Regulation of Adipsin and Body Composition in the Monosodium Glutamate (MSG)-Treated Mouse

Ph}sinlugy & Bcluvior, Vol. 60, No. 5, pp. 1217– 1221, 1996 Cnpyright O 1996 Elsevier Science Inc. Printed in the USA. All rights reserved

0031-9384/96$15.00+ .00 ELSEVIER

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Regulation of Adipsin and Body Composition in the MonosodiumGlutamate (MSG) -Treated Mouse MICHAEL

E. SPURLOCK,*l

KIM J. HAHN~

AND JESS L. MINER~2

*Research Center, Purina Mills, Inc., 100 Dan{?)rthDrive, Gray Summit, MO 63039 USA and fThe Monsanto Co., St. Louis, MO, USA Received

1 December

1995

SPURLOCK, M. E., K. J. HAHN AND J. L. MINER. Re,gukfionoj’adipsit]and bmf} [wtposifion in the monosodiumglutamate (M.YG)-/reutedJrIoLL\e. PHYSIOL BEHAV 60( 5 ) 1217– 12’21, [996.—Changes in food intake, serum adipsin, and obesity were evaluated in the MSG-treated mouse In Experiment 1, mice treated with MSG had 50% lower serum adipsin and over 2-fold higher percentage of bndy fat than the lean cnnt]-ols. Both feeding caffeine and restricting intake normalized serum adipsin and caused weight loss, but did not norrmdize the percentage of bndy fat. No additional effect was gained by feeding isopmterenol or ephedrine in combination with c~ffeine. In Experiment 2. we separated the direct effect of’caffeine from the associated depression in intake using a paired feeding design, and also determined the effects of selected adrenergic agents and somatotropin ( S). Somatotmpin increased weight gain and reduced the percentage uf bndy fat in healthy and obese mice, and tended to lower serum ufipsin. Caffeine clearly depressed intake, caused weight loss, and increased serum adipsin, but similar results were achieved by restricting intake. None of the adrenergic drugs tested changed serum adipsin. Ephedrine depressed food intakeand causedweight loss, but reducedthe percentageof body fat only at the higbest dietary concentration ( 2000 mg per kg of diet). Phenylephrine reducedweightgain withouta concomitanteffect on the percentagenf budy fat, and isoprotererml did not influence weight gain right O 1996Elset,ierScience [n<’. or body fat. Cor).y Adipsin

Monnsudium glutamate

Obesity

Adrenergic agnnist

ADIPSIN is a developtnentally regulated serine protease (4) that is synthesized and secreted constitutively by tissues associated with lipid metabolism. In mice, adipose tissue, and nerve, albeit to a lesser extent than adipose tissue, were reported to be the principle sources of circulating adipsin (5). Depressed adipsin expression has been associated with obesity of genetic ( ob/ob and rib/db models) and cberniczd ( MSG ) origin (7). This potential relationship of adipsin to energy balance and obesity, and the possible linkage of obesity to immune function via the complement factor D activity ( 14) of adipsin, has provided the impetus for studies of the regulation of adipsin in multiple models of obesity. The genetic and chemical models of obesity in which adipsin expression is downregulated are also characterized by decreased sympathetic nervous system activity and thermogenesis. Therefore, in addition to adrenal steroids (8,15), insulin (6,12), and S ( 12), sympathomimetic and thermogenic agents are potential regulators of adipsin expression. The sympatbomi metic-thermogenic combination of ephedrine (a mixed a- and ,fadrenoceptor agonist) and caffeine has been shown to normalize adipsin mRNA levels and reduce abdominal fat pad size in the MSG model of obesity ( 1I ). It is not clear if caffeine was es-

Caffeine

Fnod intake

Somatotropin

sential to achieve these results, or if the response was consecfttential to the a- or ~-adrenergic activity of ephedrine. An atypical ~-adrenoceptor agonist, BRL26K30A, has been reported to have no impact on adipsin in genetically obese (ob/ob genotype) mice, and to actually depress adipsin in lean controls ( 13). Accordingly, we have extended the studies of the regulation of adipsin expression in MSG-treated mice to include isoproterenol, a well-characterized typical &adrenoceptor agonist, and phenylephrine, an a-adrenoceptor agonist. Initially, we evaluated the effects of’ephedrine and isoproterenol in the presence of caffeine, and assessed the independent effects of caffeine and intake restriction. Finally, we determined the effect of the adrenergic agents, independently of caffeine, and also evaluated S and the effect of lactation. METHODS

Arrimds All animals were cared for in keeping with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For botb experiments, outbred gestating mice (CD-1 )

‘ To whomreprint requestsshouldbe ~ddressed 2 Present addr&s: Department of’Animal Sciences, University nf Nebraska, Lincoln. NE. 1217

1218

SPURLOCK, HAHN AND MINER TABLE 1 EFFECT OF OI
Serum adipsin-lBody fat, %$ Weight change, g$

NC

MSG

104 12.2 0.7

50 37.4 0.4

cd

100 30.9 – 14.1

INTAKE RESTRICTIONON SELECTED VARIABLES*

RF

E20

El(x)

Elooo

120

1100

11000

SE

1lg 36.() – 14.2

87 27.9 – 14.()

112 29.3 – 13.6

127 30.() – 14,9

103 29.7 – 13.8

88 24.1 –11 .6

88 30.5 – 14.3

26 2.5 1.2

* Obesity was induced via neonatal injections of monosodium glutamate (MSG). At 9 weeks of zge, treatment groups were fed diets containing caffeine alone (Caf, 1400 mg per kg of diet), or ephedrine (E) or isoproterenol (I) at 100, 500, or 1000mg per kg of diet in combinationwith caffeine for 3 weeks. Drug treatmentsare indicatedalphanumerically(E20indicatesephedrineat 20 mg per kg of diet). Both normal (NC) and MSG control groups were used in this study. The intake of the restricted-fed (RF) group was limited to 40% of that of the MSG control group, and was divided into 2 (a.m. and p.m. ) feedings. Data are reported as least-squares means. 7 Volume units determined by phnsphorimage analysis of Western blots. The MSG mean is different (p = 0.06) from the NC mean, whereas Caf and RF are not (p ~ 0.87). Contrasts of Cat’vs. ephedrine and Caf vs. isopmterenol diets are not different (p = 0.80). $ The MSG, Cat’,and RF means are different (p s 0.01) from the NC mean. Contrasts of Caf vs. ephedrine and Caf vs. isoproterenol diets are not different (p = 0.67). $ The MSG mean is not different (p = 0.88) from the NC mean. Both the Caf and RF means are different (p = O.01) from the NC mean, and contrasts of Caf vs. ephedrine and Caf vs. isoproteremrl diets are not different (p = 0.87).

were obtained from Harlan Sprague –Dawley, Indianapolis, IN. at 7– 10 d postdating and placed in individual cages in an environmentally-controlled room. Daily injections of MSG ( 3 mg/ g of body weight, SC) were initiated in neonates at approximately 12– 18 h of age and continued for 7 consecutive days. The mice were weaned to Certified Rodent Chow TMpellets (Purina Mills, Inc., St. Louis, MO) at 21 days of age. Also at weaning in experiment 2, blood was obtained from 26 lactating and 15 nonlactating females for determination of serum adipsin. For both experiments, the mice were maintained in individual wiremesh cages in an environmentally-controlled room maintained at approximately 25”C. The light:dark cycle was also regulated at 12 h each. Experiments in offspring were initiated at 9 weeks old and were of 3- and 4-week duration for experiments 1 and 2, respectively. Each treatment contained a minimum of 15 replicates. At the completion of the studies, the mice were anesthetized with Telezol T~ (teletamine HCI and xylazine, Fort Dodge Laboratories, Fort Dodge, IA) and blood collected by cardiac puncture or decapitation. Carcasses were stored frozen and either submitted to a commercial laboratory for determination of body fat by ether extraction or by near-infrared spectroscopy (NIRS ), as previously described (9). Drugs and Somatotropin The adrenergic drugs and caffeine were purchased from Sigma Chemical, St. Louis, MO. Certified Rodent Chow meal was obtained from Purina Mills, Inc.. Drugs were mixed with the meal diet weekly and fresh feed dispensed 3 times per week. In the first study, ephedrine and isoproterenol were added to a basal diet containing 1400 mg caffeine per kg to achieve the final concentrations of 20, 100, and 1000mg per kg. Additional treatments included healthy (lean) and MSG-treated (obese) controls, caffeine only (the basal diet), and a group in which intake was restricted to 40’ZOof that of the MSG-treated control group fed ad lib. In the second study, the effect of ephedrine, phenylephrine, and isoproterenol ( 100, 500, and 2000 mg/kg diet) were determined independently of caffeine. Caffeine was, again, evaluated as a single entity ( 1400 mglkg diet) and the effect of the caffeine was separated from that of the caffeine-induced reduction in intake by limiting the intake of one group to that of the caffeine-fed mice. This daily ration was divided into equal a.m. and p.m. rations. Additionally, the impact of S was evaluated in normal and MSG-treated mice. These mice were injected twice

daily with 250 Kg ,bovine S. Food intake was measured by disappearance. Special cup-type feeders designed to minimize spi]lage and contamination with excreta were used. Spilled and contaminated feed was collected and quantified at the end of the study and intake records adjusted accordingly. Ektrophoresis

and Immunoblotting

Sample proteins were separated by discontinuous SDS-PAGE ( 10) using 3.75% stacking and 12% resolving gels. Two ~1 of serum were diluted in 10 pl of reducing SDS sample buffer and heated to 90”C. Each sample was evaluated on 3–4 independent gels. Proteins were transferred from the gels to nitrocelltdose filters ( 16), and immunoblotting was accomplished using a polyclonal Imbbit antiserum (400-082090) raised against a synthetic peptide (mouse adipsin~s.1~~,SLSAPEPYKRWYDVQS ) conjugated to thyroglobttlin as described previously (5). The antiserum was diluted 1:1000 for immunoblotting. Immunoreactive adipsin was quantified via autoradiography after incubation with ‘z51-radiolabeledprotein A using a PhosphorimagerTM (Molecular Dynamics, Sunnyvale, CA). In preliminary immunoblots, this antiserum detected a doublet (M, of 37,000–44,000) in 3T3-L1 adipocyte conditioned media, and a single band of approximately the same size in murine serum. No bands were detected in media conditioned by preadipocytes or in the serum of several other mammalian and avian species. The data were evaluated based on analysis of variance (ANOVA) with mouse (treatment) used as the error term. Specific comparisons of interest were accomplished using preplanned orthogonal contrasts. RESULTS

Experiment 1 Serum adipsin, body-weight change, and body-fat data are summarized in Table 1. Circulating adipsin was reduced approximately 50% (p s 0.06) by treatment of neonates with MSG and percent body fat was approximately 3 times that of the lean controls (p s 0.0 I ) at the end of the study. Both feeding caffeine and restricting food intake normalized serum adipsin in MSGtreated mice. Neither isoproterenol nor ephedrine, fed in combination with caffeine, increased adipsin concentrations beyond that achievable with caffeine alone (p s 0.90).

ADIPSIN AND OBESITY

1219 TABLE 2

EFFECTOF MONOSODIUM GLUTAMATE (MSG) AND SOMATOTROPIN [S) ON SELECTED VARIABLES* Cmrtml

MSG

s

MSG+s

SE

Serum adipsint

632

Body fat (%) Weight change (g) Feed intake (g)

17.1 2.8 136

503 38.8 4,5 127

610 I3.7

433 25.4

32 2.3 0.6 4

Statisticvd Significance

(1) 9.0

12.1

148

134

(2)

0.0001

().0001 0.0002 0.0022

(3)

0.1341 0.0076 0.0001 ().0125

0.4290

0.0011 0.2803 0.4984

* Obesity was induced via neonatal injections of MSG. At 9 weeks of age, the mice designated for treatment with S were injected SC twice daily

for 28 days with 0.25 mg recombinantbovine somatotropin. t Data were analyzed w a completely randomized design with treatments arranged as ~ 2 X 2 factorial (3 MSG, t S), and are reported as Ieastsqtmres means. Probability wdues are reported for the main effects of MSG (1) and S (2), as well as the interaction of MSG and S (3). ~ Volume units determined by phosphorimage analysis of Western blots.

TABLE 3 J+FE(’T OF DRUG TREATMENT AND INTAKE RESTRICTION ON SELECTED VARIABLES*

Serum udipsin-lBody fat (%)$ Weight change (g)$ Feed intake (g)ll

MSG

(-a,

RF

El(lo

Esoo”

E20()()

PI()(I

P500

503 38,8 4.5 127

744 23.7 –4.5 90

664 26.7 –2.6 90

512 42.6 4.5 120

554 36.9 0.3 113

558 27.6 –5.6 85

481 41.6 3.7 120

432 43.0 3.1 121

P2000

448 40.5 1.8 113

I 100”

1500

12000

SE

481 42.3 4.9 121

449 40.1 4.0 125

513 36.3 3.7 117

32 2.25 0.6 4

* Obesity was induced via neonatal injections of monosodium glutamate (MSG). At 9 weeks of age, treatment groups were fed diets containing caffeine (Caf, 1400 mg per kg of diet), m ephedrine (E), phenylephrine (P), or isoproteremrl (I) at 100, 500, or 2000 mg per kg of diet for 4 weeks. The daily intake of the restricted-ted (RF) group was matched to that of the Cat’ group and was divided into 2 (a.m. and p.m.) feedings. t Serum adipsin is reported as volume units (least-squares means) determined by phcrsphorimage analysis of Western blots. The MSG mean is different (p s 0.01) from the Caf and RF means. Contmstsof MSG vs. ephedrine,MSG vs. phenylephrine,and MSG vs. isoproterenol-containing diet are not different (p = O.16). Contrasts of the highest dietary concentration of each drug vs. the 2 lower concentrations are not different (p =

0.20). ~ The MSG mean is different (,, s ().()1) from the Caf and RF

means. contrasts of MSG vs. ephedrine, MSG vs. phenylephrine, and MSG vs. isopmterenol-containing diet are not different (p = 0.22). Contrasts of tbe highest dietary concentration of ephedrine and isoproterenol vs. the 2 lower concentrations are different (p s 0.06). For phenylephrinc, P2000 vs. Pl 00, 500 was not different (p = 0.50). $ The MSG mean is different (p s 0.01) from the Caf and RF means. Contrasts of MSG vs. epbedrine and MSG vs. pbenylephtine are different of ephedrineand phenylephrinevs. the 2 lower (P = 0.02), but isoproterenol bad no effect (p ~ 0.65).contrasts of the highestdietaryc~ncentmtion concentrationsare different (p = 0.04). For isoproterenol, 12000 vs. 1100, 500 was not different (p = 0.35). IIThe MSG mean is different (p = 0.01) from the Cat’mean. Contrasts of MSG vs. ephedrine and MSG vs. phenylephrine are different (p s 0.02), but isoproterenol had no effect (p = O.12). Contrasts of the highest dietary concentration of ephedrine and phenylephrine vs. the 2 lower concentrations are different (p = 0.07). For isopmterenol, 12000 vs. 1100, 500 was not different (p = 0. 18).

Weight loss was considerable ( 12–15 g) in mice fed the sympathomimetic and thermogenic agents, and in those with restricted intake. As with serum adipsin, restricting intake and feeding caffeine caused similar weight losses, with no additional effect achieved by feeding isoproterenol or ephedrine in combination with caffeine. Although the weight loss in the mice fed caffeine was accompanied by a reduction in the percentage of body fat from 37.44 to 30.88 (p s 0.01 ), these mice still had 2.5 times the body fat of the lean controls. The percentage of body fat was not reduced further by adding ephedrine and ism proterenol to the diets (p = 0.67). Experiment 2 Adipsin concentration was higher (p s 0.08) in the serum of nonlactating than in lactating f’emales ( 113 <4 vs. 90 t 3, mean t SE). As for the offspring, the effects of MSG and S are presented in Table 2. As in experiment 1, MSG treatment of’neonates depressed serum adipsin and increased weight gain. The increased weight gain was associated with an increase in the per-

centage of body fat from 17.11 to 38.82 (p s 0.01 ) at the termination of the study. Somatotropin also increased weight gain, but reduced body fat (p s 0.01), with the percentage reduction in body fat being greater in the MSG-treated mice ( SXMSG, p s 0.01 ). However, percent body fat in the obese mice treated with S was still 50% higher than that of the lean control group. Somatotropin also tended (p s 0.13) to lower serum adipsin and increased (p s 0.01 ) food intake. Food intake was actually lower (p ~ 0.01) in the MSG-treated than in nontreated mice. As shown in Table 3, both feeding caffeine and restricting food intake alleviated the depression in serum adipsin caused by MSG, caused weight loss, and reduced the percentage of body fat (p s 0.01). None of the ztdrenergic agents tested changed ~erum adipsin in MSG.treated mice (p ~ 0.16). Ephedtine caused weight loss (p s 0.01 ) and phenylephrine reduced (p s 0.02) weight gain, but the effect of isoproterenol on weight change was not significant (p s 0.65). Both ephedrine and isoproterenol (both @-adrenergic agonists) at 2000 mg/kg of’ diet showed a significant (p s 0.0 I and 0.06, respectively) reduction in body fat vs. the lower 2 dietary concentrations of each drug.

1220

SPURLOCK, HAHN AND MINER

Caffeine clearly depressed intake (p s ().()1), as did ephedrine and phenylephrine (p s 0.02). This effect of ephedrine and phenylephrine was lu-gely due to the impact of the high concentrations ( 2000 mg/kg of diet). The reduction in intake caused by isoproterenol was less notable (p s ().12). OISCUS.S1ON

Because adipsin expression has been shown in rats and mice to respond positively to fasting ( 7), the previously reported ( I 1) increase in adipsin expression and reduction in obesity attributed to the combination of caffeine and ephedrine might have been achieved by the reduced intake caused by caffeine and(or) ephedrine. Indeed, in Experiment 1, caffeine alone normalized circulating adipsin, caused weight loss, and reduced percent body fat in the MSG-treated mice, with no additional response attributable to either ephedrine ( a mixed a- and ~-adrenergic agonist) or isoprotereno] ( a typical ~-adrenergic agonist). Additionally, restricting intake also normalized adipsin and caused weight loss, but did not significantly improve the percentage of body fat. In Experiment 2, we separated the effect of caffeine from its anorcctic effect using a group paired-feeding design, and also evolaated selected adrenergic agents independently of caffeine. Although caffeine increased serum adipsin and reduced the percentage of body fat, limiting the daily intake of the restricted-fed group to that of the caffeine-fed partners was also effective. These data, coupled with the fact that none of the adrencrgic agents tested increased adipsin expression, even at 2000 mg per kg of diet, indicates that neither a- nor typical ~-adrenoceptor agonists have direct regulatory control of adipsin expression. Though it is possible that some degradation of the adrenergic agents in the feed, or perhaps even pmtabsorpti ve metabolism, may have li]mited adrenergic activity, data from o[her laboratories also contraindicate a role for adrenergic drugs in the regulation of circulating adipsin. The adipsin gene cont~ins no cAMP-responsive element, and neither chemical sympathectomy nor cold exposure, a potent stimulator of’ sympathetic nervous system activity, augment adipsin expression ( 13). Furthermore, although Iipolysis is readily induced in cultured adipocytes by the caffeine-ephedrine combination, adipsin expression is unchanged ( 1I). Given the clear inability 01 exogenous adrenoceptor agc~nists to increase serum adipsin concentrations, and therefore

the unlikeliness of regulation by endogenous agonists, an explanation of the increase in serum adipsin caused by restricting intake is not obvious. However, it is possible that the reduction in circulating insulin that normally occurs concomitantly with reduced intake is responsible, in part. Insulin has been repeatedly implicated in the regulation of adipsin. The downregulation of adipsin expression by adrenal steroids has been linked to the hyperinsulinemia of the animal (8,15), and regulation by S and placental Iactogen has been shown to be dependent on the ability of the animal to secrete insulin, in that neither hormone downregulated adipsin expression in diabetic tnice ( 12). Although other laboratories have shown that a 3day fasting period failed to normalize adipsin mRNA levels in genetically obese (db/db and ob/ob ) mice (7), it should be noted that there is a marked difference in insulin status between the genetic and chemically-induced models of obesity. The genetic models are characterized by dramatic hyperinsuline]mia, whereas insulin status is normal in the MSG mouse ( 2,1 I ). In the present study, it is clear that caffeine depressed intake, Given the apparently normal insulin physiology in the MSG mouse, one might anticipate a decline in circulating insulin, and therefore an increase in adipsin, concomitant with a reduction in intake. The endocrine regulation of adipsin and the physiological relationship between circulating adipsin and adiposity remain unclear. The decrease in adiposity caused by S and ephedrine without a concomitant increase in serum adipsin indicate that there is not a concrete physiological linkage between body fat and circulating adipsin. It is interesting that the proteolytic activity of adipsin results in the formation of a potent stimulator of triacylglycerol (TG ) synthesis and mobilizer of intracellular glucose transport proteins called acylation stimulating protein (ASP, I ). Furthermore, adipocytes express the proximal components of the alternate complement pathway, including adipsin ( factor D), necessary for the formation of ASP ( 3). It is conceivable that the reduced serum adipsin common to genetic and chemically induced obesity is reflective of a biochemical mechanism, independent of insulin and endogenous catecholamines, that attempts to compensate for the excessive lipid accumulation by limiting glucose uptake and TG synthesis. Such a mechanism is compatible with the normal insulin status of the MSG mouse and the lack of adrenergic regulation of adipsin.

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