Hepatotrophic effects of insulin on glucose, glycogen, and adenine nucleotides in hepatocytes isolated from fasted adult rats

GASTROENTEROLOGY

78:558-570,

1980

Hepatotrophic Effects of Insulin on Glucose, Glycogen, and Adenine Nucleotides in Hepatocytes Isolated from Fasted Adult Rats KHURSHEED N. JEEJEEBHOY, JOSEPH HO, RAJNI and ALAN BRUCE-ROBERTSON Department

of Medicine,

University

of Toronto,

Previous evidence that portal blood insulin is an hepatotrophic factor led to this study of its effect on hepatocytes, isolated from fasted rats, in suspension culture. Control hepatocytes (C), noninsulin-treated, and those infused continuously at low (LJ) and high (HI) levels of insulin were compared concurrently with regard to their survival, glucose transport, and intracellular concentrations of glucose, glycogen, and adenine nucleotides, over a 48-hr period of incubation. Low insulin was adjudged to be comparable to portal insulin concentrations in fasted animals and HI to those in fed animals. ;AJJ hepatocytes had been depleted of glucose, glycogen, and adenine nucleotides at the start of the study by prior fasting of the rat. For the first 6 hr of culture, there was little difference between C, LJ, and HI with reference to the above parameters. In contrast, after 48 hr of incubation, cePJ survival, as judged by the DNA content, was significantly lower in C compared with LJ and HI. The transport of 3-O-[methyl-‘H] Dglucose was also significantly lower in C compared with LJ and HI. The higher uptakes in both LJ and HI were reduced by phloridzin, which had little effect on C. Correspondingly, the intracellular glucose concentrations in C were significantly lower than the extracellular glucose concentrations in contrast to those in LJ and HI, which were comparable. For inReceived January 3, 1979. Accepted October 31, 1979. Address requests for reprints to: Dr. K. N. Jeejeebhoy, Room 6352, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S lA8. The authors sincerely thank the Medical Research Council of Canada for support of these studies (grant number MT 3204), Dr. Mitchell Halperin for very helpful discussions, and Mrs. Janet Chrupala for expert typing and associated work. 0 1980 by the American Gastroenterological Association 0016-5085/80/030556-15$02.25

Toronto,

MEHRA,

Canada

tracellular concentrations of glycogen and adenine nucleotides, the results of LJ and HI were amalgamated as they were not significantly different from each other at 48 hr. Upon analysis, glycogen values were significantly higher for insulin-treated cells. Similarly, the total adenine nucleotide pool (ATP + ADP + AMP) also was clearly higher in HI + LJ than in C. These results indicate that contrary to findings in studies with the perfused liver that have been of a short-term nature, insulin is necessary in the longer term (>12 hr) for maintaining the transport of glucose into hepatocytes; thereby insulin promotes the maintenance of intracellular glucose, glycogen, and adenine nucleotide concentrations, and also enhances cell survival.

In vivo, the hepatocyte is continuously exposed to insulin in the portal blood. The level of this portal insulin may vary from low, fasting values to high ones amounting to about 4 mu/ml after a carbohydrate meal in the dog.’ Thus the liver, via the portal vein, may receive as little as 0.2 mU/kg/min during the fasting state of 4 mU/kg/min or more, after ingestion of 50 g of glucose.’ The importance of this process in the maintenance of differentiated hepatocyte function is indicated by the observation that diversion of portal blood results in hepatocyte atrophy, which can be prevented by insulin infusion into the isolated portal radicle.’ These in vivo studies have not indicated the mechanism by which insulin infusion influences hepatocyte metabolism and integrity.3 Hitherto, in vitro studies of the effect of insulin on hepatic metabolism have been carried out by using freshly isolated, perfused liver and were generally performed over the first

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INSULIN EFFECT ON GLUCOSE

2-4 hr of this organ’s maintenance. Such studies, however, would not be comparable to the phenomenon observed in vivo with insulin both because of the brevity of observation and because of the carryover of the influence of insulin from the in vivo state in freshly isolated organs-r even in cells when these are studied during the initial period following isolation. The effects of insulin have been studied in hepatocyte cultures by a variety of investigators, some of whom have shown increased protein synthesis,4-6 better attachment to culture plates’,’ and/or improved viability.’ Using similar culture techniques, others have indicated that there was an absence of insulin effect.“’ However, none of these studies was especially directed towards systematically examining the earlier, in vivo findings of the hepatotrophic effects of insulin. Concerning hepatotrophic phenomena, most work using isolated hepatocytes has utilized either cells derived from fetal liver (cells that in culture have developed along epithelial lines), or cells derived from hepatectomized rats. Usually such work has been concerned with regenerative phenomena, such as DNA synthesis. More recently, however, a study of Junge and Creutzfeldt’” that used adult hepatocytes has been directed towards the hepatotrophic effects of insulin. They showed that insulin enhanced thymidine, leucine, and uridine uptakes, but did not influence cell survival. No other metabolic parameters were reported. The present studies, therefore, were designed to explore the hepatotrophic effect of insulin over a longer period in vitro, and used a steady perfusion of the hormone with isolated hepatocytes maintained in suspension culture for 48 hr. In each case, insulintreated cells were compared with those from the same batch maintained in noninsulin-treated suspension, thus avoiding problems arising from variance in donor rat liver cells and permitting better control of experimental variability. Furthermore, such controls indicated the metabolic changes that might be observed in cells in the absence of insulin throughout this period of time. This paper reports studies of the effect of infusing high and low doses of insulin-corresponding to those observed in portal blood in conditions similar to the fed and fasted states-into the suspension medium for hepatocytes that were derived from fasted animals. The results show-we believe for the first time-that insulin is necessary for maintaining membrane permeability to glucose and also for maintaining intracellular glucose levels. These effects, in turn, are necessary for promotion of glycogen synthesis and are associated with maintenance of adenine nucleotide levels and higher DNA concentrations in the suspension.

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Materials and Methods Materials Animals. Male Wistar rats, weighing 132-220 g, used for these studies. Purina rat chow was allowed ad libitum until it was removed 24 hr before the start of the experiment so that, although given water, the rats were starved for 24 hr before the liver perfusion that was undertaken for the isolation of the hepatocytes. Radioactive isotopes and chemicals. [“C(U)] sucrose, [l-‘4C]-D-glucose, 3-O-[methyJ-3H]-D-glucose and inulin-[carboxy-‘%] were obtained from either New England Nuclear Canada (Lachine, Quebec) or ICN Canada (Montreal, Quebec). Phloridzin was purchased from Sigma Chemical Company (St. Louis, Missouri). Medium and enzymes. Concentrated Waymouth’s medium MB 752/l and heat-inactivated horse serum were obtained as before from the Grand Island Biological Company (Grand Island, New York). For use in incubation, horse serum was added to unit-strength MB 752/l to a final concentration of 17.8%. Collagenase (Type 1: 125 U/ mg) was obtained from either Sigma Chemical Company (St. Louis, Missouri) or Worthington Biochemical (Freehold, New Jersey). Bovine serum albumin also was obtained from Sigma Chemical Company (Cat. No. A-5128). The collagenase and media solutions were filtered through 0.45 and 0.20 pm disposable Nalgene filters (Nalge Co., Sybron Corp., Rochester, New York) before use. Radioactivity assay. *“C- and ‘H-radioactivity were measured in a Mark I Nuclear-Chicago scintillation counter as previously,4 correcting for quenching by the channels-ratio method using an external standard. Nuclear-Chicago Solubilizer (NCS) continued to be used as the solubilizing agent in measurement of radioactivity. Hormone. The insulin used was Insulin-Toronto, made from zinc-insulin crystals of porcine origin. It was supplied for human use by Connaught Laboratories, Ltd. (Willowdale, Ontario). The concentration received was 100 U/cc, and its potency was 23.8 $J/ng. were

Methods Preparation of isolated hepatocytes.

The method

used was a modification (as reported by us previously4) of the method of Berry and Friend.” The pretreatment of animals, anesthetization, and perfusion techniques were essentially as described earlier,4 except for the flow rate of the infusate, which was increased to 20 ml/min, and for the omission of hyaluronidase, only collagenase 0.05% being perfused. Great care was taken to enzymatically disperse the hepatocytes so that mechanical dispersion was avoided. Incubation of cells. A concentration of cells reduced to 3-5 mg of hepatocytes per milliliter of suspension has been found to produce better results generally than the 9-5 mg/ml of our previously reported experiments.4 Suitable quantities of this whole cell suspension (usually 150-200 ml) were distributed in equal amounts in each of the special Pyrex spinner flasks (usually 3 in number) of 250-ml capacity fitted with magnetically stirred Teflon spinners and reflux condensers as described in the past.4

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Oxygenation of the suspensions was carried out more simply than previously, using only 100% oxygen, at a flow rate of about 1 liter/min. Stirring was at a rate of approximately 400 rpm. These conditions were found to maintain the partial pressure of oxygen in the range required.“ The pH was monitored repeatedly to maintain it at a slightly lower level than previously (7.2-7.3). In practice it was found that no adjustment to pH was required when the cells were “healthy.” Insulin in heat-treated horse serum, diluted with 0.9% saline to 17.8% (the same concentration as used for the incubating medium), and control diluent as required were delivered continuously via polyethylene tubing (ID 0.38 mm) to the appropriate incubation flask by means of 5.0ml syringes mounted in a Harvard variable speed infusion pump. The effects of two concentrations of insulin were compared with those of an insulin-free control (C) infusion during each experiment. For the first concentration, hereinafter referred to as high insulin (HI), the medium was primed with 12.5 mu/ml of suspension and infused with 12.5 mu/ml of suspension/hr. For the second, referred to hereinafter as low insulin (LI), the medium was primed with 27.8 $J/ml of suspension and infused with 27.8 pU/ml of suspension/hr. The control flask (C) received the same volume of insulin-free diluent. The total volume of insulin and insulin-free solutions was 3.5 ml, and they were infused at a rate that was in proportion to the volume of incubation medium remaining (by slightly reducing the pump speed after each sampling) so that the increase in volume of this last was less than 2.5% at any time. Glucagon assays of the incubation mixture during insulin infusion revealed negligible glucagon levels that did not rise above that of the uninfused medium. When sampling the suspension, care was taken to obtain the aliquots at mid-depth. Determination of intracellular glucose. Two methods were used and found to give comparable results. In the first, a 3.0-ml aliquot of a suspension was transferred to a tube containing 0.5 PCi of [“C(U)] sucrose (used as a marker for trapped extracellular water), mixed, and centrifuged. The suspension supernatant was saved by decanting it into another tube, and to the remaining cell pellet was added 1.0 ml of 0.3 N perchloric acid (PCA), with good mixing. The supernatant of this PCA-treated pellet was separated by centrifugation (5000 g, 10 min) and its glucose content and that of the suspension supernatant determined by a glucose oxidase method.12 14C-radioactivities were determined in both the supernatant obtained from the whole suspension culture and that from the PCA extraction of the pellet. The ratio of ‘X-radioactivity to glucose concentration in the suspension supernatant was used to determine, from the “C-radioactivity of the PCA supernatant, the amount of extracellular glucose trapped in the pellet. This value was subtracted from the total pellet glucose to indicate the amount of intracellular glucose. The second method was designed to obtain a PCA extract of the cells rapidly by a modification of the method of Hems et a1.13Here 10 X 75-mm test tubes were given a central constriction 1.5-2.0 cm long with a bore just large enough to admit a Pasteur pipette. Thus there was a reser-

GASTROENTEROLOGY Vol. 78, No. 3

4 r

C4C]Sucrose Marker Figure 1. Correlation of measurements of extracellular water using [Wlsucrose and [‘4C]inulin with cells from the same batch.

voir below the contriction (of 0.6-0.7 ml capacity). This reservoir was filled with 10% PCA, the constriction with 4% NaCl, and a 2.0-ml aliquot of the whole suspension culture mixed with 2 1.11of [“‘C]sucrose (about 0.5 PCi), was gently placed in the upper reservoir and the tube briefly centrifuged (160 g, 1 min) in order to drive the cells into the PCA. This method had been employed in other studiesX3 to precipitate cells from their suspending medium with rapidity and relative freedom from surrounding medium. Further measurements and calculations were carried out as in the first method. In order to validate the use of [‘4C]sucrose as an extracellular marker, the results of using it to measure intracellular glucose were compared, for this second method, with those of using [‘4C]inulin as a marker with cells from the same batch. To ensure that the relationship held in different situations, comparisons were made in C and LI- and HI- treated cells, and after different periods of incubation. Measurements using the two different markers showed excellent correlation, the coefficient being r = 0.92 (P < 0.001) (Figure 1). Determination of glycogen. To the pellet obtained by simple centrifugation (5000 g, 5 min) of 2.0 ml of suspension, 1.0 ml of 30% potassium hydroxide (KOH) was added. The tube was placed in a boiling water bath for about 10 min with occasional agitation until the pellet was completely solubilized. To this, 1.1 ml of absolute ethanol were added, the contents mixed, and the tube again placed in a water bath until the water just began to boil. The tubes were cooled to room temperature, and the precipitated glycogen was harvested by centrifugation (5000 g, 10 min) and washed twice with 15% KOH in 55% ethanol. TO hydrolyze the glycogen to glucose, 0.5 ml of 0.6 N HCl was added and the suspension heated for 1 hr in a boiling ter bath. The solution was then neutralized to phenol

wa-

red

March 1980

INSULIN EFFECT ON GLUCOSE

with 0.6 N NaOH and the glycogen assayed as glucose using the glucose oxidase method” noted earlier. Weight of pellet and the intracellular content of water and deoxyribonucleic acid (DNA). The cell mass during incubation was monitored by several means, described below. (The care in aliquoting, mentioned earlier, is pertinent.) WEIGHT

OF CELL PELLET OBTAINED

BY SIMPLE CENTRI-

Two milliliters of the whole suspension culture were centrifuged in previously weighed 12 x 75-mm tubes (5000 g, 10 min). The supernatant was decanted, the tubes were inverted for 5 min to drain the remaining fluid; the inside of the inverted tube below the pellet was wiped dry, and the tube and pellet were then weighed. GRAVITY-SEDIMENTED (1 g) WET WEIGHT. Two milliliters of suspension culture were transferred to previously weighed 12 X 75-cm tubes that were placed vertically for lo-20 min to allow cells to sediment by gravity alone (i.e., with a force of 1 g). Then the supernatant was aspirated (the line of demarcation was usually definite) and the l-g (gravity-sedimented) cells were packed into a hard pellet by centrifugation (5000 g, 10 min). The tubes were then inverted and allowed to drain for 5 min before wiping the inside of the tube below the pellet and weighing. DRY WEIGHT. After being weighed when wet, the sedimented cells (above) were dried overnight in an oven at 90°C and the tube and pellet were reweighed when they were dry. INTRACELLULAR WATER CONTENT. The intracellular water content was derived from the total water content of the sedimented cells (obtained by subtracting the dry weight from the wet weight), which was then corrected for trapped water by the [“Cl sucrose method, as noted under Determination of intracellular glucose. DETERMINATI~N~FDNAINTHE~ELLPELLET. The cell pellet obtained by centrifugation (5000 g, 10 min) of the whole suspension as indicated above was vigorously mixed with l-2 ml of 0.3 N PCA. The precipitate was separated (5000 g, 20 min), and the supernatant being decanted and discarded was resuspended in a known volume (0.5-0.8 ml) of 2.0 N PCA and heated at 70°C for 15 min to extract DNA.lS An equal volume of water was added, and the whole was then recentrifuged (5000 g, 20 min). DNA was estimated in 1.0 ml of this supernatant by the diphenylamine method of Dische.‘” Trypan blue staining, and ultrastructure of centrifuged and sedimented cells, and of supernatant material. These were undertaken by methods already published.‘,” FUGATION

OF AN ALIQUOT.

Determination of adenosine triphosphate (ATP), odenosine diphosphate (ADP) and adenosine monophosphate (AMP). Twenty-milliliter aliquots were taken at different time intervals, and the cells were briefly and gently centrifuged (160 g, 1 min). The supernatant was quickly decanted, and 2.0 ml of 5% PCA were added to the pellet. The supernatant resulting from another centrifugation (5000 g, 10 min) was carefully neutralized with saturated K,HCO, before being assayed for the three nucle-

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otides according to the methods of Lamprect and Trautschold” and Jaworek et al.‘” Determination of the transport of 3-6-[methyl-3H]D-glucose into hepatocytes. Five 4.0-ml aliquots of the hepatocyte suspensions were gently centrifuged (160 g, 1 min) in 12 X 75-mm tubes. The supernatants were discarded and 1.0 ml of glucose-free Waymouth’s medium, supplemented with 5% heat-inactivated horse serum and containing 1.5 pCi of the transported, but not metabolized, 3-O-[methyl-3H]-n-glucose (plus 0.06 pCi of [“C(U)] sucrose as an extracellular marker for trapped water), was added to each of the cell pellets that was then shaken for 0.5, 1, 2, 5, and 10 min, respectively, at room temperature (2l’C). The final hexose concentration was 0.6 mM. Transport of the tracer was effectively stopped by addition to the suspension of one drop of saturated glucose solution. In separate experiments it was shown that the amount of glucose added inhibited uptake by 69.1 + 1.1% (5). (It had been shown by others that “cold” glucose greatly inhibits transport of 3-O-[methyl-“HI-u-glucose in isolated fat cells,‘” and that, correspondingly, 3-O-methyl-D-glucose inhibits transport of [‘“Cl glucose in isolated hepatocytes,21 thus supporting our finding that there is effective cross inhibition of transport of the labeled material by “cold” hexose.) The suspension was then chilled in ice, diluted with 0.5 ml of ice-cold saline, overlaid on 1.6 ml of a cold solution containing 2.5% NaCl and 0.1% glucose, and centrifuged (5000 g, 10 min). This supernatant was removed and the pellet dissolved in 1.0 ml of NCS (Nuclear Chicago Solubilizer) for radioactivity determination. The ratio of “H- to 14C-radioactivities in the supernatant was used to correct the total pellet’s 3H-radioactivity for that portion of it that was extracellular. From the value at each time interval was subtracted the “0” time value. Since the uptake was linear for 2 min and then plateaued, the rates were calculated from the first 2 min of uptake. Effect of phloridzin. In a separate set of transport experiments, the procedures just noted were repeated with 100 PM phloridzin being added to all tubes at “0” time of the analytic incubation. Determination of insulin. The immunoreactive insulin concentration in the medium was determined using antipork insulin antiserum, purified pork insulin standard, and ‘2”I-labeled pork insulin (supplied by Novo Research Institute, Copenhagen, Denmark) and a charcoal separation of free from bound hormone as published previously.”

Expression

of Results

For reasons noted shortly under Results of Preliminary Experiments, measurements were expressed in relation to the DNA content of the suspension with the exceptions of intracellular glucose (which is related to the intracellular water content as well as DNA content), and the uptake of 3-0-[methyl-“HI-D-glucose (which is expressed in relation to the sedimented cell wet weight). In the preliminary experiments to be presented, the sedimented weight, especially the dry weight, corresponded more closely to that of viable and anatomically normal cells than did the ordinarily centrifuged pellet weight.

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ET AL.

Statistical

Analysis

In all instances, hepatocytes from a single animal were divided into C, LI-, and HI-treated suspensions, all studied concurrently. Hence statistical analyses were done using the paired t-test. Values are reported as mean f SEM; n is the number of observations; P < 0.05 was considered significant.

Results Preliminary

Experiments

Preliminary experiments were done to determine the best manner of expressing metabolic results in relation to the hepatocyte content of the medium. These preliminary experiments were undertaken in fed as well as starved rats before the current set of experiments. Changes in the weight of 5000-g-centrifuged and l-g- or gravity-sedimented cells, and of the latter after drying, and of the DNA content of 5000gccentrifuged) cells during incubation. The results are depicted in Figure 2. It is clear that the weight of the total pellet (as judged by the weight of the pellet obtained by 5000 g centrifugation of an aliquot of the suspension) did not decrease below 90% of the starting mass. By contrast, the weight of l-g sedimented cells fell to 71% of the initial weight over the 48 hr of incubation. Although the rate of decline was not linear, the initial loss (in these preliminary experi21% (from 100% to 79%) over ments) was greater-i.e, the first 24 hr while the later decline was only a fur-

Vol. 78, No. 3

ther 8% over the second 24 hr. To elucidate this difference between the weights of cell pellets obtained by centrifugation and by sedimentation, the degree of trypan blue staining and the ultrastructure of the cell pellets were examined. It was found that the cell pellet obtained by ordinary centrifugation (5006 g, 10 min) contained a mixture of cells stained and not stained with trypan blue, with considerable variability of ultrastructure. The cells obtained by 1 g sedimentation, however, were almost uniformally unstained and had excellent ultrastructure. In contrast, cells remaining in the supernatant after 1 g sedimentation were almost all stained with trypan blue (Figure 3). Both the dry weight of l-g cells and the DNA content of 5000-g cells changed in an indistinguishable manner, and by 48 hr they had fallen to a mean of about 60% of the starting value. This decline also was not linear, the initial decline being faster (Figure 2). The measured intracellular water as a percentage of cell weight, using [‘4C]sucrose as a marker, is shown in Table 1. This analysis showed that the difference between the results of the l-g cell wet weight and the dry weight or DNA content could be accounted for in part by an increase of about 4% in the intracellular water content of hepatocytes from starved rats, from a mean of some 78% of total cell weight at “0” time to some 82% (Table 2) after 48 hr of incubation. From these studies it became obvious that the DNA content of 5000-g cells or the dry weight of l-g cells were the best indices of the solid mass of unstained and ultrastructurally normal hepatocytes

Figure 2.

Variations during the course of incubation in (a) wet weight of pellets obtained from a given volume of suspension by (i) simple centrifugation (5000 g, 10 min) and (ii) by gravitysedimentation only (1 g, 10 min), in (b) dry weight of pellet from (a) (ii) and in (c) the DNA mass of the pellet from (a) (i). These were preliminary studies with “full” hormonal supplementation as published,4 but administered at intervals (0, 8, 12,18, 24, and 48 hr) and not infused continuously.

5olT

I

I

I

I

5

12

24

48

Hours

of Incubation

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Figure

INSULIN EFFECT ON GLUCOSE

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3. Trypan blue staining after gravity-sedimentation of cells for 10 min following a 24-hr incubation with a hormone mixture, which included insulin, added at intervals. A. Supernatant: There is much debris and the hepatocytes generally are still isolated. The great majority of these show some uptake of dye and, together with most of the remaining (unstained) cells, bear visible stigmata of cellular damage. B Sediment: Vast numbers of the hepatocytes are clumped (suggestive of more normal function of the cells17) and dyefree. Occasional hepatocytes, mostly isolated, have taken up dye, usually substantially.

and, presumably, of the truly functioning, viable cell population remaining. For this reason it was decided to express most results in relation to the DNA content of the cell pellet. The only exceptions were that, in addition to this, intracellular glucose content was expressed also in relation to the intracellular water content and the transport of 3-O-[methyl-“HI-D-glucase was expressed also in terms of the l-g pellet wet weight, representing the wet weight of healthy cells. Studies in Hepatocytes From Fasted Rats, and the Effect of Insulin Treatment Deoxyribonucleic acid (DNA) content of hepatocytes. The DNA content of cells from C, LI,

and HI suspensions (Table 2) fell over the 48 hr of study, but there was no significant difference between those treated with LI and HI doses. In contrast, C cells (no insulin) showed a significantly reduced DNA content (and a correspondingly lower survival) after 6 hr of incubation, but the difference between these and LI- and HI-treated cells was in excess of 10% only after 12 hr. Intra- and extracellular glucose concentrations. The results are set out in Table 3 and Figures 4A and 4B. The concentrations of intracellular glucose were well below those of the medium (23 mM) at “0” time, which is within 20 min of adding the hepatocytes to the medium. Although the intracellular glucose concentration rose rapidly for the first 12 hr of incubation of both C, and LI-, and HI-treated

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Table

1.

Intracellular Weight”

Water

GASTROENTEROLOGY

as Percentage

of Cell Wet

Treatment Time of incubation (hr) 0

5 12 24 48

Control (no insulin) 77.91f 1.09 75.82 f 0.11 76.47 f 1.04 76.70 zt 2.04 84.59 f 2.02

Low insulin 78.01 f 76.38 f 74.01 f 76.77 f 80.16 t-

0.72 0.67 1.03 1.23 1.92

High insulin 78.01 + 75.76 f 72.79 f 76.91 f 81.29 f

0.97 1.01 0.94 0.70 1.54

o Mean f SEM, n = 4.

cells, it was significantly higher (P < 0.01) in the last two compared with C from 6 to 48 hr of incubation. The HI and LI cells did not differ significantly from each other with regard to the intracellular glucose concentration or with regard to the mean intra- to extracellular glucose concentration ratio (I/E), although the mean HI values were numerically higher than the LI ones. As for the C cells, intracellular glucose concentrations in them fell quickly after 12 hr of incubation and were below those found in the medium at 24 and 48 hr. By contrast, intracellular glucose concentrations in LI and HI cells were maintained from 12 to 24 hr of incubation and then fell, but never dropped below those found in the medium. From Figure 4A it may be seen that even the C hepatocytes in this model have shown intracellular concentrations of glucose above those found in the medium (i.e., an I/E ratio > l), a phenomenon apparently not hitherto observed in the perfused liver. This concentration phenomenon was enhanced in the presence of insulin. The difference between C and LI or HI cells was not due to a difference in “cell survival” as judged by

Table

2.

DNA Content of Hepatocytes in Suspension” @g/ml of Whole Suspension) Treatment

Time of incubation (hr) 0

5 12 24 48

Control (no insulin) 13.72 1- 0.08 13.01 f0.09 10.51 f 0.21d 7.74 * 0.35” 3.28 f 0.63”

Low insulinb”

High insulin’”

13.66 -c 0.08 13.20 + 0.09 11.00 f 0.20 9.05 f 0.34 7.00 + 0.65

13.73 zt 0.08 13.28 f 0.09 11.53 f 0.20 9.50 f 0.35 7.55 k 0.61

a Mean of 7 experiments each with control, low, and high insulintreated suspensions. b The amount of insulin added initially and then subsequently infused is given in the text (page 558). c No statistical difference between LI and HI suspensions at all times of incubation. d Significantly lower than insulin-treated suspensions, P c 0.01. ’ Significantly lower than insulin-treated suspensions, P < 0.001.

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the DNA content of the suspensions because a similar difference is observed even if the results are calculated as intracellular glucose per microgram of DNA (Figure 4B). So expressed, the intracellular glucose content continued to increase to 24 hr in both LI and HI cells, whereas by this time it had definitely fallen in C cells. Glycogen concentrations. The concentrations of glycogen in fasted rat hepatocytes were very low (Figure 5). The levels rose rapidly in LI and HI cells as a whole to a mean of 6.50 & 0.96 (10 rats) pg/ pg DNA after which it was comparable to that seen at 0 hr in hepatocytes from fed animals, namely 6.03 +- 0.61 (16 rats) pg/pg DNA (unpublished observations). In C cells, by contrast, the levels rose only to a maximum of 2.09 & 0.39 pg/pg (10 rats). There was no significant difference between the glycogen levels of HI and LI groups. Ratio of intracellular glucose to glycogen. The intracellular glucose and glycogen concentrations rose and fell proportionately over the 48 hr of study in both C, LI, and HI cells. From Figures 6A, 6B, and 6C it can be seen that intracellular glucose correlated significantly with glycogen in both insulin-treated and noninsulin-treated cells. The regressions of these relationships were not statistically different. Expressing this in another way, the mean ratio of intracellular glucose was not significantly different amongst C, LI, and HI cells observed from 0 to 48 hr of incubation. Glucose transport. It can be seen from Figure 7A, 7B, and 7C that the rate of uptake of 3-0[methyl-3H]-D-glucose into hepatocytes was linear for a 2-min period of observation. The statistically fitted lines to the points showed a highly significant correlation (r = 0.92, P < 0.001) with time (Figure 7). The rates of uptake of 3-O-[methyl-3H]- D-glUCOse were almost identical at 24 and 48 hr of incubation for the respective C, LI, and HI cells, attesting to the reproducibility of the method (Figure 7A and 78). Also from Figure 7A and 7B it can be seen that the rate of uptake for the LI and HI cells was clearly greater than that for the corresponding Cs. From slopes of individual experiments, the rates of uptake were calculated and are shown in Figure 7C. In Figure 7C, the rate of uptake over the 2-min period of observation is expressed as dpm/mg hepatocytes/ min and the apparent hexose uptake as pmoles/mg cells/min calculated by assuming that glucose and 30-[methyl-“HI-D-glucose enter at comparable rates and in the proportion noted in the extracellular incubation medium used to study transport. It is clear that the rate of uptake of 3-0-[methyl-3H]-D-glucose was significantly higher for LI (P < 0.05) and for HI (P < 0.001) than for C cells. LI cells had a numerically lower rate of uptake when compared with HI

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IN HEPATOCYTES

Table 3. Intra- and Extracellular Concentrations of Glucose in Control Intracellular Duration of incubation (hr)

Control (no insulin) (n =lO)

0 5 12 24 48

9.03 -t 2.50 36.33 15.87 43.88 +- 3.34 13.95 2 2.14 5.39 & 3.37

7.88 f 39.53 + 68.58 + 67.92 + 28.47 +

and Insulin-Treated

(water)

Low insulinb,” (n = 4) 3.15 9.83 11.51 18.08 14.57

ISOLATED

Extracellular High insulinb,” (n = 10)

Control (no insulin) (n = 10)

9.89 +- 3.04 56.29 z!z9.37d 91.74 + 9JBd 81.37 + 8.6gd 36.17 + 8.65”

23.17 + 23.36 + 23.32 + 23.47 + 24.08 f

0.27 0.25 0.27 0.40 0.41

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(mM”)

563

water (i.e., medium) Low insuIinb (n = 4)

High insulinb (n = 10)

22.64 f. 0.46 22.80 + 0.62 22.43 f 0.59 22.59 +- 0.48 22.76 + 0.23

23.22 + 0.26 23.20 & 0.31 23.12 zt 0.14 22.52 -+ 0.20 22.33 + 0.31

” Glucose concentrations are expressed as millimoles/kilogram of intra- or extracellular water, obtained by measuring the weight of intracellular water (see text, page 559) and obtaining the weight of extracellular water from density measurements. bThe terms low and high insulin refer to given priming doses and constant infusion rates over the 48 hr of observation calculated to result in near-fasting and high feeding concentrations of insulin respectively in the medium. Exact amounts are given in the text (page 558). c Although the intracellular glucose concentrations were numerically higher in the “high” insulin compared with the “low” insulin-treated groups, the differences were not statistically significant. d Significantly different from control, P < 0.005. e Significantly different from control, P <

cells. In contrast to the significant effects of insulin after 24 and 48 hr of incubation, glucose transport after only 3 hr of incubation was uniformly low and comparable in C, LI, and HI cells, amounting to 182 + 7.5, 207 + 19.3, and 203 t- 12.2 dpm/mg/hepatocyte/min of incubation, respectively. In Table 4 are set out the effects on the uptake of 3-0-[methyl-“HI-D-glucose of adding phloridzin (100 PM) to the analytical incubation medium. It can be seen that there is significant inhibition of uptake in the LI and HI cells and although the same trend is seen for C cells, it is not statistically significant. Adenine nucleotide concentrations. The results are shown in Figure 8A and 8B. The total adenine nucleotide pool (ATP + ADP + AMP) as well as ATP, ADP, and AMP levels were comparable in C, LI, and HI suspensions at the start of the incubation. With incubation, both the adenine nucleotide pool and the concentrations of ATP and ADP in C cells initially rose (first 12 hr) and then decreased over the next 36 hr of observation. The adenine nucleotide pool fell to below the initial level after 48 hr of incubation, although the energy charge-a measure of the cell high-energy phosphate groups-was maintained (data not given). In LI and HI cells, no significant difference was noted between HI and LI groups, and hence these were amalgamated for further analysis. In LI + HI cells, the adenine nucleotide pool and ATP levels rose to a maximum after 24 hr of incubation and then dropped, but only gradually, over the next 24 hr. At 6 hr and onwards, the LI + HI cells has significantly higher levels of total adenine nucleotide pool, ATP and ADP levels compared with C. AMP levels were high initially and fell progressively during incubation in both C and LI + HI cells. Again however, at 12, 24, and 48 hr the AMP levels were higher in LI + HI cells.

These insulin concentrations in the medium. are shown in Table 5, and demonstrate negligible insulin concentrations in C suspensions and very low levels in LI suspensions. There were high, but still physiologic levels (in relation to in vivo portal vein concentrations), in HI suspensions.

Discussion The effect of insulin was studied in fasted rats so that the hepatocytes would be derived from an in vivo situation featuring low insulin and deprivation of intracellular glucose and glycogen. A priori it seems likely that the in vitro conditions necessary to restore protein synthesis and intracellular concentrations of glucose, glycogen, and adenine nucleotides to normal could be determined better in such a situation. Also bearing on this decision to use fasted donor rats were the facts that previous studies with the perfused liver had shown insulin effects could be not striking, and even insignificant at times, under the conditions we used (where glycogen was depleted),“” and that in situations where the liver was from fasted, unstressed rats or where the liver used was in a state of glucose uptake,24 similar results had been obtained. Thus the effects of insulin reported here are different from those previously observed and are not associated with conditions known to facilitate the demonstration of these effects. It is seen that insulin infusion has profound effects on the continuing ability of the hepatocyte to maintain intracellular glucose levels, and for reasons to be discussed below, the observations may all be explained on the need for insulin to sustain the ability of the hepatocyte to transport glucose into itself. In the absence of in-

564

GASTROENTEROLOGY

JEEJEEBHOY ET AL.

HI HI LI vs vs vs c LI c f t ++ - PeO.05 * - P
A-A A-A o----o

high insulin(HI) low insulin(Ll) control (C)

state to a high of about 200 @J/ml upon feeding glucose, observations of portal blood plasma in the dog have shown much higher levels of insulin-as much as about 400 III/ml following a saline infusion after an overnight fast, with up to 4-9 mu/ml after intravenous administration of 1.0 g/kg of glucose.z6 It has been calculated that our LI infusion of 27.8 pU/ ml/hr and HI infusion of 12.5 mU/ml/hr should approach the sort of insulin input that the liver might have received during the fasting and fed states in vivo, using data published earlier for dogs.’ In actual practice, the mean observed concentration of insulin increased slowly over the 48 hr of incubation from 0.14 to 8.3 /.dJ/ml for the LI groupmuch below portal blood levels observed in the dog during fastinglz6 and from 24.5 to 1620 pU/ml for the HI group-corresponding to those that would be seen during moderate glucose feeding.‘”

A-A A----A

4-B

$

2- /*

insulin-treated cells control cells

T

** /

3-

Vol. 78, No. 3

??

*-

??

- P40.05 P-=0.005

*

.* k

l-

,g’ /

‘\\ “L____

00

--a_

4

48

Hours of Incubation Figure 4. A. Effect of insulin on the ratio of intra- to extracellular glucose concentration (I/E) in hepatocytes with length of incubation. B. Effect of insulin on the glucose content of hepatocytes with length of incubation.

sulin, the hepatocyte became slowly less permeable to glucose over a period of several hours, resulting in low intracellular glucose and glycogen and this was associated with low adenine nucleotide concentrations. This effect was a gradually developing phenomenon in contradistinction to the changes noted in muscle or adipose tissue, which are immediate.z5 Dose of Insulin and Concentration in the Medium

of Glucose

Although the peripheral plasma insulin levels may vary from a low of about 6 $J/ml in the fasting

0612 24 Hours of Incubation

48

Figure 5. Concentration of glycogen in hepatocytes from fasted rats, incubated with and without added insulin.

March 1980

INSULIN EFFECT ON GLUCOSE

IN HEPATOCYTES

ISOLATED

FROM FASTED

RATS

565

Control Cells (50)

Minutes of Analytical Incubation Insulin-Treated cells (48)

’

Insulin-Treated and Control Cells (98)

?? / Minutes of Analytical Incubation

..

..

BI 4shrlncubM&n ??‘ R8.06 ??* ‘ hmos

T T

-: .o*

*.

?? I

’

4

lntraCe)IuIar

I

I

8

I

I 12

t

I 16

1

’

20

Glucose mg/g sedimented wt.

Figure 6. Relationship of intracellular glucose to intracellular glycogen in hepatocytes: A. not treated with insulin; B. treated with insulin; and C. all considered together regardless of treatment. There is no statistically significant difference between the slopes in A and in B.

The glucose concentrations in the suspension medium were high, and they were equivalent to values observed in portal blood during the feeding of large amounts of glucose. By ensuring abundant availability of this substrate, any effects noted could not be ascribed to lack of glucose in the medium. DNA Content of the Hepatocyte

Suspension

The drop in DNA content of the hepatocyte suspension was most rapid for the first 24 hr of in-

Figure 7. Uptake (i.e., amount with time of analytical incubation) of 3-O-[methyJ-3H]-D-glucose as a measure of glucose transport into hepatocytes following their incubation for 24 and 48 hr in the absence (C), and presence of low (LI) and high (HI) concentrations of porcine insulin. LI and HI were equivalent to fasting and fed state concentrations of portal plasma insulin. The results for LI and C are shown in (A). and HI and C in (B). In both instances the lower set of each pair of points is the value at 24 hr, and the upper set of each pair, at 48 hr of incubation. Figure 7C shows the rates of uptakes for HI incubation. Figure 7C shows the rates of uptakes for HI and LI cells to be significantly greater than those for C cells. It is noted that for the respective groups, the rates are very comparable at both 24 and 48 hr of incubation. Correlation coefficient for the points with time: r = 0.92.

566

GASTROENTEROLOGY

JEEJEEBHOY ET AL.

Table 4. Reduction by Phloridzin of Uptake of 3-O[methyI-3H]-D-GIucose by Hepatocytes (dpm 3H/C18DNA) Insulin Treatment

Without phloridzin With phloridzin

Control (no insulin) (n = 5)

Low insulin (n = 5)

High insulin (n = 4)

108.5 f 20.4

145.2 f 15.1

175.9 f 29.3

73.1 f 13.1

94.3 +- 12.8

125.0 f 21.2

P < 0.001

P < 0.025

Vol. 78, No. 3

(P < 0.01) higher at 12 hr in both LI and HI cells compared with C. In comparison with the glucose concentration of the medium at this time, these LI and HI cells demonstrated a 2.3-3.0-fold increase in their intracellular concentration, and yet this increase was very slight for C cells. What seems even more significant is that the fall in the intracellular glucose

TOTAL

A-A A---%

insulin-treated cells control cells

* - PcO.05 P=NS

a*- P
1.6

Uptake (in a separate set of experiments) during a 2-min incubation at room temperature of 3-O-[methyl-3H]-D-glucose by hepatocytes that had been incubated for 24 hr in the absence and presence of low and of high concentrations of insulin (as detailed in text). Phloridzin had been added to the insulin-containing flasks at “0” time, to a concentration of “100 PM”.

cubation in both C and LI + HI cells. Subsequently the DNA content of insulin-treated suspensions fell more slowly. (Over the same period, in contrast, the metabolic effects were more marked and disproportionate to the DNA content of the hepatocytes.) The overall changes and survival were comparable to those reported for cells in primary monolayer culture10.27,2Rusing cells from fed donor rats. In contrast, our control cells-obtained from fasted animals-did not survive as well after 24 hr of incubation. This may be pertinent since the lack of insulin might affect cells depleted by fasting in such a manner as to result in poorer survival.

lA

\

\\

P Z 5 l-

\\

0.6-

0.4 I-, 0612

JntraceJJuJar Water

AMP o----o

1.0

\\ \

\\

40

24 control ATP &----A ADP o---_-O

It is clear that the percentages noted in Table 1 are very close to a previously published ranged for rat hepatocytes of 70-i’% of cell weight” at the start and up to 24 hr of study. Later increases of about 4% were not different in C, compared with LI and HI cells.

\\

1

B ..

insulin-treated .-. 0-O

.-.

*-P
JntraceJJuJar Glucose Concentration The levels were very low, as would be expected in the fasted animal, but were still about twice the expected plasma level of the fasted rat, probably because the hepatocytes at “0” time did not strictly reflect their in vivo milieu since they had already been in the medium (containing 23 mM glucose) for 15-20 min, this being the time interval between adding hepatocytes to the medium and the ability to sample all individual flasks. Subsequently, glucose for up to 12 hr of incubation intracellular concentration rose above the “0” time value in C and LI and HI cells, but the rise was significantly

I

0612

I

1

24

48

Hours of incubation Figure

8. Adenine nucleotide concentrations in hepatocytes treated with and without insulin. A. total adenine nucleotide pool. B. ATP, AMP, and ADP individually.

March 1980

Table

5.

insulin

INSULIN EFFECT ON GLUCOSE

Concentration

in Suspension

IN HEPATOCYTES

ISOLATED

FROM FASTED RATS

567

Medium (Mean f SEM)” Treatment

Time of incubation (hr) 0 5 12 24 48

(n&ml) 0.008-t0.009 0.02 -+0.009 0.02 + 0.009 0.02 f 0.003 0.01 + 0.009

“n = 5 unless otherwise

W/ml) 0.19 + 0.47 + 0.47 + 0.47+ 0.24f

High insulinh

Low insuIinb

Control (no insulin)

0.21 0.21 0.21 0.07" 0.21

(ng/mI)

W/ml)

(WmJ) 0.006f 0.03 + 0.06 + 0.10 + 0.35 f

0.004 0.02 0.03 0.05 0.15

0.14-t0.09 0.71+ 0.47 1.42+ 0.71 2.38+ 1.31" 8.33+ 3.57

1.03+ 0.07 3.24+ 0.29 17.70f 1.10 32.85-t4.91 68.06f 9.20

W/m4

-

24.5-t1.66 73.78+ 6.90 421.26+ 26.18" 781.83f 116.86" 1619.83+ 213.96

noted by an a when n = 4. bFor details of insulin addition see page 558.

concentration of C cells was to below that of the medium concentration subsequently. In contrast, the LI and HI cells maintained their intracellular concentrations for up to 24 hr, and even by 48 hr the intracellular concentrations had not fallen below that of the medium. These findings suggest that in the absence of insulin the cells become impermeable to glucose after 12 hr of incubation. There are two points of note. First, in C cells the fall in intracellular glucose to below the concentration found in the medium did not occur until 24 hr of incubation so that studies that used a perfused liver and that lasted a maximum of about 12 hr would not show this phenomenon. Second, even a low rate of insulin infusion, comparable to that seen in the fasting animal,‘.“i prevented this phenomenon. Increasing the insulin infusion even 45O-fold increased intracellular glucose only modestly. The unexpected finding was that the intracellular concentration of glucose was higher than that of the medium in these studies. To us this seems to be due most likely to selective increase in intracellular glucose and not the result of artifacts. It could not have been due to glycogenolysis, because glycogenesis was observed to be occurring during the same time (Figure 5). It also seems unlikely to have been the result of artifacts caused by differential permeability of the marker used for estimating trapped extracellular glucose for the following reasons. In estimating intracellular glucose, the key assumption is that the [‘4C]sucrose used to estimate trapped extracellular glucose does not enter any cell or, alternatively, it is important to exclude differential permeability of control and insulintreated cells. Now the r4C]sucrose trapping after 3-, 24-, and 48-hr incubations by control and insulin-treated cells showed no consistent difference, taking the period of incubation as a whole. The values, as dpm/mg hepatocytes, were 74,093 + 8711 and 75,891 + 8535 (NS) at 0 hr, 87,132 f 9,512 and 78,043 rfr 8830 (P < 0.05) at 24 hr, and 75,lOOO + 7556 and 83,324 +- 11,239 (NS) at 48 hr for control and insulintreated cells, respectively (n = 10). Although the difference at 24 hr was significant statistically, it was

not consistent in direction, this being reversed at 48 hr at which time the difference was not significant. In addition, its magnitude of 12% is much less than increases in intracellular glucose (Table 3) of LI cells above those of C cells, these being 4.5- and 5-fold at 24 and 48 hr, respectively. As well, measurements of intracellular water content give results comparable to those published by others and there was no significant difference between LI and HI and C cells. Furthermore, at the time when the intracellular glucose concentration was at its highest (12 hr of incubation) the intracellular water content was close to previously observed values.14 Finally, results of intracellular glucose, measured using both [“Clinulin and [‘“Clglucose were comparable indicating that these unexpected results were most unlikely to be due to a faulty extracellular marker. Furthermore it should be noted that the difference in intracellular glucose content between C, LI, and HI cells was significant even when the results were expressed in terms of cellular DNA levels, indicating that these differences were not due to altered cell survival. It is of interest that in another but in vivo study’” using streptozotocin diabetic rats, untreated animals had a mean glucose content in liver of 19.7 + 2.7 (3) pmol/g wet weight at a time when the mean blood concentration was 20.7 + 2.4 mM [mean + SEM (number of observations)]. In insulin-treated animals by contrast the hepatic glucose was higher than the blood glucose by almost 4O%, the respective values being 8.3 f 1.9 (7) and 5.7 + 1.5. The mechanism of this process needs further elucidation.

Glucose

Transport

Earlier studies” that suggested that the hepatocyte of the isolated perfused liver was freely permeable to glucose were performed over the relatively short period of survival of the liver after its isolation. In this respect it is of interest that in our studies, too, there was no detectable difference in glucose transport of freshly isolated cells for at least the first 3 hr of incubation. However, when the cells

568

JEEJEEBHOY

GASTROENTEROLOGY

ET AL.

had been incubated for 24 and 46 hr, their ability to transport glucose was consistently lower in C suspensions as compared with LI and HI suspensions. Concerning the 3-0-[methyI-3H]-D-glucose method, the question may be raised as to whether the differences between C, and LI and HI cells were due to differential permeability of the extracellular marker [‘4C]sucrose. This was clearly not the case as shown in the results. Furthermore using another larger molecular marker, [“Clinulin, we had noted identical results for intracellular glucose measurements (Figure 1) and the same was found in transport studies (data not given). Also too, the addition of phloridzin, a known inhibitor of transport,20~*1~30 clearly reduced the uptake by LI and HI cells (Table 4), indicating that the enhancement of transport was still subject to the action of a specific inhibitor. Hepatic Glycogen In the fasted rat, hepatic glycogen is depleted and in these studies there is clear evidence of in vivo glycogenesis over a 1%hr period. The level of hepatic glycogen attained in the LI and HI cells was comparable to that noted (unpublished observations) in hepatocytes of fed rats at “0” time. Hence the presence of even near-fasting amounts of insulin in the medium was associated with glycogenesis sufficient to attain glycogen concentrations found in hepatocytes from fed animals under the same circumstances. Although glycogenesis also occurred in control hepatocytes (not treated with added insulin), the levels attained were much lower. The lower glycogen synthesis in control cells could be due either to differences in the availability of substrate in the cell, or to a specific action of insulin on the glycogenetic enzymes. The results of our studies favor the former explanation because a linear and significant correlation of a comparable magnitude (Figure 6A, 6B, and 6C) was noted between intracellular glucose and glycogen concentrations, irrespective of the presence or absence of insulin. Our findings would support the contention of DeWulf and Hers? that insulin may not directly influence liver synthase conversion. Indeed, from recent work, glucose alone seems to influence synthase interconversion in the perfused liver by influencing the level of glucose-B phosphate.“’ Adenine Nucleotide

Levels

The energy charges of the hepatocytes were comparable in C and LI and HI cells. Furthermore, the LI and HI cells attained ATP levels comparable with those peak levels (at 12 hr) seen in hepatocytes from fed rats, which averaged 0.899 f 0.080 pmol/ However mg DNA (13) (unpublished observations).

Vol. 78. No. 3

the total adenine nucleotide pool and ATP levels were much lower in C compared with LI and HI cells after 6 hr of incubation. Similarly, ADP and even AMP levels were lower in C, compared with LI and HI cells. Hence it seems that the ability to maintain a normal level of ATP depends on the availability of insulin. Although a cause-and-effect relationship of intracellular glucose to the adenine nucleotide pool of that cell cannot be proven, nevertheless it appears that the two processes occur concurrently, and the ability to maintain a normal adenine nucleotide pool may be related to the availability of energy substrate and insulin. However, further experiments using varying concentrations of glucose are required to test this hypothesis. In the in vivo experiments of Johnston et al.,29 the adenine pool was higher in insulin-treated, mildly diabetic (streptozocin) rats in which the blood glucose was well controlled (compared with untreated, mildly diabetic rats), the values being 5.35 & 0.31 (3) and 4.61 f 0.39 (7) [mean & SEM (number of observations)] micromoles per gram of wet weight of liver. Role of Insulin in the Maintenance of Differentiated Hepatocyte Function When portal blood is diverted, the liver, in addition to other factors, is deprived of a periodic surge of insulin with each meal. Although there is some insulin in systemic blood, it may be inadequate because of the high insulinase activity of the liver.33 In our experiment, we have maintained a high substrate content of the medium comparable to that likely to be seen with a high-carbohydrate meal. Despite this abundance of substrate, it was noted that within a few hours of being deprived of insulin the hepatocyte had a reduced ability to transport glucose and also had a low intracellular concentration of glucose. This was associated with a fall in intracellular glycogen and adenine nucleotide levels. Hence there was a profound disturbance in the ability of hepatocytes to take up glucose and to maintain glycogen concentrations. In the main, the supportive effects of insulin were maximum with near-fasting levels of this hormone, and even when its levels were enhanced up to 20@fold, the uptake of 3-0[methyl-“HI-D-glucose was only increased by 16%. These insulin effects (demonstrated above) are ones, among others, that could explain the marked atrophy of hepatocytes observed during portal blood diversion and that can be prevented by insulin. These longer-term effects of insulin should be clearly differentiated from short-term ones as seen in perfused liver preparations. For example, insulin is a glucagon antagonist, and in short-term experiments, may reduce adenyl cyclase.24 However, in

March

1980

INSULIN

EFFECT

ON

GLUCOSE

contrast to this reduction, Starzl et al.” found that in portal blood-deprived hepatocytes, the adenyl cyclase activity was low and found further that this enzyme’s activity was enhanced by perfusing insulin into the liver. It seems likely that here one is observing an hepatotrophic effect of insulin in promoting protein synthesis. Confronted with these differences between short- and long-term experiments, we believe the latter may be more representative and revealing of that class of insulin effect termed hepatotrophic, which has been little studied in detail in vitro hitherto. To further delineate these effects it would be desirable to study: (a) plasma membrane protein changes with insulin infusion, and (b) hepatocytes from fed rats that have been exposed to a milieu of greater glucose and insulin, but lower free fatty acid concentrations than those from fasted animals. Under the latter, detailed studies would include: (a) uptake kinetics, with and without glucose, (b) concurrent effects of insulin on protein metabolism, and (c) the effects of insulin on hepatocytes from insulindeficient, diabetic rats. It is hoped that these will be the subjects of future study. References 1. Field

JB: Insulin extraction by the liver. In: Handbook of Physiology, Chapter 32, section 7 (Endocrinology), Volume I (Endocrine pancreas). Edited by DF Steiner, N Freinkel. Washington DC, Am Physiol Sot, 1972, p 505-513 2. Starzl TE, Porter KA, Watanabe K, et al: The effects of insulin, glucagon, and insulin/glucagon infusions upon liver morphology and cell division after complete portacaval shunt in dogs. Lancet 1:821-825, 1976 3. Starzl TE. Porter KA, Francarilla JA, et al: A hundred years of the hepatic controversy. In: Hepatotrophic Factors. Edited by R Porter, J Whelan. Amsterdam, Elsevier, 1978, p 111-138 4. Jeejeebhoy KN, Ho J, Greenberg GR, et al: Albumin, fibrinogen and transferrin synthesis in isolated rate hepatocyte suspensions. A model for the study of plasma protein synthesis. Biochem J 146:141-155, 1975 5. Crane LJ, Miller DL: Synthesis and secretion of fibrinogen and albumin by isolated rat hepatocytes. Biochem Biophys Res Commun 60:1269-1277, 1974 6. Tanaka K, Sato M, Tomita Y, et al: Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. I. Hormonal effects on cell viability and protein synthesis. J Biochem 84:937-946, 1978 7. Michalopoulos G, Pitot C: Primary culture of parenchymal liver cells on collagen membranes. Exp Cell Res 94:70-78, 1975 8. Gebhard R, Bellemann P, Mecke D: Metabolic and enzymatic characteristics of adult rat liver parenchymal cells in nonproliferating primary monolayer cultures. Exp Cell Res 112:431-441, 1978 9. Lin RC, Snodgrass PJ: Primary culture of normal adult rat liver cells which maintain stable urea cycle enzymes. Biothem Biophys Res Commun 84:725-734, 1975 10. Junge U, Creutzfeld W: Hepatotrophic effects of pancreatic and gastrointestinal hormones in the rat in vivo and in vitro. In: Hepatotrophic Factors. Edited by R Porter, J Whelan. Amsterdam, Elsevier, 1978, p 267-297

IN HEPATOCYTES

11.Berry

12.

13.

14.

15.

16.

17.

18.

19.

20. 21.

22.

23.

ISOLATED

MN, Friend

FROM

DS: High-yield

FASTED

preparation

RATS

of isolated

569

rat

liver parenchymal cells. A biochemical and fine structural study. J Cell Biol 43:506-520, 1969 Determination of glucose based on use of glucose oxidase, peroxidase and o-dianisidine. Sigma Technical Bulletin No. 510. Sigma Chemical Co, St Louis, MO., revised July 1973 Hems R, Lund P, Krebs HA: Rapid separation of isolated hepatocytes or similar tissue fragments for analysis of cell constituents. Biochem J 150:47-50, 1975 LeCam A, Freychet P: Neutral amino acid transport characterisation of the A and L systems in isolated rat hepatocytes. J Biol Chem 252148-156, 1977 Hutchison WC, Downie ED, Munro HN: Factors affecting the Schneider procedure for estimation of nucleic acids. Biochim Biophys Acta 55:561-570, 1962 Dische Z: Spectrophotometric method for the determination of free pentose and pentose in nucleotides. J Biol Chem 181:379-392, 1949 Phillips MJ, Oda M, Edwards VD, et al: Ultrastructural and functional studies of cultured hepatocytes. Lab Invest 31:533542, 1974 Lamprect W, Trautschold I: Adenosine-5’-triphosphate. Determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Methods of Enzymatic Analysis, Volume 4. 2nd Edition. Edited by HU Bergmeyer. New York, Academic Press Inc, 1974, p 2101-2110 Jaworek D, Gruber W, Bergmeyer HU: Adenosine-S’-diphosphate and adenosine-5’-monophosphate. In: Methods of Enzymatic Analysis, Volume 4.2nd Edition. Edited by HU Bergmeyer. New York, Academic Press, Inc., 1974, p 2127-2131 Czech MP: Regulation of the D-glucose transport system in isolated fat cells. Mol Cell Biochem 11:51-63, 1976 Baur H, Heldt HW: Glucose transport by isolated hepatocytes. In: Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies. Edited by JM Tager, HD Soling, JR Williamson. Amsterdam, North-Holland Publishing Co, 1976, p 357-363 Jeejeebhoy KN, Anderson GH, Nakhooda AF, et al: Metabolic studies in total parenteral nutrition with lipid in man: comparison with glucose. J Clin Invest 57:125-136, 1976 Exton JH, Park CR: Interaction of insulin and glucagon in the control of liver metabolism. In: Handbook of Physiology, Chapter 28 Section 7 (Endocrinology), Volume I (Endocrine pancreas). Edited by DF Steiner, N Freinkel. Washington DC, Am Physiol Sot, 1972, p 437-455

GE: The influence of insulin on the hepatic uptake 24. Mortimore and release of glucose and amino acids. In: Handbook of Physiology, Chapter 31, Section 7 (Endocrinology), Volume I (Endocrine pancreas). Edited by DF Steiner, N Freinkel. Washington DC, American Physiology Society, 1972, p 495504 25. Morgan HE, Neely JR: Insulin and membrane transport. In: Handbook of Physiology, Chapter 20, Section 7 (Endocrinology), Volume I (Endocrine pancreas). Edited by DF Steiner, N Freinkel. Washington DC, Am Physiol Sot, 1972, p 323-331 26. Kanazawa Y, Kuzvya T, Ide T: Insulin output via the pancreatic vein and plasma insulin response to glucose in dogs. Am J Physiol 215:620-626, 1968 GM, Bermudez E, Scaramuzzind D: Rat hepatocyte 27. Williams primary cell cultures. III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro 13:809-817, 1977 28. Ho J, Mehra R, Jeejeebhoy J, et al: Glucose transport in isolated hepatocytes (abstr). Clin Res XXIV:(5), 681A, 1976 29. Johnston DG, Johnson GA, Alberti KGMM: Hepatotrophic factors: implications for diabetes mellitus. In: Hepatotrophic Factors. Edited by R Porter, J Whelan. Amsterdam, Elsevier, 1978, p 357-379

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ET AL.

30. Williams TF, Exton JH, Park CR, et al: Stereospecific transport of glucose in the perfused liver. Am J Physiol 215:12001209, 1968 31. DeWulf H. Hers HG: The role of glucose, glucagon and glucocorticoids in the regulation of liver glycogen synthesis. Eur J Biochem 6:558-5&l, 1968 32. Larner J, Villar-Palasi C: Glycogen synthase and its control.

GASTROENTEROLOGY

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In: Current Topics in Cellular Regulation, Volume 3. Edited by B Horecker, ED Stadman. New York, Academic Press, 1971, p 195-236 33. Tomizawa HH, Halsey YD: Isolation of an insulin-degrading enzyme from beef liver. J Biol Chem 243:307-310, 1959 34. Lehninger AL: Biochemistry. 2nd Edition. New York, Worth Publishers, 1975