (AUC) was calculated using the trapezoidal rule.
10
Glucose Tolerance Test and Body Composition
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Body weight was monitored regularly until 14 weeks of age. The mice were then individually housed for a week for acclimation and then their daily food intake was measured over 3 consecutive days with normal chow supplied ad libitum. The males were fasted for 18 h (5:00 P.M.-11:00 A.M.) and subjected to an intraperitoneal glucose tolerance test (GTT). Glucose was measured at time 0 using a OneTouch Ultra2 glucometer after cutting off the tip of the tail. D-glucose (1 g/kg; G5767, Sigma; i.p.) was administered and their blood glucose measured at 15, 30, 60, and 120 min. At week 16, male and female mice were euthanized via cervical dislocation. Body length was measured from the nose to the tail base. Bilateral subcutaneous (inguinal) and visceral (retroperitoneal and gonadal) white adipose tissue, interscapular brown adipose tissue, and livers were dissected and weighed.
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