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From GH to Billy Ghrelin
Cell Metabolism
Previews From GH to Billy Ghrelin Roy G. Smith1,* 1Department of Metabolism and Aging, The Scripps Research Institute Florida, 130 Scripps Way 3B3, Jupiter, FL 33458, USA *Correspondence: [email protected] DOI 10.1016/j.cmet.2009.07.009
The octanoylated peptide hormone ghrelin regulates episodic growth hormone release and energy balance. Work in genetically modified mice (Kirchner et al., 2009) now shows that in vivo activity of ghrelin O-acyltransferase (GOAT), responsible for ghrelin octanoylation, decreases during fasting but increases after ingesting medium-chain fatty acid triglycerides (MCT). Identification of an orphan receptor (GHSR1a) that mediates the action of synthetic molecules designed to rejuvenate the growth hormone (GH) axis in elderly humans subsequently led to identification of a natural GHS-R1a agonist called ghrelin (Smith et al., 1997; Kojima et al., 1999). Ghrelin is an octanoylated 28residue peptide that modulates important physiological functions, including appetite, glucose homeostasis, aging of the GH axis, immune function, and energy balance. Without the octanoate modification on serine-3, the ghrelin peptide neither binds to nor activates its receptor (GHS-R1a); at this time, no other biological function has been ascribed to this unique modification. All attempts to increase ghrelin levels by overexpressing the ghrelin gene (Ghrl) in transgenic mice produced high levels of des-acyl ghrelin (DAG) without increasing active ghrelin; hence, the in vivo concentration of the acylating enzyme appeared rate limiting. Last year, two groups independently identified the acyltransferase that selectively esterifies DAG to produce ghrelin (Yang et al., 2008; Gutierrez et al., 2008). The enzyme MBOAT4, or ghrelin O-acyltransferase (GOAT), is 1 of a 16 member family of membrane-bound O-acyltransferases (MBOATs). New work from Kirschner et al. extends these findings by demonstrating in genetically modified mice that ghrelin production in vivo is dependent upon the concentration of Mboat4 and that food intake regulates Mboat4 expression. The investigators first asked what effect feeding and fasting (12–36 hr) would have on Mboat4 and Ghrl gene expression. Intriguingly, expression of Mboat4 was found to be highest during ad lib feeding and declined to a minimum following a 12 hr fast. This was a surprise because
ghrelin levels surge shortly before a meal, and it was anticipated that GOAT expression would parallel ghrelin secretion. An increase in DAG/ghrelin ratio accompanied fasting, consistent with the reduction in Mboat4 mRNA levels. The increase in Mboat4 expression following ingestion of food explains the delay in observing increases in circulating ghrelin after the first meal following a fast. In fasted mice that were fed the GOAT substrate glyceryl trioctanoate, 3 hr passed before active ghrelin was significantly increased in the stomach (Nishi et al., 2005). Based on reports that ghrelin administration increases food intake and fat accumulation in rodents, the authors tested whether Mboat4 / mice, which cannot produce active ghrelin, are resistant to diet-induced obesity. During ad lib feeding, Mboat4 / mice and wild-type mice consumed identical amounts of normal chow and exhibited identical body weights and body composition. Similarly, when fed a high-fat diet for 8 weeks, the Mboat4 / mice again exhibited food intake and body composition identical to wild-types. Although the lack of resistance to dietinduced obesity may seem surprising, it is consistent with results from studies in congenic ghrelin / and Ghsr / mice that were fed normal chow and high-fat diets (Sun et al., 2008). However, when wildtype and Mboat4 / mice were fed a diet enriched in MCT, wild-type mice ate less, became heavier, and deposited more fat compared to Mboat4 / mice. This result indicates that inhibiting fat accumulation by blocking ghrelin action is highly dependent upon composition of the diet, and medium-chain fatty acid triglycerides play a unique role. To investigate the effects of producing supraphysiologic levels of ghrelin, trans-
82 Cell Metabolism 10, August 6, 2009 ª2009 Elsevier Inc.
genic mice (Tg) were generated containing human GHRL and human MBOAT4 under the control of the human APOE promoter in the liver. Surprisingly, these mice did not produce active human ghrelin (GHRL) until they were fed a diet supplemented with MCT. On the MCT diet, the Tg mice produced 30-fold higher levels of active human GHRL compared to levels of endogenous mouse Ghrl in wild-type mice. This resulted in higher body weight and greater fat mass. Food intake was unaffected in the Tg mice, but energy expenditure was reduced. Overall, the differences were consistent with reduced fat oxidation in the Tg mice. However, when the Tg mice were switched back to normal chow, despite overexpression of GHRL and MBOAT4, body weight and energy expenditure differences disappeared, further supporting the notion that preventing fat accumulation by blocking ghrelin action is dependent upon dietary composition. This new work by Kirchner et al. adds significantly to our understanding of the conversion of DAG to active ghrelin in vivo. The authors clearly show codependence upon MBOAT4 and dietary MCT substrate for the production of active ghrelin. Although these new data explain the lack of ghrelin production during fasting, the spontaneous rhythmic parallel rise and fall of both ghrelin and DAG occurring during ad lib feeding remains something of a mystery (Liu et al., 2008). The data do, however, reinforce the notion that circadian rhythmicity is regulated by cephalic input and not simply by ingestion of food. Studies in humans have shown that, during the first 24 hr of a fast, under conditions in which GOAT activity is likely reduced, the rhythmic rise and fall of ghrelin and DAG levels in the blood continues at declining
Cell Metabolism
Previews amplitude (Natalucci et al., 2005). Furthermore, when rats were allowed to see or smell but not eat food, ghrelin and DAG levels fell as if they had ingested food (Seoane et al., 2007). This response was blocked by atropine, suggesting that the parallel rise and fall in circulating ghrelin and DAG levels is likely controlled by a cephalic cholinergic signal at customary meal times, rather than by ingestion of food. It is exciting to anticipate the inclusion of additional parameters into the model. Future studies will undoubtedly include quantitation of MBOAT4 protein for making correlations with Mboat4 gene expression, as well as a consideration of increased GHS-R1a mRNA levels that have been observed during fasting. As with all good papers, the results beg answers to new questions. Now that we recognize the interdependence of GOAT activity and dietary MCT substrates for production of ghrelin, we must ask how independent actions of DAG fit into the puzzle? Administered centrally, DAG
stimulates feeding by activating orexin neurons, whereas ghrelin’s effect is through NPY/AGRP neurons. How do the ghrelin and DAG pathways complement each other? Is GOAT expressed in ghrelin neurons of the CNS? Effects on tissues that do not express GHS-R1a have been described for active ghrelin and DAG, but elucidation of these pathways awaits identification of the DAG receptor. Though questions remain, the new data from Kirchner et al. (2009) refute the popular belief that ghrelin production is highest during fasting. The fact that neither Mboat4 expression nor Ghrl expression were augmented during fasting also argues that ghrelin production is not critical for generation of a hunger signal. REFERENCES Gutierrez, J.A., Solenberg, P.J., Perkins, D.R., Willency, J.A., Knierman, M.D., Jin, Z., Witcher, D.R., Luo, S., Onyia, J.E., et al. (2008). Proc. Natl. Acad. Sci. USA 105, 6320–6325.
Kirchner, H., Gutierrez, J.A., Solenberg, P.J., Pfluger, P.T., Czyzyk, T.A., Willency, J.A., Schu¨rmann, A., Joost, H.G., Jandacek, R.J., Hale, J.E., et al. (2009). Nat. Med. 15, 741–745. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., and Kangawa, K. (1999). Nature 402, 656–660. Liu, J., Prudom, C.E., Nass, R., Pezzoli, S.S., Oliveri, M.C., Johnson, M.L., Veldhuis, P., Gordon, D.A., Howard, A.D., Witcher, D.R., et al. (2008). J. Clin. Endocrinol. Metab. 93, 1980–1987. Natalucci, G., Riedl, S., Gleiss, A., Zidek, T., and Frisch, H. (2005). Eur. J. Endocrinol. 152, 845–850. Nishi, Y., Hiejima, H., Hosoda, H., Kaiya, H., Mori, K., Fukue, Y., Yanase, T., Nawata, H., Kangawa, K., and Kojima, M. (2005). Endocrinology 146, 2255–2264. Seoane, L.M., Al-Massadi, O., Caminos, J.E., Tovar, S.A., Dieguez, C., and Casanueva, F.F. (2007). Endocrinology 148, 3998–4006. Smith, R.G., Van der Ploeg, L.H., Howard, A.D., Feighner, S.D., Cheng, K., Hickey, G.J., Wyvratt, M.J., Jr., Fisher, M.H., Nargund, R.P., and Patchett, A.A. (1997). Endocr. Rev. 18, 621–645. Sun, Y., Butte, N.F., Garcia, J.M., and Smith, R.G. (2008). Endocrinology 149, 843–850. Yang, J., Brown, M.S., Liang, G., Grishin, N.V., and Goldstein, J.L. (2008). Cell 132, 387–396.
Cell Metabolism 10, August 6, 2009 ª2009 Elsevier Inc. 83
Previews From GH to Billy Ghrelin Roy G. Smith1,* 1Department of Metabolism and Aging, The Scripps Research Institute Florida, 130 Scripps Way 3B3, Jupiter, FL 33458, USA *Correspondence: [email protected] DOI 10.1016/j.cmet.2009.07.009
The octanoylated peptide hormone ghrelin regulates episodic growth hormone release and energy balance. Work in genetically modified mice (Kirchner et al., 2009) now shows that in vivo activity of ghrelin O-acyltransferase (GOAT), responsible for ghrelin octanoylation, decreases during fasting but increases after ingesting medium-chain fatty acid triglycerides (MCT). Identification of an orphan receptor (GHSR1a) that mediates the action of synthetic molecules designed to rejuvenate the growth hormone (GH) axis in elderly humans subsequently led to identification of a natural GHS-R1a agonist called ghrelin (Smith et al., 1997; Kojima et al., 1999). Ghrelin is an octanoylated 28residue peptide that modulates important physiological functions, including appetite, glucose homeostasis, aging of the GH axis, immune function, and energy balance. Without the octanoate modification on serine-3, the ghrelin peptide neither binds to nor activates its receptor (GHS-R1a); at this time, no other biological function has been ascribed to this unique modification. All attempts to increase ghrelin levels by overexpressing the ghrelin gene (Ghrl) in transgenic mice produced high levels of des-acyl ghrelin (DAG) without increasing active ghrelin; hence, the in vivo concentration of the acylating enzyme appeared rate limiting. Last year, two groups independently identified the acyltransferase that selectively esterifies DAG to produce ghrelin (Yang et al., 2008; Gutierrez et al., 2008). The enzyme MBOAT4, or ghrelin O-acyltransferase (GOAT), is 1 of a 16 member family of membrane-bound O-acyltransferases (MBOATs). New work from Kirschner et al. extends these findings by demonstrating in genetically modified mice that ghrelin production in vivo is dependent upon the concentration of Mboat4 and that food intake regulates Mboat4 expression. The investigators first asked what effect feeding and fasting (12–36 hr) would have on Mboat4 and Ghrl gene expression. Intriguingly, expression of Mboat4 was found to be highest during ad lib feeding and declined to a minimum following a 12 hr fast. This was a surprise because
ghrelin levels surge shortly before a meal, and it was anticipated that GOAT expression would parallel ghrelin secretion. An increase in DAG/ghrelin ratio accompanied fasting, consistent with the reduction in Mboat4 mRNA levels. The increase in Mboat4 expression following ingestion of food explains the delay in observing increases in circulating ghrelin after the first meal following a fast. In fasted mice that were fed the GOAT substrate glyceryl trioctanoate, 3 hr passed before active ghrelin was significantly increased in the stomach (Nishi et al., 2005). Based on reports that ghrelin administration increases food intake and fat accumulation in rodents, the authors tested whether Mboat4 / mice, which cannot produce active ghrelin, are resistant to diet-induced obesity. During ad lib feeding, Mboat4 / mice and wild-type mice consumed identical amounts of normal chow and exhibited identical body weights and body composition. Similarly, when fed a high-fat diet for 8 weeks, the Mboat4 / mice again exhibited food intake and body composition identical to wild-types. Although the lack of resistance to dietinduced obesity may seem surprising, it is consistent with results from studies in congenic ghrelin / and Ghsr / mice that were fed normal chow and high-fat diets (Sun et al., 2008). However, when wildtype and Mboat4 / mice were fed a diet enriched in MCT, wild-type mice ate less, became heavier, and deposited more fat compared to Mboat4 / mice. This result indicates that inhibiting fat accumulation by blocking ghrelin action is highly dependent upon composition of the diet, and medium-chain fatty acid triglycerides play a unique role. To investigate the effects of producing supraphysiologic levels of ghrelin, trans-
82 Cell Metabolism 10, August 6, 2009 ª2009 Elsevier Inc.
genic mice (Tg) were generated containing human GHRL and human MBOAT4 under the control of the human APOE promoter in the liver. Surprisingly, these mice did not produce active human ghrelin (GHRL) until they were fed a diet supplemented with MCT. On the MCT diet, the Tg mice produced 30-fold higher levels of active human GHRL compared to levels of endogenous mouse Ghrl in wild-type mice. This resulted in higher body weight and greater fat mass. Food intake was unaffected in the Tg mice, but energy expenditure was reduced. Overall, the differences were consistent with reduced fat oxidation in the Tg mice. However, when the Tg mice were switched back to normal chow, despite overexpression of GHRL and MBOAT4, body weight and energy expenditure differences disappeared, further supporting the notion that preventing fat accumulation by blocking ghrelin action is dependent upon dietary composition. This new work by Kirchner et al. adds significantly to our understanding of the conversion of DAG to active ghrelin in vivo. The authors clearly show codependence upon MBOAT4 and dietary MCT substrate for the production of active ghrelin. Although these new data explain the lack of ghrelin production during fasting, the spontaneous rhythmic parallel rise and fall of both ghrelin and DAG occurring during ad lib feeding remains something of a mystery (Liu et al., 2008). The data do, however, reinforce the notion that circadian rhythmicity is regulated by cephalic input and not simply by ingestion of food. Studies in humans have shown that, during the first 24 hr of a fast, under conditions in which GOAT activity is likely reduced, the rhythmic rise and fall of ghrelin and DAG levels in the blood continues at declining
Cell Metabolism
Previews amplitude (Natalucci et al., 2005). Furthermore, when rats were allowed to see or smell but not eat food, ghrelin and DAG levels fell as if they had ingested food (Seoane et al., 2007). This response was blocked by atropine, suggesting that the parallel rise and fall in circulating ghrelin and DAG levels is likely controlled by a cephalic cholinergic signal at customary meal times, rather than by ingestion of food. It is exciting to anticipate the inclusion of additional parameters into the model. Future studies will undoubtedly include quantitation of MBOAT4 protein for making correlations with Mboat4 gene expression, as well as a consideration of increased GHS-R1a mRNA levels that have been observed during fasting. As with all good papers, the results beg answers to new questions. Now that we recognize the interdependence of GOAT activity and dietary MCT substrates for production of ghrelin, we must ask how independent actions of DAG fit into the puzzle? Administered centrally, DAG
stimulates feeding by activating orexin neurons, whereas ghrelin’s effect is through NPY/AGRP neurons. How do the ghrelin and DAG pathways complement each other? Is GOAT expressed in ghrelin neurons of the CNS? Effects on tissues that do not express GHS-R1a have been described for active ghrelin and DAG, but elucidation of these pathways awaits identification of the DAG receptor. Though questions remain, the new data from Kirchner et al. (2009) refute the popular belief that ghrelin production is highest during fasting. The fact that neither Mboat4 expression nor Ghrl expression were augmented during fasting also argues that ghrelin production is not critical for generation of a hunger signal. REFERENCES Gutierrez, J.A., Solenberg, P.J., Perkins, D.R., Willency, J.A., Knierman, M.D., Jin, Z., Witcher, D.R., Luo, S., Onyia, J.E., et al. (2008). Proc. Natl. Acad. Sci. USA 105, 6320–6325.
Kirchner, H., Gutierrez, J.A., Solenberg, P.J., Pfluger, P.T., Czyzyk, T.A., Willency, J.A., Schu¨rmann, A., Joost, H.G., Jandacek, R.J., Hale, J.E., et al. (2009). Nat. Med. 15, 741–745. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., and Kangawa, K. (1999). Nature 402, 656–660. Liu, J., Prudom, C.E., Nass, R., Pezzoli, S.S., Oliveri, M.C., Johnson, M.L., Veldhuis, P., Gordon, D.A., Howard, A.D., Witcher, D.R., et al. (2008). J. Clin. Endocrinol. Metab. 93, 1980–1987. Natalucci, G., Riedl, S., Gleiss, A., Zidek, T., and Frisch, H. (2005). Eur. J. Endocrinol. 152, 845–850. Nishi, Y., Hiejima, H., Hosoda, H., Kaiya, H., Mori, K., Fukue, Y., Yanase, T., Nawata, H., Kangawa, K., and Kojima, M. (2005). Endocrinology 146, 2255–2264. Seoane, L.M., Al-Massadi, O., Caminos, J.E., Tovar, S.A., Dieguez, C., and Casanueva, F.F. (2007). Endocrinology 148, 3998–4006. Smith, R.G., Van der Ploeg, L.H., Howard, A.D., Feighner, S.D., Cheng, K., Hickey, G.J., Wyvratt, M.J., Jr., Fisher, M.H., Nargund, R.P., and Patchett, A.A. (1997). Endocr. Rev. 18, 621–645. Sun, Y., Butte, N.F., Garcia, J.M., and Smith, R.G. (2008). Endocrinology 149, 843–850. Yang, J., Brown, M.S., Liang, G., Grishin, N.V., and Goldstein, J.L. (2008). Cell 132, 387–396.
Cell Metabolism 10, August 6, 2009 ª2009 Elsevier Inc. 83