Short Communications Amplitude Smoothing and Phase Shifting Effect of the Isolated Perfused Rat Liver on Oscillating Insulin and Glucagon W. Hildebrandt1, W. Blech1 and K.-D. Kohnert1 Institut fur Biochemie, Bereich Medizin, Martin-Luther-Universitat Halle-Wittenberg, Halle 2 Institut fur Diabetes, Karlsburg, Germany
8.2 ±1.1 g, a maximum portal mass flow of 6 ng/min insulin (0.75 + mg/1) was monitored. With glucagon, this value amounted to 4.5 ng/min (0.56 ug/1) at a mean liver mass of 7.9 ± 1.2 g. For measurNumerous studies in man and animals have shown ing the hormone radioactivity, 0.5-ml-samples were drawn synchronithat the peripheral concentrations of insulin and glucagon are oscillating (Lefebvre, Paolisso, Scheen and Henquin 1987; Stagner 1991). cally from the portal inflow and the caval outflow of the perfused liver. The measuring values were converted into the appropriate hormone Direct portal measurements suggest that these oscillations are primass flows by means of the actual flow rates. For reasons of better repmarily a secretory phenomenon (Jaspan, Lever, Polonsky and Van Cauter 1986; Chou and Ipp 1990). Recent studies indicate that beside resentation, we have dispersed with the standard deviations or stanthe top wave period of 8 to 14 min. for insulin (Chou and Ipp 1990) and dard errors of means. The relative errors of means amounted to 8 to 20 min. for glucagon (Weigle and Goodner 1986; Hildebrandt, 4.3 ±1.1% (portal insulin) and 5.2 ± 1.3% (caval insulin, n = 6), reBlech and Kohnert 1991a), the secretion pattern of pancreas hormones spectively, and 3.9±1.3% (portal glucagon) and 5.4±1.7% (caval contains also short-term oscillations between 0.5 and 3 min. with glucagon, n = 6), respectively. amplitude fluctuations of 20 to 75 % of the mean hormone concen125 tration (Hildebrandt, Blech and Kohnert 1991a). For this reason, the I-Na was purchased from IZINTA, Budapest present study was designed to investigate the quantitative influence of (Hungary), glucagon was from Novo Industrie GmbH Pharthe isolated perfused rat liver on rapidly changing portal insulin and maceutic, Mainz; insulin was from Serva, Heidelberg, and buffyglucagon concentrations. coat free red cell concentrate from human blood was obtained from the Institut fur Blutspende-und Transfusionswesen (Martin-LutherUniversitat), Halle (Germany).
Materials and Methods
125
As liver donors, 12 week old female Lewis rats (1A Max K) with a mean body mass of 215±12g were used. Methodological details, such as animal breeding, liver mobilization and non-recirculating in vitro perfusion of the organ, have been published recently (Hildebrandt, Bromme, Kohnert and Blech 1989). Criterions of vitality of the in vitro perfused liver (oxygen consumption, ionic balance or lactate-pyruvate-ratio) have been documented by Hildebrandt, Blech and Kohnert (1991b). The livers were perfused portocavally for 45 min with Krebs-Ringer-bicarbonate buffer (pH 7.32 ± 0.06) which contained 2.8 mM glucose, 0.06 mM human serum albumin and freshly washed human erythrocytes (final hematocrit 0.1). To avoid pressure waves the oxygenized buffer was continuously pumped into a storage vessel from which it was streamed into the liver by means of gravitation (18 cm water column). The flow rate was adjusted to 8.0 ±0.5 ml/min. After an adaptation period of 15 min. to the in vitro system, insulin or glucagon from stock solutions (40 ng/ml insulin or 30 ng/ml glucagon) were infused by means of a peristaltic pump via a 2-ml-mixing chamber into the portal main circulation. Previously, the hormone concentrations of stock solutions were controlled by radioimmunoassay (ref. in: Bromme, Hahn, Lucke, Hildebrandt and Blech 1989).
I-Glucagon and 125I-insulin were prepared according to Greenwood, Hunter and Glover (1963) yielding a specific radioactivity of 8.8 MBq/ug glucagon and 15.3 MBq/ug insulin, respectively.
Results and Discussion
The isolated perfused rat liver was able to respond to rapid changes of portal insulin and glucagon concentrations just as fast (Figs. 1 and 2). However, the average portal glucagon amplitude fluctuation of 54.0 ± 3.7% was reduced by 57.4% (residual amplitude fluctuation 23.1 ±3.8%). Moreover, the portal insulin amplitude fluctuation of 37.0 + 4.2% was reduced even by 63.8% giving a residual fluctuation of 13.4 ± 1.3 %. This effect must be attributed to the much higher hepatic extraction rate for insulin which has been well described on conditions of a continuous insulin infusion (literature in: Bromme and Blech 1984). Although previous investigations, accomplished with radio-labeled insulin and glucagon, showed that the hormone passage time was only 3 sec (Hildebrandt, Blech and Kohnert 1991a), both hormones appear in the caval outflow with a shifting of 180°. Thus, the liver seems to be stimulated for an enhanced hormone extraction always during the short period of the hormone pulse maximum in the portal inflow. Especially within the first minutes of horThe hormone pulses were generated by a multistage velocity gradient (hormone stock solution: 100 ul/5 sec — 50 ul/25 sec mone infusion, the hepatic extraction of insulin and glucagon took — none/30 sec) giving a frequency of portal oscillation of 1 pulse/min. place to a high degree. On the other hand, the livers released a great portion of the hormone extracted previously when the pulse was interWith these small infusion rates there was no danger of pressure waves rupted giving a maximum pulse in the caval outflow at a time at which within the main circulation. The amplitude fluctuation was adjusted the portal hormone mass flow had just its minimum. Finally, after cutbetween 37 and 54%. To allow a rapid and sensitive measurement of ting off the pulsatile hormone infusion, most of the hormone radioacthe portocaval difference in the hormone mass flows, the infusions tivity extracted during the infusion appeared again within the outflow. were accomplished with a mixture of glucagon and tracer amounts of 125I-glucagon or insulin and 125I-insulin. At a mean liver mass ofDepending on the hepatic hormone degradation rate, 96.0 ± 2.1 % of
Horm. metab. Res. 24 (1992) 345 - 346 © Georg Thieme Verlag Stuttgart • New York
Received: 5 Nov. 1991
Accepted: 22 Apr. 1992
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Introduction
Fig. 1 Hepatic response to oscillating infused glucagon (frequency 1 min). The hormone amplitude fluctuation within the portal inflow (thin line) amounted to 54.2±3.7% and within the caval outflow (fat line) 23.1 ±3.8% (n = 6).
the glucagon radioactivity which was flowing out of the liver was composed of trichloracetic acid precipitable hormone. In the case of insulin, the portion of trichloracetic acid precipitable hormone amounted to 85.0 ±3.1% indicating a clear preference of the liver to degrade insulin (not shown). By agreement with the results of trichloracetic acid precipitation, in control perfusions (without radiolabeled hormone) the portions of insulin and glucagon which remained immunologically intact according to measurement by radioimmunoassay were 86.7 + 3.3% and 95.1 ±3.2%, respectively (results not shown). On the other hand, during the pulsatile hormone infusion, the average hepatic glucagon and insulin degradation amounted to 0.9 ±0.6% and 3.2 ±0.9%, respectively, indicating that the majority of insulin and glucagon that passed the liver or were released by it again was of immunologically intact composition (results not shown). Nevertheless, the phenomenon of hormone amplitude smoothing and phase shifting could not be seen in control perfusions accomplished with trypan blue (MW 961) which is a strange component for the liver {Hildebrandt, Blech and Kohnert 1991b) suggesting that the premise for these processes is specific biochemical performances, such as hormone-receptor-association and -dissociation, internalization, degradation as well as externalization of degraded and probably of intact hormone. On the other hand, without the peculiar construction of the liver vascular system, by which both hormones are allowed to get into direct contact with their specific receptors at the hepatocyte membranes via the fenestrated sinusoidals (at least within the central liver region), the fast reaction power of the organ to respond to oscillating insulin and glucagon would not be imaginable.
W. Hildebrandt, W. Blech and K.-D. Kohnert
Fig. 2 Hepatic response to oscillating infused insulin (frequency 1 min). The hormone amplitude fluctuation within the portal inflow (thin line) amounted to 37.0 ± 1.3% (n = 6).
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