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Regulation of neuropeptide Y mRNA and peptide concentrations by copper in rat olfactory bulb
Molecular Brain Research 65 Ž1999. 80–86
Research report
Regulation of neuropeptide Y mRNA and peptide concentrations by copper in rat olfactory bulb Nancy J. Rutkoski a , Cheryl A. Fitch a , E. Carden Yeiser a , Janice Dodge a , Paul Q. Trombley b, Cathy W. Levenson a,b,) a
Department of Nutrition, Food and Exercise Sciences, Florida State UniÕersity, 237 Biomedical Research Facility, Tallahassee, FL 32308-4340, USA b Program in Neuroscience, Florida State UniÕersity, 237 Biomedical Research Facility, Tallahassee, FL 32308-4340, USA Accepted 8 December 1998
Abstract Neuropeptide Y is highly abundant in both the peripheral and central nervous systems and is known to have diverse functions including regulation of feeding behavior, blood pressure, circadian rhythms, reproductive behavior and the response to stress. Northern analysis showed that copper deficiency increased brain NPY mRNA abundance particularly in the olfactory bulb ŽOB.. These increases were not accompanied by alterations in food intake or blood pressure. After 4 weeks of a copper-restricted diet, OB copper concentrations decreased to 44% of control and NPY mRNA increased 1.5-fold. Addition of a copper chelator to the restricted diet, resulted in a two-fold increase in OB NPY mRNA over copper adequate controls. These results were confirmed in primary cultures of OB neurons suggesting that the regulation of NPY mRNA is at the level of the bulb rather than by a hormonal or other copper-regulated factor external to the OB. Immunoreactive NPY ŽIR-NPY. levels were not, however, increased following the 4 weeks of copper deficiency. Addition of the chelator resulted in a 1.4-fold increase in IR-NPY that, while statistically significant, was not proportional to the two-fold increase in NPY mRNA in the same study. This may suggest that copper deficiency inhibits the translational mechanisms responsible for the synthesis of NPY or that NPY is exported from the bulb in copper deficiency. q 1999 Elsevier Science B.V. All rights reserved. Keywords: NPY; Olfactory; Neuron; Brain; Copper; Deficiency
1. Introduction Neuropeptide Y ŽNPY., the most abundant known neuropeptide w1x, is a powerful orexigenic agent that induces feeding when injected centrally, even in satiated animals w11x. This function appears to be most closely associated with its action in the paraventricular nuclei ŽPVN. of the hypothalamus w43x. However, NPY expression is widely distributed throughout the central and peripheral nervous systems with high levels in olfactory bulb, cortex, and spinal cord, as well as the PVN w23,30x. Given this wide distribution, it is not surprising that NPY is now known to participate in diverse functions including regulation of neuroendocrine secretion w13x, blood pressure w14x, circadian rhythms w5x, and sexual behavior w11x. NPY has also
) Corresponding author. Florida State University, 237 Biomedical Research Facility, Tallahassee, FL 32308-4340, USA. Fax: q1-850-6440989; E-mail: [email protected]
been shown to play a role in the response to stress w26x, and the control of anxiety and depression w44x. A number of factors have been shown to regulate the expression of NPY. Nerve growth factor ŽNGF. w37x and epidermal growth factor ŽEGF. w13x increase NPY mRNA abundance. Insulin w18x, glucocorticoid hormones w31x and leptin w39x all decrease NPY mRNA abundance. Recently there have been two reports of hypothalamic NPY mRNA regulation by zinc deficiency w24,40x. In both studies, zinc deficiency resulted in a significant reduction in food intake, that is characteristic of zinc deficiency w9x, and a 1.5 to two-fold increase in hypothalamic NPY mRNA abundance w24,40x. The reported zinc regulation of NPY mRNA led us to test the possibility that NPY mRNA is also regulated by the trace metal copper. While copper deficiency has not been reported to reduce food intake, there are well known antagonistic interactions between these two essential trace metals w32x. Furthermore, copper is clearly needed for normal brain function as an essential cofactor for a number
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 3 4 5 - 3
N.J. Rutkoski et al.r Molecular Brain Research 65 (1999) 80–86
of essential enzymes w41x including cytochrome c oxidase which is an integral part of the electron transport chain w12x, superoxide dismutase ŽSOD., which acts as a free radical scavenger w7x, and dopamine b-monooxygenase ŽDBM., the rate limiting enzyme in catecholamine synthesis w20x. Copper is also essential for the activity of the enzyme peptidylglycine a-amidating monooxygenase ŽPAM. that is responsible for the post-translational modification of dozens of neurohormones and neuropeptides including NPY w34,38x.
2. Materials and methods 2.1. Animal care and housing The Animal Care and Use Committee ŽACUC. at Florida State University approved all animal protocols. Five-weekold male Sprague–Dawley rats were housed in hanging stainless steel cages in temperature-controlled rooms on a 12 h light–dark cycle with free access to distilled water. Rats in the copper-restricted groups Ž –Cu. received a casein-based, copper deficient diet Ž0.4 mg Curkg. ŽHarlan Teklad, Madison, WI. ad libitum. Control rats were fed a similarly composed diet containing adequate copper Ž18 mg Curkg.. Other animals Ž –Cu q trien. were fed the copper-deficient diet supplemented with the copper chelator triethylenetetramine Ž0.6% wrw. w4x. Food intake was evaluated throughout the study. After 4 weeks, rats were anesthetized with methoxyflurorane and brain or olfactory bulbs were immediately removed for isolation of total cellular RNA, radioimmunoassay, or measurement of copper and zinc concentrations. Trunk blood was also collected for preparation of serum and measurement of ceruloplasmin activity using the p-phenylenediamine oxidase ŽPPD. assay w19,36x.
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and serum extender. Additional plates were treated with 1 mM carnosine. Carnosine has been reported to act as an endogenous copper chelator w8x. Control cells were grown in the supplemented serum-free medium. After 7 days of treatment, cells were harvested for isolation of total cellular RNA and Northern analysis or measurement of total cellular copper. 2.3. Northern analysis The acid guanidinium thiocyanate–phenol–chloroform extraction method was used to isolate total cellular RNA w10x from whole brain Ž n s 6. or brain regions Žolfactory bulb, cortex, brain stem, hypothalamus, thalamus, cerebellum and pituitary, n s 4. from rats fed either the control or –Cu q trien diets for 4 weeks. Olfactory bulb RNA was also collected from control Ž n s 5. and copper-restricted Ž –Cu, n s 5. rats. Purity of the RNA as verified by A 260rA 280 ratio. Equal amounts of total RNA Ž15 mg. were fractionated by size on a 1% Žwrv. agarose gel containing 0.66 M formaldehyde. RNA was then transferred to a nylon membrane ŽGeneScreen, NEN, Boston, MA. by capillary blotting. RNA transfer was confirmed by visualization of ethidium bromide stained RNA under UV light. Blots were UV crosslinked and stored at 48C until hybridization with a random primed 32 P-labeled preproNPY cDNA probe ŽRadPrime DNA Labeling System, Gibco BRL, Gaithersburg, MD.. Equal loading of lanes was confirmed by hybridization to 32 P-labeled 28S rRNA probe. Filters were exposed to Kodak X-OMAT AR film at y808C. Relative amounts of bound cDNA probe were determined by computer evaluated densitometry ŽQuantity One quantification program. created by Protein and DNA Imaging ŽPDI, Boston, MA. and expressed as a function of 28S rRNA abundance. 2.4. Radioimmunoassay
2.2. Cell culture Using sterile technique, olfactory bulbs were excised from 7-day-old rat pups. The dura was removed and the tissue minced and placed in dissection buffer ŽCarEDTA with 20 Urml papain, Collaborative Research, Bedford, MA. at 378C for 1 h. Following enzyme digestion, the tissue was gently disrupted with a polished pasteur pipette, and olfactory bulb neurons ŽOBN. released into the media were transferred to 15 ml conical centrifuge tubes, pelleted at low speed, and resuspended in Minimum Eagles Media ŽMEM, Gibco USA. with 4% glucose, fetal calf serum ŽFCS. and a nutrient supplement ŽSerum Extender, Collagen, Palo Alto, CA.. Cells in media Ž3.66 = 10 6 . were plated on olfactory bulb astrocytes grown to confluence in 60 mm dishes. Following one week in culture, the copper chelator tetraethylene pentamine ŽTEPA, 100 mM. w33x was added to cells in serum free media with 4% glucose
A modification of the radioimmunoassay developed by Beck et al. w3x was used to measure olfactory bulb NPY in control Ž n s 8. and copper-restricted Ž n s 10. rats. After the 4 weeks of copper restriction, olfactory bulbs were removed and immediately sonicated at 48C in 0.3 ml buffer Ž0.2N HClr0.1% EDTA, containing 0.5% Titron X-100 and Aprotinin 100 mgrml.. Sonicated samples Ž100 ml. were incubated overnight at 48C with a rabbit antirat-NPY antibody Ž1:8000, Sigma, St. Louis, MO.. Synthetic NPY ŽSigma. was used as the standard. The tracer, I 125 NPY Ž0.25 ngr100 ml, Amersham, Arlington Heights, IL., was added and incubated overnight at 48C. Bound NPY was separated from free using a 0.4% dextranr0.4% charcoal suspension. All tubes were centrifuged at 2000 = g for 20 min and the 125 I in the supernatant detected using a Micromedic 4r600 Plus gamma counter ŽMicromedic Systems, Huntsville, AL.. The range of the standard curve
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Table 1 Effect of copper restriction on brain and olfactory bulb copper Žmgrg wet wt.
Control
–Cu
–Cuqtrien
Whole brain Olfactory bulb
2.15"0.13 3.18"0.79
1.51"0.16) 1.43"0.36)
1.32"0.16) 1.45"0.11)
–Cu, 4-week copper restriction. –Cuqtrien, 4-week copper restriction with chelator. )Significant at pF 0.05.
was 1 ng–128 ng NPY. Concentrations were estimated for each sample using weighted linear regression. The inter-assay coefficient of variation was - 10% and the intra-assay coefficient of variation was - 5% for duplicate measurements. Data is expressed as ng NPYrmg total protein as determined by Lowry protein assay w27x. 2.5. Measurement of brain copper concentration Whole brain Ž n s 5 in control, –Cu, and –Cu q trien groups. or olfactory bulbs Ž n s 4 in control, –Cu, and –Cu q trien. were digested with nitric acid and hydrogen peroxide as previously described w25x. Flame atomic absorption spectroscopy ŽPerkin Elmer 3100. was used to determine whole brain copper concentrations Žmg Curg wet weight.. Graphite furnace atomic absorption spectrophotometry ŽZeeman 5100, Perkin Elmer, Norwalk, CT. was used to measure copper concentration in rat olfactory bulb Žmg Curwet wt. and cultured bulb neurons Žmg Curmg protein.. Copper concentrations were determined by automated analysis based on a standard curve developed using Fisher copper reference solution. Repeated measures of the same sample revealed an intra-sample variation of - 1%.
ŽGraphPad Software, San Diego, CA.. Values were considered statistically significant at p F 0.05.
3. Results 3.1. Copper status Copper restriction resulted in a significant reduction in the activity of the copper-dependent serum enzyme ceruloplasmin ŽCp.. After 4 weeks of copper restriction, Cp activity decreased to 8% of control Ž p F 0.001, n s 5.. Table 1 shows the effect of dietary copper restriction and the addition of the chelator trien on brain and olfactory bulb ŽOB. copper. Four weeks of restriction Ž –Cu. significantly reduced both whole brain and OB copper levels Ž p s 0.05, n s 4–5.. Neither the –Cu or –Cu q trien diets significantly altered zinc levels in OB or whole brain Žcontrol 12.1 " 0.7; –Cu 11.2 " 1.12; –Cu q trien 13.1 " 1.8 mgrg wet wt.. Copper chelation reduced copper levels in primary cultures of neurons by approximately 25% Ž11.7 " 0.7 vs. 8.9 " 1.0 mg Curmg protein.. Addition of the endogenous chelator carnosine did not significantly alter OB cell copper Ž13.9 " 0.8 mg Curmg protein. compared to controls. 3.2. Effect of copper-restriction on feeding and blood pressure Given the role of NPY in controlling feeding behavior and vasoconstriction, measurements were made to detect changes in food intake or blood pressure following copper restriction. Copper restriction did not result in significant differences in daily food intake, nor did it alter food intake in response to a 24–48 h fast. Additionally, there was no
2.6. Blood pressure Blood pressure was assessed in an additional group of copper deficient and copper sufficient rats Ž n s 3.. Under halothane anesthesia, intravascular catheters were chronically implanted into the descending aorta via the left carotid artery and tunnelled to the nape of the neck, exteriorized, and sealed with a 28-gauge stainless steel pin. Catheters were flushed daily with heparinized saline Ž100 Urml.. After one week of recovery, blood pressure was measured in conscious, unrestrained rats on three successive days using transducers connected to amplifiers interfaced with an AT-CODAS data acquisition card ŽDATAQ Instruments, Akron, OH.. Blood pressure was sampled at 200 Hz and written to a computer hard drive for subsequent analysis. 2.7. Statistical analysis Data were analyzed using ANOVA with a Tukey’s multiple group post-hoc test and unpaired Students t-test
Fig. 1. Northern analysis of NPY mRNA in brain regions. Total cellular RNA Ž15 mg. from each adult rat brain region was subjected to Northern analysis using a 32 P-labeled NPY cDNA probe. Relative abundance of NPY mRNA in each region is shown in this representative autoradiograph.
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control Ž118 " 3 mmHg. and copper deficient Ž122 " 3 mmHg. rats did not reveal significant differences. 3.3. Effect of copper-restriction on NPY and NPY mRNA
Fig. 2. Effect of copper-restriction on brain NPY mRNA abundance. Following 4 weeks of copper restriction and supplementation with a copper chelator Ž –Cuqtrien., 15 mg whole brain total cellular RNA was subjected to Northern analysis. NPY mRNA was normalized to 28S rRNA abundance for each sample Ž ns6.. Bars represent relative NPY mRNA Žmean"S.E.M... )))Statistically different from control values at pF 0.001.
difference in body weight of rats in the –Cu group Ž284 " 29 g. as compared to control group Ž277 " 30 g.. Direct measurement of mean arterial blood pressure ŽMAP. in
Northern analysis of adult rat brain regions revealed that NPY mRNA abundance is highest in olfactory bulb ŽFig. 1.. Other brain regions tested had significantly lower NPY mRNA levels with cortex) brain stem ) hypothalamus) thalamus) cerebellum. Copper restriction with chelation increased whole brain NPY mRNA 1.8-fold Ž p F 0.001, n s 6. compared to animals on the copper-adequate control diet ŽFig. 2.. Analysis of individual brain regions showed that the greatest increases Ž) 2-fold. were found in olfactory bulb and cerebellum ŽFig. 3.. While there was a clear trend toward an increase in NPY mRNA in other regions such as cortex, brain stem and thalamus, changes in these regions did not reach statistical significance ŽFig. 3.. Following this observation, the regulation of OB NPY mRNA by copper was investigated in a separate experiment without the addition of the chelator trien to insure that there was no independent effect of trien on NPY mRNA. Following 4 weeks on the copper restricted diet,
Fig. 3. Effect of copper restriction on brain region NPY mRNA abundance. Following 4 weeks of copper restriction and supplementation with a copper chelator Ž –Cu q trien., 15 mg whole brain total cellular RNA was subjected to Northern analysis. NPY mRNA was normalized to 28S rRNA abundance for each sample Ž n s 6.. Bars represent mean relative NPY mRNA. Inset shows representative Northern of olfactory bulb NPY mRNA and 28S rRNA in control and copper-restricted conditions Ž –Cu q trien.. )Statistically different from control values at p F 0.05.
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N.J. Rutkoski et al.r Molecular Brain Research 65 (1999) 80–86
Fig. 4. Effect of copper-restriction on olfactory bulb NPY mRNA abundance. Following 4 weeks of copper restriction Ž –Cu., 15 mg total cellular RNA was subjected to Northern analysis. NPY mRNA was normalized to 28S rRNA abundance for each sample Ž ns 5.. Bars represent relative NPY mRNA Žmean"S.E.M... )Statistically different from copper-adequate controls at pF 0.05.
NPY mRNA abundance was approximately 1.5-fold higher in –Cu rats compared to control rats Ž p F 0.05, n s 5. ŽFig. 4.. Measurement of NPY in rat OB by RIA revealed no significant changes following 4 weeks on the copper deficient diet Ž –Cu, Fig. 5.. However, the addition of trien to this diet Ž –Cu q trien, n s 3. resulted in a 1.4-fold increase in OB NPY Ž p F 0.05, Fig. 5.. NPY mRNA was also measured in primary cultures of OB neurons plated on astrocytes. NPY mRNA was not detected in cultures of OB astrocytes alone establishing that all NPY mRNA in these cultures was of neuronal origin Ždata not shown.. Treatment with the copper chelator TEPA not only reduced total cellular copper, but also increased NPY mRNA approximately two-fold over untreated control cells Ž p F 0.01, Fig. 6.. Treatment with
Fig. 5. Effect of copper-restriction on olfactory bulb NPY. Following 4 weeks of copper-restricted diet Ž –Cu. or a copper-restricted diet supplemented with a copper chelator Ž –Cuqtrien., NPY levels were measured in rat olfactory bulb by radioimmunoassay. )Statistically different from copper-adequate controls at pF 0.05.
Fig. 6. Effect of copper chelation on NPY mRNA abundance in primary cultures of olfactory bulb neurons. Olfactory bulb neurons from neonatal rats were plated on olfactory bulb astrocytes and treated with 100 mM TEPA or 1 mM carnosine. After 7 days of treatment, RNA was harvested for Northern analysis. ))Significantly different from control cells at pF 0.01.
carnosine did not alter copper concentrations or NPY mRNA abundance.
4. Discussion To date, a majority of the work on NPY has focused on its role as a hypothalamic stimulator of appetite and feeding behavior w11,29x. While deficiencies of the trace metal zinc on feeding behavior and NPY gene expression has been explored w24,40x, the role of copper in the regulation of this multifunctional neuropeptide has not been previously examined. Thus, we first examined the possible regulation of brain NPY mRNA in copper restricted rats. While there appeared to be a significant increase in NPY mRNA, functional measures of NPY alterations such as food intake and blood pressure were not changed by copper deficiency. This led to the hypothesis that NPY was not altered by copper deficiency in the hypothalamus, and the subsequent examination of the region specificity of the copper effect. Not only was NPY mRNA abundance highest in olfactory bulb ŽOB., this was a region that exhibited significant copper regulation. Given that the OB also contains high concentrations of copper compared to other regions w15x, we focused our remaining studies on the regulation of OB NPY by copper restriction. The similarities between the regulation of NPY mRNA by copper and zinc are striking. Zinc restriction for 3–6 weeks resulted in a 1.5 to two-fold increase in hypothalamic NPY mRNA w40,24x. Copper deficiency also resulted in a 1.5 to two-fold increase in the OB. We have also confirmed the regulation of NPY mRNA by copper restriction in primary cultures of OB neurons. The culture model suggests that the regulation of NPY mRNA is the result of copper deficiency in the OB and not due to hormonal or other factors originating outside of the bulb. Carnosine,
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while previously reported to act as an endogenous chelator of copper w8x, did not alter NPY mRNA abundance suggesting that copper–carnosine complexes may be available for the copper-dependent mechanisms involved in the regulation of NPY. The increases in NPY mRNA seen with copper restriction were accompanied to a lesser degree by increases in immunoreactive NPY ŽIR-NPY.. Likewise, one of the studies of zinc deficiency showed that up to 3 weeks of zinc restriction did not result in significant increases in hypothalamic IR-NPY, even though there was a two-fold increase in mRNA w40x. Subsequent studies examining the paraventricular region of the hypothalamus found a 1.4-fold increase in IR-NPY after 6 weeks of zinc deficiency Žcompared to an approximately two-fold increase in mRNA. and in a separate experiment a nearly two-fold increase in IR-NPY w24x. Unlike the increases in IR-NPY in the zinc deficiency studies w24x the 1.4-fold increase in IR-NPY in our copper deficiency studies cannot be explained by a reduction in food intake because feeding behavior was not altered in the copper deficient rats. While the exact mechanisms responsible for the increases in OB NPY mRNA are not known, the fact that the increases in NPY mRNA and IR-NPY are not proportional suggest that a deficiency of trace metals such as copper may interfere with translational mechanisms responsible for the synthesis of NPY. Copper restriction may also influence the export of NPY out of the bulb or the degradation rate of NPY. The current data do not rule out the possibility that copper plays a role in the stability of NPY mRNA. Furthermore, peptidylglycine a-amidating monooxygenase ŽPAM. is a copper dependent enzyme responsible for the post-translational modification of NPY w6x. While the antibody used in this study did not distinguish between these different forms of NPY, copper deficiency would be expected to reduce the post-translational amidation of NPY that has been shown to be required for NPY receptor binding w2x. Finally, there is a known antagonism between zinc and copper at the absorptive level w32x. This might lead to the hypothesis that the changes seen in the copper deficient rats were simply due to alterations in zinc status. However, there were no changes in OB zinc concentrations compared to controls in the current study. Furthermore, zinc and copper deficiency both increase NPY mRNA which is not consistent with a simple antagonism between the metals. The significance of the copper regulation of NPY in the OB requires further exploration. Proposals for NPY function in the OB include the regulation of aggression w21x, regulation of anxiety and depression w22,42,44x modulation of OB output w17x, and regulation of the auditory startle reflex w16,42x. Rats born to copper deficient dams and weaned to copper deficient diets had diminished auditory startle reflexes, even after 5 months of copper repletion w35x. Olfactory NPY may be involved in this behavior because surgical removal of the olfactory bulb resulted in
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an increase in the auditory startle reflex w28x. Furthermore, behavioral abnormalities seen after bulbectomy can be reversed by central administration of NPY w42x.
5. Conclusion In summary, the present results demonstrate that copper restriction increases NPY mRNA levels in the rat olfactory bulb and in primary cultures of rat olfactory bulb neurons. This increase is not accompanied by a proportional increase in NPY in the bulb suggesting that either export of NPY from the bulb is increased or translational mechanisms are impaired.
Acknowledgements The authors express their appreciation to Dr. Steven Sabol ŽLaboratory of Biochemical Genetics National Heart, Lung and Blood Institute, Bethesda, MD USA. for kindly providing the NPY clone used in this work and to Dr. J. Mark VanNess for his help collecting the blood pressure data. This work was supported by NIH Grant DK 50472.
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tion of the acoustic startle reflex produced by acute or repeated stress, Behav. Neurosci. 111 Ž1997. 80–91. M.S. Medeiros, A.J. Turner, Metabolism and functions of neuropeptide Y, Neurochem. Res. 21 Ž1996. 1125–1132. C.D. Minth, P.C. Andrews, J.E. Dixon, Characterization, sequence and expression of the cloned human neuropeptide Y gene, J. Biol. Chem. 261 Ž1986. 11974–11979. N. Misaki, H. Higuchi, K. Yamagata, N. Miki, Identification of glucocorticoid responsive elements ŽGREs. at far upstream of rat NPY gene, Neurochem. Int. 21 Ž1992. 185–189. P. Oestereicher, R.J. Cousins, Copper and zinc absorption in the rat, mechanism of mutual antagonism, J. Nutr. 115 Ž1985. 2159–2166. S.S. Percival, M. Layden-Patrice, HL-60 cells can be made copper deficient by incubating with tetraethylenepentamine, J. Nutr. 122 Ž122. Ž1992. 2424–2429. J.R. Prohaska, W.R. Bailey, Alterations of rat brain peptidylglycine a-amidating monooxygenase and other cuproenzyme activities following perinatal copper deficiency, Proc. Soc. Exp. Biol. Med. 210 Ž1995. 107–116. J.R. Prohaska, R.G. Hoffman, Auditory startle response is diminished in rats after recovery from perinatal copper deficiency, J. Nutr. 126 Ž1996. 618–627. E.W. Rice, Standardization of ceruloplasmin activity in terms of international enzyme units. Oxidative formation of ‘Brandrowski’s Base’ from p-phenylenediamine by ceruloplasmin, Anal. Biochem. 3 Ž1962. 452–456. S.L. Sabol, H. Higuchi, Transcriptional regulation of the neuropeptide Y gene by nerve growth factor: antagonism by glucocorticoids X X and potentiation by adenosine 3 5 -monophosphate and phorbol ester, Mol. Endocrinol. 4 Ž1990. 384–392. M.K. Schafer, D.A. Stoffers, B.A. Eipper, S.J. Watson, Expression of peptidylglycine a-amidating monooxygenase in the rat central nervous system, J. Neurosci 12 Ž1992. 222–234. M.W. Schwartz, R.J. Seeley, L.A. Campfield, P. Burn, D.G. Baskin, Identification of targets of leptin action in rat hypothalamus, J. Clin. Invest. 98 Ž1996. 1101–1106. P.L. Selvais, C. Labuche, X.N. Nguyen, J.M. Ketelslegers, J.F. Denef, D.M. Maiter, Cyclic feeding behaviour and changes in hypothalamic galanin and neuropeptide Y gene expression induced by zinc deficiency in the rat, J. Neuroendocrinol. 9 Ž1997. 55–62. R.M. Smith, Copper in the developing brain. In: I.E. Dreosti, R.M. Smith ŽEds.., Neurobiology of the Trace Elements, Humana Press, Clifton, NJ, 1983, pp. 1–31. C. Song, B. Earley, B.E. Leonard, The effect of central administration of NPY on behavior, neurotransmitter, and immune functions in the olfactory bulbectomized rat model of depression, Brain Behav. Immun. 10 Ž1996. 1–16. B.G. Stanley, S.F. Leibowitz, Neuropeptide Y injected in the paraventricular hypothalamus, a powerful stimulant of feeding behavior, Proc. Natl. Acad. Sci. USA 82 Ž1985. 3940–3943. C. Wahlestedt, E.M. Pich, G.F. Koob, F. Yee, M. Heilig, Modulation of anxiety and neuropeptide Y–Y1 receptors by antisense oligodeoxynucleotides, Science 259 Ž1993. 528–531.
Research report
Regulation of neuropeptide Y mRNA and peptide concentrations by copper in rat olfactory bulb Nancy J. Rutkoski a , Cheryl A. Fitch a , E. Carden Yeiser a , Janice Dodge a , Paul Q. Trombley b, Cathy W. Levenson a,b,) a
Department of Nutrition, Food and Exercise Sciences, Florida State UniÕersity, 237 Biomedical Research Facility, Tallahassee, FL 32308-4340, USA b Program in Neuroscience, Florida State UniÕersity, 237 Biomedical Research Facility, Tallahassee, FL 32308-4340, USA Accepted 8 December 1998
Abstract Neuropeptide Y is highly abundant in both the peripheral and central nervous systems and is known to have diverse functions including regulation of feeding behavior, blood pressure, circadian rhythms, reproductive behavior and the response to stress. Northern analysis showed that copper deficiency increased brain NPY mRNA abundance particularly in the olfactory bulb ŽOB.. These increases were not accompanied by alterations in food intake or blood pressure. After 4 weeks of a copper-restricted diet, OB copper concentrations decreased to 44% of control and NPY mRNA increased 1.5-fold. Addition of a copper chelator to the restricted diet, resulted in a two-fold increase in OB NPY mRNA over copper adequate controls. These results were confirmed in primary cultures of OB neurons suggesting that the regulation of NPY mRNA is at the level of the bulb rather than by a hormonal or other copper-regulated factor external to the OB. Immunoreactive NPY ŽIR-NPY. levels were not, however, increased following the 4 weeks of copper deficiency. Addition of the chelator resulted in a 1.4-fold increase in IR-NPY that, while statistically significant, was not proportional to the two-fold increase in NPY mRNA in the same study. This may suggest that copper deficiency inhibits the translational mechanisms responsible for the synthesis of NPY or that NPY is exported from the bulb in copper deficiency. q 1999 Elsevier Science B.V. All rights reserved. Keywords: NPY; Olfactory; Neuron; Brain; Copper; Deficiency
1. Introduction Neuropeptide Y ŽNPY., the most abundant known neuropeptide w1x, is a powerful orexigenic agent that induces feeding when injected centrally, even in satiated animals w11x. This function appears to be most closely associated with its action in the paraventricular nuclei ŽPVN. of the hypothalamus w43x. However, NPY expression is widely distributed throughout the central and peripheral nervous systems with high levels in olfactory bulb, cortex, and spinal cord, as well as the PVN w23,30x. Given this wide distribution, it is not surprising that NPY is now known to participate in diverse functions including regulation of neuroendocrine secretion w13x, blood pressure w14x, circadian rhythms w5x, and sexual behavior w11x. NPY has also
) Corresponding author. Florida State University, 237 Biomedical Research Facility, Tallahassee, FL 32308-4340, USA. Fax: q1-850-6440989; E-mail: [email protected]
been shown to play a role in the response to stress w26x, and the control of anxiety and depression w44x. A number of factors have been shown to regulate the expression of NPY. Nerve growth factor ŽNGF. w37x and epidermal growth factor ŽEGF. w13x increase NPY mRNA abundance. Insulin w18x, glucocorticoid hormones w31x and leptin w39x all decrease NPY mRNA abundance. Recently there have been two reports of hypothalamic NPY mRNA regulation by zinc deficiency w24,40x. In both studies, zinc deficiency resulted in a significant reduction in food intake, that is characteristic of zinc deficiency w9x, and a 1.5 to two-fold increase in hypothalamic NPY mRNA abundance w24,40x. The reported zinc regulation of NPY mRNA led us to test the possibility that NPY mRNA is also regulated by the trace metal copper. While copper deficiency has not been reported to reduce food intake, there are well known antagonistic interactions between these two essential trace metals w32x. Furthermore, copper is clearly needed for normal brain function as an essential cofactor for a number
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 3 4 5 - 3
N.J. Rutkoski et al.r Molecular Brain Research 65 (1999) 80–86
of essential enzymes w41x including cytochrome c oxidase which is an integral part of the electron transport chain w12x, superoxide dismutase ŽSOD., which acts as a free radical scavenger w7x, and dopamine b-monooxygenase ŽDBM., the rate limiting enzyme in catecholamine synthesis w20x. Copper is also essential for the activity of the enzyme peptidylglycine a-amidating monooxygenase ŽPAM. that is responsible for the post-translational modification of dozens of neurohormones and neuropeptides including NPY w34,38x.
2. Materials and methods 2.1. Animal care and housing The Animal Care and Use Committee ŽACUC. at Florida State University approved all animal protocols. Five-weekold male Sprague–Dawley rats were housed in hanging stainless steel cages in temperature-controlled rooms on a 12 h light–dark cycle with free access to distilled water. Rats in the copper-restricted groups Ž –Cu. received a casein-based, copper deficient diet Ž0.4 mg Curkg. ŽHarlan Teklad, Madison, WI. ad libitum. Control rats were fed a similarly composed diet containing adequate copper Ž18 mg Curkg.. Other animals Ž –Cu q trien. were fed the copper-deficient diet supplemented with the copper chelator triethylenetetramine Ž0.6% wrw. w4x. Food intake was evaluated throughout the study. After 4 weeks, rats were anesthetized with methoxyflurorane and brain or olfactory bulbs were immediately removed for isolation of total cellular RNA, radioimmunoassay, or measurement of copper and zinc concentrations. Trunk blood was also collected for preparation of serum and measurement of ceruloplasmin activity using the p-phenylenediamine oxidase ŽPPD. assay w19,36x.
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and serum extender. Additional plates were treated with 1 mM carnosine. Carnosine has been reported to act as an endogenous copper chelator w8x. Control cells were grown in the supplemented serum-free medium. After 7 days of treatment, cells were harvested for isolation of total cellular RNA and Northern analysis or measurement of total cellular copper. 2.3. Northern analysis The acid guanidinium thiocyanate–phenol–chloroform extraction method was used to isolate total cellular RNA w10x from whole brain Ž n s 6. or brain regions Žolfactory bulb, cortex, brain stem, hypothalamus, thalamus, cerebellum and pituitary, n s 4. from rats fed either the control or –Cu q trien diets for 4 weeks. Olfactory bulb RNA was also collected from control Ž n s 5. and copper-restricted Ž –Cu, n s 5. rats. Purity of the RNA as verified by A 260rA 280 ratio. Equal amounts of total RNA Ž15 mg. were fractionated by size on a 1% Žwrv. agarose gel containing 0.66 M formaldehyde. RNA was then transferred to a nylon membrane ŽGeneScreen, NEN, Boston, MA. by capillary blotting. RNA transfer was confirmed by visualization of ethidium bromide stained RNA under UV light. Blots were UV crosslinked and stored at 48C until hybridization with a random primed 32 P-labeled preproNPY cDNA probe ŽRadPrime DNA Labeling System, Gibco BRL, Gaithersburg, MD.. Equal loading of lanes was confirmed by hybridization to 32 P-labeled 28S rRNA probe. Filters were exposed to Kodak X-OMAT AR film at y808C. Relative amounts of bound cDNA probe were determined by computer evaluated densitometry ŽQuantity One quantification program. created by Protein and DNA Imaging ŽPDI, Boston, MA. and expressed as a function of 28S rRNA abundance. 2.4. Radioimmunoassay
2.2. Cell culture Using sterile technique, olfactory bulbs were excised from 7-day-old rat pups. The dura was removed and the tissue minced and placed in dissection buffer ŽCarEDTA with 20 Urml papain, Collaborative Research, Bedford, MA. at 378C for 1 h. Following enzyme digestion, the tissue was gently disrupted with a polished pasteur pipette, and olfactory bulb neurons ŽOBN. released into the media were transferred to 15 ml conical centrifuge tubes, pelleted at low speed, and resuspended in Minimum Eagles Media ŽMEM, Gibco USA. with 4% glucose, fetal calf serum ŽFCS. and a nutrient supplement ŽSerum Extender, Collagen, Palo Alto, CA.. Cells in media Ž3.66 = 10 6 . were plated on olfactory bulb astrocytes grown to confluence in 60 mm dishes. Following one week in culture, the copper chelator tetraethylene pentamine ŽTEPA, 100 mM. w33x was added to cells in serum free media with 4% glucose
A modification of the radioimmunoassay developed by Beck et al. w3x was used to measure olfactory bulb NPY in control Ž n s 8. and copper-restricted Ž n s 10. rats. After the 4 weeks of copper restriction, olfactory bulbs were removed and immediately sonicated at 48C in 0.3 ml buffer Ž0.2N HClr0.1% EDTA, containing 0.5% Titron X-100 and Aprotinin 100 mgrml.. Sonicated samples Ž100 ml. were incubated overnight at 48C with a rabbit antirat-NPY antibody Ž1:8000, Sigma, St. Louis, MO.. Synthetic NPY ŽSigma. was used as the standard. The tracer, I 125 NPY Ž0.25 ngr100 ml, Amersham, Arlington Heights, IL., was added and incubated overnight at 48C. Bound NPY was separated from free using a 0.4% dextranr0.4% charcoal suspension. All tubes were centrifuged at 2000 = g for 20 min and the 125 I in the supernatant detected using a Micromedic 4r600 Plus gamma counter ŽMicromedic Systems, Huntsville, AL.. The range of the standard curve
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Table 1 Effect of copper restriction on brain and olfactory bulb copper Žmgrg wet wt.
Control
–Cu
–Cuqtrien
Whole brain Olfactory bulb
2.15"0.13 3.18"0.79
1.51"0.16) 1.43"0.36)
1.32"0.16) 1.45"0.11)
–Cu, 4-week copper restriction. –Cuqtrien, 4-week copper restriction with chelator. )Significant at pF 0.05.
was 1 ng–128 ng NPY. Concentrations were estimated for each sample using weighted linear regression. The inter-assay coefficient of variation was - 10% and the intra-assay coefficient of variation was - 5% for duplicate measurements. Data is expressed as ng NPYrmg total protein as determined by Lowry protein assay w27x. 2.5. Measurement of brain copper concentration Whole brain Ž n s 5 in control, –Cu, and –Cu q trien groups. or olfactory bulbs Ž n s 4 in control, –Cu, and –Cu q trien. were digested with nitric acid and hydrogen peroxide as previously described w25x. Flame atomic absorption spectroscopy ŽPerkin Elmer 3100. was used to determine whole brain copper concentrations Žmg Curg wet weight.. Graphite furnace atomic absorption spectrophotometry ŽZeeman 5100, Perkin Elmer, Norwalk, CT. was used to measure copper concentration in rat olfactory bulb Žmg Curwet wt. and cultured bulb neurons Žmg Curmg protein.. Copper concentrations were determined by automated analysis based on a standard curve developed using Fisher copper reference solution. Repeated measures of the same sample revealed an intra-sample variation of - 1%.
ŽGraphPad Software, San Diego, CA.. Values were considered statistically significant at p F 0.05.
3. Results 3.1. Copper status Copper restriction resulted in a significant reduction in the activity of the copper-dependent serum enzyme ceruloplasmin ŽCp.. After 4 weeks of copper restriction, Cp activity decreased to 8% of control Ž p F 0.001, n s 5.. Table 1 shows the effect of dietary copper restriction and the addition of the chelator trien on brain and olfactory bulb ŽOB. copper. Four weeks of restriction Ž –Cu. significantly reduced both whole brain and OB copper levels Ž p s 0.05, n s 4–5.. Neither the –Cu or –Cu q trien diets significantly altered zinc levels in OB or whole brain Žcontrol 12.1 " 0.7; –Cu 11.2 " 1.12; –Cu q trien 13.1 " 1.8 mgrg wet wt.. Copper chelation reduced copper levels in primary cultures of neurons by approximately 25% Ž11.7 " 0.7 vs. 8.9 " 1.0 mg Curmg protein.. Addition of the endogenous chelator carnosine did not significantly alter OB cell copper Ž13.9 " 0.8 mg Curmg protein. compared to controls. 3.2. Effect of copper-restriction on feeding and blood pressure Given the role of NPY in controlling feeding behavior and vasoconstriction, measurements were made to detect changes in food intake or blood pressure following copper restriction. Copper restriction did not result in significant differences in daily food intake, nor did it alter food intake in response to a 24–48 h fast. Additionally, there was no
2.6. Blood pressure Blood pressure was assessed in an additional group of copper deficient and copper sufficient rats Ž n s 3.. Under halothane anesthesia, intravascular catheters were chronically implanted into the descending aorta via the left carotid artery and tunnelled to the nape of the neck, exteriorized, and sealed with a 28-gauge stainless steel pin. Catheters were flushed daily with heparinized saline Ž100 Urml.. After one week of recovery, blood pressure was measured in conscious, unrestrained rats on three successive days using transducers connected to amplifiers interfaced with an AT-CODAS data acquisition card ŽDATAQ Instruments, Akron, OH.. Blood pressure was sampled at 200 Hz and written to a computer hard drive for subsequent analysis. 2.7. Statistical analysis Data were analyzed using ANOVA with a Tukey’s multiple group post-hoc test and unpaired Students t-test
Fig. 1. Northern analysis of NPY mRNA in brain regions. Total cellular RNA Ž15 mg. from each adult rat brain region was subjected to Northern analysis using a 32 P-labeled NPY cDNA probe. Relative abundance of NPY mRNA in each region is shown in this representative autoradiograph.
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control Ž118 " 3 mmHg. and copper deficient Ž122 " 3 mmHg. rats did not reveal significant differences. 3.3. Effect of copper-restriction on NPY and NPY mRNA
Fig. 2. Effect of copper-restriction on brain NPY mRNA abundance. Following 4 weeks of copper restriction and supplementation with a copper chelator Ž –Cuqtrien., 15 mg whole brain total cellular RNA was subjected to Northern analysis. NPY mRNA was normalized to 28S rRNA abundance for each sample Ž ns6.. Bars represent relative NPY mRNA Žmean"S.E.M... )))Statistically different from control values at pF 0.001.
difference in body weight of rats in the –Cu group Ž284 " 29 g. as compared to control group Ž277 " 30 g.. Direct measurement of mean arterial blood pressure ŽMAP. in
Northern analysis of adult rat brain regions revealed that NPY mRNA abundance is highest in olfactory bulb ŽFig. 1.. Other brain regions tested had significantly lower NPY mRNA levels with cortex) brain stem ) hypothalamus) thalamus) cerebellum. Copper restriction with chelation increased whole brain NPY mRNA 1.8-fold Ž p F 0.001, n s 6. compared to animals on the copper-adequate control diet ŽFig. 2.. Analysis of individual brain regions showed that the greatest increases Ž) 2-fold. were found in olfactory bulb and cerebellum ŽFig. 3.. While there was a clear trend toward an increase in NPY mRNA in other regions such as cortex, brain stem and thalamus, changes in these regions did not reach statistical significance ŽFig. 3.. Following this observation, the regulation of OB NPY mRNA by copper was investigated in a separate experiment without the addition of the chelator trien to insure that there was no independent effect of trien on NPY mRNA. Following 4 weeks on the copper restricted diet,
Fig. 3. Effect of copper restriction on brain region NPY mRNA abundance. Following 4 weeks of copper restriction and supplementation with a copper chelator Ž –Cu q trien., 15 mg whole brain total cellular RNA was subjected to Northern analysis. NPY mRNA was normalized to 28S rRNA abundance for each sample Ž n s 6.. Bars represent mean relative NPY mRNA. Inset shows representative Northern of olfactory bulb NPY mRNA and 28S rRNA in control and copper-restricted conditions Ž –Cu q trien.. )Statistically different from control values at p F 0.05.
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Fig. 4. Effect of copper-restriction on olfactory bulb NPY mRNA abundance. Following 4 weeks of copper restriction Ž –Cu., 15 mg total cellular RNA was subjected to Northern analysis. NPY mRNA was normalized to 28S rRNA abundance for each sample Ž ns 5.. Bars represent relative NPY mRNA Žmean"S.E.M... )Statistically different from copper-adequate controls at pF 0.05.
NPY mRNA abundance was approximately 1.5-fold higher in –Cu rats compared to control rats Ž p F 0.05, n s 5. ŽFig. 4.. Measurement of NPY in rat OB by RIA revealed no significant changes following 4 weeks on the copper deficient diet Ž –Cu, Fig. 5.. However, the addition of trien to this diet Ž –Cu q trien, n s 3. resulted in a 1.4-fold increase in OB NPY Ž p F 0.05, Fig. 5.. NPY mRNA was also measured in primary cultures of OB neurons plated on astrocytes. NPY mRNA was not detected in cultures of OB astrocytes alone establishing that all NPY mRNA in these cultures was of neuronal origin Ždata not shown.. Treatment with the copper chelator TEPA not only reduced total cellular copper, but also increased NPY mRNA approximately two-fold over untreated control cells Ž p F 0.01, Fig. 6.. Treatment with
Fig. 5. Effect of copper-restriction on olfactory bulb NPY. Following 4 weeks of copper-restricted diet Ž –Cu. or a copper-restricted diet supplemented with a copper chelator Ž –Cuqtrien., NPY levels were measured in rat olfactory bulb by radioimmunoassay. )Statistically different from copper-adequate controls at pF 0.05.
Fig. 6. Effect of copper chelation on NPY mRNA abundance in primary cultures of olfactory bulb neurons. Olfactory bulb neurons from neonatal rats were plated on olfactory bulb astrocytes and treated with 100 mM TEPA or 1 mM carnosine. After 7 days of treatment, RNA was harvested for Northern analysis. ))Significantly different from control cells at pF 0.01.
carnosine did not alter copper concentrations or NPY mRNA abundance.
4. Discussion To date, a majority of the work on NPY has focused on its role as a hypothalamic stimulator of appetite and feeding behavior w11,29x. While deficiencies of the trace metal zinc on feeding behavior and NPY gene expression has been explored w24,40x, the role of copper in the regulation of this multifunctional neuropeptide has not been previously examined. Thus, we first examined the possible regulation of brain NPY mRNA in copper restricted rats. While there appeared to be a significant increase in NPY mRNA, functional measures of NPY alterations such as food intake and blood pressure were not changed by copper deficiency. This led to the hypothesis that NPY was not altered by copper deficiency in the hypothalamus, and the subsequent examination of the region specificity of the copper effect. Not only was NPY mRNA abundance highest in olfactory bulb ŽOB., this was a region that exhibited significant copper regulation. Given that the OB also contains high concentrations of copper compared to other regions w15x, we focused our remaining studies on the regulation of OB NPY by copper restriction. The similarities between the regulation of NPY mRNA by copper and zinc are striking. Zinc restriction for 3–6 weeks resulted in a 1.5 to two-fold increase in hypothalamic NPY mRNA w40,24x. Copper deficiency also resulted in a 1.5 to two-fold increase in the OB. We have also confirmed the regulation of NPY mRNA by copper restriction in primary cultures of OB neurons. The culture model suggests that the regulation of NPY mRNA is the result of copper deficiency in the OB and not due to hormonal or other factors originating outside of the bulb. Carnosine,
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while previously reported to act as an endogenous chelator of copper w8x, did not alter NPY mRNA abundance suggesting that copper–carnosine complexes may be available for the copper-dependent mechanisms involved in the regulation of NPY. The increases in NPY mRNA seen with copper restriction were accompanied to a lesser degree by increases in immunoreactive NPY ŽIR-NPY.. Likewise, one of the studies of zinc deficiency showed that up to 3 weeks of zinc restriction did not result in significant increases in hypothalamic IR-NPY, even though there was a two-fold increase in mRNA w40x. Subsequent studies examining the paraventricular region of the hypothalamus found a 1.4-fold increase in IR-NPY after 6 weeks of zinc deficiency Žcompared to an approximately two-fold increase in mRNA. and in a separate experiment a nearly two-fold increase in IR-NPY w24x. Unlike the increases in IR-NPY in the zinc deficiency studies w24x the 1.4-fold increase in IR-NPY in our copper deficiency studies cannot be explained by a reduction in food intake because feeding behavior was not altered in the copper deficient rats. While the exact mechanisms responsible for the increases in OB NPY mRNA are not known, the fact that the increases in NPY mRNA and IR-NPY are not proportional suggest that a deficiency of trace metals such as copper may interfere with translational mechanisms responsible for the synthesis of NPY. Copper restriction may also influence the export of NPY out of the bulb or the degradation rate of NPY. The current data do not rule out the possibility that copper plays a role in the stability of NPY mRNA. Furthermore, peptidylglycine a-amidating monooxygenase ŽPAM. is a copper dependent enzyme responsible for the post-translational modification of NPY w6x. While the antibody used in this study did not distinguish between these different forms of NPY, copper deficiency would be expected to reduce the post-translational amidation of NPY that has been shown to be required for NPY receptor binding w2x. Finally, there is a known antagonism between zinc and copper at the absorptive level w32x. This might lead to the hypothesis that the changes seen in the copper deficient rats were simply due to alterations in zinc status. However, there were no changes in OB zinc concentrations compared to controls in the current study. Furthermore, zinc and copper deficiency both increase NPY mRNA which is not consistent with a simple antagonism between the metals. The significance of the copper regulation of NPY in the OB requires further exploration. Proposals for NPY function in the OB include the regulation of aggression w21x, regulation of anxiety and depression w22,42,44x modulation of OB output w17x, and regulation of the auditory startle reflex w16,42x. Rats born to copper deficient dams and weaned to copper deficient diets had diminished auditory startle reflexes, even after 5 months of copper repletion w35x. Olfactory NPY may be involved in this behavior because surgical removal of the olfactory bulb resulted in
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an increase in the auditory startle reflex w28x. Furthermore, behavioral abnormalities seen after bulbectomy can be reversed by central administration of NPY w42x.
5. Conclusion In summary, the present results demonstrate that copper restriction increases NPY mRNA levels in the rat olfactory bulb and in primary cultures of rat olfactory bulb neurons. This increase is not accompanied by a proportional increase in NPY in the bulb suggesting that either export of NPY from the bulb is increased or translational mechanisms are impaired.
Acknowledgements The authors express their appreciation to Dr. Steven Sabol ŽLaboratory of Biochemical Genetics National Heart, Lung and Blood Institute, Bethesda, MD USA. for kindly providing the NPY clone used in this work and to Dr. J. Mark VanNess for his help collecting the blood pressure data. This work was supported by NIH Grant DK 50472.
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