Increased orexin and melanin-concentrating hormone expression in the perifornical lateral hypothalamus of rats prone to overconsuming a fat-rich diet

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Abstract

The goal of this study is to examine the expression pattern of orexigenic peptides, orexin (OX) and melanin-concentrating hormone (MCH), in the perifornical lateral hypothalamus (PFLH) in subpopulations of Sprague–Dawley rats differing in their propensity to overconsume a high-fat diet. Immediately after an initial 5-day screening test that predicts long-term consumption, rats identified as high-fat consumers (HFC), ingesting 35% more calories of a high-fat relative to low-fat chow diet, had significantly elevated mRNA expression of OX in the perifornical but not lateral hypothalamic area and of MCH mRNA in both areas, when compared to control rats that consume similar amounts of these diets. This same OX and MCH expression pattern was seen in HFC rats maintained for two weeks on a low-fat chow diet, indicating that increased expression of these orexigenic peptides, occurring independently of the high-fat diet, may be an inherent characteristic of these rats. These HFC rats were also more active and slightly more anxious than controls, as measured by line crossings and time spent in the periphery or middle segments of an open field. Together, these results demonstrate that animals prone to overeating a high-fat diet show a baseline increase in orexigenic peptide expression in the PFLH along with higher behavioral arousal, which together may contribute to their increased consummatory behavior.

Research Highlights

►Rats prone to fat overconsumption have increased OX and MCH peptide expression ►Rats prone to fat overconsumption exhibit increased activity levels ►Increased orexigenic peptide expression and behavioral arousal may lead to fat overconsumption

Introduction

The consumption of palatable diets, such as those containing high amounts of fat, has increased significantly during the past several decades (WHO, 2003). This may be due, in part, to increased availability of more palatable, fatty products, such as fast foods or junk foods (Bowman & Vinyard, 2004, Jeffery et al., 2006, Paeratakul et al., 2003). However, the vulnerability to overconsume these foods varies markedly across individuals. Human and animal studies show that consummatory behavior and food preferences exhibited at a young age can predict adult eating patterns, with measures of these preferences providing useful tools for identifying individuals prone to overconsumption (Johnson et al., 1991, Serdula et al., 1993). Also, in adult animals first exposed to a high-fat diet, their initial intake of or natural preference for fat is found to be positively related to their long-term measures of caloric and fat intake, in addition to their ultimate weight gain and body fat accrual (Dourmashkin et al., 2006, Lauterio et al., 1994, Pagliassotti et al., 1994, Shor-Posner et al., 1991, Shor-Posner et al., 1994, Wang et al., 1998). Such measures allowing early identification of subpopulations prone versus resistant to overeating and weight gain on a high-fat diet permits one to examine disturbances in brain mechanisms that may be causally related to these differential phenotypes.

Studies of brain neurochemicals in genetically altered animal models with varying propensity to become obese have revealed marked differences between animals that are already obese compared to those that are lean (Alexander et al., 2006, Cai et al., 2000, Qu et al., 1996, Stricker-Krongrad et al., 2001, Yamamoto et al., 2002). Since these differences may simply be a consequence of the obesity, other studies have examined rats selectively bred for their propensity to become obese on a high-fat diet and observed early disturbances in brain systems that precede and thus may be causally related to the obesity (Barnes et al., 2006, Levin et al., 1997, White et al., 2005). While it is difficult to predict the propensity of outbred rats towards dietary obesity, studies in these animals have shown weight gain during the first few days on a high-fat diet to be a strong predictor (Chang et al., Submitted for publication, Dourmashkin et al., 2006, Lauterio et al., 1994, Leibowitz et al., 2007, Pagliassotti et al., 1994, Shor-Posner et al., 1991) and revealed an increased expression of hypothalamic orexigenic peptides in obesity-prone animals that are still at normal body weight (Dourmashkin et al., 2006, Leibowitz et al., 2007). These investigations provide new information on neurochemical systems that are disturbed in early stages and may contribute to the development of obesity on a high-fat diet. It remains unclear, however, whether these neurochemical changes in outbred rats are related to the obesity per se or to the rats' behavioral pattern of high-fat diet overconsumption that invariably accompanies the obesity.

To address this question, we have recently established a method in Sprague–Dawley rats for reliably predicting, with a measure of initial acute consumption of a fat-rich diet, animals that are prone versus resistant to overconsuming this diet in a chronic situation (Chang et al., in press). Using this animal model, we have chosen to investigate a possible role of the hypothalamic orexigenic peptides, orexin (OX) and melanin-concentration hormone (MCH), which are both expressed predominantly in the perifornical lateral hypothalamus (PFLH) and have widespread projections throughout the brain (Bittencourt et al., 1992, de Lecea et al., 1998, Nahon et al., 1989, Peyron et al., 1998, Sakurai et al., 1998, Skofitsch et al., 1985). There is evidence showing OX to preferentially stimulate consumption of a high-fat diet (Clegg et al., 2002), mediate the reinforcing properties of fat-rich foods (Nair et al., 2008), and to be elevated with anticipation of or in response to consumption of a fat-rich meal (Choi et al., 2010, Wortley et al., 2003). Further, like OX, MCH stimulates food intake (Abbott et al., 2003, Rossi et al., 1999, Sakurai et al., 1998) and plays a distinct role in the consumption of different reinforcing substances (Benoit et al., 2005, Duncan et al., 2005, Duncan et al., 2006, Richards et al., 2008, Schneider et al., 2007). Evidence implicating both OX and MCH in locomotor activity (Chung et al., 2009, Novak et al., 2006, Smith et al., 2006, Thorpe & Kotz, 2005) suggests that these peptides may have a broader function in mediating behavioral activation that accompanies the consumption of preferred substances. While studies have revealed disturbances in OX and MCH expression in already obese rats (Cai et al., 2000, Elliott et al., 2004, Qu et al., 1996, Stricker-Krongrad et al., 2001, Yamamoto et al., 2002), it is unknown whether these peptides are disturbed and possibly contribute to the differential propensity of individual rats to overconsume a high-fat diet.

The present study was aimed at measuring the expression of OX and MCH mRNA in the PFLH of Sprague–Dawley rats characterized as prone or resistant to overeating a high-fat diet based on their initial 5-day intake of this diet. In these subgroups, OX and MCH peptides were first analyzed immediately after their few days of access to a high-fat diet. Then, to determine whether any observed differences can be seen independently of the high-fat diet, the peptides were measured in the subgroups after being returned to a lower-fat chow diet for two weeks. To gain a broader understanding of these peptides' functions, additional measurements were taken of locomotor activity in these subgroups and also of peptide expression in the sub-regions of the PFLH, which have been differentially related to the arousing and rewarding aspects of often overconsumed substances (Harris and Aston-Jones, 2006). The goal of this study was to determine whether the PFLH OX and MCH peptide systems are disturbed and possibly contribute to an increased propensity to overconsume a diet high in fat.

Section snippets

Subjects

Adult, male Sprague–Dawley rats weighing on average 250 g at the start of all experiments (Charles River Breeding Labs, Kingston, NY) were individually housed (22 °C, with lights off at 3:30 p.m. for 12 h) in a fully accredited American Association for the Accreditation of Laboratory Animal Care facility, according to institutionally approved protocols as specified in the NIH Guide to the Use and Care of Animals and also with the approval of the Rockefeller University Animal Care and Use Committee.

Experiment 1: Increased OX and MCH mRNA in HFC rats as measured by qRT-PCR

With the rats rank-ordered based on intake values on the high-fat compared to chow diet and separated into two subgroups (n = 6/group), the HFC rats were found to consume 35% more calories of the high-fat diet compared to the chow diet (p < 0.05), in contrast to the control rats that consumed similar amounts of the two diets (Table 1). After the 5-day access to the high-fat diet, the HFC compared to control rats, consuming 29% more kcal/day, became slightly but not significantly higher in their

Discussion

The present study measured differences in orexigenic peptide expression of animals classified based on their intake of a high-fat relative to lower-fat chow diet. Compared to control rats, animals characterized as HFC, at normal body weight, showed increased mRNA expression of OX and MCH in the PFLH. These peptide changes, observed while the animals were consuming the high-fat diet, persisted even after the animals were switched for 2 weeks to a lower-fat chow diet and allowed to resume normal

Conclusion

The HFC animal model described here is one of the first to characterize animals based specifically on varying degrees of acute consummatory behavior, a few days of high-fat diet intake compared to baseline chow intake that predicts long-term eating patterns. Our results suggest that animals prone to increased consumption of a diet rich in fat may have specific disturbances in the OX and MCH peptide systems in the PFLH. Evidence suggests that these peptides, in addition to responding to fat

Acknowledgements

This research was supported by USPHS Grant DA21518. We would like to thank Valerie Weed, Si-Yi Chang and Zhiyu Ye for their technical assistance. We would also like to thank Dr. Luis de Lecea (Stanford University) and Dr. Nicholas A. Tritos (Joslin Diabetes Center) for their generosity in providing the RNA probes used for this study.

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