GIP Overexpression and Diet-Induced Obesity Figure 6. Decreased energy intake in Het GIP Tg mice. After 11–12 weeks of LF or HF diet feeding, WT and Het GIP Tg mice (n = 4,5/group) were placed in individual cages, and energy intake was assessed during the light and dark cycle. A and B. Cumulative energy intake in LF (A)- and HF (B) diet-fed Het GIP Tg and WT littermates. C–D. Histological analyses of brain tissue from Het GIP Tg and WT littermates following 13 weeks of LF and HF feedings. C. Representative images of GIP immunoreactivity in the hypothalamus. Brain sections were stained with GIP (green), PACAP (red) and DAPI (blue). The scale bar indicates 100 mm. D. Percentage of positive cells. GIP-, PACAP-, and GIP/PACAP double positive cells were expressed as the percentage of positive cells, respectively. 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 indicated group; NS represents not significant. doi:10.1371/journal.pone.0040156.g006 strongly co-expressed in this region in Het Tg mice on both LF and HF diets, with significantly increased numbers of GIP/ PACAP double positive neurons on HF feeding (Figures 6C and 6D). Although VMN application of GIP does not appear to have been performed to date, there is therefore a high possibility that changes in central expression of GIP contributed to the reduced food intake in the GIP overexpressing mice. One of the remaining questions arising from this study is the relative contribution of central and peripheral GIP to the phenotype of the overexpressing mice. Subcutaneous administra- tion of D-Ala2-GIP1–30 to Zucker diabetic fatty rats resulted in significantly improved glucose tolerance and insulin sensitivity, with a mild effect on food intake [13]. However, in carefully controlled studies, subcutaneous administration of GIP1-42 or GIP1-30 in HF fed mice was not found to have any major effects on food intake or body weight, despite significantly improved metabolic profiles [11]. Additionally, administration of a range of long acting GIP analogues, including N-acGIP, GIP (Lys37MYR), N-AcGIP(Lys37MYR) and N-AcGIP(LysPAL37), had no effect on body weight and food intake when compared to saline-treated controls [52–53]. This suggests that reduced food intake in GIP Tg mice resulted mainly in response to the hypothalamic expression and action of GIP. However, peripherally administrated GIP analogues have recently been reported to cross the blood-brain- barrier and exert central effects in mice [46,54]. It is therefore possible that the high circulating levels of GIP in the transgenic mice and the duration of GIP overexpression contributed to the reduced food intake. The almost complete ablation of steatosis was probably secondary to the reduction in adiposity. However, in view of the more robust changes in the pancreatic b-cells and adipose tissue (,68.5% decrease of TUNEL+ b-cells, ,58.6% decrease of crown-like structure content vs ,6.1% decrease in fat mass in Het GIP Tg mice), the improved b-cell function, altered WAT gene expression pattern as well as the decreased macro- phage infiltration likely involved direct effects of GIP on these tissues. Further expanded studies will be required to clearly delineate central vs peripheral effects of GIP, and their potential direct/indirect contribution to GIPR agonist action. In summary, we have shown that GIP Tg mice exhibit enhanced b-cell function, resulting in improved glucose tolerance and insulin sensitivity. Lower body weight was linked to reduced energy intake, that probably resulted mainly from overexpression of hypothalamic GIP in the Tg mice. They were also characterized by altered expression of adipocyte genes involved in the regulation of mitochondrial function and inflammation. Together, our findings support chronically elevating transgenic GIP expression exerts beneficial effects on a number of metabolic factors that are linked to the pathology of obesity and type 2 diabetes, and suggest that potential hypothalamic effects of transgenic GIP on food intake are worthy of further study. Experimental Procedures Animals and Genotyping Male GIP-overexpressing transgenic mice developed at the Medical College of Georgia, Augusta, GA [29] were crossed with female C57BL/6, and rederived in the Centre for Disease Modeling (CDM), University of British Columbia. GIP-over- expressing transgenic mice were generated using a gene construct containing the GIP coding sequence flanked by regulatory regions from the endogenous metallothionein locus (MTI and MTII) [55], resulting in a construct that retains proper regulation with limited artifactual effects derived from the site of integration. The metallothionein promoter drives ubiquitous gene expression but levels of transgene expression differ depending on the target tissue [55]. Genotyping was performed at weaning with genomic DNA obtained from ear notched biopsies. The duplex PCR primer sequences used for the genotyping were as follows: forward primer for GIP, 59-TCT GTT GCT GGT GCT CCT GTT-39; reverse primer for GIP, 59-ATC ACT GAG GCT CTT GGG CAA-39; forward primer for INS-2, 59-AGT GGC ACA ACT GGA GCT GG-39; reverse primer for INS-2, 59-TAT TCA TTG CAG AGG GGT AGG C-39. Heterozygous offspring of the founders (Het, Tg/+) was further crossed with nontransgenic wild-type (WT, +/+) littermates. At 5 weeks of age, resulting Het GIP transgenic (GIP Tg) mice and non-transgenic WT littermates offspring were placed on either a low fat (LF) diet (D12450B, Research Diets Inc., New Brunswick, NJ) composed of 10, 70 and 20 kcal% of fat, carbohydrate and protein or high fat (HF) diet (D12492, Research Diets Inc) composed of 60, 20 and 20 kcal% of fat, carbohydrate and protein. For induction of GIP expression, 25 mM ZnSO4 was added to the drinking water of Het and non-transgenic WT littermates. For the second part of the experiment, (Figures 7), Het mice were further crossed with Hets to generate homozygous transgenic mice (Homo, Tg/Tg). Progeny testing was performed and homozygosity confirmed by outcrossing presumptive Homo mice to nontransgenic WT littermates and checking progeny for 100% transmission of the transgene. Circulating plasma GIP levels were determined using a Rat/Mouse total GIP ELISA kit (Millipore). Resulting Het, Homo GIP Tg mice and non- transgenic WT littermates (5 weeks old) were placed on HF diet in the presence or absence of 25 mM ZnSO4 in the drinking water, as indicated in the Figure legends. All animal experiments were specifically approved by the University of British Columbia Committee on Animal Care and Canadian Council on Animal Care. DEXA (Dual Energy X-ray Absorptiometry) Scanning Tissue composition of the mice was determined at 5 and 14 weeks, using a Lunar PIXImus2 densitometer (Outside Sales, Madison WI). Shortly before scanning, mice were anesthetized by isoflurane inhalation, placed on the plastic tray of the scanner bed in a prone position and a nose cone was fitted over the face to maintain isoflurane anesthesia throughout the procedure. DEXA scans were analyzed based on manufacturer’s algorithms, using the PIXImus2 software. Values for total tissue mass (g), fat content (g), lean content (total tissue mass minus fat content, g) and percentage fat (fat content divided by total tissue mass) were obtained. PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e40156