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Hypothalamic Phosphatidylinositol 3-Kinase Pathway of Leptin Signaling Is Impaired during the Development of Diet-Induced Obesity in FVB/N Mice

Anantha S. Metlakunta, Maitrayee Sahu, and Abhiram Sahu

OBESITY IS ONE of the major health problems throughout the world, particularly in the United States (1). A large body of evidence suggests that leptin, a product of the obese gene (2), signals nutritional status to key regulatory centers in the hypothalamus, and it has emerged as a major signal regulating energy homeostasis by decreasing food intake and increasing energy expenditure (3,4,5,6). Paradoxically, in the majority of cases, human obesity cannot be attributed to defects in leptin or its receptor (7,8,9,10,11,12,13). Serum leptin levels are significantly higher in obese humans relative to nonobese humans (6,14), and leptin administration shows very limited response in obese people (15), suggesting a state of leptin-resistance in obese individuals. Thus, understanding the mechanism of leptin resistance is quite significant in developing a new approach to prevent or treat obesity and associated disorders.

It has been evident that humans or rodents made obese by dietary manipulation have elevated levels of circulating leptin but maintain a normal food intake (6,14,16). Although a defective leptin transport may be one of the many factors behind the development of leptin resistance (17,18,19), available data from diet-induced obese (DIO) rodents, which may represent the form of obesity seen in most humans, strongly suggest that central leptin resistance also contributes to the development of obesity. Thus, anorectic effect of central leptin is reduced in DIO rats (20,21) and DIO mice (22); nutritional regulation of leptin receptor gene expression in the hypothalamus is defective in DIO rats (23), and leptin signaling in the hypothalamus through signal transducer and activator of transcription 3 (STAT3) is reduced in DIO mice (24). In obesity prone rats, gene expression of the long form of the leptin receptor (Ob-Rb) and leptin-induced STAT3 activation in the hypothalamus is compromised before the development of obesity or exposure to a high-energy diet (25). In addition, DIO in rodents is associated with increased expression of suppressor of cytokine signaling 3 (SOCS3), a negative regulator of the leptin signaling pathway (26,27), in the hypothalamus (28). Furthermore, hypothalamic AMP-activated protein kinase (AMPK) pathway of leptin signaling is also defective in DIO mice (29). Therefore, it is possible that those signaling pathways, which have been implicated to play a significant role in leptin action in the hypothalamus but not yet studied for their role in DIO, could be defective and contribute to the development of DIO.

In this regard, we have demonstrated that leptin signaling through the phosphatidylinositol 3-kinase (PI3K)-phosphodiesterase 3B-cAMP pathway plays a critical role in transducing leptin action in the hypothalamus (30). Other investigators have shown that leptin increases PI3K in the hypothalamus, and PI3K inhibitor reverses the anorectic effect of leptin (31). In addition, PI3K is localized in the hypothalamus (32); leptin induces PI3K in proopiomelanocortin (POMC) neurons, and leptin withdrawal activates PI3K in AgRP neurons in hypothalamic slice preparation (33). PI3K inhibitors reverse the effect of leptin on NPY and AgRP gene expression (34). Leptin also increases the activity of K-ATP channels in arcuate nucleus (ARC) neurons in a PI3K-dependent manner in vitro (35). Furthermore, more recently Kim et al. (36) showed PI3K signaling pathway to be upstream of forkhead transcriptional factor subfamily forkhead box O1 (Foxo1) in hypothalamic neurons, and PI3K-Akt-Foxo1 signaling pathway to mediate the effects of leptin and insulin on the transcriptional regulation of NPY/AgRP and POMC neurons. Thus, regulation of Foxo1 by PI3K in the hypothalamus appears to play an important role in food intake and energy homeostasis (36,37). Altogether, these lines of evidence clearly establish an important role of PI3K signaling in transducing leptin action in the hypothalamus. Thus, it is most likely that central leptin resistance seen during the development of DIO is due, at least in part, to an impaired PI3K pathway of leptin signaling in the hypothalamus. The present study tested this hypothesis in FVB/N mice that developed DIO in response to high-fat diet (HFD) feeding. Although many studies have used FVB/N background for developing transgenic mice (38,39,40,41,42), studies related to DIO in FVB/N mice are relatively few with the report of either an increase (40,29) or no change (39,41) in body weight after a HFD feeding. Although these discrepancies may be related to percentage of fat, diet composition, and duration of HFD feeding, hypothalamic mechanisms of leptin signaling during the development of DIO are relatively unknown in these mice (29). Therefore, we examined if the PI3K pathway of leptin signaling in the hypothalamus was altered during the development of DIO in FVB/N mice.

Changes in food intake, body weight, fat pad weight, and metabolic parameters after long-term HFD feeding in FVB/N mice

Male FVB/N mice were randomly assigned to either a LFD or HFD at 4 wk of age, when body weight was not different between the groups. The mice achieved significant body weight gain when they were put on a HFD. As shown in Fig. 1​1,, the body weight of HFD-fed mice was significantly (P = 0.016) increased within 1-wk dieting compared with LFD-fed mice. Body weight gain remained significantly higher and continued to diverge throughout 19 wk on the HFD, and at the end, the body weight difference between the groups reached approximately 11.5 g. Cumulative food intake (at 16 wk on the diet) was significantly increased (P = 0.0067) in the HFD group compared with the LFD group (Fig. 2A​2A).). The HFD group showed hyperglycemia (tested at 17 wk, LFD: 102.5 ± 6.91 mg/dl; HFD: 164.87 mg/dl; P < 0.0001; n = 8 per group), hyperinsulinemia (Fig. 2D​2D;; P = 0.0001), and hyperleptinemia (Fig. 2E​2E;; P = 0.003), and reduced insulin sensitivity (assessed by GTT and ITT at 14 and 15 wk, respectively) when compared with the LFD group (Fig. 2​2,, B and C). In addition, fat pad weights (E fat, RP fat, and BAT) were significantly increased (P < 0.001) in the HFD compared with those in the LFD group (Fig. 2F​2F).

Figure 1Figure 2