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Endocrinology Vol. 143, No. 9 3449-3453
Copyright © 2002 by The Endocrine Society

ARTICLE

Low Density Lipoprotein Can Cause Death of Islet ß-Cells by Its Cellular Uptake and Oxidative Modification

Miriam Cnop, Jean Claude Hannaert, Annick Y. Grupping and Daniel G. Pipeleers

Diabetes Research Center, Brussels Free University—Vrije Universiteit Brussel, Brussels 1090, Belgium

Address all correspondence and requests for reprints to: D. G. Pipeleers, Diabetes Research Center, Brussels Free University—Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium. E-mail: dpip{at}mebo.vub.ac.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Islet ß-cells express receptors for low density (LDL) and very low density (VLDL) lipoproteins that are internalized by receptor-mediated endocytosis. The present study examined whether this process can affect the viability of isolated rat islet ß-cells. Culture with LDL (from 6 µg/ml on), but not VLDL, causes necrosis of ß-cells within 2 d. No toxicity was observed when LDL binding and/or endocytosis was prevented by low temperature (8 C), or by addition of heparin or an excess of VLDL. The LDL toxicity did not occur in the presence of antioxidants (probucol or a mixture of glutathion, vitamins A, C, E plus dithiothreitol) or of the radical scavenger butylated hydroxytoluene. The degree of LDL-induced toxicity was correlated with an increase in the electrophoretic mobility of LDL, an index for its oxidative modification. Both LDL toxicity and oxidation were suppressed by omission or chelation of copper and iron in the medium. Addition of oxidized LDL was not cytotoxic to ß-cells, which lack oxidized LDL receptors. It is concluded that uptake of LDL by islet ß-cells and subsequent oxidative reactions can be damaging for the cells. This process can be counteracted by HDL and VLDL, and by antioxidants.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A RISE IN fat consumption is an established risk factor for type 2 diabetes (1, 2). Its pathogenic role is probably multifactorial, affecting peripheral metabolism as well as ß-cell functions.

Elevated fatty acid levels can reduce peripheral sensitivity to insulin (3, 4, 5), as well as the ß-cell capacity to compensate (6). Studies in diabetes-prone rodents have suggested that prolonged elevation of fatty acids can cause ß-cell dysfunction and death (7, 8).

Elevated low density lipoprotein (LDL) and very low density lipoprotein (VLDL) levels are known to modify peripheral metabolism but it is not yet known whether they can exert deteriorations in ß-cell functions. We have previously shown that both rat and human ß-cells express high affinity receptors for LDL and VLDL, which can internalize both lipoproteins (9). These receptors and associated endocytosis were not observed in islet {alpha}-cells (9). In human ß-cells, the process of LDL and VLDL uptake may contribute to the intracellular lipid accumulation, which is found to occur in the aging ß-cell population (10). It is not yet known whether prolonged exposure to LDL or VLDL can influence the functional ß-cell mass. In the present study, we have examined whether these lipoproteins can affect the survival of rat ß-cells. Previous studies in other cell types have indeed shown that LDL can exert cytotoxic effects through formation of oxidized lipids (11).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of lipoproteins
Human lipoproteins were obtained from serum of healthy volunteers, after an overnight fast. The VLDL, LDL, and high density lipoprotein (HDL) fractions were isolated by ultracentrifugation, as previously described (9, 12). The lipoproteins were dialyzed overnight against PBS containing 1 mM ethylenediamine tetraacetic acid and stored at 4 C.

For copper-oxidation of LDL, the native lipoprotein was extensively dialyzed against PBS containing 10 µM EDTA and incubated with 5 µM Cu2+ for 20 h at 37 C (13). Oxidation was stopped at 4 C by increasing the EDTA concentration to 200 µM, followed by centrifugation in a Centricon 100 concentrator (Amicon Inc., Beverly, MA).

The lipoprotein fractions were filtered through a 22-µm filter (Millipore Corp.) before use. Their protein concentration was determined by the Pierce Chemical Co. (Rockford, IL) BCA kit using BSA as a standard.

Isolation and culture of rat ß-cells
Adult male Wistar rats were housed according to the guidelines of the Belgian Regulations for Animal Care. The protocol was approved by the Ethical Committee for Animal Experiments of the Brussels Free University. Rats were sedated and killed with CO2, followed by decapitation. Pancreatic islets were isolated by collagenase digestion and dissociated into single cells in calcium-free medium containing trypsin and deoxyribonuclease (14). Single ß-cells (more than 90% pure) were purified by autofluorescence-activated sorting using cellular light-scatter and flavin adenine dinucleotide-autofluorescence as discriminating parameters (14).

For viability testing, cells were cultured in polylysine-coated microtiter plates in Ham’s F10 medium containing 10 mmol/liter glucose, 1% BSA pretreated with charcoal (fraction V, RIA grade, Sigma, St. Louis, MO), 2 mmol/liter L-glutamine, 50 µmol/liter 3-isobutyl-1-methylxanthine, 0.075 mg/ml penicillin, and 0.1 mg/ml streptomycin (15, 16). Lipoprotein fractions were added to the culture medium in absence or presence of the following compounds: a mixture of retinoic acid (13-cis retinoic acid, Sigma), dithiothreitol (dl-dithiothreitol, Sigma), vitamin E acetate ({alpha} tocopherol acetate, Sigma), glutathion (glutathion reduced form, Sigma) and vitamin C (L-ascorbic acid, Sigma) (17, 18), butylated hydroxytoluene (BHT, 2, (6)-Di-tert-butyl-p-cresol, Sigma) (19, 20), probucol (Sigma) (21), selenium (sodium selenite, Sigma) (22), Desferal (desferoxamine mesylate, Sigma) or heparin (170 U/mg sodium salt, Sigma) (13). After 1–8 d of culture, the percent of living cells was counted after staining with neutral red (16). The mode of cell death was determined by a fluorescent assay using Hoechst 33342 and propidium iodide DNA binding dyes (15). Hoechst 33342 readily enters intact and damaged cells and stains DNA blue, whereas propidium iodide, a highly polar dye, only penetrates cells with damaged membranes and stains DNA orange. After 5-min incubation with these dyes, viable or necrotic ß-cells were identified by an intact nucleus, stained blue or orange, respectively, while apoptotic cells exhibited a fragmented nucleus, stained either blue or orange, depending on the stage of the apoptotic process (15). Percentages of living, necrotic and apoptotic cells were counted after 2–8 d of culture with LDL.

Samples for electron microscopy were fixed in cacodylate-buffered glutaraldehyde (4.5%, pH 7.3), postfixed in osmium tetroxide (1%), and embedded in Spurr’s resin. Ultrathin sections were stained with uranylacetate and lead citrate and examined for features of necrosis or apoptosis in a Carl Zeiss EM 109 electron microscope (Oberkochen, Germany).

Determination of nitrite formation and induction of NO synthase (iNOS) expression
Single ß-cells were cultured in multiwell plates (100,000 cells per condition) with or without LDL. After 1 d, medium was collected for nitrite determination (23, 24) and cells were harvested for RT-PCR analysis of iNOS and glyceraldehyde 3-phosphate dehydrogenase mRNA expression (25). As a positive control for iNOS expression and NO production, ß-cells were exposed in parallel to the cytokine IL-1ß (30 U/ml).

Determination of LDL electrophoretic mobility
Electrophoresis of LDL was carried out in a 0.75% agarose gel in 0.08 M Tris hippuric acid buffer at pH 8.8 (26). After capillary blotting of the gel onto Protran nitrocellulose membrane (Schleicher \|[amp ]\| Schuell, Dassel, Germany), LDL was visualized by Protogold (BioCell Research Laboratories, Cardiff, UK) total protein staining to determine the relative electrophoretic mobility of the LDL particle. Both native lipoproteins and LDL obtained after a 48-h incubation in culture medium were examined.

Data analysis
Results are presented as means ± SEM. Single comparisons were performed by Student’s paired t test. For multiple comparisons, data were analyzed by ANOVA, followed by group comparisons using Student’s paired or unpaired t test, as indicated, with correction of the P values for multiple comparisons by the Bonferroni method (27).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cytotoxicity of LDL
The LDL fraction was toxic for cultured rat ß-cells from a concentration of 6 µg/ml on (Table 1Go). Its cytotoxic effect was dose dependent, causing 70% cell death at 25 µg/ml. It was detected after 2 d of exposure—sometimes within 16 h (data not shown)—with little further increases during the following 6 d (Table 1Go).


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  		Table 1. Effect of lipoproteins on viability of cultured rat ß-cells

 
Using a Hoechst 33342-propidium iodide fluorescence staining, LDL-induced ß-cell death was identified as necrosis; no apoptosis was detected under these conditions (Table 2Go). The mode of LDL-induced cell death was not different during culture at 6 mM glucose (data not shown). In electron microscopy, dead ß-cells exhibited picnotic nuclei and disrupted organelles and membranes, which is characteristic for necrosis.


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  		Table 2. LDL-induced apoptosis and necrosis in rat ß-cells

 
No toxicity was measured when ß-cells were exposed to LDL at 8 C, a condition in which the lipoprotein is not internalized (Table 3Go). Heparin, which is known to inhibit LDL internalization at 37 C, also prevented LDL toxicity (Table 3Go).


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  		Table 3. Effect of low temperature and heparin on LDL-induced ß-cell death

 
Culture of ß-cells in the presence of comparable concentrations of VLDL or HDL did not cause cell death (Table 1Go). When these lipoproteins were added to medium containing LDL (3–25 µg/ml), a protective effect was noticed. The toxicity of LDL 25 µg/ml was completely prevented by a high concentration of VLDL. In the presence of 250 µg/ml VLDL, LDL (25 µg/ml) did not induce ß-cell death after 48 h (8 ± 2% cell death; n = 5; P < 0.01 vs. LDL alone).

Addition of a low concentration of HDL (10 µg/ml) partially prevented LDL-induced ß-cell damage (P < 0.01; Table 4Go), but this protective effect was lost after heating HDL to 56 C (data not shown), which suggests that it involves protein interactions.


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  		Table 4. Effect of antioxidants, chelating agents and HDL on LDL-induced ß-cell death

 
Role of oxidative reactions
No LDL toxicity occurred in culture media without copper, iron, and zinc ions (P < 0.01; Table 4Go), or in media containing the iron scavenger desferoxamine (200 µM) (P < 0.01; Table 4Go), also an inhibitor of hydroxyl radical formation, or EDTA, known for its high association constant for Fe2+, Cu2+, and Zn2+ (P < 0.01; Table 4Go) (20). Protection was conferred by compounds that counteract oxidative reactions, such as 1) a mixture of antioxidants, including vitamin A (1 µM), vitamin C (57 µM), vitamin E acetate (0.5 µM), dithiothreitol (10 µM) and glutathion (1 µM) (P < 0.01; Table 4Go); 2) the lipid-soluble free radical scavenger butylated hydroxytoluene (10 µM) (P < 0.01; Table 4Go); 3) probucol (5 µM), a hypocholesterolemic drug with strong antioxidant properties (P < 0.01; Table 4Go); and 4) selenium (10 ng/ml) (P < 0.01; Table 4Go).

To assess whether the LDL toxicity is conferred by its oxidative end product (LDLox), we tested the effect of LDLox on ß-cell survival. This oxidative form was prepared by incubating the LDL fraction for 24 h with 5 µM Cu2+ at 37 C. When ß-cells were now exposed to LDLox, no cytotoxic effect was noticed (Table 1Go).

Role of nitric oxide
LDL-induced ß-cell necrosis was not attributable to an excessive NO production. After 24-h exposure to LDL (25–200 µg/ml), medium nitrite levels did not raise above the background levels as measured in the control condition (P > 0.05; n=3). On the other hand, addition of the cytokine IL-1ß (30 U/ml) induced a high nitrite production (control, 0.2 ± 0.2 pmol nitrite/103 ß-cells/24 h; IL-1 ß, 44.4 ± 2.4 pmol nitrite/103 ß-cells/24 h; P < 0.005; n = 3). Similarly, no induction of iNOS expression was detected after a 24 h exposure to LDL (25 or 200 µg/ml), whereas a clear induction occurred after IL-1ß treatment for 24 h (data not shown; n = 3).

Alterations in LDL particle
The relative electrophoretic mobility (Rf) of LDL on agarose gels was used to detect alterations in this lipoprotein as induced by the experimental conditions. Standards were the LDL fraction after isolation, with an Rf of 0.26, and after oxidative modification by copper treatment, with an Rf of 0.58 (Table 5Go). Culture for 2 d also increased the Rf of LDL but this effect represented only 21% of the copper effect (Table 5Go). This increase was less pronounced in conditions that prevent or counteract oxidative reactions (only a 4–8% increase in Rf; Table 5Go).


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  		Table 5. Oxidative modification of LDL following different ß-cell culture conditions

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that prolonged exposure of isolated rat ß-cells to LDL can induce their necrosis. Toxic concentrations are in the physiologic range and induce cell death within 1–2 d. This effect was dependent on cellular binding and internalization. Islet ß-cells exhibit LDL receptors that mediate endocytosis of the lipoprotein (9, 10). Up-regulation of LDL receptors occurs during the first days of serum-free culture which is expected to enhance the susceptibility of ß-cells to LDL toxicity; down-regulation may occur during prolonged culture and thus explain fading of LDL-induced cell death. No cytotoxicity was seen in the presence of heparin or of an excess of VLDL, two conditions that are known to inhibit LDL binding to its receptor (9), or at 8 C, when receptor binding occurs but not its internalization.

The LDL-induced necrosis of ß-cells is not attributable to iNOS and production of toxic NO levels as in the case of IL 1ß-induced necrosis (28). Instead, LDL toxicity appears to be attributable to an oxidative process with production of free radicals. It was indeed absent in media wherein the potent oxidation catalyzers copper, iron, and zinc were omitted or chelated, and was suppressed by addition of antioxidants or free radical scavengers. Protection by the various antioxidants might result from effects at, or within, the LDL particle. BHT and probucol, a drug with two BHT groups (29), are lipophilic compounds that bind to the lipoprotein core. Vitamin C is known to prevent initiation of lipid peroxidation in LDL by sparing endogenous antioxidants and by inhibiting copper binding to the apo B protein (18, 30). Both reduced glutathion and selenium are involved in the inactivation of lipid hydroperoxides by intracellular selenoperoxidases (31). That these conditions indeed reduce the oxidative state of LDL is supported by analysis of its electrophoretic mobility on agarose gels. The relative mobility of LDL was increased after its oxidation to LDLox by copper treatment. An increase was also seen in the extracellular LDL fraction after culture, suggesting its oxidation in the culture medium, be it to a much lower extent (only 20% of the increase seen with LDLox). This oxidation-related increase in electrophoretic mobility was counteracted by the antioxidant compounds that were found to be cytoprotective in the present experiments, suggesting that a similar phenomenon takes place at the ß-cell level. Direct proof for the latter mechanism might come from TBARS analysis of intracellular LDL, a study that could not be performed with the presently available cell numbers.

It is unlikely that the LDL toxicity is mediated by LDLox particles that are formed in the culture medium. We have previously found that rat ß-cells do not exhibit LDLox receptors (9) so that this particle is an unlikely component in the LDL cytotoxic process that depends on receptor-mediated endocytosis. More direct evidence against this possibility comes from the observation that no ß-cell death occurred when the cells were exposed to LDLox that was produced by prior copper treatment.

Because LDL toxicity is correlated to cellular uptake of the lipoprotein, it is conceivable that the LDL-associated oxidative reactions occur intracellularly, and likewise the protective actions of antioxidative conditions. Intracellular oxidation of the LDL lipid moiety is expected to form reactive peroxides of cholesterol and fatty acids and to propagate complex radical reactions (11) with the generation of aldehydic products of decomposition (32). Pancreatic ß-cells are considered to be particularly susceptible to reactive oxygen species and radical lipid hydroperoxides as they exhibit a relatively low content in scavenging enzymes, such as catalase, superoxide dismutase and glutathion selenoperoxidases (33, 34). This could explain why necrosis occurs in isolated ß-cells that are exposed to physiologic LDL concentrations while this is not the case in other cell types (35, 36, 37, 38, 39). Overexpression of catalase, Cu/Zn superoxide dismutase and glutathione peroxidase has been shown to reduce the susceptibility of insulin-producing cells to agents that cause their necrosis (40).

There is not yet evidence that the presently observed LDL toxicity for isolated ß-cells can be responsible for ß-cell death in vivo. Little is known about LDL receptor number and occupancy of ß-cells in situ, nor on LDL levels in the islet interstitium. In more general terms, LDL is thought to penetrate in the extravascular space where it might shift more easily to a more oxidative state. The extent of such oxidative process will probably vary with the lipoprotein’s content in antioxidants (41), in lipid hydroperoxides and in polyunsaturated fatty acids (42). Any in vivo effect will also depend on the presence of protective factors in their microenvironment. Plasma contains a variety of antioxidant compounds, some of which may be active in the islet interstitium. Furthermore, as shown by our data, the presence of HDL and VLDL can interfere with a deleterious effect of LDL. The protective effect of VLDL can be explained by its competition for the LDL receptor (9), whereas HDL is known to enzymatically inactivate reactive fatty acyl species that are generated during LDL oxidation (43, 44). Finally, as for other causes of ß-cell death, the functional state of the ß-cells should be considered as an active participant in the ultimate effects that LDL might induce on their viability (45). ß-Cells can play this role at different levels, from the process of LDL-binding to membrane receptors to the intracellular susceptibility to oxidative compounds (34). Further in vitro studies are needed to identify key steps in the cellular handling of LDL.

We have previously shown that rat and human ß-cells exhibit similar LDL binding and uptake (9), and that, in human ß-cells, the process of LDL and VLDL uptake may contribute to the intracellular lipid accumulation, which is found to occur in the aging ß-cell population (10). Experiments are now undertaken to investigate the effects of lipoproteins on the viability of human ß-cells, and to examine whether LDL uptake in lipid-storing vesicles represents a defense or a threat for aging human ß-cells.


   Acknowledgments
 
We thank the staff of the Diabetes Research Center for preparing rat islet cells and Geert Stangé and Ruth Leeman for excellent technical assistance. We are grateful to Dr. A. Hoorens for help with the Hoechst 342-PI assay and to Dr. D. L. Eizirik for advice on the iNOS pathway.


   Footnotes
 
This study was supported by grants from the Juvenile Diabetes Foundation International (JDF 995004), the Belgian Fonds voor Wetenschappelijk Onderzoek (F.W.O.0376.97), the services of the Prime Minister (Interuniversity Attraction Pole P4/21). M.C. was Aspirant of the Fund for Scientific Research-Flanders (F.W.O.).

Abbreviations: BHT, Butylated hydroxytoluene; HDL, high density lipoprotein; iNOS, inducible NO synthase; LDL, low density lipoprotein; LDLox, oxidized LDL; Rf, electrophoretic mobility; VLDL, very low density lipoprotein.

Received March 6, 2002.

Accepted for publication May 7, 2002.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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