Ghrelin and its receptor play an important role in the regulation of glucose homeostasis. Both ghrelin KO and GhrR KO mice demonstrate a lower fasting blood glucose with reduced corresponding plasma insulin relative to wild type littermates when fed a HFD [2, 5], suggesting that ablation of ghrelin signaling improves insulin sensitivity. In the present studies, we have further characterized the degree and nature of the insulin sensitivity in GhrR KO mice in both hyperglycemic and hyperinsulinemic-euglycemic clamp assays. Obviously, decreases in body weight will lead to improvements in glucose homeostasis. However, while there was a tendency for the HFD-fed GhrR KO mice to have reduced BW (~5 g) relative to WT controls, this was not always the case. Our lab has evaluated dozens of HFD-fed GhrRKO mouse cohorts over the course of roughly 5 years of experimenting with this model. In so doing we have documented a degree of variability in the BW response to HFD from cohort to cohort such that we occasionally observe non-significant differences between groups. That variability is reflected in the data described in the present work and most likely indicates physiological variability one would expect to see upon repeated testing over the course of a year. However, despite the variability of the BW responses in GhrR KO mouse cohorts, the reported pattern of insulin sensitivity characterized by significantly reduced insulin release coupled with improved glucose disposal is always present. Thus, while not investigated explicitly in these experiments, our data reveal that there is a BW-independent component to the improved insulin sensitivity of GhrR KO mice. Further, we provide evidence from HI-clamp, PTT and expression data that suggests an hepatic mechanism for GhrR KO insulin sensitivity. Clearly, decreases in BW contribute to the overall improved insulin sensitivity of HFD-fed GhrR KO mice, but it is not the only factor.
Our lab has demonstrated previously that GhrR KO mice fed HFD require less insulin for glucose disposal relative to WT controls during a GTT . We therefore conducted HG clamps in order to examine GSIS in these animals in more detail. The GhrR KO mice fed a HFD responded to the initial glucose priming dose with a robust first-phase release of insulin that was not significantly different than WT controls, indicating no deficit in their ability to secrete insulin upon challenge . However, a relative trend of decreased insulin requirement of GhrR KO mice was maintained throughout the 2nd phase of insulin release , and reaching its maximal effect at the point of HG clamp. This pattern is consistent with previous data obtained during a GTT in HFD-fed GhrR KO mice (2). Indeed, GhrR KO mice fed a HFD are more insulin sensitive compared to WT mice as determined by their significantly increased GIR during the HG clamp. Furthermore GhrR KO mice showed decreased HGP, increased glucose disposal and increased tissue glucose uptake in HI-E clamp studies. Remarkably, these effects could be discerned regardless of whether mice were fed a LFD or HFD. In contrast, LFD-fed ghrelin-deficient mice responded to a glucose challenge with increased insulin secretion . The reasons for this apparent dichotomy may be a reflection of differences in islet glucose sensing as a result of chronic exposure to high plasma FFA  caused by the HFD. Thus, unlike glucose responsiveness, the underlying improvements of insulin sensitivity resulting from blockade of GhrR signaling do not appear to be affected by diet.
In order to understand the mechanism underlying improved hepatic insulin sensitivity, we evaluated hepatic insulin sensitivity of HFD-fed GhrR KO mice with a pyruvate tolerance test (PTT), which measures the capacity of the liver to convert pyruvate to glucose, a process that would normally be inhibited by insulin. In comparison to WT control mice, GhrR KO mice had a significantly lower gluconeogenic response to the pyruvate challenge, consistent with their relative insulin sensitivity. Therefore, blockade of GhrR signaling does not appear to interfere with gluconeogenesis, but rather may decrease HGP via an alternative mechanism, possibly as a secondary result of decreased hepatic lipid burden (2).
Hyperinsulinemia is associated with hepatic steatosis and hyperlipidemia in humans and animal models [20–22], and treatment with insulin-sensitizing drugs ameliorates these conditions [21, 23]. Increased fatty acid levels lead to elevations in hepatic UCP2 expression in rats , and the circulating free fatty acid concentration correlates with UCP2 expression in white fat and skeletal muscle , suggesting that UCP2 is important for the metabolic adaptation of these tissues to excessive fatty acid. Both GhrR and ghrelin KO mice have improved plasma lipid profiles relative to WT mice on a HFD [2, 5]. GhrR KO mice have higher fatty acid oxidation and lower lipogenesis, as evidenced by improved hepatic steatosis and lower intestinal triglyceride secretion rate when exposed to HFD . Ghrelin deficiency has been associated previously with a decrease in mitochondrial UCP2 mRNA expression in the livers of chow-fed mice . Likewise, in the present study hepatic expression of UCP2 mRNA was lower in HFD-fed GhrR KO mice. Taken together, our results suggest that lower expression of UCP2 mRNA of liver reflects improved lipid metabolism in HFD-fed GhrR KO mice and likely contributes to the overall insulin sensitivity in these animals.
Our observation that GhrR KO mice had increased glucose uptake into BAT suggested that increased glucose utilization in this tissue may contribute to the improved metabolic phenotype of these mice via increased fatty acid oxidation. BAT is a thermogenic organ in which increased expression of UCP1 decreases mitochondrial energy efficiency, leading to the generation of heat . However, the impact of GhrR signaling on thermogenesis is not yet clear. To investigate whether the improved glucose uptake in BAT is associated with increased thermogenesis in GhrR KO mice consuming HFD, we measured UCP1 mRNA expression in BAT. We previously reported that GhrR deletion in mice had no effect on total energy expenditure , which was consistent with the finding that injection of ghrelin had no effect on energy expenditure in rats . In another study, however, central administration of ghrelin suppressed energy expenditure and thermogenesis in BAT via an inhibitory effect on BAT sympathetic nerve activity [26, 27]. In the current study, BAT UCP1 mRNA expression was unaffected by genotype in HFD-fed mice, which indicated that diet-induced thermogenesis was not impacted by the loss of ghrelin signaling.