Normalization of Intracellular Lysosomal Hydrolases in I-cell Disease Fibroblasts with Sucrose Loading*

I-cell disease (ICD) is an hereditary inborn error of metabolism by lysosomal storage due to the multiple lysosomal hydrolases deficiency. Many inclusion ma- terials are seen by phase contrast microscopy in cultured skin fibroblasts from the patients with ICD. We recently reported that the addition of 88 mM sucrose to the medium of cultured human skin fibroblasts from normal subjects induced several lysosomal hydrolases, but did not induce deficient hydrolases in lyososomal enzyme deficiencies T., Okada, S., Ohshima, T., Inui, K., Yutaka, T., and Yabuuchi, H. (1981) Biochem Znt. 3,551-556). This time sucrose loading was applied to the cultured skin fibroblasts from the patients with ICD. Incubation with 88 m~ sucrose for more than 10 days exhibited significant effects. Biochemically, the activities of deficient hydrolases reached their normal levels, and morphologically, typical inclusion materials disappeared. These results in- dicate that sucrose enhanced synthesis of normal lysosomal enzymes and lysosome functions were nor- malized in ICD fibroblasts.

I-cell disease (Mucolipidosis 11) is a generalized neurodegenerative disease in childhood, transmitted by an autosomal recessive trait, clinically similar to the mucopolysaccharidoses, but usually with normal urinary excretion of mucopolysaccharides (1-4). The disease owes its name to many inclusions that are seen by phase contrast microscopy in the cultured skin fibroblasts from the patients with ICD' (1,2). These inclusions have been identified electron microscopically as lysosomes fiied with membrane fragments and other debris (2, 5, 6). ICD is characterized by decreased activities of most of acid hydrolases in cultured fibroblasts, with concomitant increases of many of these enzyme activities in the fibroblast medium (7, 8). ICD offers a unique opportunity to study the kinetics of lysosomal hydrolases (9-13).
Since De Duve and Wattiaux (14) described sucrose-induced vacuolation of cells, many studies have been published on the effects of sucrose on the cultured mammalian cells (15-17). The experimental period was relatively short in their studies, i.e. 3 days at the longest (16). Therefore, it is assumed that they have not fully investigated the effects of sucrose on the induction of lysosomal hydrolases. In a recent paper (18), we reported that the addition of 88 m~ sucrose to the medium * Part of this work was supported by grants from the Ministry of Education and the Ministry of Health, and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Present address, Department of Pediatrics, University of Colorado, Health Science Center, Denver, CO 80262.
The abbreviation used is: ICD, I-cell disease.
of cultured normal human skin fibroblasts as long as 13 days caused a higher increase in the intracellular activities of hydrolases than those reported in the literature with shorter experimental period. In addition, we observed that the lysosomal hydrolases were sigdicantly stimulated in the cultured skin fibroblasts of ICD and the enzymic activities reached the normal range after sucrose loading. Here we report the details of the induction and the normalization of several lysosomal hydrolases in ICD fibroblasts.

MATERIALS AND METHODS
Fibroblast Cell Strains-Cultured skin fibroblasts used in the experiments were derived from normal subjects and two cases of ICD (Nos. 186 and 224). Diagnosis of E D was made on the basis of both typical clinical features and biochemical characteristics (2)(3)(4). The latter is summarized in Table I. Skin biopsies were obtained by pinch technique after obtaining informed parental consent; fibroblast cultures were initiated and maintained in Eagle's minimum essential medium (Nissui Seiyaku Co., Tokyo, Japan), supplemented with 10% fetal bovine serum (Flow Laboratories McLean, VA) as described elsewhere (19). Cells were dissociated with 0.25% trypsin, 0.02% EDTA and subcultured with 1:4 split ratio unless otherwise stated.
All experiments were performed in 10-cm Corning plastic dishes with 10 ml medium.
Sucrose Loading Experiments-Details of the sucrose loading experiments were described in the previous paper (18). Essentially, for the sucrose loading, culture medium supplemented with 3 g/dl(88 mM) of sucrose was not changed for 13 days (intracellular lysosomal hydrolases were measured on the specified days). For the recovery experiment, the fibroblasts were cultured with sucrose for 12 days, and then were trypsinized and subcultured afterwards with the conventional medium without sucrose. Intracellular lysosomal hydrolases were measured on the day of subculture and once every 3 days thereafter.
NH,Cl Loading Experiments-On the 12th day of culture (cultured with 88 m~ sucrose in the medium, SUC+; or without, SUC-), after equal numbers of cells were seeded, old medium was removed and rinsed with fresh serum-free medium (with or without sucrose) three times. Such SUC' or SUC-group which consisted of two 10-cm dishes with 10 ml of medium was then divided into two groups and incubated with 10 m~ NH4C1 (NH4+) or without NH&l (NH4-). As a result, intracellular and extracellular hydrolases were assayed after 24 h of incubation in four groups; i.e. SUC-NH4-(control group), SUC-NH4+, SUC'NR-, SUC+NH,+ groups. Extracellular enzyme assays were performed with 20 pl of medium and 50 pl of buffered 4methylumbelliferyl glycoside substrate solution like the intracellular hydrolase assays.
Lysosomal Hydrolase Assays-Cells were rinsed with 0.9% NaCl and detached by rubber policeman. The details of the methods were described in the previous report (20). Protein was measured by the method of Lowry et al. (21).

Normalization of I-cell Fibroblasts by Sucrose 7815
crose Loading Experiments-ICD fibroblast strains (Nos. 186 and 224) were incubated in the same medium for 13 days with (SUC') or without (SUC-) 88 m~ sucrose. Five intracellular lysosomal hydrolases, ,&galactosidase, P-N-acetylglucosaminidase, a-mannosidase, a-fucosidase, and /?-glucuronidase, were measured. Data are shown in Fig. 1. In ICD strains, most hydrolase activities of SUC' except /?-glucuronidase were several times as high as those of SUC-. The induction ratios of these enzymes were much greater in ICD than in normal fibroblasts in which the ratios were 3.5 at the largest (for amannosidase) (18). Induced enzymic activity levels of ICD well reached the normal activities (indicated as shaded areas in the SUC-columns in Fig. 1). Induction of P-glucuronidase was clearly observed in ICD (SUC'/SUC-= 1.48), whereas no clear change was found in normal fibroblasts (SUC'/SUC-= 0.95) (18). However, in SUC', the activity of /?-glucuroni-dase in ICD fibroblasts did not reach the normal enzymic level.
The same degree of induction was observed in the experiment with heat-inactivated serum (70 "C, 30 min). Two ICD strains demonstrated reproducible induction by sucrose loading between different passages. (Tables I1 and 111; Figs. 1 to 4 showed the data from different experiments).
The changes of intracellular hydrolase activities during the sucrose loading experiment for 13 days are shown in Fig. 2. The effect of sucrose was not found in the fist one week. As normal SUC' fibroblasts showed somewhat decreased enzymic activities at this period, it is supposed that subculture shock may be a main factor to depress the effects of sucrose (22). In both ICD SUC' and normal SUC' fibroblasts induction became clear and big after 9th day of incubation.
Enzymic induction rate of intracellular hydrolase in ICD

Normalization of I-cell Fibroblasts by Sucrose
fibroblasts seemed dose-dependent on sucrose, and the highest rate was observed at 88 m~ sucrose concentration (Table 11).
On the other hand, in normal fibroblasts, dose dependency was not clearly found, and even with lower sucrose concentra- /?-glucuronidase was not so large as that of other lysosomal hydrolases examined (Table 11).
Several saccharides other than sucrose were tested for the loading experiments. Concentration of every saccharide was 88 mM. We observed that sucrose caused the highest effect of inducing intracellular hydrolase activities (39.3 times control for ,&galactosidase, 9.22 times for a-mannosidase, and 1.75 times for P-glucuronidase), although maltose, lactose, mannose, and glucose showed relatively good inductive effects (Table 111). Thus, it is concluded that except for sucrose, mono-or disaccharides examined were found either poor inducer of hydrolases or inhibitor of cell growth.
Cultured skin fibroblasts (SUC' and SUC-on the 12th day) in 10-cm dishes were trypsinized with split ration of 1 5 and maintained with the conventional medium (without sucrose) afterwards. The intracellular hydrolase activities were measured on the day of subculture and once every 3 days during the subsequent incubation, and a clear decrease and complete recovery of the activities were found within 10 days after subculture (Fig. 3).
If sucrose-containing medium was changed every 3 days (SUC'. Change+), the induction of intracellular hydrolases was sigdkantly retarded. P-galactosidase and a-mannosidase increased less slowly in the SUC+-Change+ than SUC'. Change-. P-N-Acetylglucosaminidase was not induced with Growth of cell was checked under phase contrast microscopy. Specific activity is expressed as nanomoles per mg of cell protein per h. e Ratio is expressed in specific enzyme activities at different saccharide of 88 mM concentration against the value in the control condition.

Effects of sucrose concentration in medium on the lysosomal hydrolase activities in ICD fibroblast (No. 224)
Confluent ICD (No. 224) cells were trypsinized and subcultured at 1:6 split ratio with different concentration of sucrose. After 12 days of incubation, intracellular lysosomal hydrolase activities were determined. Abbreviations used are same as in Table I (Fig. 4). In SUC-group, the changes of the intracellular hydrolases were not detected by the change of medium (without sucrose) every 3 days. Therefore, the results of SUC--Change+ group were not indicated in Fig. 4.
Extracellular Lysosomal Hydrolases Excreted from Normal and ICD Fibroblasts by the Addition of NH4C1- Fig. 5 shows the acid a-mannosidase activities excreted from normal and ICD fibroblasts in the sucrose loading and/or the NH4Cl loading experiments. P-N-Acetylglucosaminidase and P-glucuronidase were also assayed in those media. Determination of the enzyme activities in the medium was performed directly without dialysis. The results were very similar to those of acid a-mannosidase.
In the control condition (SUC--NH4-), the hydrolase activities in the media of ICD fibroblasts was from 3 (P-N-acetylglucosaminidase) to 5 (a-mannosidase) times as high as that of control fibroblasts. Changes of lysosomal hydrolase activities in ICD fibroblast (No. 224) after removal of sucrose from medium. On day 0, equal numbers of cell were subcultured with or without 88 mM sucrose. After 12 days of incubation with (SUC') or without (SUC-), cells were trypsinized and subcultured with 1:5 split ratio with conventional medium (SUC-). Intracellular lysosomal hydrolase activities were determined on the day of subculture and once every 3 days thereafter. 0, ICD, SUC' + SUC-; 0, ICD, SUC-+ SUC-. Enzyme activity is expressed as nanomoles per mg of cell protein per h. Abbreviations as in Fig. 1.  (Fig. 5b).

DISCUSSION
The present data demonstrate that the intracellular hydrolases were induced more actively in ICD fibroblasts by 88 mM sucrose loading for 13 days than in normal fibroblasts. In ICD fibroblasts, most hydrolase activities reached their normal ranges (Fig. 1). Besides a-galactosidase and arylsulfatase A also reached their normal ranges (data not shown). Probable subculture shock and reversibility of the induction were found in both normal and ICD strains (Figs. 2 and 3). The degree of induction was different between normal and ICD strains. In low concentration of sucrose (18 m) normal cells reached nearly the maximal induction, whereas ICD cells seemed to respond to the higher concentration of sucrose (up to 88 m ) (Table 11). Extracellular hydrolase activities increased linearly in ICD with sucrose loading as in control (data not shown). It seems unlikely that ICD cells with sucrose loading internalize their own secreted hydrolases because it was recognized that a rate of fluid endocytosis was not induced by the addition of 80 m sucrose in cultured cells (15,23).
The characteristic inclusion disappeared after sucrose loading when cytoplasm of ICD fibroblasts was investigated by phase contrast (Fig. 6), transmission, and scanning electron microscopies. Subcellular fraction experiments of both ICD and normal fibroblasts revealed that hydrolase activities increased by sucrose loading were detected in latency positive particles, probably packed in intact lysosomes (data not shown). Morphological study on ICD fibroblasts with sucrose loading w i l l be reported elsewhere. At this time, the precise factor to trigger the hydrolase induction still remains unclear. As several saccharides other than sucrose were found to be poor enzyme inducer (Table  111), the change of the plasma membrane by the endocytosis of sucrose (15, 24) may mediate the induction. However, the response of intracellular hydrolase activities to the addition ( Fig. 2) or the removal (Fig. 3) of 88 m sucrose in the medium needed a rather long preparing period. Moreover, the frequent changes of medium with sucrose caused the retarded induction as indicated in Fig. 4 (in SUC'Change'). Thus, it is very likely that the important stimulating factor is not the extracellular sucrose in the medium but the intracellular sucrose or its metabolites. No biologically active factor seems to occur in the supplemented fetal bovine serum because the same results were obtained when heat-inactivated serum (70 "C, 30 min) was used instead of fresh one.
The changes of /3-glucuronidase were apparently different from those of other hydrolases such as B-galactosidase, /3-Nacetylglucosaminidase, a-mannosidase, etc. (Fig. 1 and Table  I). However, this does not indicate that /3-glucuronidase is an exception among lysosomal hydrolases, since the induction was detected after sucrose loading in ICD SUC' cells ( Fig. 1) and bigger extracellular hydrolase accumulation in SUC'NH,' normal cells (data not shown). Probably, the turnover rate of /3-glucuronidase in sucrose loading is higher than others. Warburton and Wynn reported that sucrose loading increased in the rate of degradation (25).
In the NKC1 loading experiment, the release of newly synthesized enzymes into extracellular space was measured as shown in Fig. 5. The amount of released hydrolase in serumfree medium in our experiment seems slightly less than that in albumin-supplemented medium used by most investigators (26,27). The degree of increase in enzymic release by NH4Cl was much bigger in SUC' of normal and ICD cells than in SUC-. Again from this experiment, the synthesis of enzyme in both ICD and normal fibroblasts seems to be stimulated by sucrose loading.
Recently, it has been found that ICD fibroblasts are deficient in UDP-N-acetylg1ucosamine:glycoprotein N-acetylglucosaminyl phosphotransferase activity and that a pathologically excessive amount of extracellular hydrolases is due to the defect of the signal of hydrolase to react with the transfer proteins (28, 29). For this reason, it appears reasonable that the extracellular hydrolase activities in ICD cells were not increased after NKCl loading (SUC-). However, if sucrose stimulates the production of only abnormal enzyme molecules, this theory can not explain the reason for the increased release of hydrolases from ICD cells in SUC'NH,' (Fig. 5b). This finding seems important. In ICD cells, there may be some residual function to synthesize normal lysosomal hydrolases which can be normally packed in lysosomes (30). Probably the sucrose loading also stimulates this function and produces more receptors and enzymes with normal structure, which could be well shown by the results of NKCl loading in Fig.  5b, because released hydrolases by NKCl loading were originally to be packed in lysosomes.
Accordingly, sucrose stimulates the production of lysosomal hydrolases and probably of receptor protein, and normalizes the intracellular lysosomes in ICD fibroblasts. Since sucrose normalizes both biochemical and morphological abnormalities in ICD cells, it is hoped that this approach w i l l give an idea to the treatment of ICD.