Catecholamine synthesizing enzymes in various brain regions of the genetically obese Zucker rat

560

Brain Research, 171 (1979) 560-566 J(~)Elsevier/North-HollandBiomedicalPress

Catecholamine synthesizing enzymes in various brain regions of the genetically obese Zucker rat

BARRY E. LEVIN and ANN C. SULLIVAN The Department of Neurosciences of the New Jersey Medical School, College of Medicine and Dentistry of New Jersey and the Department of Neurology, V.A. Hospital, East Orange, N.J. 07019 and ( A.C.S.) The Department of Biochemical Nutrition, Roche Research Center, Hoffmann-La Roche Inc., Nutley, N.J. 07110 (U.S.A,)

(Accepted April 12th, 1979)

The Zucker rat is an animal model of obesity and this characteristic is inherited as an autosomal recessive trait z9 which is associated with a number of physiological and endocrine abnormalities. These include hyperphagia2,8, hypercellular adiposity t2, hyperinsulinemiazS, hyperlipogenesisa6, 23, gonadal dysfunction22 and abnormalities m temperature regulation9. It is now generally accepted that hypothalamic catecholamines probably play some role in the regulation of feeding1°,18,17 and gonadalendocrine control 1~ in the normal rat. Obese Zucker rats have norepinephrine levels in the paraventricular nucleus and median eminence which are different from their lean counterparts, and these levels vary somewhat according to sex, and possibly ageZ,6. No differences have been found in dopamine levels5;6, and epinephrine levels have not been examined, although both may play a role in the modulation of feedingxz and neuroendocrine function. Noradrenergic cell bodies lie in nuclear groups within the pons and medulla and pass rostrally to innervate the hypothalamus via dorsal (originating primarily in the locus coeruleus) and ventral bundles which unite in the posterior hypothalamus to enter the median forebrain bundle z4. The majority of dopamine-containing terminals in the hypothalamus originate from endogenous dopaminergic cells 24. Epinephrine cell bodies lie primarily in the medulla and ascend in close proximity to the noradrenergic fibers to supply hypothalamic sites H. Therefore, norepinephrine and epinephrine neuronal cell bodies lie primarily outside the hypothalamus, while dopaminergic cell bodies are found within the hypothalamus along with fibers of passage and terminals of all 3 catecholamine neurons. The present study examined the activity of 3 catecholamine synthesizing enzymes in the hypothalamus, brain stem and striatum in the Zucker rat to further characterize differences in catecholamine metabolism between lean and obese animals. Tyrosine hydroxylase (TH:EC I. 14.3a) is the rate limiting enzyme in catecholamine synthesis and its activity correlates well with dopamine levels2L Dopamine-/3-hydroxylase (DBH:EC 1.14.2.1) converts dopamine to norepinephrine and its activity

561

MEDIAL HYPOTHALAMUS

0.4 0.2

LATERAL HYPOTHALAMUS

~ O.4

LI 5

° 2.0

3.25

MEDULLA

I~ 1.6 j t

"

0.8

~

Female Obese

Female Lean

Male Obese

Mole Leon

Fig. 1. PN MT activity in brain regions of various Zucker rat groups : open bars represent 13-14-weekand stippled bars, 30-32-week-old rats, respectively. Vertical lines above bars give the S.E.M. ; * significant difference (P < 0.05, two-tailed Student's t-test) between 13-14 week and 30-32 week rats of the same sex and genotype; t = significant difference (P < 0.05) between lean and obese animals of the same sex and age; "~ ~ significant difference (P < 0.05) between male and female animals of the same age and genotype. Units = nmol [3HlN-methylphenylethanolamine formed/g wet weight tissue/h.

correlates well with n o r e p i n e p h r i n e levels in the h y p o t h a l a m u s zl. P h e n y l e t h a n o l a m i n e - N - m e t h y l - t r a n s f e r a s e ( P N M T : E C 2.1.1) converts n o r e p i n e p h r i n e to epinephrine a n d is found exclusively in epinephrine c o n t a i n i n g cells, but m a y n o t correlate well with epinephrine levels 25. Eight sets of animals, at two different ages, were studied in groups o f 4 - 6 male o r female rats o f b o t h h o m o z y g o u s lean ( F a / F a ) and obese (fa/fa) genotypes. R a t s were h o u s e d 1--2 per cage on ad libitum P u r i n a rat chow with 12 h l i g h t - d a r k cycles for at least 3 weeks p r i o r to study at 13-14 and 30-32 weeks o f age, respectively. A n i m a l s were killed by decapitation, the brains quickly removed and dissected on ice into 5 sections: (1) s t r i a t u m and h y p o t h a l a m u s were dissected by the m e t h o d o f G l o w i n k s i a n d Iversen 8, and the h y p o t h a l a m u s was further divided into medial and lateral p o r t i o n s by p a r a s a g i t t a l cuts m i d w a y between the third ventricle and lateral margin o f the section; (2) locus coeruleus was dissected by the m e t h o d o f Z i g m o n d et al. zv in the 13-14 week rats and that of Reis a n d Ross 2° in 30-32 week animals; and ( 3 ) t h e C1 a n d C2 m e d u l l a r y groups o f P N M T - c o n t a i n i n g cell bodies

562 MEDIAL HYPOTHALAMUS

I0

40i

LATERAL HYPOTHALAMUS

~20

o -r

I--

otli II I l l ! 40

STRIATUM

Fete

Ol~,e

Femele

Leon

Mele

Obe,,e

Mele

Lean

Fig. 2. TH activity in brain regions of various Zucker rat groups: legend is the same as Fig. 1. Units ~ nmol [3H]dihydroxyphenylalanineformed/g wet weight tissue/h. were dissected in wedge-shaped sections corresponding to the diagrams of H6kfelt et al. 11 and combined as 'medulla'. All sections were weighed, diluted 10-fold with 5 mM Tris buffer, pH 7.5, containing 0.1 ~ Triton XI00 and son±cared. Aliquots of the 11,000 × g supernatants were assayed for T H on day 1 (method of Black1), DBH on day 2 (method of Reis and Ross 2° as modified from Molinoff et a1.19), and P N M T on day 3 (method of Lew et a1.14). The body weights in grams (mean ± S.E.) for all artimals studied were as follows: 13-14 week female obese and lean, 284 ± 17 and 212 ± 7, respectively; 30-32 week female obese and lean, 495 ± 9 and 269 ± 8, respectively; 13-14 week male obese and lean, 361 ± 31 and 325 ± 7, respectively; 30-32 week male obese and lean, 629 ± 10 and 489 ± 8, respectively. Some significant differences in weights of dissected brain areas did occur, primarily related to the two techniques used for the locus coeruleus where the sections from older rats weighed up to two times that of the comparable younger groups. Smaller, but significant differences were also present in the medulla sections for the young vs old female obese (149 ~o increase) and male tear, (129 ~ increase) groups. However, none of the age- or genotype.retated changes appeared to be influenced by differences in section weights since units calculated on a 'per nuclear group' basis showed the same changes.

563 MEDIAL HYPOTHALAMU$

c

LATERAL HYPOTHALAMUS

LOCUS COERULEUS

)

6

iio

r l Female

Obese

Femal Lean

Male

Obese

Male

Loan

Fig. 3. DBH activity in brain regions of various Zucker rat groups : legend is the same as Pig. 1. Units = nmol [14C]octopamine formed/g wet weight tissue/h for lateral and medial hypothalamus or per locus coeruleus.

Several changes in enzyme activities occurred which were related to sex, genotype and age differences. A significant sex effect in P N M T activity was observed by analysis of variance in both the lateral hypothalamus [F(1,29) -- 4.89, P < 0.035] and medulla [F(1,28) = 9.00, P < 0.006], where activities in lean males were higher than females in both areas at 3 months of age (Fig. 1). Although no other overall differences by sex were present by analysis of variance, individual comparisons by ttest revealed higher T H activity in 3-month-old obese males in the medial hypothalamus and higher activity in 7-month-old lean females in the lateral hypothalamus (Fig. 2). Medial hypothalamic and locus coeruleus D B H activity was higher in 7month-old lean females, while lateral hypothalamic activity was higher in the 7month-old female obese than male rats (Fig. 3). Although no overall gene effect was seen in the 3 brain areas, individual comparisons demonstrated significant changes in P N M T activity in the medulla where levels in 13-14 week obese males were 41 ~ of lean, and in the 30-32 week obese female rats were 1 6 2 ~ of lean. A significant age effect in P N M T activity was seen in medullary P N M T , [F(1,28) = 93.54, P < 0.001] where activities decreased significantly in all groups from 13-14 to 30-32 weeks of age.

564 This was comparable to the decreases seen in nerve cell bodies for DBH '~ and T H ~ during early brain development, although no accompanying increase was seen for P N M T in the terminal areas of the hypothalamus as occurs with DBH and TH. The only significant genotype related TH change was a 29 ~i decrease in mediat hypothalamic activity in the 30-32 week male obese rats compared to the lean (Fig. 2). By analysis of variance, TH activity decreased significantly with age in the medial hypothalamus [F(1,31) -- 7.44, P .< 0.01]. In the lean males, TH activity decreased with age in both hypothalamic sections, while in the obese males it was decreased in the medial hypothalamus. No significant changes were present in the striatum. DBH activities increased significantly from 13-14 weeks to 30~32 weeks of age in the medial hypothalamus by analysis of variance [F(1,28) := 25.50, P < 0.001], lateral hypothalamus [F(1,27) ~- 13.80, P ~- 0.001] and in the locus coeruleus [F(1,25) 10.88, P < 0.003] (Fig. 3). However, the change in dissection techniques could account for the latter differences in the locus coeruleus. No overall sex or genotype changes were seen although DBH activity in the 30 32 week female obese was 135 °~i of the female lean in the lateral hypothalamus. The significant changes reported in this study, therefore, were increases in DBH with age in the medial and lateral hypothalamus and locus coeruleus, and decreases in activities of both T H in the medial hypothalamus and P N M T in the medulla. Males showed the only genotype related differences between lean and obese in the 13-14 weeks age groups, with lower PNMT in the medulla of the obese rats. At 30-32 weeks, T H was also decreased in obese males in the medial hypothalamus, while PNMT in the medulla and DBH in the lateral hypothalamus were increased in the female obese rats compared to the lean. Sex-related changes occurred at 3 months for P N M T (lateral hypothalamus and medulla) and T H (medial hypothalamus) where males had higher levels than females, while at 7 months, females had higher T H (lateral hypothalamus) and DBH (medial and lateral hypothalamus, and locus coeruleus) activities than males. The major problems in interpreting these data are first, whether catecholamine or enzyme activity levels are a cause or effect of the differences in the metabolic and behavioral parameters previously observed between the lean and obese Zucker rat. For example, the acts of feeding 26 or drinking 7 are associated with changes in brain catecholamine uptake and metabolism. Second, there is the difficulty of relating changes in enzyme activities to abnormalities in specific functions. While it is tempting to try to correlate the present findings to differences in ingestive behavior in the Zucker rat because of the extensive literature relating catecholamines to feeding behavior, the differences in enzyme activities might just as plausibly be related to abnormalities in gonadal or endocrine function, temperature regulation, or even to possible differences in learning ability or behavioral responses. Third, while most attention has been focused upon the role of hypothalamic catecholamines in regard to feeding behavior, another possibility is that the norepinephrine, epinephrine and dopaminergic innervations of the vagal and other medullary centers, rather than their hypothalamic connections, could be responsible for differences in ingestive behavior or other metabolic parameters known to differ between the obese and lean Zucker rat. Finally,

565 neither enzyme activity nor catecholamine levels give an adequate understanding of the actual m e t a b o l i c events occurring within a given area. Clearly, c a t e c h o l a m i n e turnover, r e - u p t a k e a n d receptor mechanisms m u s t also be considered before specific predictions can be m a d e a b o u t the meaning o f the changes f o u n d in this and similar studies. The i m p o r t a n t findings o f the present study were t h a t considerable changes in c a t e c h o l a m i n e synthesizing enzymes continue to take place as the Z u c k e r rat ages, n o t j u s t in early development. In m a n y ways, the age related changes o v e r s h a d o w the differences in enzyme activities between the lean a n d obese animals, a n d in fact, m a y be responsible for the o b s e r v a t i o n t h a t all b u t one o f the differences between the genotypes occurred in the older animals. The reasons t h a t such differences were only evident in the 30-32-week-old rats are speculative, b u t these results suggest t h a t future studies o f brain catecholamines in the Z u c k e r rat should be carried out in older, as well as y o u n g e r animals. This w o r k was s u p p o r t e d in part by a grant from the F o u n d a t i o n o f the College o f Medicine and Dentistry of New Jersey a n d by the Medical Research Service o f the Veterans A d m i n i s t r a t i o n . We gratefully acknowledge the expert technical assistance of Victoria H u n t a n d Jean A d a m u s . B a r b a r a Van Ness provided excellent assistance in the p r e p a r a t i o n of this manuscript.

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