Reproductive Tissues Maintain Insulin Sensitivity in Diet-induced Obesity

Sheng Wu; Sara Divall; Fredric Wondisford; Andrew Wolfe

Disclosures

Diabetes. 2012;61(1):114-123. 

In This Article

Research Design and Methods

Animals and Diets

Mice were maintained on a mixed background: CD1/129SvJ/C57BL6. Pituitary-specific insulin receptor KO mice (PITIRKO) were produced by our group as described previously.[2] αGSU-Cre-negative littermates were used as controls (wild type [WT]). Female PITIRKO mice and controls were fed a high-fat diet (HFD) or regular chow as described previously.[2] Mice used in this study were female and were maintained with food and water ad libitum under a 14-h/10-h dark/light cycle. Procedures were approved by the Johns Hopkins Animal Care and Use Committee. Experiments were conducted with 5.5- to 6.5-month-old female mice (lean and DIO): lean WT (30.6 ± 1.0 g), lean PITIRKO (29.6 ± 1.8 g), WT DIO (44.3 ± 6.9 g), and PITIRKO-DIO (43.4 ± 6.4 g). Mice were tested for estrous cyclicity, and WT DIO mice were acyclic as reported previously.[2]

Hormonal Assays

Mice were fasted overnight, and blood was collected from mice via mandibular bleed at 10 A.M All samples were collected by 10:30 A.M. Insulin, leptin, and glucagon were measured with the Milliplex Map Mouse Serum Adipokine Panel (Millipore, Billerica, MA), and insulin-like growth factor-I (IGF-I) was measured with a single-plex IGF-I panel (Millipore) on a Luminex 200IS platform (Luminex Corporation, Austin, TX). All samples were performed in one plate. The intra-assay coefficient of variation for each assay was between 5 and 9%.

Glucose and Insulin Tolerance Tests

Mice were fasted overnight and injected intraperitoneally with 2 g/kg body wt dextrose. Glucose was measured from tail blood at the times indicated using a One Touch Ultra glucometer. After insulin injection (Lilly, Indianapolis, IN) in lean and WT DIO mice, glucose was measured as described above.

Insulin-signaling Assay

Mice were fasted overnight, and insulin or 0.9% saline was injected intraperitoneally with different doses. Tissues were collected at 10, 15, 30, or 45 min after insulin injection and snap-frozen in liquid nitrogen. Protein was obtained and measured as described previously.[2] To quantify the levels of phosphorylated AKT (pAKT), phosphorylated extracellular signal–related kinase (pERK), and AKT for different tissues of each individual animal after insulin or saline injection, Bio-Plex Phosphoprotein Detection Multiplex assays were used (Bio-Rad Laboratories, Hercules, CA. For quantification of phosphorylated Tyr (pTyr)-IRS1 (or total IRS1) levels in cell lysates, Milliplex Map Phospho IRS1 Mapmates (or Total IRS1 Mapmates) kits were used. Protein (10 μg) from each tissue was loaded into a 96-well microplate. Tissues from four to eight mice were measured independently and were not pooled. Assays were conducted using the Luminex 200 system. Values were assessed as mean fluorescent intensity. For quantification of the pTyr-IRS2 level, a PathScan Phospho-IRS-2 (panTyr) ELISA kit (Cell Signaling Technology) was applied according to the manufacturer's instructions.

Antibodies and Immunoblotting.

Identical amounts of protein were loaded and separated by SDS-PAGE gel and blotted onto nitrocellulose membranes. Signals were detected by enhanced chemiluminescence plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, U.K.). Rabbit primary antibodies against pAKT (Ser473), AKT, pERK, ERK, phosphorylated Jun NH2-terminal kinase (pJNK), Jun NH2-terminal kinase (JNK), and actin were from Cell Signaling Technology. Rabbit anti-IRS1 was from Millipore. Goat anti-rabbit IgG (H+L)-HRP conjugate was from Bio-Rad Laboratories.

Total RNA Extraction, Reverse Transcription, and Quantitative PCR

Pituitary and ovary RNA was extracted and reverse transcribed as previously described.[4] For the expression of irs1, iQ SYBR Green quantitative PCR (Bio-Rad Laboratories) was performed, and irs1 primers were from the study by Murata et al..[5]gapdh was used as described previously.[4]irs1 values were corrected with gapdh values. Reactions were performed using an iCycler iQ5 quantitative PCR machine (Bio-Rad Laboratories) as described previously.[4]

Immunohistochemistry.

Tissues were paraffin sectioned by Johns Hopkins University Pathology Core Facility. Sections were stained with rat anti-mouse F4/80 (1:100) and Mac-2 (1:200), and markers for macrophages[6] were purchased from Cedarlane (Burlington, Ontario, Canada). Secondary antibody Alexa Fluor 594 goat anti-rat IgG (H+L) (1:200) was from Invitrogen.

Statistical Analysis

All data were analyzed with unpaired two-tailed Student t test using Prism Software (GraphPad Software, Inc.) and are expressed as means ± SEM. P < 0.05 was defined as statistically significant.

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