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Evidence for a role of the sodium pump of hepatocytes in the control of food intake
Journal of the A utonomic Nervous System, 20 (1987) 199- 205
199
Elsevier
JAN 00752
Evidence for a role of the sodium pump of hepatocytes in the control of food intake * W. Langhans and E. Scharrer lnstitut fikr Vetermiir-Physiologie, Z~rich (Switzerland) (Received 3 June 1987) (Accepted 22 June 1987)
Key words: Food intake; Appetite regulation: Sodium pump, N a + / K *-ATPase, Ouabain, Metabolic receptor, Vagal afferent, Hepatic vagotomy, Rat
Summao' To test the hypothesis that the sodium p u m p of hepatocytes is involved in the control of food intake, we investigated the effect of ouabain, an inhibitor of the scxiium pump, on feeding in intact and hepatic vagotomized rats. Ouabain (2 m g / k 8 b.wt.L injected intraperitoneally during the bright phase of the lighting cycle, stimulated feeding in intact and sham-vagotomized rats. but not in hepatic vagotomized rats. Atropinization did not block ouabain's hyperphagic effect. Ouabain did not affect portal blood glucose level. Rats started to eat sooner than normal when ouabain was injected, while their meal size and duration was unchanged. The results are consistent with a role of the sodium pump of hepatocytes in the control of food intake.
Introduction More than 20 years ago, Russek first suggested that hepatic glucoreceptors and their vagal afferents are involved in the control of food intake [23]. According to several recent findings [8-10,12,13, 27], vagally mediated hepatic satiety signals are not only triggered by glucose [17-21,29], but are also derived from the oxidation of different metabolic fuels [8-10,12,13,27]. This raises the question of how the oxidation of metabolic fuels
* A preliminary report of the results was given at the IXth International Conference on the Physiology of Food and Fluid Intake, Seattle, July 1986 (Abstract: Appetite, 7 (1986) 294. Correspondence: W. Langhans, lnstitut fiir Veteriniir-Physiologie, Winterthurerstr. 260, CH-8057 Ziirich, Switzerland.
in the liver is transformed into a neural signal. Various metabolites (e.g. lactate, pyruvate, palmitate) have been shown to hyperpolarize hepatocyte membranes in the perfused rat liver [5]. Pyruvate also decreases the discharge frequency of hepatic vagal afferents [19] as does glucose [17-- 19]. All these effects are blocked by ouabain [5,17-19] which potently inhibits the sodium pump [28] and hence decreases hepatocyte membrane potential [22]. Thus, the sodium pump activity and hepatocyte membrane potential may link the oxidation of metabolites within hepatocytes to the discharge frequency in hepatic vagal afferents and may thereby mediate the effect of hepatic oxidative metabolism on food intake. The present study investigates the role of the hepatic sodium pump in control of food intake. We tested the effect of ouabain on food intake in intact rats and in rats with selective hepatic
0165-1838/87/$03.50 '~ 1987 Elsevier Science Publishers B.V. (Biomedical Division)
200 vagotomy or sham-vagotomy. Besides the possibility that ouabain stimulates feeding through a direct inhibition of the hepatic sodium pump, ouabain may indirectly trigger a hepatic glucostatic hunger signal [16,21], by decreasing portal blood glucose levels due to its inhibitory effect on intestinal glucose absorption [4]. Therefore, we also investigated the effect of ouabain on portal blood glucose level. Finally, in order to further characterize the effect of ouabain on food intake, meal patterns after ouabain or control injection were recorded. All experiments were performed during the bright phase of the lighting cycle, because a possible attenuation of hepatic satiety signals by ouabain might be more effective during the day [12,13,27], when rates normally eat less than during the night.
Materials and Methods Animals and maintenance Adult male Sprague-Dawley rats (KFM, Fiillinsdorf) were used for all experiments. The rats were individually housed in a temperaturecontrolled (23 _+ 2°C) colony room and kept on an artificial dark-light cycle of 12 h each. Except where noted otherwise, the rats were fed a powdered commercial laboratory diet (NAFAG, Gossau, Diet No. 890), with about 70, 20, and 10% of the metabolizable energy (12.2 kJ/g) derived from carbohydrates, proteins, and fat respectively. Food and tap water were available ad libiturn. Effect of ouabain on food intake in intact rats Twenty-eight rats (272-319 g b.wt.) were divided in 2 groups (n = 14, each), matched for food intake measured during the two preceding bright phases and for body weight. At the onset of the bright phase of the lighting cycle, the rats of one group were injected i.p. with 2 m g / k g b.wt. ouabain dissolved in isotonic saline (1 mg ouabain/ml saline). Control rats (n = 14) were injected with the same volume ( - 0.6 ml) of saline. Cumulative food intake was measured by weighing the cups ( + 0.1 g) just before injections and at 3, 6, 12, and 24 h thereafter.
Effect of ouabain on food intake in vagotomized rats Twenty-eight rats (377-472 g b.wt.) were used. which had been vagotomized (n = 14) or shamvagotomized (n = 14) about 8 weeks before. Our method for selective hepatic vagotomy and shamvagotomy has been described in detail elsewhere [9]. Briefly, at least 12 h fasted, anesthetized rats were laparotomized and the hepatic branch of the vagus was identified under a binocular microscope (12 x ) and lesioned between two sutures. All connections between the esophagus and liver were transected. For sham-vagotomies, the vagus was similarly exposed as for hepatic vagotomy but not sutured or cut. The vagotomies were anatomically verified during postmortem examinations. All vagotomies were regarded as complete, because in no rat, an ambiguous connection between the fight trunk of the vagus and the liver was found under the microscope (8-24 × ~. For behavioral testing, two groups (n = 7. each) of vagotomized and two groups (n = 7. each) of sham°vagotomized rats were matched for food intake measured during the preceding bright phase and for body weight. As the effect of ouabain on food intake in intact rats (see Results) was always acute and independent of the injection time within the bright phase of the lighting cycle, the experimental design was modified as follows: 3 h after light onset, vagotomized and sham-vagotomized rats (one group of 7 rats, each) were injected i.p. with ouabain (2 m g / k g b.wt.I. Vagotomized and sham-vagotomized control rats (one group of 7 rats, each), received the same volume of isotonic saline. Cumulative food intake was measured at 2 h after the injections. Two days later, the experiment was repeated using a counterbalanced design. The results of the two trials have been combined for presentation. In an additional experiment, the effect of atropinization (pharmacological blockade of peripheral postganghonic muscarine receptors) on the stimulation of feeding by ouabain was tested under similar conditions in 28 intact rats (426-530 g b.wt.). Two hours 30 rain after light onset. 14 rats were injected i.p. with 5 m g / k g b.wt. atropine methylhitrate dissolved in isotonic saline (2.5 mg atropine methylnitrate/ml saline) and 14 with saline, Thirty rain later, 7 atropinized rats and 7
201
control rats were injected with ouabain and 7 each
with saline. Cumulative food intake was measured at 2 h after the second injection, Two days later, the experiment was repeated using a counterbalanced design for the second injection (ouabain or saline). The results of both trials have been combined for presentation.
t",ffect qf ouabam on portal blood glucose level Twenty-four intact rats were used. Ouabain (2 m g / k g b.wt.) or control ( = saline) injections were administered 3 h after light onset as described. Thirty rain after the injections, rats were etherized and laparotomized. To prevent in vitro glycolysis, portal blood samples were drawn into sodium-fluoride monovettes (Sarstedt, Sevelen). Blood glucose was assayed enzymatically [11]. Effect of ouabain on meal pattern Meal patterns after ouabain or control injection were recorded in 12 rats (mean b.wt. 416 g), adapted to a medium fat diet [27] with about 46, 41, and 13% of the total energy ( - 1 6 . 5 kJ/g) derived from carbohydrate, fat, and protein respectively. This diet was used because it is better suited for the feeding cups of the recording device than the powdered diet used before. Rats' cages were constructed so rats were forced to feed out of spill-resistant feeding cups, which were fixed on scales (Mettler PE 300). Output from each scale was continuously monitored and analyzed for meal patterns (size, duration and intermeal interval) by a Hewlett Packard personal computer (HP 85B). Meals were defined as food removals exceeding 0.3 g, separated by at least 15 rain of non-feeding. The experiment started at light onset. Two groups of rats (n = 6, each) were approximately matched for meal frequency and meal size during the preceding 24 h and for body weight, lntraperitoneal injections of 2 m g / k g b.wt. ouabain or saline were administered on two consecutive days in counterbalanced order. Recording of meal patterns and cumulative food intake for 24 h began immediateh' after injections. The data of both trials have been combined for presentation. Statistical evaluation Differences between group means were tested
using t-tests. P values less than 0.05 were considered significant.
Results
kbod intake In intact rats, ouabain (2 m g / k g b.wt.), injected i.p. at light onset, increased 3 h and 6 h cumulative food intake dramatically (Fig. 1), Twelve and 24 h (not shown) after the injections, food intake of ouabain-injected rats and control rats did not differ. In an additional experiment, which is not reported in detail, 0.5 and 1.0 m g / k g b.wt. ouabain slightly increased 3 h cumulative food intake under the same conditions from 0.7 _+ 0.3 (X + S.E.M., control rats) to 0.9 _+ 0.2 and 1.1 _+ 0.3 g, respectively. These increases were not statistically significant. Further experiments in rats with varying body weights (250-550 g) revealed that ouabain (2 m g / k g b.wt.) had always a similar, acute stimulating effect on feeding, whether injected at light
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Fig. 1. Effect of i.p. o u a b a i n (2 m g / k g b.wt.) on food i n t a k e in rats. O u a b a i n values are significantly (t-test: * P < 0.05, ** P < 0.01 ) higher than control values.
202
onset or 2, 3 or 6 h thereafter (data not shown). Ouabain stimulated feeding significantly also in sham-vagotomized rats (Fig. 2, left), but not in vagotomized rats (Fig. 2, right). Pretreatment with atropine methylnitrate did not block ouabain's hyperphagic effect in intact rats (Fig. 3). Although overt behavioral effects of atropine were not observed, atropinized rats generally ate somewhat less (not statistically significant) than controls. However, the relative potency of ouabain to increase food intake was similar in both groups (Fig. 3). These results indicate that intact hepatic vagal afferents are necessary for the hyperphagic effect of i.p. injected ouabain. This implies that ouabain exerts its hyperphagic effect via the liver. In general, the stimulation of feeding after ouabain is consistent with the idea that sodium pump activity plays a role in the control of food intake. Portal blood glucose As shown in Table I, ouabain (2 m g / k g b.wt.) did not affect portal blood glucose levels. Therefore, ouabain probably does not increase food intake by inhibiting intestinal glucose absorption. Further support for this conclusion is derived from additional experiments, which are not reported in detail. In these experiments, ouabain stimulated feeding also in rats fed a carbohydrate-
Vagotomlzed rats
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Fig. 3. Effect of ouabain (OUA, 2 m g / k g b.wt., i.p.) on food intake in atrophinized rats (5 m g / k g b.wt. atropine methylnitrate, i.p.). Each column represents the mean _+S.E.M. of 14 rats. Significantly higher (paired t-test: * P < 0.05, ** P < 0.01 ) than the corresponding control values,
free, high fat diet. It must be mentioned, however, that in rats kept on the high fat diet, a higher dose of ouabain (2.8 m g / k g b.wt.) was necessary to stimulate feeding reliably. When rats were fed the high fat diet, 2.0 and 2.8 m g / k g b.wt. ouabain increased food intake 27 and 100%, respectively, compared to control rats. The reason for this apparent shift in the dose-response curve of ouabain with fat-rich feeding is unknown. However, an increased bile production due to fat-rich feeding may partly explain the decreased ouabain sensitivity of rats fed the high fat diet, because ouabain is effectively excreted into the bile of rats [30] and because an increased bile production has been correlated with greater ouabain resistance I301.
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Fig. 2. Effect of selective hepatic vagotomy on the stimulation of feeding induced by ouabain (OUA, 2 m g / k g b.wt., i.p.). Each colutrm represents the mean±S.E.M, of t4 rats. * Significantly (paired t-test: P < 0.001) higher than sham vagotomized control value.
PortM blood glucose (rag~all) Saline ( n = 12) Ouabain (n = 12)
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Fig. 4. Latency to eat and size and duration of the first meal after ouabain (()UA. 2 m g / k g b.wt.) or saline injection. Each column represents the mean : S.E.M. of 12 rats. * Significantl~ (paired t-test: P < 0.01 ) different from control value.
food intake transiently. Four hour cumulative food intake was 1.1 + 0.4 g (X _+ S.E.M.) after saline and 2.5 _+ 0.5 g after ouabain (paired t-test: P < 0.01), while 12 h food intake did not differ between both groups (ouabain: 5.6 _4-0.9 g; ,X + S.E.M.) controls: 5.3 _+ 0.7 g). Therefore, feeding rats the medium fat diet instead of the commercial laboratory diet, obviously did not influence ouabain's hyperphagic effect. The stimulation of feeding by ouabain was entirely due to a shorter latency to eat (Fig. 4). The size and duration of the first meal were not affected by ouabain (Fig. 4). Thereafter, ouabain-injected rats compensated for the initial hyperphagia by eating somewhat smaller meals than controls with longer intermeal intervals (not shown). However, the respective differences were not statistically significant. Injected ouabain initially accumulates in the liver [3]. In rats it is then mostly eliminated within one hour [3]. This may explain why only the latency to eat was shortened by ouabain while subsequent intermeal intervals were not. The results indicate that inhibition of the sodium pump in the liver by ouabain triggers initiation of a meal. This implies that a high sodium pump activity is important in the maintenance of post-prandial satiety. Discussion
The present results demonstrate that ouabain stimulates feeding in rats and that this effect is
blocked by selective hepatic vagotomy. Ouabain potently inhibits the sodium pump [28] and decreases hepatocyte membrane potential [22]. Taken together, our findings are therefore consistent with the hypothesis that the hepatic sodium pump is involved in the control of food intake. The failure of atropinization to block the stimulation of feeding by ouabain strengthens the above interpretation, because it suggests that the effect of ouabain is mediated by hepatic vagal afferents. Furthermore, the portal glucose concentration was not decreased by ouabain under the conditions tested. Thus, ouabain most likely stimulates feeding directly bv inhibiting the sodium pump of hepatocytes. Rats started to eat sooner than normal when ouabain was injected. In previous experiments [12], i.p. injected mercaptoacetate, which inhibits fatty acid oxidation [2], also stimulated feeding by shortening intermeal intervals [12]. The same has been reported for 2-deoxyglucose [6,14]. Therefore, oxidative metabolism and sodium pump activity+ both, apparently affect meal frequency rather than meal size. Similarly, mercaptoacetate's hyperphagic effect was also blocked by selective hepatic vagotomy [13]. Together with the hyperpolarizing effect of various metabolites, including palmitic acid, on liver cell membrane [5], the similarities between the hyperphagic effects of ouabain and mercaptoacetate support the idea that hepatocyte sodium pump activity and membrane potential link hepatic oxidative metabolism to hunger and satiety. Thus, innervated hepatocytes [15,26] may act as metabolic receptors. Fluctuations in the hepatic oxidation of different metabolic fuels seem to modulate the activity of the hepatic sodium pump and affect hepatocyte membrane potential, which seems to be inversely related to the discharge frequency of hepatic vagal afferents. Hepatocyte membrane potential has been implicated in the generation of hepatic hunger and satiety signals for more than 20 years by Russek [23-25]. To our knowledge, the present results provide the first experimental evidence in direct support of Russek's hypothesis. The above interpretation is limited, however, because it remains to be demonstrated that changes in hepatocytc membrane potential really affect the discharge frequency of hepatic afferent nerves.
2()4
Glucose oxidation might also affect the di,,,charge frequency of hepatic vagal afferents [17 191 and perhaps also food intake [20,21,29] through the sodium pump of hepatic nerve cndings, because glucose is oxidized within neurons. This IS actualh the coding mechanism which was lirst describcd for hepatic glucoreceptors b3 Ni(lima [19]. It is unknown whether hepatic nerve endings can oxidize other metabolites than glucose, but nevertheless, ouabain presumably also inhibits the sodium pump of hepatic nerve fibers and hence affects their discharge frequency directly. Therefore. i.p. ouabain ma5 stimulate feeding also m this way. Finally. the ouabain dose in iected in the present experiments (2 m g / k g b.wt.) appears high compared to the dose (15 ~g) used in liver pcrfusion experiments [19]. It must he considered, ho~vever, that Niijima perfused livers from the guinea pig [19], a species which is about 100 times more sensitive to ouabain than the rat [11. It remains to be demonstrated that ph3siological fluctuations in hepatic sodium pump activity arc instrumental in the control of food intake. However, if, as seems likely [7,9-11.13,19,20,24], vagally mediated metabolic feedback signals derived from hepatic oxidative metabolism affect feeding, the present results indicate that the sodium pump is crucially involved,
Acknowledgements We Mr. T. ported (Grant
thank Miss C. Groier. Mrs. U. Kunz and Sydler for their help. This work was supby the Swiss National Science Foundation no. 3.944-0.85).
References I Abeywardena, M.Y., McMurchie. E.J., Russel. (3.R. and ('harnock, J.S., Species variation in the ouabain sensitivity of cardiac Na "/K +-ATPase. A possible role for membrane lipids, B~ochem. Pharmacol.. 33 (1984) 3649-.3654. 2 Bauch~, F., Sabourault, D., Giudicelli, Y., Nordmann. J. and Nordmann, R., 2-Mercaptoacetate administration depresses B-oxidation pathway through an inhibition of long chain acyI-CoA dehydrogenase activity, Btochem. J., 196 (1981) 803-809.
l~,()wmer 1..I and Yales.."d.S. l)h.llm,lcokmetJcs ar,d h h a r . c'~crelion ()t bromosulphophtalcil), 'l'l]-,~uahain and 11:t:.lurochohc .told in rat,, ~t,)tJ's g]~,~,r',d-i~Iduced adul," i¢ii~iI failure, Hr .i Pharmac,L. ~3 (I0~4~ "7~ 7~;2 ( ' r a n t , R.K. Intestinal ahnorpt~oI~ ,,~ ~;ugar~. l)Jll~t~.~, ' /¢~.~ 40 (196(}) 7~9-N25. I )alltbach, (i. ,llld |:riednlal(ll. ", % , ] [ ~ t l ' d l C - i l i d L I t ' C d ~tCll.b r a n t potential change~, ilt the pc)(~1.cd r.n li'.dr I~I~)CIlilH Ih,,gt b , . . h t,. 367 (19,74) 3fl6 :-, t.,.an,., t' and Nv....,)la~di~,. S.. S1~,,m.,.ncou,. alltJ 2 I ) ( i . m duced rnetal~(>h,, change,, and fl.-cdmg the i,;ch,,mctrk i v , p()the~,v,. Brom R('~. Bull.. ]5 ( l q ~ 5 ) 4 2 9 435. tricdm;m('~ 5.1 I. and Strid, u!. ) \ I The i")hysl,)l,.')gjc;l! I','.',,.:h,~,l,L,-.,: ,,I hungel, a I,hv,.~,,h,!_,~,-.,I l'>cr'.pecti',,.'. Pq'< h,.l R , n . ~;3 11q761 4(19 431 l.anghan~. \ \ . Damasku. t a,.: ~O~arrtr. 1"... I)dlercnl mctabolltc~, migh! reduce f()od inl~tkc h',, the mitochondriat guner;liion ~I reducing ¢qt(i'.alel(t,. Ippettt(,. ,~ i 1085) 14~ ! <" l.anghans V¢. Egh. t i. ;llld .~th,ltiu! I . Selective het',anc ~agotom~. eliminates the h'~p()ph,~gic effect of different ( n e t a ~ q i t c , . . I -lutrm. Vcr~ Sr~,'. : ~' (1985) 255 262 I() l ar~glt:m~,. W.. l:gli. (} and Scharrcr. F... Regulation of food retake I.~ hepatic oxidative metal~ohsm. Brain Re~. Bull. 15 (Iq851 425.428 t.anghan,, V¢.. (iear',. N. and SOlarrcr. 1'.., I.iver gl',cogcn coulel;t decrcasc~ during meals )n fair,..4m J Pht.~ioL. 243 (.19821 R450 453, 12 I.anghan',. ~.'. and Scharrer, 1:.. Rote el fatt 3, acid oxidation in control of meal pattern. Belm;' Veural Bwl.. 47 (Iqg7) 13 l,angham,, W, antl Scharrer, I-.. l:v~dence lor a ~,gally mediated ~,atictv signal derived from hepatic f a l t \ ~cid uxidatioi~. ,! ,|uteri. Nen" 5"v.~t.. I~ (19871 13- IS. 14 l.arue-A~.'hagiotis, ('. and [,e Magnen..I., Metabolic correlates of Ihe el'fect,~ of 2-dcoxv-D-glucose on meal size and postmcal ~,atietv in rats, Ptnswl Behav., 26 (1981t 193- 196. 15 Lautt. \V.W. Hepatic nerves: a rcvie~ of their functions and cffc~t~. Can J. t'hv.~wL Pharma~ol., 58 ( 1 0 8 0 ) 1 0 5 123 I (', Ma',cr..1.. (ilucostatic mechanb, m of regulation of ft~,~t.l ,make. ",,,w l?ngl .I Med. 249 (IOg.l) I ~, 16. 1"7 N i(jima..~,, Afferent impulse discharges from glucoreccpIor~, in the li~er of the guinea p ~ g . . I n n ,V }'. ~toad Sti, 157 (1969) (,~) 7 0 0 Niuima .'k., (}lucosc-sensnivc alferen! nerve fibres in the hepatic hra.nch of the ~agus nerve in the guinea-pig..1. I'hv~trd (l.,mdonl. 332(19821 315 ~23 19 Niijima. a,.. (.ilucose-sensnive afferent nerve fiber.,, in the lixcr :~(itl their role m food intake and blood glucose regulation. J. Auton. Nerv. Svst.. q (19831 207-. 220. 20 Novm. I ) . Robinson. K.. Culbreth. L A . and Tordoff. M ( ; . . I, there a role Ik)r the liver in the control of food r e t a k e . . I r a J ( h n .Vutr. 42!19~51 1050--1062. 21 Novin. |)., Van tier Weele, D.A. and Rezek, M., Infusion of 2-deox~-l)-gluco~e into the hepatic-portal system causes eating: evidence for peripheral glucoreceptors, Science. 181 (19731 858-860. 22 Petersen. O.H.. The effect of glucagon on the liver cell
205
membrane potential, J. Physiol, (London). 239 (1974)
647-656, 23 Russek, M.. An hypothesis on the participation of hepatic glucoreceptors in the control of food intake. Nature fl,ondon). 197 (1963) 79-80. 24 Russek, M., Current status of the hepatostatic theory of food intake, Appetite, 2 (1981) 137-143 and 157-162. 25 Russek. M. and Grinstein. S., Coding of metabolic information by hepatic glucoreceptors. In R.D. Myers and R.R. Drucker-Colin (Eds.), Neurohumoral Coding of Brain Functwn. New York. 1974, pp. 81-96. 26 Sawchenko, P.E. and Friedman. M.I., Sensory' functions of the l i v e r - a review, Am. J. Phwiol.. 236 (1979) R5-20.
27 Scharrer, E, and Langhans, W., Control of food intake by fatty acid oxidation. Am. J. Ph~'~iol, 250 I10~;t RI003-1006. 28 Skou. J.C., Enzymatic basis for basis for active transport of Na" and K ' across cell membrane, Phwiol. Re~.. 45 (1965) 596-617. 29 Tordoff, M.G. and Friedman, MI., Hepatic portal glucose infusions decrease food intake and increase foc,d preference, Am. J. Pl~vsiol.. 251 (1986) R192-196. 30 Wilson, S.A. and Hewick, D.S., A strain dependent sex difference in ouabain-induced cardiotoxicitv in rats, J. Pharm Pharmacol.. 37 (1985) 671-672.
199
Elsevier
JAN 00752
Evidence for a role of the sodium pump of hepatocytes in the control of food intake * W. Langhans and E. Scharrer lnstitut fikr Vetermiir-Physiologie, Z~rich (Switzerland) (Received 3 June 1987) (Accepted 22 June 1987)
Key words: Food intake; Appetite regulation: Sodium pump, N a + / K *-ATPase, Ouabain, Metabolic receptor, Vagal afferent, Hepatic vagotomy, Rat
Summao' To test the hypothesis that the sodium p u m p of hepatocytes is involved in the control of food intake, we investigated the effect of ouabain, an inhibitor of the scxiium pump, on feeding in intact and hepatic vagotomized rats. Ouabain (2 m g / k 8 b.wt.L injected intraperitoneally during the bright phase of the lighting cycle, stimulated feeding in intact and sham-vagotomized rats. but not in hepatic vagotomized rats. Atropinization did not block ouabain's hyperphagic effect. Ouabain did not affect portal blood glucose level. Rats started to eat sooner than normal when ouabain was injected, while their meal size and duration was unchanged. The results are consistent with a role of the sodium pump of hepatocytes in the control of food intake.
Introduction More than 20 years ago, Russek first suggested that hepatic glucoreceptors and their vagal afferents are involved in the control of food intake [23]. According to several recent findings [8-10,12,13, 27], vagally mediated hepatic satiety signals are not only triggered by glucose [17-21,29], but are also derived from the oxidation of different metabolic fuels [8-10,12,13,27]. This raises the question of how the oxidation of metabolic fuels
* A preliminary report of the results was given at the IXth International Conference on the Physiology of Food and Fluid Intake, Seattle, July 1986 (Abstract: Appetite, 7 (1986) 294. Correspondence: W. Langhans, lnstitut fiir Veteriniir-Physiologie, Winterthurerstr. 260, CH-8057 Ziirich, Switzerland.
in the liver is transformed into a neural signal. Various metabolites (e.g. lactate, pyruvate, palmitate) have been shown to hyperpolarize hepatocyte membranes in the perfused rat liver [5]. Pyruvate also decreases the discharge frequency of hepatic vagal afferents [19] as does glucose [17-- 19]. All these effects are blocked by ouabain [5,17-19] which potently inhibits the sodium pump [28] and hence decreases hepatocyte membrane potential [22]. Thus, the sodium pump activity and hepatocyte membrane potential may link the oxidation of metabolites within hepatocytes to the discharge frequency in hepatic vagal afferents and may thereby mediate the effect of hepatic oxidative metabolism on food intake. The present study investigates the role of the hepatic sodium pump in control of food intake. We tested the effect of ouabain on food intake in intact rats and in rats with selective hepatic
0165-1838/87/$03.50 '~ 1987 Elsevier Science Publishers B.V. (Biomedical Division)
200 vagotomy or sham-vagotomy. Besides the possibility that ouabain stimulates feeding through a direct inhibition of the hepatic sodium pump, ouabain may indirectly trigger a hepatic glucostatic hunger signal [16,21], by decreasing portal blood glucose levels due to its inhibitory effect on intestinal glucose absorption [4]. Therefore, we also investigated the effect of ouabain on portal blood glucose level. Finally, in order to further characterize the effect of ouabain on food intake, meal patterns after ouabain or control injection were recorded. All experiments were performed during the bright phase of the lighting cycle, because a possible attenuation of hepatic satiety signals by ouabain might be more effective during the day [12,13,27], when rates normally eat less than during the night.
Materials and Methods Animals and maintenance Adult male Sprague-Dawley rats (KFM, Fiillinsdorf) were used for all experiments. The rats were individually housed in a temperaturecontrolled (23 _+ 2°C) colony room and kept on an artificial dark-light cycle of 12 h each. Except where noted otherwise, the rats were fed a powdered commercial laboratory diet (NAFAG, Gossau, Diet No. 890), with about 70, 20, and 10% of the metabolizable energy (12.2 kJ/g) derived from carbohydrates, proteins, and fat respectively. Food and tap water were available ad libiturn. Effect of ouabain on food intake in intact rats Twenty-eight rats (272-319 g b.wt.) were divided in 2 groups (n = 14, each), matched for food intake measured during the two preceding bright phases and for body weight. At the onset of the bright phase of the lighting cycle, the rats of one group were injected i.p. with 2 m g / k g b.wt. ouabain dissolved in isotonic saline (1 mg ouabain/ml saline). Control rats (n = 14) were injected with the same volume ( - 0.6 ml) of saline. Cumulative food intake was measured by weighing the cups ( + 0.1 g) just before injections and at 3, 6, 12, and 24 h thereafter.
Effect of ouabain on food intake in vagotomized rats Twenty-eight rats (377-472 g b.wt.) were used. which had been vagotomized (n = 14) or shamvagotomized (n = 14) about 8 weeks before. Our method for selective hepatic vagotomy and shamvagotomy has been described in detail elsewhere [9]. Briefly, at least 12 h fasted, anesthetized rats were laparotomized and the hepatic branch of the vagus was identified under a binocular microscope (12 x ) and lesioned between two sutures. All connections between the esophagus and liver were transected. For sham-vagotomies, the vagus was similarly exposed as for hepatic vagotomy but not sutured or cut. The vagotomies were anatomically verified during postmortem examinations. All vagotomies were regarded as complete, because in no rat, an ambiguous connection between the fight trunk of the vagus and the liver was found under the microscope (8-24 × ~. For behavioral testing, two groups (n = 7. each) of vagotomized and two groups (n = 7. each) of sham°vagotomized rats were matched for food intake measured during the preceding bright phase and for body weight. As the effect of ouabain on food intake in intact rats (see Results) was always acute and independent of the injection time within the bright phase of the lighting cycle, the experimental design was modified as follows: 3 h after light onset, vagotomized and sham-vagotomized rats (one group of 7 rats, each) were injected i.p. with ouabain (2 m g / k g b.wt.I. Vagotomized and sham-vagotomized control rats (one group of 7 rats, each), received the same volume of isotonic saline. Cumulative food intake was measured at 2 h after the injections. Two days later, the experiment was repeated using a counterbalanced design. The results of the two trials have been combined for presentation. In an additional experiment, the effect of atropinization (pharmacological blockade of peripheral postganghonic muscarine receptors) on the stimulation of feeding by ouabain was tested under similar conditions in 28 intact rats (426-530 g b.wt.). Two hours 30 rain after light onset. 14 rats were injected i.p. with 5 m g / k g b.wt. atropine methylhitrate dissolved in isotonic saline (2.5 mg atropine methylnitrate/ml saline) and 14 with saline, Thirty rain later, 7 atropinized rats and 7
201
control rats were injected with ouabain and 7 each
with saline. Cumulative food intake was measured at 2 h after the second injection, Two days later, the experiment was repeated using a counterbalanced design for the second injection (ouabain or saline). The results of both trials have been combined for presentation.
t",ffect qf ouabam on portal blood glucose level Twenty-four intact rats were used. Ouabain (2 m g / k g b.wt.) or control ( = saline) injections were administered 3 h after light onset as described. Thirty rain after the injections, rats were etherized and laparotomized. To prevent in vitro glycolysis, portal blood samples were drawn into sodium-fluoride monovettes (Sarstedt, Sevelen). Blood glucose was assayed enzymatically [11]. Effect of ouabain on meal pattern Meal patterns after ouabain or control injection were recorded in 12 rats (mean b.wt. 416 g), adapted to a medium fat diet [27] with about 46, 41, and 13% of the total energy ( - 1 6 . 5 kJ/g) derived from carbohydrate, fat, and protein respectively. This diet was used because it is better suited for the feeding cups of the recording device than the powdered diet used before. Rats' cages were constructed so rats were forced to feed out of spill-resistant feeding cups, which were fixed on scales (Mettler PE 300). Output from each scale was continuously monitored and analyzed for meal patterns (size, duration and intermeal interval) by a Hewlett Packard personal computer (HP 85B). Meals were defined as food removals exceeding 0.3 g, separated by at least 15 rain of non-feeding. The experiment started at light onset. Two groups of rats (n = 6, each) were approximately matched for meal frequency and meal size during the preceding 24 h and for body weight, lntraperitoneal injections of 2 m g / k g b.wt. ouabain or saline were administered on two consecutive days in counterbalanced order. Recording of meal patterns and cumulative food intake for 24 h began immediateh' after injections. The data of both trials have been combined for presentation. Statistical evaluation Differences between group means were tested
using t-tests. P values less than 0.05 were considered significant.
Results
kbod intake In intact rats, ouabain (2 m g / k g b.wt.), injected i.p. at light onset, increased 3 h and 6 h cumulative food intake dramatically (Fig. 1), Twelve and 24 h (not shown) after the injections, food intake of ouabain-injected rats and control rats did not differ. In an additional experiment, which is not reported in detail, 0.5 and 1.0 m g / k g b.wt. ouabain slightly increased 3 h cumulative food intake under the same conditions from 0.7 _+ 0.3 (X + S.E.M., control rats) to 0.9 _+ 0.2 and 1.1 _+ 0.3 g, respectively. These increases were not statistically significant. Further experiments in rats with varying body weights (250-550 g) revealed that ouabain (2 m g / k g b.wt.) had always a similar, acute stimulating effect on feeding, whether injected at light
7
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.
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.
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.
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Fig. 1. Effect of i.p. o u a b a i n (2 m g / k g b.wt.) on food i n t a k e in rats. O u a b a i n values are significantly (t-test: * P < 0.05, ** P < 0.01 ) higher than control values.
202
onset or 2, 3 or 6 h thereafter (data not shown). Ouabain stimulated feeding significantly also in sham-vagotomized rats (Fig. 2, left), but not in vagotomized rats (Fig. 2, right). Pretreatment with atropine methylnitrate did not block ouabain's hyperphagic effect in intact rats (Fig. 3). Although overt behavioral effects of atropine were not observed, atropinized rats generally ate somewhat less (not statistically significant) than controls. However, the relative potency of ouabain to increase food intake was similar in both groups (Fig. 3). These results indicate that intact hepatic vagal afferents are necessary for the hyperphagic effect of i.p. injected ouabain. This implies that ouabain exerts its hyperphagic effect via the liver. In general, the stimulation of feeding after ouabain is consistent with the idea that sodium pump activity plays a role in the control of food intake. Portal blood glucose As shown in Table I, ouabain (2 m g / k g b.wt.) did not affect portal blood glucose levels. Therefore, ouabain probably does not increase food intake by inhibiting intestinal glucose absorption. Further support for this conclusion is derived from additional experiments, which are not reported in detail. In these experiments, ouabain stimulated feeding also in rats fed a carbohydrate-
Vagotomlzed rats
Sham vagotomlzed rats
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Fig. 3. Effect of ouabain (OUA, 2 m g / k g b.wt., i.p.) on food intake in atrophinized rats (5 m g / k g b.wt. atropine methylnitrate, i.p.). Each column represents the mean _+S.E.M. of 14 rats. Significantly higher (paired t-test: * P < 0.05, ** P < 0.01 ) than the corresponding control values,
free, high fat diet. It must be mentioned, however, that in rats kept on the high fat diet, a higher dose of ouabain (2.8 m g / k g b.wt.) was necessary to stimulate feeding reliably. When rats were fed the high fat diet, 2.0 and 2.8 m g / k g b.wt. ouabain increased food intake 27 and 100%, respectively, compared to control rats. The reason for this apparent shift in the dose-response curve of ouabain with fat-rich feeding is unknown. However, an increased bile production due to fat-rich feeding may partly explain the decreased ouabain sensitivity of rats fed the high fat diet, because ouabain is effectively excreted into the bile of rats [30] and because an increased bile production has been correlated with greater ouabain resistance I301.
4
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TABLE I
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Fig. 2. Effect of selective hepatic vagotomy on the stimulation of feeding induced by ouabain (OUA, 2 m g / k g b.wt., i.p.). Each colutrm represents the mean±S.E.M, of t4 rats. * Significantly (paired t-test: P < 0.001) higher than sham vagotomized control value.
PortM blood glucose (rag~all) Saline ( n = 12) Ouabain (n = 12)
58 + 4 63 ± 3
203 tO
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Fig. 4. Latency to eat and size and duration of the first meal after ouabain (()UA. 2 m g / k g b.wt.) or saline injection. Each column represents the mean : S.E.M. of 12 rats. * Significantl~ (paired t-test: P < 0.01 ) different from control value.
food intake transiently. Four hour cumulative food intake was 1.1 + 0.4 g (X _+ S.E.M.) after saline and 2.5 _+ 0.5 g after ouabain (paired t-test: P < 0.01), while 12 h food intake did not differ between both groups (ouabain: 5.6 _4-0.9 g; ,X + S.E.M.) controls: 5.3 _+ 0.7 g). Therefore, feeding rats the medium fat diet instead of the commercial laboratory diet, obviously did not influence ouabain's hyperphagic effect. The stimulation of feeding by ouabain was entirely due to a shorter latency to eat (Fig. 4). The size and duration of the first meal were not affected by ouabain (Fig. 4). Thereafter, ouabain-injected rats compensated for the initial hyperphagia by eating somewhat smaller meals than controls with longer intermeal intervals (not shown). However, the respective differences were not statistically significant. Injected ouabain initially accumulates in the liver [3]. In rats it is then mostly eliminated within one hour [3]. This may explain why only the latency to eat was shortened by ouabain while subsequent intermeal intervals were not. The results indicate that inhibition of the sodium pump in the liver by ouabain triggers initiation of a meal. This implies that a high sodium pump activity is important in the maintenance of post-prandial satiety. Discussion
The present results demonstrate that ouabain stimulates feeding in rats and that this effect is
blocked by selective hepatic vagotomy. Ouabain potently inhibits the sodium pump [28] and decreases hepatocyte membrane potential [22]. Taken together, our findings are therefore consistent with the hypothesis that the hepatic sodium pump is involved in the control of food intake. The failure of atropinization to block the stimulation of feeding by ouabain strengthens the above interpretation, because it suggests that the effect of ouabain is mediated by hepatic vagal afferents. Furthermore, the portal glucose concentration was not decreased by ouabain under the conditions tested. Thus, ouabain most likely stimulates feeding directly bv inhibiting the sodium pump of hepatocytes. Rats started to eat sooner than normal when ouabain was injected. In previous experiments [12], i.p. injected mercaptoacetate, which inhibits fatty acid oxidation [2], also stimulated feeding by shortening intermeal intervals [12]. The same has been reported for 2-deoxyglucose [6,14]. Therefore, oxidative metabolism and sodium pump activity+ both, apparently affect meal frequency rather than meal size. Similarly, mercaptoacetate's hyperphagic effect was also blocked by selective hepatic vagotomy [13]. Together with the hyperpolarizing effect of various metabolites, including palmitic acid, on liver cell membrane [5], the similarities between the hyperphagic effects of ouabain and mercaptoacetate support the idea that hepatocyte sodium pump activity and membrane potential link hepatic oxidative metabolism to hunger and satiety. Thus, innervated hepatocytes [15,26] may act as metabolic receptors. Fluctuations in the hepatic oxidation of different metabolic fuels seem to modulate the activity of the hepatic sodium pump and affect hepatocyte membrane potential, which seems to be inversely related to the discharge frequency of hepatic vagal afferents. Hepatocyte membrane potential has been implicated in the generation of hepatic hunger and satiety signals for more than 20 years by Russek [23-25]. To our knowledge, the present results provide the first experimental evidence in direct support of Russek's hypothesis. The above interpretation is limited, however, because it remains to be demonstrated that changes in hepatocytc membrane potential really affect the discharge frequency of hepatic afferent nerves.
2()4
Glucose oxidation might also affect the di,,,charge frequency of hepatic vagal afferents [17 191 and perhaps also food intake [20,21,29] through the sodium pump of hepatic nerve cndings, because glucose is oxidized within neurons. This IS actualh the coding mechanism which was lirst describcd for hepatic glucoreceptors b3 Ni(lima [19]. It is unknown whether hepatic nerve endings can oxidize other metabolites than glucose, but nevertheless, ouabain presumably also inhibits the sodium pump of hepatic nerve fibers and hence affects their discharge frequency directly. Therefore. i.p. ouabain ma5 stimulate feeding also m this way. Finally. the ouabain dose in iected in the present experiments (2 m g / k g b.wt.) appears high compared to the dose (15 ~g) used in liver pcrfusion experiments [19]. It must he considered, ho~vever, that Niijima perfused livers from the guinea pig [19], a species which is about 100 times more sensitive to ouabain than the rat [11. It remains to be demonstrated that ph3siological fluctuations in hepatic sodium pump activity arc instrumental in the control of food intake. However, if, as seems likely [7,9-11.13,19,20,24], vagally mediated metabolic feedback signals derived from hepatic oxidative metabolism affect feeding, the present results indicate that the sodium pump is crucially involved,
Acknowledgements We Mr. T. ported (Grant
thank Miss C. Groier. Mrs. U. Kunz and Sydler for their help. This work was supby the Swiss National Science Foundation no. 3.944-0.85).
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205
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