GIP Overexpression and Diet-Induced Obesity Figure 2. Het GIP Tg mice were resistant to HF diet-induced glucose intolerance. A and C. Glucose levels during a glucose tolerance test in LF (A)- and HF (C) diet-fed Het GIP Tg and WT littermates. After 10 weeks of LF and HF feeding, WT and Het GIP Tg mice (15 weeks old, n = 6/group) were fasted for 4 hours and blood glucose levels measured at 0, 15, 30, 60 and 120 min following a glucose challenge (2 g/kg). B and D. Quantification of AUC for the total glycemic excursions in A and C. E–F. Plasma insulin (E) and glucagon (F) during a glucose tolerance test. G. Representative pancreatic images from WT and Het GIP Tg mice (18 weeks old) following 13 weeks of LF and HF feedings. Pancreatic sections were stained with insulin (green), glucagon (red) and DAPI (blue). The scale bar indicates 100 mm. H. Relative b-cell area expressed as the percentage of sectional pancreas area as described in Experimental Procedures. I. The percentage of b-cells undergoing apoptosis as determined by TUNEL positive nuclei. J. Total pancreatic insulin content in WT and Het GIP Tg mice (18 weeks old) following 13 weeks of LF and HF feedings. 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 ** and ## represents p,0.05 vs indicated group or WT littermates; NS represents not significant. doi:10.1371/journal.pone.0040156.g002 Figures 4B–4E, GIP administration resulted in significantly reduced glycemic excursions relative to the saline control in both Het GIP Tg mice and WT littermates in both LF and HF feeding groups. Therefore, despite the greatly elevated GIP levels, Het GIP Tg mice still responded to GIP indicating that the reduced adiposity, improved insulin sensitivity/glucose tolerance as well as b-cell function in Het GIP Tg mice were not a result of blunted GIP responses. 5. Indirect Calorimetry Revealed no Significant Differences in Energy Expenditure Mechanisms involved in the reduced adiposity in GIP Tg mice were next assessed. Since resistance to diet-induced obesity could result from increased energy expenditure and/or decreased food intake, both were examined. Het GIP Tg mice showed a non- significant trend towards decreased energy expenditure (Figure 5A and Figures S1A and S1B) and oxygen consumption (Figures S1C and S1D), during both dark and light phases, compared to WT littermates. As expected, the respiratory exchange rate (RER) decreased to ,0.80 with mice on the HF diet, but ratios in Het GIP Tg mice did not differ from WT littermates (Figures S1E and S1F). In agreement with this conclusion, Het GIP Tg mice in both LF and HF feeding groups showed significantly decreased, rather than increased, total home cage activity, including both X and Y beam activities at dark cycle, compared to WT littermates (Figures 5B–5D). Apart from Y beam activity, physical activities of Het GIP Tg mice at light cycle did not differ from WT control mice (Figures S2A–S2F). 6. Het GIP Tg Mice Exhibited Reduced Energy Intake Het GIP Tg mice showed significant reductions in food intake during most weeks over the course of the study (Figure S3), and as shown in Figures 6A and 6B, metabolic cage studies showed that these mice demonstrated a marked reduction in energy intake (food intake in kcal) during the dark phase compared to WT littermates on both LF and HF diets. On HF diet, the intake of Tg mice reached ,54.8% of that of the WT mice following 11–12 weeks of HF feeding and the overall reduction in energy balance would likely account for the decrease in body weight. Since GIP mRNA was previously reported to be overexpressed in the brain of GIP Tg mice [29], and the hypothalamus is critical for the regulation of food intake-controlling neural circuits, we considered it possible that transgenic GIP expression in the brain could be involved in the modulation of energy intake in GIP Tg mice. Immunohistochemical analyses demonstrate that GIP expression was greatly increased in the ventromedial nucleus (VMN) of the hypothalamus of Het GIP Tg mice on both LF and HF diets, compared with WT littermates (Figures 6C and 6D), mainly co-expressed in pituitary adenylate cyclase-activating polypeptide (PACAP)-positive neurons. The number of GIP/ PACAP double positive neurons were significantly increased in Het GIP Tg mice on HF, compared to those on a LF diet (Figures 6C and 6D). Together, these results suggest that transgenic GIP overexpression in the hypothalamus contributed significantly to the reduced energy intake in GIP Tg mice. 7. GIP Effects on HF Diet–induced Insulin Resistance and Glucose Intolerance were Plasma Concentration- dependent To evaluate the effect of varying circulating GIP concentrations on metabolic phenotype, homozygous (Homo, Tg/Tg) GIP Tg mice were screened by progeny testing. One group of Homo GIP Tg mice and WT littermates placed on HF diet in the presence of 25 mM ZnSO4 (Zn2+) in the drinking water for 13 weeks beginning at 5 weeks of age. The second group of Homo and Het GIP Tg mice fed the same HF diet in the absence of Zn2. This resulted in GIP levels at 18 weeks of only 1.960.5 ng/ml in the Het GIP Tg mice in the absence of Zn2+ (Figure 7H), as compared to 14.865.1 ng/ml in the presence of Zn2+ (Figure 4A). GIP levels in Homo and Homo + Zn2+ were 16.364.8 ng/ml and 60.165.1 ng/ml, respectively (Figure 7H). In agreement with an earlier study [29], Het and Homo GIP Tg mice demonstrated significantly enhanced GIP levels even in the absence of Zn2, due to the ‘‘leaky’’ metallothionein promoter, compared to the WT littermates (Figure 7H). As shown in Figure 7A, Homo GIP Tg mice without Zn2+ demonstrated markedly reduced body weight at 13 weeks, compared to the other three groups, due to reduced adiposity (Figures 7B and 7C), but no difference in lean mass (Data not shown). Subsequent studies demonstrated that Homo GIP Tg mice without Zn2+ showed significantly improved insulin sensitiv- ity (Figures 7D and 7E) and glucose tolerance (Figures 7F and 7G) compared to WT littermates. The Homo GIP Tg mice with Zn2+ were GIP-resistant (Figures 7I and 7J). Together, these results suggest that chronically raising levels of GIP to ,15 ng/ml are necessary to achieve beneficial effects on glucose homeostasis and adiposity, but more elevated levels result in GIP resistance. Discussion Obesity and associated increases in circulating free fatty acids (FFA) have long been considered major contributors to insulin resistance and glucose intolerance. However, ectopic deposition of triglyceride (TG) is now recognized as an important contributor and adipose tissue expansion has been suggested to be an adaptive response to increased food intake, since it protects against accumulation of excess fat in sites such as the liver and muscle [30]. In a recent study, it was demonstrated that overexpression of adiponectin in mice on an ob/ob genetic background resulted in greater obesity, compared to non-transgenic animals, but with marked improvements in both glucose tolerance and insulin resistance, as well as reduced circulating TG and hepatic steatosis [31]. Storage of fat preferentially in adipose tissue and partitioning it away from other insulin-sensitive tissues is therefore probably a major determinant of insulin sensitivity and normal glucose tolerance, an effect of thiazolidinediones that has been termed: ‘lipid steal’ [32]. There may be additional advantages in adipose PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40156