GIP Overexpression and Diet-Induced Obesity Figure 1. Het GIP Tg mice exhibit reduced weight gain and insulin resistance on HF diet. Het GIP Tg (Tg/+) and non-transgenic WT littermates (+/+, 5 weeks old) were placed on either a low fat (LF) or high fat (HF) diet, and 25 mM ZnSO4 was added to the drinking water of both Het GIP Tg and WT mice (n = 6/group). A. Body weight gain in Het GIP Tg and WT mice placed on LF or HF diet. B. Percentage fat gain in WT and Het GIP Tg mice. C. Percentage fat of WT and Het GIP Tg mice on 14 weeks old. D and F. Glucose levels during an insulin tolerance test in LF (D)- and HF (F) diet-fed Het GIP Tg and WT littermates. After 8 weeks of feeding, WT and Het GIP Tg mice (13 weeks old, n = 6/group) were fasted for 4 hours and subsequently injected with 0.75 U/kg insulin. Blood glucose levels measured at 0, 15, 30, 60 and 120 min following insulin administration. E and G. Quantification of AUC for the total glycemic excursions in D and F. All data represent the mean 6 S.E.M. and significance was tested using ANOVA with a Newman-Keuls post hoc test or Student’s t test, where ** represents p,0.05 vs indicated group or WT littermates. doi:10.1371/journal.pone.0040156.g001 examined responses of transgenic overexpression of GIP (GIP Tg) mice to HF diet feeding. The results demonstrate that, in contrast to expectations, transgenic overexpression of GIP had major beneficial effects on both glucose and fat metabolism. Results 1. Mice Overexpressing GIP Exhibited Reduced HF Diet- induced Weight Gain and Adiposity To determine the effect of GIP overexpression on fat de- velopment, heterozygous (Het) GIP Tg (Tg/+) and WT littermates (+/+) were placed on low fat (LF) or high fat (HF) diets from 5 to 18 weeks of age. For induction of GIP expression, 25 mM ZnSO4 was added to the drinking water. As expected, WT mice fed a HF diet demonstrated a markedly greater increase in body weight, compared to those on a LF diet, over the course of the study. Although Het GIP Tg mice on a HF diet also gained more weight than those on a LF diet, from 7 weeks on the weight gain was significantly less than HF fed WT mice, as was the final weight at 18 weeks (38.460.9 g vs 47.161.1 g) (Figure 1A). Neither body length nor lean body mass differed between the Het GIP Tg mice and WT littermates (Data not shown), however, percent fat mass was significantly decreased compared to WT mice in 14 week old Het GIP Tg mice on both LF and HF diets (Figures 1B and 1C). 2. Improved Insulin Sensitivity, Glucose Tolerance and b- cell Function in Het GIP Tg Mice To evaluate the effect of GIP overexpression on insulin sensitivity, intraperitoneal insulin tolerance tests (IPITTs) were performed following 8 weeks of LF/HF feeding (n = 6 animals/ group). Fasting glucose in Het GIP TG mice on either LF or HF diets did not differ from WT mice. However, although the Het GIP Tg mice became markedly insulin resistant on the HF diet, they showed significantly enhanced insulin sensitivity compared to their WT littermates on both LF and HF diets (Figures 1D–1G). Intraperitoneal glucose tolerance tests (IPGTTs) were per- formed following 10 weeks of LF/HF feeding (n = 6 animals/ group). As shown in Figures 2A–2D, Het GIP Tg mice showed improved glucose tolerance compared to WT littermates on both of LF and HF diets. Het GIP Tg mice on LF diet showed significantly enhanced insulin secretory responses to glucose stimulation, compared with WT littermates. On the HF diet, the WT littermates almost lost their insulin secretory responses to glucose, whereas Het GIP Tg mice on the same diet showed robust insulin responses following the glucose challenge (Figure 2E). Glucagon levels did not differ between Het GIP Tg and WT littermates (Figure 2F). Subsequent studies, on islets collected at the termination of the experiment, demonstrated that b-cell area and total pancreatic insulin content were significantly increased in HF fed Het GIP Tg mice, compared with WT littermates (Figures 2H and 2J) and this was accompanied by significantly decreased b-cell apoptosis (Figure 2I). Mean b-cell area and pancreatic insulin content were increased in Het GIP Tg mice on LF feeding, although differences did not reach statistical signifi- cance (Figures 2G and 2H). Together these results strongly suggest that overexpression of GIP in mice exerts beneficial effects on the regulation of insulin sensitivity and glucose tolerance, and the improvement was at least partially due to b-cell compensation resulting from enhanced b-cell function. 3. GIP Overexpression Altered Hepatic and Adipose Tissue Responses to a High Fat Diet Liver sections from WT mice at 18 weeks exhibited marked steatosis following the HF diet, but the GIP overexpressing mice showed a remarkable attenuation of hepatic lipid accumulation (Figures 3A–3C). As expected, adipocytes in epididymal white adipose tissue (eWAT) from both WT and Het GIP Tg mice exhibited greatly increased size following the HF diet (Figure 3D). Adipose tissue macrophage infiltration was also greatly increased in WT mice following 13 weeks of HF feeding, but tissue from Het GIP Tg mice showed substantially less infiltration of macrophages (Figures 3E–3F). In view of the fact that we had previously shown that GIP and insulin act in a synergistic manner to regulate adipocyte function, and to gain a better understanding of the underlying molecular mechanisms involved in the reduced adiposity in GIP Het mice, we determined the relative expression levels of a panel of adipocyte-related genes using quantitative PCR arrays (RT2 Profiler PCR Array). As shown in Figures 3G and 3H, a number of genes involved in fatty acid synthesis (ATP citrate lyase, Acly), glyceroneogenesis (phosphoenolpyruvate carboxykinase, Pck1) and mitochondrial biogenesis and function (PPARc coactivator 1 a (Ppargc1a; PGC1a), endothelial nitric oxide synthase 3 (Nos3), Uncoupling protein 2 (Ucp2)) were down-regulated in Het GIP Tg mice fed the HF diet. Additionally, gene expression of proteins involved in inflammatory signaling pathways were significantly decreased, including angiotensin converting enzyme (Ace), glucose- 6-phosphate dehydrogenase 2 (G6pd2), MCP-1 (Ccl2), IKKb (Ikbkb), interleukin 4 receptor alpha (IL4ra), TNF receptor superfamily member 1b (Tnfrsf1b), and PAI-1 (serpine1). Intrigu- ingly, insulin receptor substrate (Irs-1) was also decreased in GIP Tg mice (Figure 3G). On the other hand, expression of angiotensinogen (Agt), PPARc (Pparg), soluble carrier family 14, member 2 (Slc14a2) and Mapk14 were increased (Figure 3H). Subsequently it was found that adipose tissue protein expression levels of IRS-1 were also decreased in GIP Tg mice, whereas IRS- 2 was increased (Figure 3I). 4. Het GIP Tg Mice were not Resistant to GIP After 13 weeks of diet feeding, circulating GIP levels in Het GIP Tg mice in LF and HF feeding groups were elevated ,83.5-fold and ,41.1-fold over wild type levels, respectively, (Figure 4A). Since the GIP receptor undergoes down-regulation with pro- longed exposure to elevated GIP [28], we next determined whether blunted GIP responses contributed to the phenotype demonstrated by Het GIP Tg mice. Exogenous GIP (24 nmol/kg) was therefore administrated to Het GIP Tg mice and their WT littermates with concurrent glucose tolerance tests. As shown in PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40156