palatability-induced hyperphagia increases hypothalamic dynorphin peptide and mrna levels

BRAIN RESEARCH

E L S E V I E R Brain Research 721 (1996) 126 131

R e s e a r c h r epo r t

Palatability-induced hyperphagia increases hypothalamic Dynorphin peptide and mRNA levels

C a t h e r i n e C. W e l c h ~, E u n - M e e K i m b, M a r t h a K. G r a c e a, C h a r l e s J. B i l l i ng ton ,.b,

A l l e n S. L e v i n e a,b,*

~ Research Service No. 151, Department of Medicine, VA Medical Center, 1 Veterans Drh, e, Minneapolis, MN 55417, USA b Departments of Food Science and Nutrition, Medicine, Surgery, and Psychiato,, UniL'ersiO' of Minnesota, Minneapolis St. Paul, MN 55455 and 55108,

USA

Accepted 23 January 1996

Abstract

Opioid involvement in regulating the intake of highly palatable diets was studied by examining the effect of feeding either a cornstarch-based diet (CHO) or a high fat diet containing sucrose (Fat/Sucrose) on hypothalamic opioid levels. Rats received either CHO ad libitum, Fat /Sucrose ad libitum, Fat /Sucrose pair-fed to the caloric intake of CHO, or Fat /Sucrose at 60% of ad libitum Fat/Sucrose intake. Animals receiving Fat /Sucrose ad libitum consumed more calories and gained more weight than animals receiving CHO (P < 0.001). Relative to CHO, ad libitum intake of Fat /Sucrose elevated proDynorphin mRNA levels in the arcuate and Dynorphin At 17 levels in the paraventricular nucleus (PVN) ( P < 0 . 0 5 ) , but did not affect arcuate mRNA levels of proEnkephalin or proOpiomelanocortin (POMC), or PVN levels of Met-Enkephalin or/3-Endorphin. Pair-feeding the Fat /Sucrose diet to the level of intake of the CHO diet resulted in levels of proDynorphin and Dynorphin AI_I7 that were similar in the two diet groups. Pair-feeding Fat /Sucrose reduced mRNA levels of proDynorpin, proEnkephalin and POMC, and Dynorphin A I_17 levels, relative to ad libitum feeding of Fat/Sucrose. Met-Enkephalin and /3-Endorphin were not affected by dietary treatment. Feeding Fat /Sucrose at 60% of ad libitum intake resulted in mRNA levels of proDynorphin, proEnkephalin and POMC, and Dynorphin A~_17 levels that were similar to those observed in CHO group. Hypothalamic Dynorphin A 1 17 and proDynorpbin mRNA levels are stimulated by feeding a highly palatable diet rich in fat and sucrose. The increased synthesis may be due in part to a palatability-induced overconsumption of calories. Caloric restriction of the same diet decreases mRNA levels of proDynorphin, proEnkephalin and POMC, as well as levels of Dynorphin

AI_I7.

Keywords: Opioid; Fat; Sucrose; Highly palatable diet; Opioid peptide; Opioid mRNA

1. Introduction

While opioid involvement in food intake regulation is strongly supported in the literature, the precise role opioids have in feeding behavior is unclear. One possibility is that opioids mediate the rewarding or palatable aspects of feeding. The ability of opioid antagonists to suppress intake is greater when palatable food items or drinks are offered. Naltrexone reduced intake to a greater extent when rats were given highly palatable food items (lard, noodles, cakes, chocolate) in addition to chow [1]. Nalox- one was more effective in suppressing ingestion when saccharine, sucrose or NaC1 were added to water [22,24,25]. In food-deprived or -restricted animals, naloxone sup-

* Corresponding author at addres a. Fax: (1) (612) 725-2093.

0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S0006-8993(96)00151-5

pressed intake to a greater extent when palatable food items or diets sweetened with sucrose were offered [15,23]. Additionally, opioid receptor-deficient (CXBK) mice did not demonstrate the preference for sweet solutions observed in control mice [38].

Conversely, administration of opioid receptor agonists stimulates the ingestion of palatable foods and drinks. Ingestion of saccharine solutions was stimulated by morphine [8] and of selective mu and delta agonists [16]. Consumption of cafeteria diet was enhanced by administration of Dynorphin and D-Ser 2-Leu-Enk-Thr 6 (DSLET) [31 ]. Likewise, kappa receptor agonists have been shown to increase consumption of sweetened condensed milk [10,18] or high fat diets [32].

In humans, opioid antagonists are most effective in decreasing consumption of sweet high fat foods [12] or foods that are rated as highly palatable [36,37]. It has been

C.C. Welch et al./ Brain Research 721 (1996) 126-131 127

suggested that opioid blockade results in altered taste perception making sweetened and salty solutions less palatable [5,14].

Biochemical evidence for opioid involvement in per- ceiving palatability is demonstrated by the observed reduction in opiate tracer binding and decrease in /3-Endorphin concentration in the hypothalamus of rats fed highly palatable foods [13]. These results were interpreted as increased occupation of opioid receptors by /3-Endorphin thereby decreasing the availability of receptors to the tracer, and an increase release and breakdown of /3-Endorphin. /3-En- dorphin like immunoreactivity was elevated in pituitary of rats made obese by prolonged maintenance on palatable foods [17]. ProDynorphin mRNA levels in the paraventricular nucleus and the supraoptic nucleus were positively correlated with consumption of a high fat diet [6].

We hypothesized that consumption of a highly palatable diet would increase endogenous opioid synthesis in the hypothalamus and wanted to determine whether the opioid response was due to an increase in caloric intake or to ingestion of a highly palatable stimulus. We evaluated opioid mRNA levels in arcuate nucleus and opioid peptide levels in paraventricular nucleus in rats fed a diet rich in fat and sucrose at ad libitum and reduced levels of intake. The arcuate nucleus and PVN were selected because cell bodies containing opioid peptides are concentrated in the arcuate nucleus with axons projecting to the PVN as well as the dorsomedial hypothalamic nuclei, centers implicated in the regulation of food intake [3].

Table 1 Composition of experimental diets

Diet(% by weight)

CHO diet Fat/sucrose diet

Cornstarch 70.0 Sucrose 23.33 Shortening 32.67 Safflower oil 1.67 Casein 20.0 28.67 D,L-Methionine 0.3 0.43 Vitamin mix 1.0 1.33 Mineral mix 3.5 4.77 Choline-C1 0.2 0.27 Cellulose 5.0 6.86 Metabolizable energy(kJ/g %ME) 15.34 21.98 CHO 77.8 18.8 Protein 22.2 22.2 Fat 59.0

Vitamin and mineral mixtures were American Institute of Nutrition vitamin mixture 76 and mineral mixture 76, respectively. Fiber was Celufil (US Biochemical, CLeveland, OH). Metabolizable energy (ME) was based on Atwater values of 16.8, 37.7 and 16.8 kJ/g for carbohydrate (CHO), fat, and protein, respectively.

contained a fourth treatment in which rats received Fat/Sucrose diet at 60% of the calories consumed by the rats receiving Fat/Sucrose diet ad libitum. The data reported herein are the combined results from these two studies. Food intake of the ad libitum groups was mea- sured daily and corrected for spillage. Body weights were determined at the beginning and end of the experiment. Rats were randomly allotted to treatment by weight.

2. Materials and methods 2.2. Tissue sample preparation

2.1. Animals

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Madison, WI) weighing 225-250 g were maintained on a 12/12 h light/dark cycle (lights off at 19.00 h) in temper- ature-controlled rooms (21-23°C). Animals were individu- ally housed in stainless steel wire cages and permitted ad libitum access to food, except where noted, and water. Rats were fed standard laboratory diet (Rodent Chow, Teklad, Indianapolis, IN) until they were placed on one of two experimental diets for 7 days. Diets were either a cornstarch-based diet (CHO diet) or a mixture of vegetable shortening and sucrose (Fat/Sucrose diet), and were nutritionally complete (see Table 1). We used fat and sucrose because both are highly palatable ingredients and previous work in our laboratory has demonstrated that rats will select diets containing these ingredients over cornstarch- based diets (unpublished observations). In the first experiment, rats received either CHO diet ad libitum, Fat/Sucrose diet ad libitum, or Fat/Sucrose diet pair fed to the daily average caloric intake of rats receiving CHO diet ad libitum. The second experiment included the three treatment groups described for the first experiment but

After the experimental period, rats were sacrificed by decapitation in randomized order between 09.30 and 11.00 h, on consecutive days. The brains were rapidly excised, chilled in ice-cold saline and sliced using a Stoelting tissue slicer. Brains were sectioned with cuts at - 2 , 0, + 2 and + 5.5 mm relative to the anterior commissure, corresponding to the brain atlas of Paxinos and Watson [27]. The frontal lobe was discarded; a scalpel cut of frontal cortex (CTX) was taken from the first slice ( - 2 to 0 mm); a single 3 mm punch of PVN was taken from the second slice (0 to + 2 ram); a scalpel cut was used to remove the entire arcuate nucleus (ARC) from the third slice ( + 2 to +5.5 mm). Brain tissue samples were frozen in liquid nitrogen and stored at - 70°C until analyzed.

2.3. mRNA analysis

Total mRNA was extracted from brain tissue samples using the rapid guanidine thiocyanate/phenol chloroform method of Chomczynski [9]. Tissues were homogenized using a hand-held Potter-Elvehjem homogenizer in a buffer containing 4 M guanidine thiocyanate and water-saturated molecular grade phenol. Sarcosyl (10%), 2 M sodium

128 C. C. Welch et al. / Brain Research 721 (1996) 126 131

acetate and chloroform were then added. After centrifuga- tion at 14,000 X g for 15 rain, the aqueous phase was precipitated with isopropanol overnight, resuspended in guanidine thiocyanate buffer, and reprecipitated with isopropanol. The pellet was washed twice with 75% ethanol and stored at - 2 0 ° C in 100% ethanol overnight. The mRNA pellet was reconstituted in RNA storage buffer, 0.5% sodium dodecyl sulfate (SDS), and sterile H20. The amount of total RNA was determined by absorbance at 260 nm and the quality of extracted RNA was estimated from the ability of the A 2 6 0 / A 2 8 0 ratio to approximate 2.

Aliquots of total RNA were dissolved in 7.4% formaldehyde:6 X SSC (1 x SSC = 0.15 M NaC1, 0.015 M sodium citrate) and denatured for 10 rain at 68°C. Samples were applied in duplicates of 2 /~g total RNA onto 6 × SSC soaked nylon membranes (Zeta Probe, Bio-Rad, Rich- mond, CA) using a microfiltration apparatus (Bio-Rad, Richmond, CA). Control for RNA loading onto the slot- blots was provided by the ultraviolet shadowing technique, in which total unhybridized RNA is imaged [34]. Mem- branes were crosslinked with a UV light source and stored dry between two pieces of filter paper at 25°C.

Membranes were prewashed before use and prehy- bridized overnight at 42°C in 50% formaldehyde, 5 X SSC, 10 x Denhardt 's solution, 0.2% SDS, denatured salmon sperm DNA and yeast tRNA in 50 mM NaPhosphate, pH 6.5. Hybridization was for 48 h at 42°C in 50% for- mamide, 5 X SSC, 2 X Denhardt 's solution, 0.2% SDS, denatured salmon sperm DNA and yeast tRNA in 50 mM NaPhosphate, pH 6.5 with 10 6 c p m / m l [3ep]-dCTP random primer labeled cDNA, The radiolabeled cDNA probes for proDynorphin (proDYN), proEnkephalin (proENK) or POMC were produced in our laboratory from transformed cells generously provided by Dr. James O. Douglass (Vol- lure Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, Oregon). The cDNA utilized were 1700-, 1070- and 923-base pair nucleotide sequences coding for the genes of rat proDYN, rat pre- proENK and mouse POMC, respectively. Specificity of the three opioid probes was verified using Northern blot analysis (Fig. 1). In addition, nylons were hybridized with /3-Actin which was commercially obtained (Oncor, Gaithersburg, MD). There were no treatment effects on /3-Actin mRNA levels in the ARC (Fx64 = 0.059, P = 0.9808). Following hybridization, the membrane under- went a series of high- and low-salt washings, and was exposed to X-ray film at - 8 0 ° C . Hybridization was quantitated in arbitrary optical density units by scanning den- sitometry (Bio-Rad, Richmond, CA). All RNA from a single experiment was slotted onto a single filter, hybridized in a single vessel, and autoradiographed in a single cassette to assure comparability of group treatment.

2.4. Radioimmunoassay

The concentrations of immunoreactive Dynorphin A~_ ~7, Met-Enkephalin and /3-Endorphin were quantitated

4.4- -

2 .4- -

1.4--

- - 2 . 4

- - 1 . 4 5

- - 1 . 1 5

it i !k~ii'ili!i~! 0 . 2 4 - -

1 2 3 4 5 6

Fig. 1. Northern Blot analysis of proDynorphin, proEnkephalin and POMC genes in RNA from rat pituitary (proENK, lanes 1,2 and POMC: lanes 5,6) or hippocampus (proDYN: lanes 3,4). Twelve /xg RNA and size marker RNA fragments (BRL, Gaithersburg, MD) were size fraction- ated on a 1.0% agarose gel, transferred to nylon membranes, and hybridized with random primer labeled cDNA probes, as described under Materials and Methods. Numbers at the left indicate the chain length (Base pairs) estimated by marker RNA fragments.

in the PVN following extraction in 0. l N acetic acid, using commercially available RIA kits (Peninsula Laboratories, Belmont, CA).

2.5. Statistics

Data were analyzed by one-way analysis of variance, followed by multiple comparisons of means using least significant difference test. Data are expressed as % of the response of CHO fed group and are presented as the mean + SE.

3. Results

Animals receiving the Fat /Sucrose diet ad libitum consumed significantly more calories and gained more weight than animals receiving the CHO diet (383.7 _+ 5.0 vs. 318.4 4- 4.6 kJ /day , P < 0.001; 38.1 + 1.7 vs. 25.8 _ 2.0 g body weigh t /7 days, P < 0 . 0 0 1 ) . Pair-feeding the Fat /Sucrose diet to the intake of the CHO diet resulted in caloric intakes and body weight gains that were 82.8% and 64.7%, respectively, of the Fat /Sucrose diet ad libitum group and were not significantly different from the CHO diet group ( P > 0.05). Ad libitum intake of the Fat /Sucrose diet significantly elevated arcuate mRNA levels of proDYN ( P < 0.05), but not proENK or POMC, relative to the CHO diet (Fig. 2). Pair-feeding the Fat /Sucrose diet significantly depressed mRNA levels of proENK and POMC ( P < 0.05), but not proDYN, relative to the CHO diet. Pair-feeding the Fat /Sucrose diet reduced mRNA levels for proDYN, proENK and POMC relative to the ad libitum feeding of the Fat /Sucrose diet.

C.C. Welch et a l . /Brain Research 721 (1996) 126-131 129

~'125

0 -r- 100 U "6

Q

d d 25

Oplo ld mRNA Leve ls In Arcuate Nuc leus

[~ CliO D~et ad Ilbltum Fat/Sucrose Diet ad hbltum Fet/Sucrose Diet Pair-Fed

b

a :'," a .,,

proDynorphln proEnkephalln POMC

Fig. 2. Effect of feeding, for 7 days, CHO diet, Fat/Sucrose diet ad libitum, or Fat/Sucrose diet pair-fed to the caloric intake of the CHO diet, on mRNA levels (expressed as % of CHO diet) for proDynorphin, proEnkephalin and POMC in the arcuate nucleus. Values are means _+ SE. a,b: means within mRNA type with different superscripts differ significantly (P < 0.01).

Dynorphin A fl 17 levels in the PVN were elevated in the Fat/Sucrose diet ad libitum group relative to the CHO diet (P < 0.05), while pair-feeding the Fat/Sucrose diet resulted in Dynorphin Al_I7 levels that were lower than those observed in the Fat/Sucrose diet ad libitum group (P < 0.05) (Fig. 3). Met-Enkephalin and/3-Endorphin were not affected by dietary treatment. Feeding the Fat/Sucrose diet at 60% of the ad libitum intake resulted in mRNA levels of proDYN, proENK and POMC and Dynorphin AI_I7 levels that were similar to those observed in the CHO diet group (103.4 +_ 5.9, 95.6 _+ 8.0, 97.5 + 6.4 and 114.8 _+ 13.5% of CHO diet group, respectively, P > 0.05). No differences were observed in proDYN, proENK or POMC mRNA levels in the CTX (data not shown).

¢3 o "1- (,I 125"

100"

~. 75" 2

~ 5 0 ' E "0 ~. 25"

~ 0 c

Oplo id Pept lde Levels in PVN

b

T

a it

Dynorphln A

[71 CHO Diet ad I lbltum Fet~ucrose Diet ad I lbl tum

• Fat/Sucrose Diet Pair-Fed

Met-Enkephalln 8-Endol )hln

Fig. 3. Effect of feeding, for 7 days, CHO diet, Fat/Sucrose diet ad libitum, or Fat/Sucrose diet pair-fed to the caloric intake of the CHO diet, on peptide levels (expressed as % of CHO diet) for Dynorphin A1 17, Met-Enkephalin and /3-Endorphin in the paraventricular nucleus. Values are means _+ SE. a,b: means within peptide type with different superscripts differ significantly (P < 0.05).

4. Discuss ion

The Fat/Sucrose diet utilized in this study stimulated overconsumption of calories by 20.7% relative to the carbohydrate diet that was nutritionally adequate but was not as calorically dense. The caloric overconsumption resulted in 47.4% greater weight gains during the 7-day period animals were fed the two experimental diets. In this model of dietary-induced obesity, proDYN mRNA levels in the ARC and Dynorphin Al_~7 levels in the PVN were elevated while levels of proENK and POMC mRNA in the ARC and Met-Enkephalin and /3-Endorphin levels in the PVN did not appear to be affected. Similar elevations in proDYN mRNA levels in response to high fat diet consumption have been reported in the supraoptic nucleus and the PVN [6]. However, sucrose drinking reduced /3-En- dorphin levels in the dorsal hypothalamus in spontaneously hypertensive rats [39]. Similarly, a reduction in /3-En- dorphin but not Dynorphin was observed in whole hypothalamus of rats receiving limited exposure to highly palatable food items (20 min for 14 days) [13]. The feeding paradigm employed by Dum et al. permitted examination of opioid release under conditions in which there were no differences in body weight gains. In both of these studies, the authors interpreted the results as indications of increased /3-Endorphin release following ingestion of a palatable stimuli. From our data, it is possible to suggest that the changes in hypothalamic Dynorphin levels are a reflection of changes in body weight. Yeomans observed an increase in sensitivity to the opioid antagonist naloxone in animals with greater weight gains resulting from palatability-induced overconsumption of calories [35]. Others have observed elevated plasma concentrations of /3-En- dorphin which may be associated with elevations in pituitary /3-Endorphin levels in both genetically obese rats [26] and mice [29] and dietary-induced obese rats [17].

Reductions in mRNA levels of proDYN, proENK and POMC in ARC following caloric restrictions of the same diet have been previously reported by our laboratory [19]. These results do not indicate whether the lower opioid mRNA levels observed in calorically restricted animals are due to the reduced food intake or are secondary to the reduced body weight gains. The observed depression in opioid gene expression with decreasing food consumption may be explained by the theory that central release of opioids mediates the rewarding aspects of ingestion [20,30]. Evidence for the involvement of opioids in orosensory reward is the ability of antagonists to suppress consumption of highly preferred foods and drinks [15,23,25]. It is possible that under conditions of food restriction there are fewer rewarding stimuli and, therefore, less central synthesis and release of opioids. Enhanced food intake observed with central opioid administration would then be explained as intake in response to heightened rewarding properties of feeding. Diminished food intake with opioid blockade would be a manifestation of reduced reward from feeding.

130 c. c. Welch et al. / Brain Research 721 (1996) 126-131

In the current study, reducing intake of the F a t / S u c r o s e diet did result in lower opioid mRNA levels in the ARC. However, the absolute intake of the CHO diet in grams was higher than that of the F a t / S u c r o s e diet ad libitum intake (20.8 + 0.3 vs. 17.5 + 0.2 g d i e t / d a y , P < 0.0001), while opioid mRNA levels in the ARC were lower. Addi- tionally, pair-feeding the F a t / S u c r o s e diet to the caloric intake of the CHO diet abolished the elevations in ARC proDYN mRNA levels and PVN levels of Dynorphin A l-17 observed in the F a t / S u c r o s e ad libitum fed animals. This suggests that these elevations are a manifestation of the overconsumption of calories in ad libitum fed animals. However, it is possible that by restricting calories animals were deprived of a palatabil i ty-induced stimulus inherent in the F a t / S u c r o s e diet itself. Further research is needed to determine whether the effects of calories can be separated from those of palatabil i ty-induced reward.

It should be noted that hypothalamic opioid peptide levels demonstrate a circadian rhythm, rising during the time of day when meals are consumed [28,33]. It is possible that the pair-feeding regimen results in an alter- ation in meal patterns relative to the ad libitum fed regimen that could potentially influence hypothalamic opioid peptide levels. The authors are unable to resolve this confound at present.

In this study, the caloric response observed in proDYN mRNA and Dynorphin A 1 i7 levels suggests that the K opioid receptor is involved. Others have found that K agonists stimulate intake of highly palatable [1 1,18] or high fat [32] diets. Conversely, administration of opioid antagonists specific for the K receptor reduces consumption of sweetened solutions [4,7,16] and high fat diets [2]. Recently, it was demonstrated that the K agonist nor-bi- naltorphamine greatly inhibited sucrose intake in sham- feeding rats with their gastric fistulas open, suggesting that opioids are involved in detecting the orosensory character- istics of an ingestate [21]. This evidence suggests that the Dynorphin gene family may be more important than the Endorphin or Enkephalin gene families in regulating the palatabili ty or rewarding aspects of food.

In summary, overconsumption of a highly palatable diet increased proDYN mRNA levels in the ARC and Dynor- phin A~_~7 levels in the PVN while not affecting proENK or POMC mRNA levels, or Met-Enkephalin or /3-En- dorphin levels. When consumption of the highly palatable diet was restricted to the level of intake of the cornstarch- based diet, levels of proDYN mRNA and Dynorphin A l_ 17 were similar in the two diet groups. Restricting consumption of the highly palatable diet reduced mRNA levels of proDYN, proENK and POMC, and Dynorphin Aj_E7 levels relative to feeding the highly palatable diet ad libitum. In conclusion, hypothalamic Dynorphin Al_17 and pro- DYN mRNA levels appear to be stimulated by feeding a highly palatable diet rich in fat and sucrose. The increased synthesis may be due in part to a palatabil i ty-induced overconsumption of calories. Caloric restriction of the

same diet decreases mRNA levels of proDYN, proENK and POMC, as well as Dynorphin A l ~7.

Acknowledgements

The authors are grateful to J.O. Douglass for generously providing the transformed cell stocks needed to produce the cDNA probes for proDynorphin, proEnkephalin and POMC. This work was supported by General Research Funds of the Department of Veterans Affairs (VA) Medi- cal Center and by National Institutes of Health Grants DA-03999, DA-07097 and DK-42698.

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palatability-induced hyperphagia increases hypothalamic dynorphin peptide and mrna levels

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