Effects of monosodium glutamate and gold thioglucose on dietary regulation of sympathetic nervous system activity in rodents

Metabolism Clinical and Eqmimental VOL 40, NO 2

FEBRUARY 1991

Effects of Monosodium Glutamate and Gold Thioglucose on Dietary Regulation of Sympathetic Nervous System Activity in Rodents Abdul

G. Dulloo

and James

B. Young

Neonatal administration of monosodium glutamate (MSG) disrupts hypothalamic regulation of a number of neuroendocrine systems. Studies described in this report using techniques of norepinephrine (NE) turnover examined sympathetic nervous system (SNS) activity in heart and interscapular brown adipose tissue (IBAT) of animals given MSG as neonates. Although in every experiment overall rates of NE turnover were lower in MSG-treated mice and rats, the differences were due exclusively to diminished tissue NE content, especially in IBAT. Fractional rates of NE turnover did not differ between groups. In contrast to animals with lesions in the ventromedial hypothalamus produced by gold thioglucose (AuTG) or electric current, MSG-treated mice and rats varied SNS activity in heart and IBAT in accord with changes in nutrient intake. Thus, SNS activity, both at baseline and in resoonse to dietarv maniuulation. is urobablv not affected by neonatal MSG administration. Copyright 8 19916y WA Saundak Company

. .

E

VIDENCE OF neurotoxic effects of monosodium glutamate (MSG) following injection into neonatal animals was reported 20 years ago.’ In addition to causing destructive lesions in specific neural structures (arcuate, preoptic, and ventromedial nuclei of the hypothalamus, circumventricular organs and retina), MSG also induced an obesity syndrome, which occurred in the absence of hyperphagia.2e4 Since that time, numerous deficits in hypothalamic function have been identified in MSG-treated animals involving regulation of feeding behavior,’ body temperature: and release of various neuropeptides, including growth hormone releasing factor,’ &endorphin/ACTH,* and vasopressin’ among others. However, the impact of MSG on the regulation of sympathetic nervous system (SNS) activity has not yet been determined. Several independent sets of observations raised the possibility that dietary regulation of the SNS might be abnormal in MSG-treated rats or mice. First, SNS activity in mice fed ad libitum was reportedly diminished in MSG-treated mice,“‘.‘3 implying an effect of MSG administration on SNS regulation. Second, lesions in the hypothalamus induced by other means, gold thioglucose (AuTG) or electric current, abolished dietary regulation of SNS activity.‘+” Third, the reduction in MSG-treated rats of binding by blood-borne insulin to receptors in median eminence (presumably located on axonal projections of neurons in arcuate nucleus)16 and the attenuation of behavioral responses to insulin administration” suggested that MSG might disrupt other hypothalamic responses to carbohydrate or insulin, such as stimulation of SNS activity. And Metabolism,

Vol40,No 2 (Februaryj.1991: pp 113-121

fourth, in spontaneously hypertensive rats the antihypertensive effect of fasting, like that induced by the sympatholytic agents, clonidine, a-methyl DOPA, and propranolol, is mediated, in part, by an opiate-dependent mechanism.“.” Since MSG treatment eliminates these cardiovascular responses to clonidine,” central MSG-sensitive opiate mechanisms might also participate in the sympatholytic response to fasting. The following studies were, therefore, undertaken to test the hypothesis that neonatal treatment with MSG impairs dietary regulation of the SNS. Measurements of norepinephrine (NE) turnover in heart and interscapular brown adipose tissue (IBAT) of unanesthetized animals served as the index of SNS activity. Results obtained in both mice and rats indicated that, contrary previous reports,‘“‘3 MSG treatment does not affect SNS activity either at baseline or in response to alterations in nutrient intake. In contrast, nutrient regulation of SNS activity, particularly in IBAT, is markedly impaired in mice that become obese following AuTG administration.

From rhe Charles A. Dana Research Institute and Thorndike Laboratory, Department of Medicine, Harvard Medical School, Beth Israel Hospital, Boston, MA. Supported in part by US Public Health Service Grants No. DK 20378 and AG 00599. Address reprint requests to James B. Young, MD, Department of Medicine, Beth Israel Hospital, 330 Brookhne Ave, Boston, MA 0221.5. Copyright 0 1991 by WB. Saunders Company 00260495l91l4002-0001$03.00/0 113

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114

METHODS

Animals Pregnant CD-l mice (Charles River Breeding Laboratories, Wilmington, MA) were housed singly in plastic cages prior to delivery. On days 1 to 7 after birth, the offspring (litters of 10 to 12 pups) were injected subcutaneously with either monosodium L-glutamate (Sigma Chemical, St Louis, MO) as described previously,2’ or 0.9% NaCI. The dose of MSG employed was 3 mg/g body weight (300 mg MSG/mL 0.9% NaCl x 0.01 mL/g body weight). On the other hand, rat pups (CD male, Charles River) were obtained within 24 hours after birth; each litter of 10 to 12 pups was accompanied by a foster mother. The newborn rats were injected with MSG or saline according to the same schedule as employed for mice. Both mice and rats were weaned at 4 weeks of age. Mice were then housed in groups of five to seven of similar sex and treatment, while the rats were housed in pairs. All MSG- or saline-treated animals were kept in metal-suspended cages in rooms maintained at 21 2 2°C (with a 12-hour light/dark cycle). Studies were performed in both mice and rats at approximately 8 weeks of age. AuTG was administered to adult female mice, (20 to 25 g). Animals were fasted overnight before being injected with AuTG (800 mg/kg intraperitoneally). Studies were performed 9 weeks after AuTG administration. At the time of turnover measurement, AuTG mice were separated into one of two groups for study. Those which weighed 24 SD above the mean of control animals were referred to as AuTG-Fat mice (the same criterion used to determine eligibility for study in the previous report from this laboratory”‘); those that weighed 13 SD above the control mean were referred to as AuTG-Lean mice. Animals used in these studies were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and those prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHHS publication no. [NIH] 78-23, revised 1978). Diets

AND YOUNG

adsorbed onto alumina at pH 8.6 and eluted in 0.2N acetic acid. For each experiment the [‘H]NE was diluted to an appropriate concentration with 0.9% NaCl and injected intravenously into the tail veins of unanesthetized animals in a total volume of 1.O mL. The dose of [‘HJNE used in these studies varied between 25 and 100 @/kg (-0.1 to 0.4 ug NE/kg). The animals were killed at preselected times by cervical dislocation. For each time point in the studies of NE turnover, four to eight animals were killed from each experimental group. The tissues were rapidly removed, frozen on dry ice, and stored at -20°C for later processing (usually within 2 weeks). Extraction and Isolation of Catecholamines For NE analysis, the organs were weighed and homogenized in iced 0.2N perchloric acid in a ground glass homogenizer (DuallKontes Glass, Vineland, NJ) to extract the catecholamines and precipitate the proteins. After volume adjustment, the precipitated protein was removed by low-speed centrifugation. Following addition of the internal standard, 3,4-dihydroxy-benzylamine (DHBA; Aldrich Chemical, Milwaukee, WI), catecholamines were isolated from the perchloric acid extract by adsorption onto alumina (Woelm neutral, ICN Nutritional Biochemicals, Cleveland, OH) in the presence of 2 mol/L Tris(hydroxymethyl)aminomethane buffer (pH 8.7; Sigma, St Louis, MO) containing 2% EDTA. Catecholamines were eluted from the alumina with 0.2N perchloric acid. After removal of alumina fines by filtration (Microfilter: Bioanalytical Systems, West Lafayette, IN) aliquots of the alumina eluate were injected onto a liquid chromatographic system for catecholamine analysis. Unless otherwise specified, all chemicals were obtained from Fisher Scientific (Fairlawn, NJ). Measurement of [‘H]NE Aliquots of the alumina eluates were counted for [‘H]NE by scintillation spectrometry in a Packard 460C liquid scintillation counter (Packard Instrument, Downers Grove, IL). Efficiency for ‘H in this system is 30% to 35%. Determination of Endogenous NE Levels

NE turnover was computed from the rate of disappearance of [‘H]NE after labeling of the endogenous stores with tracer quantities of [jH]NE and from the decline in tissue NE content following administration of cy-methyl-p-tyrosine ((Y-MPT), an inhibitor of tyrosine hydroxylase. 23These techniques use a monoexponential model of NE kinetics in the calculation of a fractional NE turnover rate (k), which permits comparison of turnover rates in different groups of animals within the same experiment. Because physiological variation in NE turnover is principally dependent on changes in sympathetic impulse traffic, higher NE turnover rates reflect increased sympathetic activity and lower rates of diminished sympathetic activity.

Analysis of catecholamines in the alumina eluates was performed by a slight modification of the method of Eriksson and Persson.‘5 The chromatographic system was composed of a pump (M45; Waters Chromatography Division, Millipore, Milford, MA), an automatic sample injector (Waters Intelligent Sample Processor, WISP; Millipore), a reverse-phase column (250 x 4.6 mm) packed with ODSHypersil(5 urn, Shandon Southern Instruments, Pittsburgh, PA), and a glassy carbon amperometric detector (LC4A/17; Bioanalytical Systems). The mobile phase was an acetate-citrate buffer composed of sodium acetate (100 mmol/L), sodium hydroxide (60 mmol/L), and citric acid (40 mmol/L, all three from Mallinkrodt, Paris, KY) at pH 5.3 containing 10% methanol and 1.0 mmol/L sodium octyl sulfate (Aldrich or Eastman Kodak, Rochester, NY) flowing at a rate of 1.0 mL/min. The detector potential was set at +0.65 V versus AgiAgCl reference electrode. Detector response was quantitated by peak height using an integrating recorder (339OA. Hewlett Packard, Avondale, PA). Intraassay coefficients of variation for replicate determinations of NE specific activity in the same tissue sample are routinely 1% to 2%.

Turnover Procedure

Data Analysis

I-[ring-2,5,6-‘H]NE (40 to 60 Ci/mmol specific activity; DupontNew England Nuclear, Boston, MA) was purified before use by column chromatography with alumina previously prepared according to the method of Anton and Sayre”; the labeled NE was

Data are presented as means + SEM. Statistical analyses of tissue NE content used ANOVA, but where differences in tissue weight between control and MSG-treated animals were also noted, comparisons were repeated after adjustment of NE content for

Animals were allowed free access to water and Charles River chow (R-M-H-3200; Agway-Country Food, Syracuse, NY) except as noted. While fasting, the animals were given a hypotonic saline solution (50 mmol/L) to drink, and during sucrose feeding a 10% sucrose solution was provided to supplement the lab chow diet. Principle of NE Turnover Techniques

115

NE TURNOVER IN MSG-OBESE MICE AND RATS

weight by ANCOVA.B In studies of [“H]NE turnover, NE specific activity was plotted semilogarithmically and the slope of decline over time after 13H]NE administration (fractional NE turnover rate, k) was calculated by the method of least squares.” In all measurements of NE turnover using [“H]NE, no significant variation in endogenous NE was observed over the 24 hours of the experiment. The statistical significance of each computed regression line was assessed by ANOVA, and comparison of fractional turnover rates was made with ANCOVA. Simultaneous comparisons of fractional turnover rates among four or six experimental groups (experiments presented in Tables 4, 5, and 6) employed indicator variables for animal treatment and diet and used a covariance form of the general linear model.” NE turnover rates were calculated as the product of the fractional turnover rate and the endogenous NE content.”

Heart

RESULTS

Effect of MSG on NE Turnover in Heart and IBAT of Mice and Rats Fed Ad Libitum [3H]NE turnover rates were measured in heart and IBAT

of MSG-treated and control mice. The results in male mice are presented in Table 1 and Fig 1. In these animals, body weight was 8% less, and heart weight 33% less, but IBAT weight over 200% greater in the MSG-mice than in controls (at least P < .Ol for all three comparisons). Fractional NE turnover rates in both tissues were similar in treated and control mice. Cardiac NE content was reduced 40% in MSG-treated mice (P < .OOl); after adjustment for differences in heart weight, NE content was still 17% less in the MSG group (P < .02). NE content in IBAT of MSGtreated mice was also 30% lower than control (P < .OOl). (No adjustment for group differences in tissue weight was made in IBAT since the relationship between NE content and weight was not statistically significant in this tissue.) Overall rates of NE turnover (unadjusted for tissue weight) were, thus, 28% lower in the MSG-mice in heart and 18% lower in IBAT compared with those found in control animals. Female mice treated with MSG (Table 2) were 12% heavier than untreated controls (P < .005); otherwise, results were similar to those obtained in the male mice. Heart weights were lower, while IBAT weights were higher Table 1. Effect of MSG on pH]NE Turnover in Male Mice CWltrOl (n = 18) Body weight (g)

MSG (n = 18)

P

31.1 + 0.8

< .Ol

70 + 4

< .OOl

55.9 f 2.8

34.2 + 1.8

< .OOl

49.4 2 2.1

40.8 k 2.1

< .02

33.9 zrz0.5

Heart Tissue weight (mg) NE content (ng) Adjusted for heart weight Fractional NE turnover (%/h) Total NE turnover (ng/h)

1042

5

8.9-cl.O 5.0

10.4-tO.8 3.6

NS -28%

IBAT 102 2 6

325 k 14

< .OOl

NE content (ng)

53.7 + 2.4

37.7 -c 1.8

< .OOl

Fractional NE turnover (%/h)

15.5 * 1.7

18.0 k 1.5

8.3

6.8

Tissue weight (mg)

Total NE turnover (nglh)

NS -18%

NOTE. Data are presented as mean 2 SEM. Results are illustrated in Fig 1.

1000

3

11 0

IBAT

6

12

18

24

Time (h) Fig 1. Effect of MSG treatment on PH]NE turnover in heart and IBAT of male mice. Mice were injected neonatalfy with either MSG or a control aalhte solution and, following weaning, were fed a lab chow diet ad libiium until study. For the measurement of PH]NE turnover, radiolabeled NE (100 FCi/kg) was injected intravenously and mice were killed 2,6.12, and 24 hours later. Data are plotted as means 2 SEM for NE specific activity. Control animals, 0; MSG-treated mfce, 0. Statistical significance of all regression lines was P < .Wl. Fractional NE turnover rates did not differ between control and MSG-treated mice. Additional data from this experiment are presented in Table 1.

in MSG-treated than in controls animals. While fractional rates of NE turnover in neither heart nor IBAT were affected by MSG pretreatment, tissue NE content was reduced by MSG in both, by 35% (P < .OOl) in heart (15% following weight adjustment, P < .06) and by 28% (P < .OOl) in IBAT (again no weight adjustment was warranted due to lack of a significant relationship between weight and NE). Therefore, total rates for NE turnover were 20% to 23% less in the MSG-injected mice than in controls. Additional studies in male and female mice (data not shown) employing an alternative method for measuring NE turnover (inhibition of NE biosynthesis with cx-MPT) yielded results similar to these obtained with [3H]NE.

116

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Table 2. Effect of MSG on IfH]NE Turnover in Female Mice Control

Body weight (g)

MSG

(n = 19)

(n = 198)

P

26.3 r 0.4

29.5 + 0.9

< .005

AND YOUNG

male rats, neonatal treatment with MSG lowered NE turnover rates in heart and IBAT of freely feeding animals, a reduction due, not to slower fractional NE turnover, but rather to diminished tissue NE content.

Heart Tissue weight (mg) NE content (ng) Adjusted for heart weight

63 -c 4

43 + 4

< ,002

40.0 2 2.7

26.0 c 2.1

< ,001 < .06

35.6 + 1.8

30.4 + 1.8

Fractional NE turnover (%/h)

8.3 2 0.6

10.2 r 0.8

Total NE turnover (nglh)

3.3

2.6

NS -21%

BAT 66 + 4

222 f 17

< ,001

NE content (ng)

71.3 + 2.5

51.3 rt 3.4

< ,001

Fractional NE turnover (%/h)

15.6 + 1.7

16.6 -c 1.5

Tissue weight (mg)

Total NE turnover (q/h)

11.1

8.5

NS -23%

NOTE. Data are presented as mean + SEM.

Studies were also performed in control and MSG-treated male rats (Table 3). As in the male mice, body weights were 21% lower in the MSG-treated group (P < .OOl). Additional effects of MSG treatment included evidence of stunted linear growth (12% reduction in nasoanal length) and of increased body fatness (higher Lee index and expansion of epididymal fat depot relative to body weight). However, estimated ad libitum food intake did not differ between groups. Despite these changes in body size and body composition secondary to MSG treatment, fractional [‘H]NE turnover rates did not differ in either heart or IBAT between the two groups of rats. Tissue NE content was reduced 9% in heart (P < .05, but NS following adjustment for heart weight) and 17% in IBAT (P < .002) in the MSG-treated animals. Overall NE turnover rates (unadjusted for tissue weight) were, therefore, slightly lower in heart (-6%) and markedly reduced in IBAT (-44%) of MSG-treated rats compared with rates obtained in control animals. Thus, in both male and female mice and also in Table 3. Effect of MSG on [‘H]NE Turnover in Male Rats Control (n = 18)

MSG p

(n = 18)

Body weight (g)

332 + 6

263 t 4

< .OOl

Naso/anal length (cm)

21.5 2 0.2

19.0 & 0.3

< .OOl

Lee index*

321 +3

339 2 5

Gonadal adipose tissue weight(g) (as % body weight) Food intake (g chow/ratid)

<.Ol

3.46 r 0.23

3.21 2 0.09

NS

1.04 2 0.05

1.22 -c 0.06

< .05

20.5 * 1.1

19.9 2 0.7

Heart Tissue weight (mg) NE content (ng) Adjusted for heart weight Fractional NE turnover (%/h) Total NE turnover (ng/h)

1,070 -t 20

822 ? 20

< ,001

724 2 24

660 + 30

< .05

658237

726?

37

NS

4.6 f 0.5

4.7 -c 0.4

NS

31.0

-7%

33.3

IBAT Tissue weight (mg)

434 2 15

411215

NS

NE content (ng)

415 + 13

348~16

< ,002

fractional NE turnover (%/h) Total NE turnover (ng/h)

5.1 * 1.2 21.2

3.3 & 0.9

NS

11.5

-46%

NOTE. Data are presented as mean + SEM. *Lee index = (body weight (g)]“‘/nasoanal length (cm) x 1,000.

Effect of MSG on Diet-Induced Changes in 13H]NE Turnover in Heart and IBAT The possibility that MSG-treated mice might exhibit an impairment in dietaIy regulation of SNS activity similar to that previously reported in AuTG-treated rni& was examined by comparing [-‘H]NE turnover in fasted and sucrosefed MSG-treated mice with rates obtained in similarly fed control animals. (Food was withdrawn from fasting mice 1 day before and during turnover measurement and a 10% sucrose solution to drink was provided to feeding animals 3 days before and during turnover study, a design similar to that previously employed with AuTG-treated mice.“) Turnover measurements in control and MSG-treated mice were performed on consecutive days. Results of this experiment in male mice are presented in Table 4 and Fig 2. Although body weights did not differ between groups, MSG-treated mice displayed reduced linear growth and increased body fatness as did the male rats in the preceding study: however, body weights in both groups of mice were reduced by fasting. NE content in heart and IBAT was again lower in MSG-treated mice, although following adjustment for heart weight, NE content was higher in the treated mice. NE content in both tissues was also slightly higher in fasted, than sucrose-fed animals. Fractional NE turnover rates were similar in control and MSG-treated mice and were lower in fasting animals from both groups. Covariance analysis raised the possibility that fractional NE turnover in hearts of MSG-treated and control mice might differ slightly in their response to dietary alteration (Rx x Diet interaction, P < .025); data derived from IBAT contained no such implication. Total NE turnover rates in fasted mice were 26% lower in hearts of MSG-treated mice and 32% lower in controls; the patterns in IBAT were qualitatively similar to, but quantitatively less than, those observed in heart. However in both tissues, the differences in NE turnover associated with nutrient status reflected alterations in fractional NE turnover, not in tissue NE content. Thus, SNS responsiveness to alterations in nutrient intake is essentially intact following MSG treatment. Rats with electrolytic lesions in the ventromedial hypothalamus (VMH) also exhibit evidence consistent with impaired suppression of the SNS by fasting.” The influence of a 2-day fast on cardiac [‘H]NE turnover was examined in both male and female MSG-treated and intact rats (Table 5). In all groups, fasting slowed cardiac NE turnover 40% to 70% by diminishing fractional NE turnover, not by lowering NE content. In both male and female rats, as above, cardiac NE content was less in MSG-treated animals. Moreover, covariance analysis provided no suggestion that the effect of diet on fractional NE turnover differed between control and MSG-treated animals, although in this experiment in female rats fractional NE turnover was slightly faster overall in MSG-treated than in control animals. Thus in

NE TURNOVER IN MSG-OBESE MICE AND RATS

117

Table 4. Effect of MSG on Diet-Induced Changes in PHJNE Turnover in Male Mice Control

MSG

(n = 24)

In = 30)

8.3 k 0.04

Nasoanal length (cm) Lee index

339 + 2

Gonadal adipose tissue weight (mg)

480243

(as % body weight)

Body weight (9)

383 f 4

< .OOl

1,190 ? 62

< ,001

3.96 f 0.15

Fed

Fasted

Fed

Fasted

(n = 13)

(n = 11)

(n = 15)

(n = 14)

29.8 + 0.5

140 2 4

145 + 4

< .OOl

8.1 2 0.09

1.51 2 0.11

32.4 f 0.8

P

30.2 ? 1.0

-

< ,001 Rx x -

RX

-

Diet

-

Diet

29.1 2 1.1

NS

94 z! 7

88 -t 5

< .OOl

NS

NS

81.3 ? 6.8

85.9 2 6.3

<.Ol

< ,025

NS

.05

NS

Heart Tissue weight (mg) NE content (ng)

89.1 + 4.3

112 + 3.5

adjusted for heart weight

67.2

98.4

108.3

< ,001

< .OOl

NS

Fractional NE turnover (%/h)

12.1 t 1.5

6.6 -t 0.8

12.7 + 0.9

8.8 + 0.8

NS

< .OOl

< ,025

10.8

7.4

10.3

7.6

NS

Total NE turnover (ng/h)

85.7

IBAT 94 + 6

58 ? 8

240 + 27

238 ? 19

< .OOl

NS

NE content (ng)

45.4 * 4.0

51.7 f 3.4

32.9 + 3.7

38.0 f 3.0

< .005

< .075

NS

Fractional NE turnover (%/h)

14.5 + 3.1

12.1 f 2.2

21.1 * 2.4

17.7 2 1.8

NS

< .05

NS

6.6

6.2

6.9

6.7

Total weight (mg)

Total NE turnover (nglh)

NOTE. Data are presented as mean ? SEM. Fed mice received 10% sucrose to drink in addition to lab chow for 3 days before study, while fasted mice were given 50 mmol/L NaCl to drink during the 1 day of total fasting before study. Studies in control and MSG-treated mice were performed on consecutive days. Results are illustrated in Fig 2. “Rx” refers to neonatal treatment with either MSG or saline; ‘Diet” refers to fasted v sucrose-fed; “Rx x Diet” refers to the first-order interaction between treatment and diet. For comparisons among fractional NE turnover rates, the model included terms for “Time” and for the interactions between “Time and Rx, ” “Time and Diet,” and “Time, Rx and Diet”; statistical significance refers to the appropriate interactions with “Time.”

as in the mice, neonatal MSG administration does not abolish dietary regulation of SNS activity in heart.

rats,

Effect of Au TG on Diet-Induced Changes in [3H]NE Turnover in Heart and IBAT

Since treatment with AuTG diminished the difference in cardiac [3H]NE turnover between fasted and sucrose-fed mice,14 a study was performed, similar to those in MSGtreated animals presented above, examining the effect of AuTG administration on dietary regulation of NE turnover in IBAT. In the previous report,r4 the subsequent development of obesity was employed as a physiological marker for the presence of the AuTG lesion. This experiment was designed to examine the effect of AuTG administration not just in obese mice, but also in treated animals that did not become obese. Consequently, AuTG-treated mice were divided into an obese group (AuTG-Fat; body weights 2 4 SD above controls) and a lean group (AuTG-Lean; body weights 13 SD above controls). All three groups were further subdivided into fasted and sucrose-fed groups. The results of this experiment are presented in Table 6. By definition, AuTG-Fat mice were substantially heavier than controls, while AuTG-Lean mice were only slightly larger. Cardiac NE content was lowest overall in the AuTG-Lean mice, but following adjustment for cardiac weight NE content in both AuTG-treated groups was slightly less than in controls (P = .05). NE content in all groups was higher in the fasted mice. On the other hand, NE content in IBAT was lower in AuTG-Lean mice than controls and lower still in the AuTG-Fat animals; no

adjustment for differences in IBAT weight was made since NE content was inversely related to IBAT weight (P = .065). Due to the presence of three treatment groups in this analysis, fractional NE turnover rates in the two AuTGtreated groups were separately compared with those obtained in controls. In both heart and IBAT, fractional NE turnover rates differed as a function of diet (P < .OOl and P = .006, respectively). While in heart the effect of diet was diminished in both AuTG-treated groups (Rx x Diet interactoin, P < .02 and P < .005 in AuTG-Lean and AuTGFat mice, respectively), in IBAT the AuTG-Fat mice, but not the AuTG-Lean animals, displayed a statistically significant reduction in dietary variation of fractional NE turnover (Rx x Diet interaction, P = NS andP < .OOlin AuTGLean and AuTG-Fat mice, respectively). Calculated rates for total NE turnover exhibited a similar pattern. While total NE turnover was 34% lower in heart and 39% lower in IBAT of fasting mice (compared with sucrose-fed controls), the respective differences were only 14% and 22% in AuTG-Lean animals and 8% and 0% in AuTG-Fat mice. Thus, administration of AuTG induces a loss of dietary variation in NE turnover in heart and IBAT that is roughly correlated with the degree of obesity produced by treatment. This response is quite different from that which occurs following neonatal MSG. DISCUSSION

examined dietary regulation of SNS activity in mice and rats treated neonatally with MSG and in adult mice treated with AuTG. In all experiThe

current

investigations

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118

Control 10000

AND YOUNG

MSG

3

Heart

Heart

1000 : Fasted

10 0

6

I 12

Fasted

I 24

18

10 ! 0

6

12

18

24

1000 IBAT

IBAT

(tt/2=4.6h)

1 , 0

6

12

18

I 24

Time(h)

-1

0

6

12

18

24

Tlme (h)

Fig 2. Effect of MSG treatment on [WINE turnover in heart and IBAT of fed and fasted male mice. Mice were injected neonataily with either MSG or a control saline solution and, following weaning, werefed a lab chow diet ad libitum until study. Fed mice recrlved 10% sucrose to drink in addition to lab chow for 3 days before study, while fasted mice were given 50 mmol/L NeCI to drink during 1 day oi total fasting before study. Turnover experiments were performed in control and MSG-treated mice on consecutive days. For the measurement of f’H]NE turnover, radiolabeled NE (100 @/kg) was Injected Intravenously and mice were kllled 2,5,12. and 24 hours later. Data are plotted as means 2 SEM for NE specific actlvlty. Fasted animals, 0; fed mice, G. Statistkal significance of all regression llnee was P < .oOl. Fractional NE turnover rates were significantly lower in hearts of fasted mice In both control and MSG-treated groups. Additional data from this experiment are presented in Table 4.

ments described here, the overall NE turnover rate was less in MSG-treated mice and rats than in control animals, but in contrast to previous reports,‘b’3 the reduction in NE turnover was due exclusively to lower tissue NE content in the obese animals, not to any difference in fractional NE turnover. Moreover, while AuTG administration led to a reduction in dietary variation in NE turnover, especially in IBAT, MSG treatment did not. Thus, the effect of neonatal MSG on regulation of SNS activity is functionally distinct from that of AuTG. Analysis of the four experiments examining dietary regulation of NE turnover in MSG and AuTG-treated mice and MSG-treated rats relied upon the use of indicator variables in a covariance form of the general linear model.z7

This statistical approach has, to our knowledge, not previously been applied to experimental data from a NE turnover study. Analysis of NE turnover data routinely employs regression techniques to compare rates of change in tissue NE specific activity (or rather the natural logarithm of tissue specific activity) over time in various experimental groups. The use of indicator variables permits the examination of interaction among experimental conditions and, thus, provides a direct statistical test of the null hypothesis that the effect of diet on fractional NE turnover rates is the same for all treatment groups. This hypothesis was rejected with high probability for both heart and IBAT in the obese, AuTG-treated mice, while in MSG-treated animals it was accepted in mouse IBAT and rat heart and only weakly

NE TURNOVER IN MSG-OBESE MICE AND RATS

119

Table 5. Effect of MSG on Diet-Induced Changes in Cardiac PH]NE Turnover in Rate Control

P

MSG

x

Rx

Male

Fed

Fasted

Fed

(n=

Fasted

12

Rx

Diet

Diet

(n = 18)

(n = 18)

Body weight (g)

353 2 13

292 -c 11

302 2 10

267 f 7

< ,002

<.OOl

NS

Tissue weight (mg)

906 + 31

792 + 21

772 f 22

702 f 21

< ,001

< ,001

NS

1,040 + 43

1,030 f 31

852 2 42

822 2 42

<.OOl

NS

977

1,040

873

888

< ,005

NS

NS

7.4 f 0.4

4.5 + 0.7

NS

<.OOl

NS

63

37

NE content (ng) Adjusted for heart weight Fractional NE turnover (%/h)

2.1 ? 0.9

6.8 ? 0.6

Total NE turnover (nglh)

71

22

(n = 13)

NS

(n = 20)

(n = 14)

Body weight(g)

248+5

202 f 5

220 f 6

185 I 6

<.OOl

<.OOl

NS

Tissue weight (mg)

697 * 13

594 + 13

580 f 15

544210

<.OOl

< .OOl

< .02

NE content (ng)

824 + 34

1,025 r 50

669 + 27

728 + 37

< .OOl

< .002

1,050

714

825

<.015

< ,001

< .OOl

7.7 f 0.8

3.8 + 1.2

< .025

< .05

NS

52

28

Female

Adjusted for heart weight

699 7.1 + 0.5

Fractional NE turnover (%/h) Total NE turnover (nglh)

(n = 16)

3.2 + 0.9

58

33

(n = 15)

<.l

NOTE. Data are presented as mean + SEM. Fed rats received only lab chow and water, while fasted rats were given 50 mmol/L NaCl to drink during the 2 days of total fasting before study. Additional notes as in Table 4.

ful. Whether these interlaboratory differences reflect procedural and/or biological (animal) variability is unknown, but the fact that all experimental groups in this report exhibited evidence of MSG effects (ie, stunted linear growth and increased body fat) indicates that a reduction in fractional NE turnover is not a universal consequence of neonatal administration of MSG. All previous reports from this laboratory describing the influence of diet and other environmental factors on SNS activity in tissues of experimental animals using measurements of NE turnover methods were based on changes in

rejected in mouse heart. Thus, this analytical method supports the contention that AuTG, but not MSG, treatment impairs dietary regulation in experimental animals. A satisfactory explanation for the discrepancy between our present results and previously published data concerning the effects of MSG on SNS activity’@‘3is not immediately apparent. Although in our laboratory the [3H]NE technique for measuring NE turnover is clearly superior to the synthesis inhibition method, our attempts to confirm the findings of these other laboratories using the tyrosine hydroxylase inhibitor (Y-MPT were also entirely unsuccess-

Table 6. Effect of AuTG on Diet-Induced Changes in PH]NE Turnover in Female Mice AuTG-Lean

Control

Body weight (9)

P

AuTG-Fat

Rx x

Fed

Fasted

Fed

Fasted

Fed

Fasted

In = 21)

(n = 20)

(n = 22

In = 22)

(n = 22)

(n = 21)

26.9 2 0.4

24.6 + 0.5

29.8 2 0.7

27.8 + 0.6

41.2 + 1.0

39.5 k

1.0

Rx

Diet

Diet

< .OOl

< .002

NS

< ,001

< ,001

NS

< .Ol

< ,001

NS

Heart 104 + 2

97 f 2

107 2 3

99 * 2

87.2 + 3.1

96.9 ? 2.6

82.6 2 3.0

92.6 2 2.5

90.0 f 3.2

104.8 2 3.8

Adjusted for heart weight

88.3

103.6

81.3

97.7

83.2

100.4

Fractional NE turnover (%/h)*

11.1 -c 1.0

Tissue weight (mg) NE content (ng)

Total NE turnover (ng/h)

9.7

6.6 + 0.7 6.4

9.1 2 0.8 7.5

7.0 2 0.5 6.5

114*

2

10.4 f 0.6 9.4

11122

8.2 + 0.4 8.6

.05 ,025 .OOl

.OOl < ,001

NS <.02 .005

IBAT 69 -c 4

60 2 4

97 2 7

75 + 5

178 2 12

143 2 16

< .OOl

NE content (ng)

78.0 + 5.5

69.5 ? 4.7

71.1 + 4.0

60.4 * 3.6

59.7 * 4.3

57.8 -+ 3.3

< ,005

Fractional NE turnover (o/o/h)*

19.3 f 1.3

13.2 2 1.5

17.0 ? 1.6

15.5 + 1.6

17.4 + 1.5

18.2 f 1.4

NS

15.0

9.2

12.1

9.4

10.4

Tissue weight (mg)

Total NE turnover (nglh)

10.5

NS

< ,005

NS

< .05

NS

.006

NS ,001

NOTE. Data are presented as mean f SEM. Fed and fasted mice were prepared for study as in Table 4. Turnover measurements in all groups were performed simultaneously. “Rx” refers to the three groups treated with either AuTG or saline; “Diet” refers to fasted versus sucrose-fed; “Rx x Diet” refers to the first-order interaction between treatment and diet. Since three groups, rather than two as in Tables 4 and 5, were involved, a slightly more complicated statistical model was employed for comparison among fractional NE turnover rates. In this analysis, the single indicator variable for “Rx” (used in Tables 4 and 5) was replaced with two others, one indicating the AuTG-Lean group and the other the AuTG-Fat group; these provide a measure of the difference between the indicated group and controls.*’ The ensuing linear model, thus, included terms for “Time” and for the interactions between “Time and Lean, ” “Time and Fat,” “Time and Diet, ” “Time, Lean and Diet,” and “Time, Fat and Diet.” *The upper line in the statistical output for fractional turnover rates refers to the appropriate interactions with “Time”for the AuTG-Lean group and the lower line for the AuTG-Fat group.

DULL00

120

fractional, as well as total, NE turnover (as illustrated by the responses to fasting in Tables 4-6, and Fig 2). Therefore, it is unclear whether group differences in total NE turnover due solely to inequality in tissue NE content imply an underlying difference in SNS activity. If tissue NE were, in fact, a single homogeneous pool, as the monoexponential kinetic model assumes, then lower NE turnover due to a reduction either in fractional NE turnover or in pool size would be functionally equivalent. However, this assumption is known not to be absolutely correct,*’ although it remains a convenient approximation under most circumstances. If NE depletion from tissues of MSG-treated animals occurred from a slowly turning over portion of the total NE pool, then the impact of such a change on sympathetic function might be relatively minor. Evidence that thermogenic responses in IBAT to overfeeding are nearly normal in MSG-obese mice6,*’ is consistent with intact dietary variation in sympathetic function in these animals. Thus, on balance, it would appear that SNS activity is probably not affected by neonatal MSG administration, although current data cannot provide a definitive answer to this question. However, in previous studies of sympathetic function in obese animals reported abnormalities were based on more substantial evidence of altered SNS activity than found here.“’ The present findings also have implications for potential

AND YOUNG

mechanisms involved in dietary regulation of the SNS. As shown in Tables 4 and 5 and in Fig 2, NE turnover in heart and IBAT varies in response to dietary manipulation to the same extent in MSG-treated rats and mice as in normal controls. (Whether the inference from the statistical analysis of data in Table 4 that a subtle abnormality exists in the response of cardiac sympathetic activity to dietary change is correct warrants further examination.) Nonetheless, these results stand in marked contrast to those obtained in AuTG-treated mice. The current observations are, thus, consistent with other evidence of behavioral differences between mice treated with either MSG or AuTG.~’ Moreover, the lack of effect of MSG on dietary variation in SNS activity does not support participation of hypothalamic insulin receptors exposed to circulating insulin in mediating sympathetic responses to dietary change. Although further comparisons of SNS responses to diet may ultimately demonstrate selective deficits in MSG-treated animals, a reasonable inference, in light of present findings, is that coordination of SNS activity with diet must either be mediated by insulin at some other (presumably nonhypothalamic) site and/or by non-insulin mechanisms. ACKNOWLEDGMENT

The expert technical assistance of Susan Fish, Martha Berardino, and Susan Gunn is gratefully acknowledged.

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