This is the discussion portion from this article: The Effects of Human GH and Its Lipolytic Fragment (AOD9604) on Lipid Metabolism Following Chronic Treatment in Obese Mice and ß3-AR Knock-Out Mice
It is well established that hGH is a lipolytic hormone (15), but the exact mechanisms used are still unclear. In this paper we present data that suggest that hGH and its lipolytic fragment (AOD9604) induce their chronic in vivo actions on lipolysis in part by modulating the expression of the ß3-AR. Human GH has been shown to affect the in vivo expression and function of ß-ARs in vivo in sheep (16). Data presented in this paper indicate that chronic administration of hGH influences expression of the ß3-AR in adipose tissue in the ob/ob mouse. In brown adipose tissue (BAT), these compounds also increase expression of ß3-AR expression in the lean C57BL/6J mouse. The increase in expression induced by chronic hGH or AOD9604 treatment correlated with the decrease in adipose tissue mass. We therefore hypothesize that treatment with either hGH or AOD9604 enhances ß3-AR expression, which has been observed in murine 3T3-F442A and human SK-N-MC cells in vitro (11).
Both AOD9604 and, to a greater extent, hGH increase body weight in lean mice, compared with saline-treated animals. This is in the absence of an increase in fat mass, which suggests an increase in lean body mass occurs with these compounds. This supports previous work with hGH in rodents and humans (17). Both compounds have also been previously shown to reduce body weight and adiposity in obese mice (11). The effects of hGH and AOD9604 occur without significant changes to caloric intake. It has been reported that hGH increases, reduces, or does not change food intake in which the differences are attributed to variations in hGH preparations, concentrations, and animals used between different laboratories.
The effects of hGH and AOD9604 on fat metabolism may be mediated by an alteration in the expression of a lipolytic/antilipogenic gene. The ß3-AR is a major lipolytic receptor identified in rodent fat cells (18) that mediates its effects through G protein coupling to adenylate cyclase, generation of cAMP, and stimulation of PKA (19). This enzyme then phosphorylates proteins in the lipolytic cascade, including hormone-sensitive lipase (20). In BAT, the ß3-AR stimulates uncoupling of the electron transport chain, enhancing the ability of mitochondria to generate heat in preference to ATP through the dissipation of the electron gradient (21). Mice that lack this receptor have lower rates of resting energy expenditure (0.0041 vs. 0.0047 kcal/min, P < 0.02) and lower rates of fat oxidation (0.00019 vs. 0.00030 g/min, P < 0.02) than control mice (data not shown).
AOD9604 and hGH appear to act in a similar manner to induce their effects on body weight regulation and adipose tissue mass in vivo. However, in vitro studies have demonstrated a number of differences suggesting that the two compounds operate via unique signaling pathways to control the regulation of the ß3-AR. These studies suggested that AOD9604 had no interaction with the ß3-AR or hGH receptors (11).
In lean animals, neither AOD9604 nor hGH had any effect on epididymal white adipose tissue mass or expression of ß3-AR RNA, indicating that in lean animals, this fat tissue is not a major target for these drugs in this study. In contrast, the mass of BAT in lean animals was reduced by both hGH and AOD9604, and ß3-AR RNA expression was increased by both these compounds. This could possibly suggest that the increased expression of ß3-ARs in brown adipocytes sensitizes catecholamines to dissipate heat.
In ob/ob mice, both AOD9604 and hGH reduced both white and brown adipose tissue mass and increased ß3-AR RNA expression. This suggested that an elevation in ß3-AR RNA expression is associated with increased fat metabolism and a reduction in the fat tissue mass in the ob/ob mouse model. Obese mice have lower levels of ß3-AR expression in their adipose tissues than lean mice, shown in this study and others (14). The ability of AOD9604 and hGH to increase the level of ß3-AR RNA expression in obese mice to a level that is comparable to those in lean mice is an exciting finding. However, it must also be considered that both hGH and AOD9604 may influence the expression of other members of the adrenergic pathway, such as the ß1-ARs, hormonesensitive lipase, and signaling proteins, which are all expressed in adipose tissue and associated with lipolysis. The importance of the change in ß3-AR expression with AOD9604 and hGH in humans is not established and will depend on the use of potent and selective ß3-AR agonists that are active at the human receptor.
From this study it appears that the ß3-AR is an important contributor to the effects observed on body weight in obese mice treated with AOD9604 and hGH. To determine whether the ß3-AR is partly responsible for this effect, we examined the effects of AOD9604 and hGH in the ß3-KO mouse. The ß3-KO mouse is not grossly obese, but female mice have increased fat depots (21) and the mice do develop late-onset obesity (Summers, R. J., personal communication). AOD9604 and hGH increased body mass and decreased BAT mass in the WT strain but had no effect in the KO animals. In WT mice, plasma glycerol was increased in response to AOD9604 and hGH treatment (4 wk). However, in the KO mice, only hGH resulted in increased levels of glycerol in the KO mice, and this effect was significantly less than that observed in the WT mice. This suggests that the regulation of the ß3-AR is essential in the ability of AOD9604 and hGH to mediate chronic effects on lipolysis and fat mass reduction.
The effect of AOD9604 and hGH on ß3-ARs in adipose tissue is believed to be a direct action of these compounds and not an effect secondary to the fat metabolism, given that both AOD9604 and hGH can influence ß3-AR expression and function in a nonadipocyte human cell line (11). Hence, the ß3-AR appears to be necessary for the chronic effectiveness of AOD9604 on lipolysis in BAT.
The acute effect of AOD9604 and BRL37344 (a ß3-AR agonist) on energy expenditure and substrate oxidation rates in WT and KO mice was also assessed. KO animals had lower energy expenditure, lower fat oxidation, and increased glucose oxidation, compared with the WT controls (data not shown). Injection of WT mice with a single dose of BRL37344 or AOD9604 increased energy expenditure and fat oxidation and decreased glucose oxidation. In the KO animals, BRL37344 failed to elicit any response in these metabolic parameters, clearly demonstrating that its effects are mediated exclusively through the ß3-AR. AOD9604 did elicit a response in the KO mice, increasing fat oxidation and energy expenditure, although the response was not as great as in WT mice, suggesting that ß3-ARs are not responsible for the acute biological response of AOD9604 on lipid metabolism. This is consistent with our previous findings in which AOD9604 was shown not to bind to the ß3-AR (11). The size and duration of the metabolic responses to AOD9604 in the ß3-AR KO animals was different from that observed in the control wild-type mice. The response was more rapid, shorter in duration, and greater in peak response. This may be because the KO animals are more acutely sensitive to lipolytic agents, a compensation for the ablation of the major lipolytic receptor.
These findings suggest that the acute effects of AOD9604 are quite different from the chronic effects. Enhanced ß3-AR expression appears to play a major role in the chronic effectiveness of the compound in terms of fat metabolism and weight loss. The acute effects observed in this study confirm that the ß3-AR is not the sole mediator of this action. The increase in ß3-AR expression in response to hGH and AOD9604 would permit enhanced lipolytic sensitivity. Identification of the components of the intracellular pathway(s) and effector(s) activated by AOD9604 are currently being investigated. The results presented in this paper suggest that the effectiveness of AOD9604 and hGH may partly rely on their ability to increase levels of ß3-AR RNA expression in models of obesity in which the numbers of the lipolytic receptor are low. These unique properties may give AOD9604 an advantage over other lipolytic agonists such as adrenergic agents and hGH, which exhibit undesirable side effects when administered chronically (22)