The unstirred water layer as a site of control of apolipoprotein B secretion.

Apolipoprotein B-100 (apoB) is a constituent of low density lipoproteins that has been implicated in the development of coronary artery disease (Editorial (1988) Lancet 1, 1141-1142). It is produced primarily in the liver, but mechanisms of secretory control are unclear. We examined the possibility that rapid reuptake of newly secreted lipoproteins regulates the net output of apoB by cultured liver cells. Polyclonal blocking antibodies to the low density lipoprotein receptor markedly increased apoB output, and varying the width of the unstirred water layer around the cells also changed apoB output, consistent with local reuptake. Labeled apoB added to the bulk fluid phase of the incubation media was not detectably taken up, implying that re-uptake is predominantly local. We conclude that a major site of apoB secretory control resides in the unstirred water layer, external to the cell. Because many cells secrete products for which they possess receptors, local re-uptake from the unstirred water layer may be a general mechanism for secretory control.


Apolipoprotein
B-100 (apoB) is a constituent of low density lipoproteins that has been implicated in the development of coronary artery disease (Editorial (1988) Lancet 1, 1141Lancet 1, -1142.Itisproducedprimarily in the liver, but mechanisms of secretory control are unclear.
We examined the possibility that rapid reuptake of newly secreted lipoproteins regulates the net output of apoB by cultured liver cells. Polyclonal blocking antibodies to the low density lipoprotein receptor markedly increased apoB output, and varying the width of the unstirred water layer around the cells also changed apoB output, consistent with local reuptake.
Labeled apoB added to the bulk fluid phase of the incubation media was not detectably taken up, implying that re-uptake is predominantly local. We conclude that a major site of apoB secretory control resides in the unstirred water layer, external to the cell. Because many cells secrete products for which they possess receptors, local re-uptake from the unstirred water layer may be a general mechanism for secretory control.
A variety of seemingly unrelated stimuli affects the output of apolipoprotein B-100 (apoB)' by cultured liver cells. These include cholesterol depletion and enrichment (2), oleic acid supplementation (2)(3)(4), insulin (4)(5)(6), and the viscosity of the culture medium (7). Similar stimuli affect hepatic output of apoB in uiuo including inhibition of cholesterol biosynthesis by the administration of lovastatin (8-10) and cholesterol delivery to the liver by high density lipoproteins (11) or synthetic substitutes (12). None of the stimuli examined to data has any effect on intracellular concentrations of apoB mRNA, indicating that regulation is post-transcriptional (4). To study mechanisms of regulation of apoB output, we used Previous studies in vitro have examined the possibility of receptor-mediated re-uptake of secreted particles as an explanation for this inverse correlation. However, all have used the indirect technique of determining cellular uptake of exogenously supplied lipoproteins and have concluded that re-uptake does not occur (2,5,7,20). In contrast to this approach, we directly blocked the uptake by HepGP cells of their own secreted particles.
Anti-LDL receptor blocking antibody (16) abolished most of the difference in apoB output between LS and HCC cells (Fig. 1). In the presence of non-immune rabbit IgG, the output of apoB from LS cells was 45.0 + 2.6% less than the output from HCC cells. In the presence of polyclonal rabbit anti-LDL receptor-blocking antibody, the output from LS cells was only 19.3 f 2.6% less than the output from HCC cells. These results indicate that most, if not all, of the difference in apoB output between LS and HCC cells depends on the presence of unblocked receptors. The difference in output was reduced because the blocking antibody was associated with a large increase in the absolute output from the LS cells (Fig.  l), suggesting substantial re-uptake of secreted apoB. The HCC cells showed a small but statistically insignificant rise in apoB output in the presence of blocking antibody (Fig. l), suggesting minimal re-uptake.
To reconcile our data with the prior literature demonstrating no re-uptake of secreted particles, we repeated a published experiment using exogenous lipoproteins (20 extensive dialysis. Aliquots of this conditioned dialyzed medium were placed directly, without further processing, onto a set of unlabeled HepG2 cells for a second incubation. The content of [3H]apoB in the conditioned dialyzed medium before and after the second incubation was assayed by ultracentrifugation and isopropyl alcohol precipitation (13). As previously reported (20), there was no statistically significant loss of labeled apoB from the media during the second incubation. Note that similar results have been reported for plasma lipoproteins; only a small fraction of the total amount typically incubated for several hours with a monolayer of cultured cells is taken up and degraded (see, for example, p. 246 of Ref. 21). Although this type of experiment seems to disprove significant re-uptake of newly secreted lipoproteins, it actually examines events only in the bulk fluid phase of the incubation media. Ongoing secretion of [3H]apoB could, however, be plausibly associated with a high local concentration of t3H] apoB in the unstirred water layer adjacent to the cultured cells. Local accumulation of secreted particles would facilitate receptor-mediated re-uptake.
To test the involvement of the unstirred water layer in the regulation of apoB secretion by HepG2 cells, we sought to vary the width of this layer (17). To narrow the unstirred water layer, HepGZ cells preincubated with LS or HCC were shaken during the labeling period (Fig. 2). Shaking increased the apparent secretion rate of [3H]apoB from LS cells by 35.5 f 3.1% (n = 4, p < 0.0005) compared with unshaken LS cells. Shaking produced only a 4.9% (not significant) rise in the output from HCC cells, again suggesting that minimal reuptake occurs in these cells under ordinary conditions (cf. Fig. 1).
To widen the unstirred water layer, we supplemented the media with 3% gelatin during the labeling period (7). Gelatin reduced the net output of apoB from LS and HCC cells ( Fig.  2 uptake could be rather widespread if local extracellular processing of secreted products occurs, particularly in constrained environments like a synapse or the space of Disse. It is also possible that intracellular receptors (36) interact with newly synthesized ligands before their release from the cell. Regulation of hepatic apoB output by local re-uptake suggests that "reverse" transport of cholesterol from peripheral tissues to the liver by high density lipoproteins (37,38) or synthetic substitutes (39) may not necessarily be anti-atherogenic. Cholesterol delivery may provoke suppression of hepatic LDL receptors (l), which would reduce re-uptake of nascent lipoproteins and thereby enhance total hepatic output of atherogenic apoB-rich particles (cf. Refs. 11 and 12). To be salutary, "reverse" transport may require concomitant cholesterol depletion of the liver.

Cont
Gel Shake Cont Gel Shake

LS HC&C
In summary, our results indicate that the unstirred water layer, though not internal to the cell, is nonetheless an integral metabolic part. It is analogous to the front porch of a house. diffusion of nascent particles allows substantial re-uptake by both sets of cells. An ability of HCC cells to take up their own secreted particles is consistent with the observation that HepG2 cells, unlike fibroblasts, only incompletely suppress their LDL receptors after supplementation with cholesterol or 25-hydroxycholesterol (22,23). Uptake independent of LDL receptors (24, 25) may also play a role. Neither shaking nor gelatin affected the secretion of total 3H-protein (Fig. 3). Gelatin was previously shown to have no effect on the secretion of albumin (7). Overall, our data indicate that there is a significant involvement of the unstirred water layer in the regulation of apoB output by cultured hepatocytes. This involvement is likely to be important in uiuo because the unstirred water layer around hepatocytes within the liver is anatomically protected in the space of Disse, a fluid-filled gap between hepatocytes and vascular endothelial cells.
Although substantial re-uptake of newly secreted lipoproteins has generally been discounted (2,5,7,20), there are several reports that nascent particles are, in fact, taken up by cells. Nascent very low density lipoprotein from one set of perfused livers is rapidly taken up by a second set of perfused livers (26). Nascent hepatic very low density lipoprotein also stimulates cholesterol esterification in cultured macrophages (27). Re-uptake of nascent lipoproteins from HepG2 cells has been demonstrated indirectly (28) and directly (29). None of these studies, however, considered re-uptake as a means to affect net apoB output.
It is possible that local re-uptake may be a general means for secretory control. A related mechanism, re-uptake inactivation of norepinephrine, has been described in adrenergic synapses (30). The action of norepinephrine is terminated by rapid re-uptake back into the presynaptic nerve terminal. Because re-uptake is faster than diffusion out of the synaptic cleft, a substantial amount of the neurotransmitter can be reused through several cycles (30).
There are many other situations in which a cell secretes a product for which it has a receptor or channel (31)(32)(33)(34)(35). Some secretory products, including lipoproteins, can interact with more than one receptor (25). Secretory regulation by re-