GIP Overexpression and Diet-Induced Obesity Figure 7. Excessive overexpression ablates GIP effects on HF diet–induced weight gain. A. Body weight gain in Het, Homo GIP Tg and WT mice placed on HF diet in the presence or absence of 25 mM ZnSO4. B. Percentage fat gain in Het, Homo GIP Tg and WT mice. C. Percentage fat of Het, Homo GIP Tg and WT mice on 14 weeks old. D. Glucose levels during an insulin tolerance test in HF diet-fed Het, Homo GIP Tg and WT mice in the presence or absence of 25 mM ZnSO4. After 7 weeks of HF feeding, Het, Homo GIP Tg and WT mice (12 weeks old, n = 4 ,6/group) were fasted for 4 hours and subsequently injected with 0.75 U/kg insulin. Blood glucose levels were measured at 0, 15, 30, 60 and 120 min following insulin administration. E. Quantification of AUC for the total glycemic excursions in D. F. Glucose levels during a glucose tolerance test in HF diet-fed Het, Homo GIP Tg and WT mice in the presence or absence of 25 mM ZnSO4. After 11 weeks of HF feeding, Het, Homo GIP Tg and WT mice (n = 4 ,6/ group) were fasted for 4 hours and blood glucose levels measured at 0, 15, 30, 60 and 120 min following the glucose challenge (2 g/kg). G. Quantification of AUC for the total glycemic excursions in F. H. Plasma GIP levels. After 13 weeks of feeding, circulating GIP levels were determined in Het, Homo GIP Tg and WT mice (n = 4 ,6/group). I. Effects of GIP on glycemic excursion in HF diet-fed Het, Homo GIP Tg and WT mice. After 13 weeks of feeding, Het, Homo GIP Tg and WT mice (n = 4 ,6/group) were fasted for 4 hours, and PBS or GIP (24 nmol/kg) was administered immediately prior to glucose loading (2 g/kg). Blood glucose levels were measured at 0, 15, 30, 60 and 120 min following the glucose challenge. J. Quantification of AUC for the total glycemic excursions in I. All data represent the mean 6 S.E.M. and significance was tested using ANOVA with a Newman-Keuls post hoc test, where ## represents p,0.05 vs WT + Zn++, ** represents p,0.05 vs indicated group or WT littermates; NS represents not significant. doi:10.1371/journal.pone.0040156.g007 Blood Glucose Determinations, Intraperitoneal Glucose Tolerance Tests (IPGTTs), Intraperitoneal Insulin Tolerance Tests (IPITT), GIP-Glucose Tolerance Tests (GIP- GTT), and Plasma Hormone Measurements Fasting and non-fasting blood glucose levels were measured in blood collected from the saphenous vein using a Freestyle Glucometer (Abbott Laboratories) at the time points indicated in Figures 1, 2, 4 and 7. For the IPGTTs and IPITTs, mice were fasted for 4 hr and blood glucose levels measured at 0, 15, 30, 60 and 120 min following the glucose (2 g/kg) or insulin challenge (0.75 U/kg). For the GIP-GTT, mice were fasted for 4 hr and PBS or GIP (24 nmol/kg) were administered immediately prior to glucose loading (2 g/kg). Blood glucose levels were measured as above. Blood samples with glucose levels $27.8 mmol/l were diluted with control solution provided by the manufacturer (Abbott Laboratories) and levels calculated. Plasma insulin, glucagon and GIP levels were determined using a multi-spotH assay kit (Meso Scale Discovery, Gaithersburg, MD) and Rat/ Mouse total GIP ELISA kit (Millipore), respectively. Morphological Analyses of Liver and Epididymal White Adipose Tissue Livers and epidydimal fat pads were harvested and fixed in 10% neutral buffered formalin. Sections obtained from paraffin- embedded tissues were stained with Hematoxylin & Eosin (H & E) using standard protocols. Fat sections were incubated with antibody against the mouse macrophage antigen MAC-2 and visualized with FITC conjugated anti-rat secondary antibody. Cell nuclei were counterstained with DAPI (49, 6- diamino-2- phenylindole) and imaged using a Zeiss laser scanning confocal microscope (Axioskop2). MAC-2 positive crown-like structures (CLS) per field were counted manually and the number of CLS per 1000 nuclei was used as a measure of adipose tissue CLS content. Liver sections were incubated with the lipid body stain BODIPY 493/503 and counterstained with DAPI. Intensity of signals was quantified by computer-based densitometric analysis using the Northern Eclipse program (ver.6). RT2 ProfilerTM PCR Arrays Total RNA was extracted from epididymal fat tissues using an RNeasy Lipid Tissue Mini Kit (Qiagen), and cDNA fragments were generated by reverse transcription using RT2 First Strand Kit (SAbiosciences). RT2 Profiler PCR Arrays were performed to simultaneously quantify mRNAs of 84 genes associated with insulin signaling plus housekeeping genes, according to the manufacturer’s protocol. Average expression of the five house- keeping genes on the array was used to normalize the gene expression by the DDCt method. Western Blot Analysis Total cellular extracts from each sample were separated on a 13% sodium dodecyl sulfate (SDS)/polyacrylamide gel and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Mississauga, ON). Probing of the membranes was performed with IRS-1, IRS-2 and b-actin antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (Millipore Millipore, Billerica, MA) using horseradish peroxidase-conjugated IgG secondary antibodies. Immunohistochemistry Pancreatic sections from GIP Tg and WT littermates were subjected to double immunostaining for insulin and glucagon, with apoptosis staining in adjacent sections. Following deparaffiniza- tion, the sections were incubated with insulin and glucagon antibodies and visualized with Texas RedH dye conjugated anti- rabbit secondary antibody and Alexa fluorH 488 conjugated anti- mouse antibody. Cell nuclei were counterstained with DAPI (49, 6- diamino-2-phenylindole) and imaged using a Zeiss laser scanning confocal microscope (Axioskop2). TUNEL assay was performed in adjacent sections to detect DNA fragmentation in apoptotic cells using the ApoptagH apoptosis detection kit (Intergen Company, Purchase, NY), according to the manufacturer’s instructions. b-cell area was measured on the sections of each pancreas stained for insulin. Morphometric evaluation of the total b-cell area was performed by computer-assisted image analysis (Northern Eclipse ver. 6 and NIH Image J) using light and immunofluorescence microscopy. The area of insulin-positive cells and the total area of the tissue section were evaluated for each section, and the percentage b-cell area was calculated by the ratio of the area occupied by insulin-positive cells to the total pancreatic area. Measurement of Total Pancreatic Insulin Content Pancreata were homogenized in 5 ml of ice-cold 2 N acetic acid, boiled and centrifuged (10 min, 15,000 rpm, 4uC). The supernatant was then assayed for insulin content and normalized for protein concentration (BCA; Pierce, Rockford, IL). Indirect Calorimetry Mice were individually housed, with free access to food and water, and acclimated in metabolic chambers. The following parameters were continuously recorded with measurements taken every 15 minutes: Gas exchange (VO2 and VCO2), homecage activity, water intake and food intake. Physical activity was measured by utilizing infrared beams to monitor an animal’s homecage movement in the X, Y, and Z axis. Following metabolic chamber recordings, DEXA scanning was performed to determine lean body mass (LBM). VO2, VCO2 and energy expenditure were PLoS ONE | www.plosone.org 12 July 2012 | Volume 7 | Issue 7 | e40156