Introduction

Ghrelin is a 28 amino-acid orexigenic peptide released in large amounts by the stomach and to a lesser extent by the pancreas, kidneys, liver, pituitary and hypothalamus.^{1, 2} To be active, ghrelin needs to be modified by addition of an n-octanoyl group on the serine 3 residue.² This post-translational modification is essential for ghrelin to produce its biological effects through the growth hormone secretagogue receptor (GHS-R),³ which is mainly expressed in the pituitary, hypothalamus, vagal nodose ganglia, heart, pancreas and liver.^{1, 4}

Downstream effects of ghrelin seem to be mediated through central pathways, since exogenous administration of ghrelin, either centrally or peripherally, leads to the activation of orexigenic peptides, such as neuropeptide Y.^{5, 6, 7} In addition to its major role in regulation of food intake, ghrelin has also been reported to regulate gastric emptying⁸ and gastric acid secretion⁹ as well as secretion of growth hormone.^{4, 10, 11} In lean rodents, ghrelin levels have been reported to be increased by fasting.^{12, 13} Similarly, circulating ghrelin levels were found to be elevated before meals in humans, suggesting an important role for ghrelin in meal initiation.^{14, 15} Chronic administration of ghrelin in rodents led to significant increase in body weight^{5, 12, 16} as well as hyperphagia.^{5, 11, 16} This increased body weight was mostly explained by the accumulation of fat and elevated respiratory quotient.^{12, 16}

Interestingly, in obese humans and rodents, circulating ghrelin levels are significantly reduced as compared to lean individuals.^{17, 18, 19} Importantly, those levels inversely correlate with BMI and fasting insulin levels²⁰ and are restored upon weight loss.²¹ These results may indicate that circulating ghrelin levels are decreased in obese individuals in order to limit its effect on food intake. To date, there are no available data on the orexigenic effects of ghrelin administration in obese when compared to lean individuals. To test this hypothesis, we examined the effect of diet-induced obesity on circulating ghrelin levels. In addition, we investigated if obesity and dietary fat may affect the ability of ghrelin to increase food intake.

Methods

Animals

Lean C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). At 4 weeks of age, mice were fed a chow diet (Purina #5001, 4.5% kcal fat; n=48), a low-fat (n=132; LFD: 10% kcal fat (D12450B) from Research Diet, NJ, USA) or a high-fat diet (n=172; HFD: 60% kcal fat (D12492) from Research Diet, NJ, USA) for 16 weeks. DIO stands for diet-induced obesity. According to the manufacturer, the chow diet provided 3.04 kcal/g as energy, of which 49.9% were from carbohydrate, 23.4% from protein and 4.5% from fat. The LFD offered 3.8 kcal/g as energy, of which 70% were from carbohydrate, 20% from protein and 10% from fat. The HFD 5.2 kcal/g consisted of 25.8% (wt/wt) protein (casein), 31.7% lard (54% of energy), 3.2% soybean oil (5.5% of energy), 8.9% sucrose (6.7% of energy), 16.2% maltodextrin (12.3% of energy) and 6.5% cellulose. The HFD was supplemented with 1.3% vitamin mixture and 1.3% mineral mix. Mice were individually housed in a regular environment with controlled temperature and humidity and 12:12 h light/dark cycle (light on at 1800). Food and water were available ad libitum except otherwise indicated. All the animal protocols were approved by the Millennium Pharmaceuticals Institutional Animal Care and Use Committee (IACUC) and were in accordance with the National Research Council.

Ghrelin secretion in dietary obese mice

Ghrelin secretion during the light/dark cycle

A total of 44 DIO and 44 lean mice were used for this experiment. All mice had free access to food. Starting at the beginning of the light phase, four mice of each group were euthanized at different time points (every 2 h for 24 h), blood was collected and plasma extracted.

Ghrelin secretion during fasting and refeeding

A total of 40 DIO and 40 lean mice were used for this experiment. All mice were food deprived for 16 h starting at the end of the light phase (1800) and then refed with their respective diet. Four mice of each group were euthanized at different time points (0, 8 and 16 h after fasting and 1, 2, 4, 8 and 24 h after refeeding), blood was collected and plasma extracted.

Ghrelin challenge in lean and DIO mice

In total, 48 lean mice fed chow diet, 48 lean mice fed on LFD and 48 DIO mice fed on HFD were used for this experiment. At the beginning of the light phase, animals were injected intraperitonealy (i.p., 200 l, n=8 per group) with saline or different doses of ghrelin (3, 10, 30, 100, 300 or 600 nmol/mouse). Food intake was recorded 30 min, 1, 2, 4, 6 and 8 h after injection. Ghrelin used for these experiments was either purchased at Biopeptide Co. (San Diego, CA, USA) or provided by Paul Richardson at Abbott Laboratories (Abbott Park, IL, USA).

Ghrelin challenge in DIO mice switched from HFD to LFD

For this experiment, 20 DIO mice were maintained on HFD and 20 DIO mice were switched to LFD, as reported previously²² with minor modifications. At the beginning of the light phase, animals were injected i.p. (200 l, n=10 per group) with saline or ghrelin (300 nmol/mouse) after 3 and 13 days on LFD. Food intake was recorded 30 min, 1, 2, 4, 6 and 8 h after injection. Ghrelin used for this experiment was provided by Paul Richardson at Abbott Laboratories (Abbott Park, IL, USA).

Body composition

Body composition was measured using dual X-ray absorptiometry (DEXA) with a Lunar Piximus (GE).

Ghrelin synthesis and measurement

The bioactive form of ghrelin (O-n-octanoyl-[Ser³]) was synthesized by BioPeptide Co. (San Diego, CA, USA). Endogenous ghrelin was measured in plasma using a commercial radioimmunoassay (Phoenix Pharmaceuticals, CA, USA). The lower limit of detection was 0.1 ng/ml and the intra-assay variability was 1%.

Statistics

Statistical analyses were carried out using analysis of variance and post hoc group comparisons (Tukey). Probability values less than 5% were considered significant.

Results

Effects of HFD feeding in mice

The effects of high-fat feeding on body weight and body fat in mice is indicated in Figure 1. After 20 weeks, mice fed a LFD gained approximately 17 g whereas mice fed a HFD gained 31 g of body weight. Body composition studies using DEXA indicated that the extra weight gain in HFD fed mice was mostly due to accumulation of body fat.

Figure 1.

Effects of HFD on body weight and body fat in mice. Mice were fed a LFD or a HFD for 20 weeks (see Materials and methods for details). Body weights were measured at 4, 8 and 20 weeks. Body fat mass was determined after 8 weeks of HFD. ^*P<0.05 vs lean.

Full figure and legend (23K)

Ghrelin secretion in dietary obese mice

Ghrelin secretion during the light/dark cycle

As shown in Figure 2a, we observed a diurnal secretion of ghrelin with two major peaks (P<0.001) occurring in free fed lean mice. The first peak occurred 2 h before light onset and the amplitude of the peak was 1.50 ng/ml. The second peak occurred during the transition from light to dark and its amplitude was more pronounced than the first one (1.82 ng/ml). A third peak was also observed between 0800 and 2400 but no significant difference was observed. In DIO mice, the first peak persisted with lower amplitude (0.79 ng/ml). However, the second peak almost disappeared. Interestingly, overall ghrelin levels during the light and the dark phases were lower in DIO mice when compared to lean animals (light phase: 1.230.24 ng/ml in lean and 0.860.18 ng/ml in DIO. Dark phase: 0.950.3 ng/ml in lean and 0.630.15 ng/ml in DIO).

Figure 2.

Ghrelin secretion in dietary obese mice. (a) Diurnal pattern of plasma ghrelin levels in low-fat (lean) and high-fat (DIO) diets fed mice. ^**P<0.001. (b) Circulating ghrelin levels in lean and DIO mice during fasting and refeeding. ^*P<0.05 vs time 1800 from respective group.

Full figure and legend (48K)

Ghrelin secretion during fasting and refeeding

The effect of fasting and refeeding on circulating ghrelin in both lean and DIO mice is shown in Figure 2b. In this experiment, we observed basal ghrelin levels to be similar between lean and DIO mice. However, 16 h fasting significantly increased ghrelin levels by 2.1-fold in lean animals. Immediately after refeeding (within 1 h), ghrelin levels returned to the initial value. Surprisingly, the same fasting conditions had no significant effect on ghrelin secretion in the DIO mice but refeeding decreased ghrelin levels in those animals with a slower onset (significant effect observed only 12 h after refeeding).

Ghrelin challenge in lean and DIO mice

When injected in 8-week old lean C57BL/6 mice fed a regular chow diet, ghrelin (i.p.) induced a dose-dependent and long-lasting increase in food intake (Figure 3a). We found that the minimal dose of ghrelin having an effect at 2 h was 100 nmol/mouse. When injected in 20 weeks old lean C57BL/6 fed on a LFD, ghrelin (i.p.) also induced a dose-dependent increase in food intake (Figure 3b). In these animals, the minimal dose of ghrelin having an effect at 2 h was similar. However, in age-matched DIO mice fed on HFD, ghrelin had no effect on food intake. EC₅₀s (half effective dose) and E_max (maximal effect) were the following: the EC₅₀s for chow lean LFD were 166 and 32 nmol, respectively. The E_max were 4.5 and 2.4 kcal, respectively. No effects on food intake were observed in the DIO mice.

Figure 3.

Ghrelin challenge in lean and DIO mice. (a) Effects of i.p. administered ghrelin (3–300 nmol/mouse) on cumulative food intake in ad lib chow fed lean mice. ^*P<0.05 as compared to saline-treated group. (b) Comparisons of the orexigenic effects of ghrelin administered i.p. in chow fed, low-fat (LF) or high-fat (DIO) diets fed mice. Cumulative food intake for the first 2 h is represented.

Full figure and legend (49K)

Ghrelin challenge in DIO mice switched to LFD

As expected, the DIO mice switched to LFD lost weight when compared to the ones maintained on HFD (Figure 4a). As indicated in Figure 4b, we observed that 3 days of LFD feeding restored ghrelin sensitivity in the DIO mice, as demonstrated by the feeding response of LFD mice to 300 nmol/mouse of ghrelin. This effect was even more pronounced after 14 days on LFD (Figure 4b; 376 vs 185%).

Figure 4.

Dietary modulation of ghrelin sensitivity in obese mice. (a) Body weight of DIO mice (HFD) and DIO switched to a low-fat diet (LFD) for 13 days. Body weights were measured at 3, 8 and 13 days. P<0.05 vs HFD. (b) Orexigenic effects of ghrelin (300 nmol/mouse) of HFD and LFD at 3 and 13 days. The Cumulative food intake for the first 2 h following ghrelin administration is represented. P<0.05 as compared to saline-treated mice from each group.

Full figure and legend (47K)

Discussion

Effects of dietary-induced obesity on circulating ghrelin in mice

In order to determine the diurnal regulation of circulating ghrelin in both lean and DIO mice, we measured ghrelin levels in free fed animals during 24 h. As expected, exposure of lean animals to HFD for 16 weeks induced obesity.^{23, 24} It has been reported previously that similar diet (60% kcal from fat) increased significantly body weight, adipose tissue mass as well as plasma leptin levels, without affecting muscle mass.²³ We observed a diurnal variation in the secretion of ghrelin in free fed lean mice with two major peaks occurring during the transition of phases. The first peak occurred between 1600 and 2000 hours, at a time during which mice normally have a large food intake.²⁵ The second peak occurred at the end of the dark period and was proposed to be responsible for gastric acid secretion.¹³ This pattern is very similar to the diurnal rhythm of ghrelin secretion observed in lean humans and animals.^{13, 15} Murakami et al have reported that ghrelin is secreted with two major peaks occurring at 1500 during the light phase and 0600 during the dark phase. Although the first peak appeared earlier in their study, the general secretion pattern was about the same, where ghrelin peaks at the end of both the light and dark periods. In the contrary, the light-to-dark transition induced peak almost disappeared in DIO mice. Interestingly, ghrelin levels during the light and the dark phases were lower in DIO mice when compared to lean animals (P<0.05), as previously described in obese humans and rats.^{13, 19, 26} These results would indicate that not only ghrelin secretion is reduced in obese mice but that its diurnal regulation is lost. It could also indicate that meal initiation is not preceded by a rise in ghrelin in DIO mice. To test this hypothesis, we investigated the regulation of circulating ghrelin by feeding states in both lean and DIO mice. We observed that basal ghrelin levels were similar between lean and DIO mice before feeding (1800 in Figure 2b). This similarity may be explained by the time at which the bleedings were performed. The basal value (before fasting) was measured at 1800 and comparable ghrelin levels between lean and DIO ad-lib fed mice were also observed at 1800 in the diurnal regulation of circulating ghrelin (Figure 2a). As recently published,^{12, 13} we found that fasting significantly increased ghrelin levels in lean mice. However, in DIO mice, we failed to observe any significant change in ghrelin secretion upon fasting. This disregulation of ghrelin secretion by fasting in obesity was also reported previously in Zucker rats.¹⁷ Ariyasu et al reported that ghrelin secretion was delayed upon fasting in Zucker fa/fa rats as compared to control rats. In fact, 24 h fasting significantly increased ghrelin levels only in lean rats whereas at least 48 h fasting was required to increase ghrelin in obese fa/fa rats. This could indicate that ghrelin levels in DIO mice would be increased upon longer fasting. The delay in ghrelin secretion upon fasting in obese animals may be partly explained by an excess of energy deposit.

Effects of dietary-induced obesity on ghrelin sensitivity in mice

It is known that exogenous ghrelin, when administered centrally or peripherally, has an orexigenic effect in rats and mice.^{5, 8, 11} We speculated that the decrease in circulating ghrelin observed in the DIO mice could be a regulatory phenomenon in order to limit its effect on food intake. To test this hypothesis, we injected ghrelin peripherally in lean and DIO animals, fed a LFD and a HFD, respectively. When injected in chow fed lean C57BL/6 mice (9-week old), ghrelin induced a dose-dependent increase in food intake, with a minimal effective dose of 100 nmol/mouse. The effect on food intake lasted for at least 8 h. When the same experiment was conducted in LFD mice (20-week old), ghrelin produced also a dose-dependent effect on food intake, with a similar minimal effective dose, albeit with a lower magnitude of the feeding response. However, when injected in age-matched DIO mice (20-week old), we found that ghrelin did not increase food intake at doses up to 600 nmol/mouse, indicating that the DIO animals had lost their sensitivity to exogenously administered ghrelin.

The efficacious doses of ghrelin used for our studies were much higher than those reported to be effective in the literature. It was shown that ghrelin as low as 10 g significantly increased cumulative food intake in mice,^{8, 27} producing an increase in food intake as small as 50 mg. In our case, higher doses of ghrelin had to be used to induce a reliable and measurable effect. Another factor that can explain the differences observed between our study and those reported in the literature is the strain of mice used in the experiment. We have conducted our experiment in C57BL/6J mice, whereas they performed their studies using ddy mice.

Dietary modulation of ghrelin sensitivity in obese mice

In order to determine whether obesity may be responsible for the observed decrease in ghrelin sensitivity in DIO mice, we measured the effect of peripherally injected ghrelin on food intake in DIO mice and DIO mice switched to a LFD for a short (3 days) and an extended period of time (13 days). As expected, when switched to LFD, the animals readjusted their body weight. Our results indicate that 3 days of LFD feeding were enough to restore the positive effect of peripherally injected ghrelin on food intake. This effect was even more pronounced after 13 days on LFD, when body weight was at a level comparable to lean mice fed on a LFD. These results indicate that dietary-induced obesity in mice is accompanied by a state of resistance to the peripheral effect of ghrelin on feeding and that weight loss can restore the sensitivity to ghrelin. Moreover, the rapid reversal of ghrelin insensitivity in DIO mice suggests that not only obesity but also dietary fat content might be responsible for the decrease in ghrelin sensitivity observed in the obese animals.

In this paper, we focused on the effects of ghrelin when injected peripherally. We hypothesized that peripheral effects of ghrelin are important since ghrelin is synthesized in the stomach and released in the circulation and also because circulating ghrelin levels are regulated under conditions of negative (fasting, anorexia) or positive (obesity, feeding) energy balance.^{6, 12, 18} It has been demonstrated that C57BL/6J mice develop a resistance to peripheral administrated leptin when fed an HFD,^{23, 28} concomitantly to an increase in food intake when compared to LFD fed mice.²³ Interestingly, in dietary obese rodents, circulating ghrelin levels are negatively correlated to those of leptin¹⁹ and both hormones levels are sensitive to the fat content of the diet.^{19, 29} Moreover, leptin administration in mice has been demonstrated to induce a decrease in ghrelin expression.⁸ There is evidence that ghrelin signals are integrated in the hypothalamus,^{30, 31} at the level of leptin-sensitive neurons,³¹ and more probably via neuropeptide Y/AGRP and/or orexin pathway.^{32, 33, 34} These two pathways are modulated by obesity in mice^{35, 36} and their orexigenic effect are modulated by leptin.^{35, 37} Moreover, the effects of ghrelin on food intake are blocked by neuropeptide Y Y1 and Y5 antagonists^{38, 39} and are partially blunted in the orexin knockout mice.³³ Altogether, these results may suggest that NPY and orexin pathways could be implicated in the reduced sensitivity of dietary obese mice to orexigenic effects of ghrelin. One possible explanation for the reduced ghrelin sensitivity observed in DIO mice could be that the increased leptin levels lead to the negative regulation of the NPY/AGRP pathway. Leptin blocks NPY/AGRP in order to reduce food intake but, by the same way, prevents ghrelin's orexigenic effects. However, after weight loss, leptin levels are reduced and NPY/AGRP pathway is no longer inhibited, thus ghrelin sensitivity is restored.

On the other hand, it has been proposed that circulating ghrelin is acting through ghrelin receptors located in nodose gangliosa of the vagal nerve.⁴ Moreover, recent data showed that peripheral, but not central, administration of ghrelin loses its orexigenic effect in vagotomized animals.^{4, 8} These results suggest that ghrelin may act through two different mechanisms to stimulate food intake: one central and one peripheral. Whether the reduced sensitivity to the effect of ghrelin in DIO mice is mediated by one or the other (or both) is not known. Further experiments are underway to investigate the respective role of these two sites in ghrelin insensitivity in the DIO mice.

In conclusion, our results indicate that HFD-induced obesity in mice is associated with a decrease in the circulating levels of ghrelin and a loss of regulation by the nycthemeral cycle and the feeding status. We also demonstrate here for the first time that these effects are associated with an unexpected decrease in ghrelin sensitivity in obese animals. Furthermore, our results indicate that the sensitivity to ghrelin can be restored during weigh loss, suggesting that dietary fat content could modulate ghrelin sensitivity in rodents.

References

1. Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, Korbonits M. The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 2002; 87: 2988. | Article | PubMed | ISI | ChemPort |
2. Hosoda H, Kojima M, Matsuo H, Kangawa K. Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res Commun 2000; 279: 909–913. | Article | PubMed | ISI | ChemPort |
3. Yoshihara F, Kojima M, Hosoda H, Nakazato M, Kangawa K. Ghrelin: a novel peptide for growth hormone release and feeding regulation. Curr Opin Clin Nutr Metab Care 2002; 5: 391–395. | Article | PubMed |
4. Date Y, Murakami N, Toshinai K, Matsukura S, Niijima A, Matsuo H, Kangawa K, Nakazato M. The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology 2002; 123: 1120–1128. | Article | PubMed | ISI | ChemPort |
5. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S. A role for ghrelin in the central regulation of feeding. Nature 2001; 409: 194–198. | Article | PubMed | ISI | ChemPort |
6. Bagnasco M, Tulipano G, Melis MR, Argiolas A, Cocchi D, Muller EE. Endogenous ghrelin is an orexigenic peptide acting in the arcuate nucleus in response to fasting. Regul Peptides 2003; 111: 161–167. | Article |
7. Shintani M, Ogawa Y, Ebihara K, Aizawa-Abe M, Miyanaga F, Takaya K, Hayashi T, Inoue G, Hosoda K, Kojima M, Kangawa K, Nakao K. Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes 2001; 50: 227–232. | Article | PubMed | ISI | ChemPort |
8. Asakawa A, Inui A, Kaga T, Yuzuriha H, Nagata T, Ueno N, Makino S, Fujimiya M, Niijima A, Fujino MA, Kasuga M. Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology 2001; 120: 337–345. | Article | PubMed | ISI | ChemPort |
9. Date Y, Nakazato M, Murakami N, Kojima M, Kangawa K, Matsukura S. Ghrelin acts in the central nervous system to stimulate gastric acid secretion. Biochem Biophys Res Commun 2001; 280: 904–907. | Article | PubMed |
10. Casanueva FF, Dieguez C. Ghrelin: the link connecting growth with metabolism and energy homeostasis. Rev Endocr Metab Disord 2002; 3: 325–338. | Article | PubMed | ChemPort |
11. Wren AM, Small CJ, Ward HL, Murphy KG, Dakin CL, Taheri S, Kennedy AR, Roberts GH, Morgan DG, Ghatei MA, Bloom SR. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology 2000; 141: 4325–4328. | Article | PubMed | ISI | ChemPort |
12. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000; 407: 908–913. | Article | PubMed | ISI | ChemPort |
13. Murakami N, Hauashida T, Kuroiwa T, Nakahara K, Ida T, Mondal MS, Nakazato M, Kojima M, Kangawa K. Role for central ghrelin in food intake and secretion profile of stomach ghrelin in rats. J Endocrinol 2002; 174: 283–288. | Article | PubMed | ChemPort |
14. Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, Bloom SR. Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab 2001; 86: 5992. | Article | PubMed | ISI | ChemPort |
15. Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 2001; 50: 1714–1719. | Article | PubMed | ISI | ChemPort |
16. Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR. Ghrelin causes hyperphagia and obesity in rats. Diabetes 2001; 50: 2540–2547. | Article | PubMed | ISI | ChemPort |
17. Ariyasu H, Takaya K, Hosoda H, Iwakura H, Ebihara K, Mori K, Ogawa Y, Hosoda K, Akamizu T, Kojima M, Kangawa K, Nakao K. Delayed short-term secretory regulation of ghrelin in obese animals: evidenced by a specific RIA for the active form of ghrelin. Endocrinology 2002; 143: 3341–3350. | Article | PubMed | ISI | ChemPort |
18. Rigamonti AE, Pincelli AI, Corra B, Viarengo R, Bonomo SM, Galimberti D, Scacchi M, Scarpini E, Cavagnini F, Muller EE. Plasma ghrelin concentrations in elderly subjects: comparison with anorexic and obese patients. J Endocrinol 2002; 175: R1–R5. | Article | PubMed | ISI | ChemPort |
19. Beck B, Musse N, Stricker-Krongrad A. Ghrelin, macronutrient intake and dietary preferences in long-evans rats. Biochem Biophys Res Commun 2002; 292: 1031–1035. | Article | PubMed |
20. Ikezaki A, Hosoda H, Ito K, Iwama S, Miura N, Matsuoka H, Kondo C, Kojima M, Kangawa K, Sugihara S. Fasting plasma ghrelin levels are negatively correlated with insulin resistance and PAI-1, but not with leptin, in obese children and adolescents. Diabetes 2002; 51: 3408–3411. | Article | PubMed | ChemPort |
21. Hansen TK, Dall R, Hosoda H, Kojima M, Kangawa K, Christiansen JS, Jorgensen JO. Weight loss increases circulating levels of ghrelin in human obesity. Clin Endocrinol (Oxford) 2002; 56: 203–206. | Article | ChemPort |
22. Bush EN, Droz B, Shaprio R, Knourek-Segel VE, Fey T, Bruen ME, Fagerland J, Jacobson PB, Collins C, Kaszubska W, Cummings DE. Effects of low-calorie or low-fat diet-induced weight loss on plasma ghrelin levels. Endocrinology 2003, Endo 2003 Endocrine society's 85th Annual Meeting, p. 398.
23. Lin S, Thomas TC, Storlien LH, Huang XF. Development of high fat diet-induced obesity and leptin resistance in C57Bl/6J mice. Int J Obes Relat Metab Disord 2000; 24: 639–646. | Article | PubMed | ChemPort |
24. Prpic V, Watson PM, Frampton IC, Sabol MA, Jezek GE, Gettys TW. Differential mechanisms and development of leptin resistance in A/J versus C57BL/6J mice during diet-induced obesity. Endocrinology 2003; 144: 1155–1163. | Article | PubMed |
25. Possidente B, Birnbaum S. Circadian rhythms for food and water consumption in the mouse, Mus musculus. Physiol Behav 1979; 22: 657–660. | Article | PubMed |
26. Cummings DE, Weigle DS, Frayo RS, Breen PA, Ma MK, Dellinger EP, Purnell JQ. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med 2002; 346: 1623–1630. | Article | PubMed | ISI |
27. Asakawa A, Inui A, Kaga T, Katsuura G, Fujjimiya M, Fujino MA, Kasuga M. Antagonism of ghrelin receptor reduces food intake and body weight gain in mice. Gut 2003; 52: 947–952. | Article | PubMed | ISI | ChemPort |
28. Van Heek M, Compton DS, France CF, Tedesco RP, Fawzi AB, Graziano MP, Sybertz EJ, Strader CD, Davis Jr. HR. Diet-induced obese mice develop peripheral, but not central, resistance to leptin. J Clin Invest 1997; 99: 385–390. | PubMed | ChemPort |
29. Lin L, Martin R, Schaffhauser AO, York DA. Acute changes in the response to peripheral leptin with alteration in the diet composition. Am J Physiol Regul Integr Comp Physiol 2001; 280: R504–R509. | PubMed | ISI | ChemPort |
30. Hewson AK, Tung LY, Connell DW, Tookman L, Dickson SL. The rat arcuate nucleus integrates peripheral signals provided by leptin, insulin, and a ghrelin mimetic. Diabetes 2002; 51: 3412–3419. | PubMed |
31. Traebert M, Riediger T, Whitebread S, Scharrer E, Schmid HA. Ghrelin acts on leptin-responsive neurones in the rat arcuate nucleus. J Neuroendocrinol 2002; 14: 580–586. | Article | PubMed |
32. Cowley MA, Cone RD, Enriori P, Louiselle I, Williams SM, Evans AE. Electrophysiological actions of peripheral hormones on melanocortin neurons. Ann NY Acad Sci 2003; 994: 175–186. | PubMed |
33. Toshinai K, Date Y, Murakami N, Shimada M, Mondal MS, Shimbara T, Guan JL, Wang QP, Funahashi H, Sakurai T, Shioda S, Matsukura S, Kangawa K, Nakazato M. Ghrelin-induced food intake is mediated via the orexin pathway. Endocrinology 2003; 144: 1506–1512. | Article | PubMed |
34. Wang L, Saint-Pierre DH, Tache Y. Peripheral ghrelin selectively increases Fos expression in neuropeptide Y—synthesizing neurons in mouse hypothalamic arcuate nucleus. Neurosci Lett 2002; 325: 47–51. | PubMed | ISI | ChemPort |
35. Stricker-Krongrad A, Richy S, Beck B. Orexins/hypocretins in the ob/ob mouse: hypothalamic gene expression, peptide content and metabolic effects. Regul Peptides 2002; 104: 11–20. | Article |
36. Ziotopoulou M, Mantzoros CS, Hileman SM, Flier JS. Differential expression of hypothalamic neuropeptides in the early phase of diet-induced obesity in mice. Am J Physiol Endocrinol Metab 2000; 279: E838–E845. | PubMed | ISI | ChemPort |
37. Smith FJ, Campfield LA, Moschera JA, Bailon PS, Burn P. Brain administration of OB protein (leptin) inhibits neuropeptide-Y-induced feeding in ob/ob mice. Regul Peptides 1998; 75–76: 433–439.
38. Lawrence CB, Snape AC, Baudoin FM, Luckman SM. Acute central ghrelin and GH secretagogues induce feeding and activate brain appetite centers. Endocrinology 2002; 143: 155–162. | Article | PubMed | ISI | ChemPort |
39. Melis MR, Mascia MS, Succu S, Torsello A, Muller EE, Denghenghi R, Argiolas A. Ghrelin injected into the paraventricular nucleus of the hypothalamus of male rats induces feeding but not penile erection. Neurosci Lett 2002; 329: 339–343. | Article | PubMed | ChemPort |

Acknowledgements

We thank Biomedical Research Model (BRM), the Animal Resource Group (ARG) and Margarita Camara as well as Sandhya Punreddy for their technical assistance. We thank Paul Richardson from Abbott Laboratories (Abbott Park, IL, USA) for ghrelin peptide supply. Finally, we address a special thank to Eugene Bush and his team at Abbott Laboratories for their effort in developing the high-fat to low-fat switch model.