Insulin

Inhibits

Apolipoprotein Arthur

I. Salhanick,

B Secretion

Seymour

I. Schwartz,

in Isolated

Human Hepatocytes

and John M. Amatruda

The effect of insulin on apolipoprotein (ape) B secretion was investigated in human hepatocytes. Freshly isolated hepatocytes, prepared by collagenase dispersion of liver specimens, were incubated in serum-free media in the absence and presence of 100 nmol/L insulin for 2 hours. The media was then assayed for apo B content by radioimmunoassay. In hepatocytes incubated without insulin, the secretion of apo B (relative to human low-density lipoprotein [LDL]) was 125 2 37 ng/lO’ cells/2 hours. In the presence of insulin, apo B secretion was reduced to 83 2 29 ng/lO’ cells/2 hours (34% inhibition, P < .05). These results using human hepatocytes are consistent with previous data from our laboratory describing insulin-dependent inhibition of apo B secretion in primary cultures of rat hepatocytes and studies by others employing the human-derived hepatoma cell line, Hap G2. We conclude that human hepatic apo B secretion is under insulin control. The role of more chronic insulin exposure requires further investigation. Copyright 0 1991 by W.B. Saunders Company

T

HE SECRETION OF very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) occurs through a complex series of events including biosynthesis of apolipoproteins and lipids, assembly of the lipid and protein moieties into lipoproteins, incorporation of the lipoprotein particle into secretory vesicles, and secretion from the sinusoidal membrane surface.‘-3 Although the liver is capable of synthesis of most of the major human apolipoproteins,‘.’ we”’ have focused our interest on the hormonal regulation of apolipoprotein (apo) B biosynthesis and secretion, since apo B (1) is the major protein component of VLDL and LDL, (2) is required for triglyceride assembly into VLDL and LDL, (3) plays an essential role in the intravascular transport of VLDL and LDL, and (4) is associated clinically, when elevated, with an increased risk of coronary artery disease.’ Insulin-dependent inhibition of lipoprotein secretion has been demonstrated with insulin administration to humans’,” and inhibition of VLDL4,“-‘4 and apo B4,7.13.‘5 secretion in primary cultures of rat hepatocytes. In contrast, stimulation of VLDL triacylglycerol secretion by insulin in perfused rat livers has been recently reported.‘” Two recent studie?.’ from our laboratory have examined the regulation of apo B secretion by insulin. In one study,4 the addition of insulin to primary cultures of hepatocytes prepared from normal rats resulted in a time-dependent inhibition of the appearance of newly synthesized apo B,, and apo B,, the respective high and low molecular weight variants of human apo B, apo E, and apo C. In a subsequent study,’ we employed pulse-chase (35S-methionine) methodology to evaluate the mechanism of the inhibitory effect of insulin on the secretion of apo B,, and apo B,,. These studies demonstrated that the inhibition of apo B,, and apo B,, secretion by insulin is due, in part, to enhanced intracellular degradation. Taken together, these studies indicate that insulin affects apo B secretion and intracellular turnover and that these effects may be time-dependent. Although liver preparations from nonhuman mammals have provided some understanding of the hormonal regulation of lipoprotein secretion, the extrapolation of data from these results to human hepatic lipoprotein metabolism may be limited. While others have recently demonstrated that cultured human hepatocytes are able to synthesize and secrete lipoproteins,‘7.‘8 no studies are available describing the hormonal regulation of these events. Extensive investiMetabolism,

Vol 40, No 3 (March), 1991: pp 275-279

gations have been performed using the human-derived hepatoma cell line, Hep G2, which has been shown to maintain many of the morphological and biochemical properties of hepatocytes’” and secrete most of the major hepatic lipoproteins,20-2* although important differences in secretory patterns (eg, deficiency in secretion of VLDLsized particles) compared with normal human hepatocytes have been described.“-‘4 Using this cell line, Dashti and Wolfbaue? have recently demonstrated that insulin inhibits the secretion of neutral lipids and apolipoproteins, especially triglycerides and apo B. To investigate whether the effects of insulin on apo B secretion can be demonstrated in human hepatocytes, we have isolated hepatocytes from human liver and evaluated apo B secretion by radioimmunoassay over a period of 2 hours in vitro.

MATERIALS

AND METHODS

Materials

Collagenase (type IV, catalog no. C5138), deoxyribonuclease I (catalog no. DN4527), bovine serum albumin (Fraction V: catalog no. A4503), and gentamicin sulfate (catalog no. G7507) were obtained from Sigma Chemical (St Louis, MO); dispase (grade I, catalog. no. 241-7.50) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN); Williams’ Medium E (catalog no. 320-25.51) and soybean trypsin inhibitor (catalog no. 840-7075) were obtained from GIBCO (Grand Island, NY); human apo B radioimmunoassay (RIA) kit (catalog no. 77055) was obtained from Ventrex Laboratories (Portland, ME); [ 1-“Clacetate (50 to 60 mCi/mmol) was obtained from Amersham (Arlington Heights, IL); and crystalline porcine insulin was a gift from Eli Lilly (Indianapolis. IN).

From the Endocrine-Metabolism Unit, Depanment of Medicine, and the Department of Surgery, University of Rochester School of Medicine and Dentistry, Rochester, NY. Suppotted by a Feasibility Grant from the American Diabetes Association and National Institutes of Health Grant No. DK-20948. Address reprint requests to Arthur I. Salhanick, PhD, EndocrineMetabolism Unit, Department of Medicine, Box 693, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave. Rochester, NY 14642. Copyright Q 1991 by WB. Saunders Cornpan? 00260495/91/4003-0010$03.00l0 275

SALHANICK, SCHWARTZ, AND AMATRUDA

276

Isolation of Hepatoqtes Specimens (5 to 20 g) of human liver were obtained from two renal organ donors and from two patients with hepatic tumors undergoing partial hepatectomy. Liver samples were maintained at 4°C in a HEPES buffer (20 mmol/L, pH 7.4) containing NaCl(l20 mmoUL), NaHCO, (3.4 mmob‘L), KC1 (4.6 mmol/L), glucose (15 mmol/L), glutamic acid (1.2 mmol/L), and pyruvic acid (2.0 mmol/L). The liver specimens were cut into l-mm slices using a cylindrical polyacrylamide gel slicer, washed repeatedly by gravity sedimentation in the above ice-cold HEPES buffer, and then minced (200x). The liver was then dissociated using a modification of the procedure of Caro et a1,26which was shown to yield hepatocytes that were insulin responsive with regard to aminoisobutyric acid uptake, lipid synthesis, and activation of insulin receptor tyrosine kinase activity. The minced liver pieces were incubated in the above HEPES buffer (equivalent to N 5 g liver per 50 mL) for 20 minutes at 37°C under ambient atmosphere in a circular water bath (100 oscillations per minute). The incubate was then centrifuged at 100 x g for 15 seconds. The supernatant was removed and discarded and the liver pieces redistributed (5 g liver per 50 mL) in the above HEPES buffer, to which was added CaCI, (5 mmol/L), bovine serum albumin (lo%, wtivol), collagenase (1 mg/mL), dispase (22 &mL), trypsin inhibitor (50 t@mL), deoxyribonuclease (75 &mL), and gentamicin sulfate (50 )Lg/mL) and incubated for 15 minutes at 37°C (220 oscillations per minute). Following incubation, the incubate was centrifuged at room temperature (100 x g, 15 seconds). The supernatant was removed and filtered through a nylon mesh. The undigested liver was redistributed in fresh HEPES buffer containing the above enzymes and the digestion sequence repeated three additional times. The filtrate after each digestion was diluted 1:l with HEPES buffer containing bovine serum albumin (3%, wt/vol) and CaCl, (5 mmol/L) and centrifuged at 100 x g for 4 minutes. The supernatant was discarded and the pelleted cells washed twice (100 x g, 2 minutes) with the same HEPES buffer. The washed cells were then resuspended in Williams’ E media containing HEPES (15 mmol/L), glutamine (2 mmol/L), bovine serum albumin (l%, wt/vol), and gentamicin sulfate (50 ug/mL), kept on ice, and then combined with the three remaining cell digests as each became available.

Incubation of Hepatocytes The freshly isolated hepatocytes were preincubated for 30 minutes at 37°C under 95% 0,/5% CO, (140 oscillations per minute). The hepatocytes were then pelleted (100 x g, 2 minutes), resuspended in the above Williams’ E media, and cell counts made in a hemocytometer. Hepatocytes were then incubated (1 to 2 x lo6 cells/ml) in Williams’ E media at 37°C for 2 hours under 95% 0,/5% CO, in the absence and presence of insulin (100 nmol/L). Following incubation, the incubation mixture was centrifuged (200 x g, 2 minutes) to remove cellular debris and the supernatant removed for immediate assay for apo B content. RlA ofApo B A commercially available polyclonal double-antibody immunoprecipitation assay was used and the procedure modified in order to be of the sensitivity needed to assay the apo B content in media samples from cell incubations. Media (250 FL) from cell incubations or fresh media containing unlabeled LDL (12.5 to 800 ng) were added to an assay mixture (final volume equalled 600 PL in a conical polystyrene centrifuge tube) containing approximately 20,000 cpm of “‘I-human LDL, 50 p,L of undiluted goat anti-apo B antisera, and diluent. Following a 2-hour incubation at room temperature, 1,400 PL of a l/10 dilution of donkey anti-goat IgG were added and the incubation continued for an additional 30

minutes before pelleting the IgG-anti IgG complex with centrifugation at 4°C (2,600 x g, 20 minutes). The supernatant was then removed by aspiration and the pellet counted in a gamma counter. In some studies, reactivity towards human VLDL was determined by including parallel assay tubes containing 100 to 800 ng VLDL. The data are expressed as ng apo B secreted/lo6 cells per 2 hours using human LDL as standard.

Lipogenesis To assess lipogenesis, hepatocytes were incubated in Williams’ E media at 37°C for 2 hours under 95% 0,/5% CO, in the absence and presence of insulin (100 nmol/L) and in the presence of [ I-“Clacetate (5 mmol/L, 20 uCi/mL). Following incubation, total lipids were extracted with chloroform:methanol as previously described.*’

Statistics Statistical analyses were performed by the paired Student’s t test. RESULTS AND DISCUSSION

Hepatocyte Yield The yield of hepatocytes ranged from 0.5 to 2 x lo6 cells/g liver, which is comparable to other studiesz8 using nonperfusion digestions of human liver fragments with collagenase and dispase. Caro et alz6 reported that with specific lots of collagenase type IV (Sigma Chemical), higher yields (> 8 x lo6 cells/g liver) could be obtained. Although we screened available lots of collagenase, we have been unable to achieve similar yields. Since the liver has been reported29 to contain approximately lo8 cells per gram, the ability to achieve high yields may be related more to the preservation of released cells rather to the degree of tissue dissociation and may account for the wide range of yields described by others.30.” While we have found that inclusion of bovine serum albumin (10%) during the digestion improves cell yield, we have no explanation as to the extensive loss (> 90%) of available hepatocytes from tissue that appears adequately digested and have not been able to devise modifications of the dissociation procedure that result in significant improvement in cell yield from limited supplies of human tissue. Quantitation of Apo B Secretion In order to detect apo B secreted during incubations of freshly isolated human hepatocytes, a commercial RIA kit was employed. By modifications of the standard procedure, a nearly lo-fold increase in sensitivity to unlabelled human LDL was achieved (Fig 1; 0, at 12.5 ng, B/Be = 0.93 and at 208 ng, B/l30 = 0.50). Since otherP have shown in primary cultures of human hepatocytes that most of the newly secreted apo B is associated with VLDL, we established the ability of the human RIA to quantiate apo B within VLDL. In a human plasma VLDL preparation, the percent apo B relative to protein by RIA was 30%, which is comparable to the 27% of protein as apo B obtained by protein staining of a 3.5% to 24% SDS-polyacrylamide gradient ge1.4 In the present study, we were unable to characterize the lipoproteins secreted by hepatocytes due to the limited number of cells available. In this regard, Bouma et al”

INSULIN INHIBITS HUMAN APO B SECRETION

277

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.

.

1

10

.

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.

.

.

.

. .

100

..I

.

1000

.

.

.

..--I

10000

APO B [maI Fig 1. RIA of human LDL. The method is as described in Materials and Methods. 0, Modified assay (mean values of three replicate assays); 0, standard assay as reported by commercial supplier.

recently demonstrated that primary cultures of human hepatocytes secrete lipoprotein particles (VLDL, LDL) similar in size and shape to plasma lipoproteins and have secretion rates comparable to that found in cultures of rat hepatocytes. Ape B Secretion in the Presence of Insulin The ability of insulin to regulate apo B secretion was examined in 2-hour incubations of freshly isolated hepatocytes. The results of these incubations are shown in Fig 2. In the absence of insulin, the secretion rate of apo B ranged from 41 to 204 @lo6 cells/2 hours (125 ~fr37, mean ? SEM). The exposure of human hepatocytes to 100 nmol/L insulin resulted in a 34% inhibition (range, 21% to 48%) of secreted apo B (83 +- 29 ngil0’ cells/2 hours, mean * SEM). Although great variability in apo B secretion was observed between different hepatocyte preparations, a statistically significant (P < .05) insulin-dependent inhibition of secreted apo B was found. Therefore, these studies demonstrate for the first time that insulin inhibits apo B secretion 250 1 I-

Suraical Age Sex

66 F

41 F

f 7k

Mean SEM

6:

Fig 2. The effect of insulin on apo B secretion in isolated human hepatocytes. Hepatocytes were incubated in the absence (Cl) and presence(M) of insulin (100 nmol/L) and the media assayed for apo B as described in Materials and Methods. Apo B content is expressed relative to human LDL. Significance: lP < .05.

by human hepatocytes. In a recent study, Kosykh et al” maintained human hepatocytes in culture in medium containing 10% fetal calf serum, 1 p,mol/L insulin, and 10 nmol/L hydrocortisone. Under this condition, he reported the apo B secretion rate (VLDL fraction, d < 0.006 g/mL) was 890 ng/mg protein/l8 hours (equivalent to - 65 ng apo B/lo6 cells/2 hours), which is comparable to the secretion rate we observed in isolated hepatocytes incubated in the presence of insulin. However, no studies have been reported from this groupI or others in which human hepatocytes were cultured in the absence of hormones. Our observations are also consistent with Vogelberg et al,“’ who showed that injection of insulin into normal humans resulted in decreased production of VLDL-triglyceride after 1 hour. In other studies, we incubated freshly-isolated human hepatocytes with [1-“Clacetate in order to evaluate insulinsensitive lipogenesis. In the absence of insulin, the rate of lipogenesis was 0.354 nmol/106 cells/2 hours. The addition of 100 nmol/L insulin resulted in a 37% stimulation of lipogenesis (0.486 nmol/lOh cells/2 hours). Similarly, Caro et alZ6have found that insulin (100 nmol/L) stimulated intracellular lipid synthesis from [?Z]acetate over a 2-hour period in human hepatocytes. Thus, the ability of insulin to acutely inhibit apo B secretion while enhancing intracellular lipid in human hepatocytes is similar to our previous observations4 with longer exposure (18 hours) of primary cultures of rat hepatocytes to insulin and supports the hypothesis that insulin uncouples triglyceride and apo B accumulation, which may influence subsequent lipoprotein assembly and secretory pathways. Furthermore, these studies support the concept that in the postprandial state while chylomicrons are being absorbed and insulin secreted, insulin promotes the intracellular accumulation of lipid in the liver while inhibiting VLDL secretion.” The mechanism of these effects of insulin are currently unknown, but could involve effects on the secretory process itself. the synthesis of apo B, or the intracellular turnover of apo B.‘.” We have previously shown that apo B from primary cultures of rat hepatocytes contain phosphoserine and phosphotyrosine residues, and that phosphorylation of apo B is increased in apo B secreted from hepatocytes isolated from hypoinsulinemic diabetic rats.’ What role apo B phosphorylation may play in insulin-dependent effects on human apo B secretion will require further study. Data from our laboratory’ indicate that insulin decreases “P-orthophosphatc incorporation into intracellular apo B, suggesting a possible role for phosphorylation in the intracellular turnover of apo B and the assembly of VLDL particles. Recently, Pullinger et al’” have examined whether the ability of insulin to regulate apo B secretion in Hep G2 cells correlates with changes in the level of apo B mRNA. Their finding that insulin-dependent inhibition of apo B secretion occurs without changes in apo B mRNA supports the concept that cotranslational or posttranslational mechanisms underly insulin’s short-term effect.” It is obvious that both the culture and subsequent characterization of the lipoproteins secreted by human hepatocytes will require methodologies in which sufficient

SALHANICK,

278

yields of hepatoqtes are obtained. Others34”6 have shown that perfusion of large liver specimens results in yields (eg, 1 to 10 x 10’ cells per liver lobe) greater than that achieved with agitation of small liver pieces in the presence of dissociating enzymes28337as employed in the present study. What remains an important issue is whether human hepatocytes can be maintained in culture in a defined media as has been achieved with rat hepatocytes4‘7.“*38.40 in order that hormonal regulation of lipoprotein secretion can be systematically evaluated. For example, a major issue that needs to addressed is the time-dependency of insulin modulation of apo B and VLDL secretion. While numerous studies43”-‘4in primary cultures of rat hepatocytes have shown that insulin exposure for less than 24 hours inhibits VLDL secretion, chronic in vitro hyperinsulinemia ( > 24 hours) stimulates VLDL secretion.41~42This apparent contradiction may result

SCHWARTZ, AND AMATRUDA

from a time-dependent resistance to the ability of insulin to inhibit VLDL secretion.42 Previous studies characterizing human lipoprotein metabolism in cultured hepatocytes17.‘8.uor organ culture” were in the presence of hormonal supplementation including insulin, “J’+~hydrocortisone,18.43 glucagon? somatotropin,43 prolactin,43 epidermal growth factor,43 thyrotropin-releasing hormone: and serum.‘7.18*43.M In preliminary experiments, we were unable to maintain human hepatocytes in defined culture media using culture conditions described previously for rat hepatocytes. This problem is under current investigation. ACKNOWLEDGMENT

The authors wish to thank the Organ Procurement Group, Strong Memorial Hospital, University of Rochester School of Medicine and Dentistry, for liver specimens.

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