The Role of Oxidized Cholesterol in Diabetes-Induced Lysosomal Dysfunction in the Brain
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Abnormalities in lysosomal function have been reported in diabetes, aging, and age-related degenerative diseases. These lysosomal abnormalities are an early manifestation of neurodegenerative diseases and often precede the onset of clinical symptoms such as learning and memory deficits; however, the mechanism underlying lysosomal dysfunction is not known. In the current study, we investigated the mechanism underlying lysosomal dysfunction in the cortex and hippocampi, key structures involved in learning and memory, of a type 2 diabetes (T2D) mouse model, the leptin receptor deficient db/db mouse. We demonstrate for the first time that diabetes leads to destabilization of lysosomes as well as alterations in the protein expression, activity, and/or trafficking of two lysosomal enzymes, hexosaminidase A and cathepsin D, in the hippocampus of db/db mice. Pioglitazone, a thiazolidinedione (TZD) commonly used in the treatment of diabetes due to its ability to improve insulin sensitivity and reverse hyperglycemia, was ineffective in reversing the diabetes-induced changes on lysosomal enzymes. Our previous work revealed that pioglitazone does not reverse hypercholesterolemia; thus, we investigated whether cholesterol plays a role in diabetes-induced lysosomal changes. In vitro, cholesterol promoted the destabilization of lysosomes, suggesting that lysosomal-related changes associated with diabetes are due to elevated levels of cholesterol. Since lysosome dysfunction precedes neurodegeneration, cognitive deficits, and Alzheimer’s disease neuropathology, our results may provide a potential mechanism that links diabetes with complications of the central nervous system.
KeywordsType 2 diabetes Brain Lysosome Cathepsin D Cholesterol Central nervous system
Diabetes mellitus, which currently affects 25.8 million Americans, is a complex metabolic disorder characterized by hyperglycemia. Type 2 diabetes (T2D) accounts for approximately 90–95 % of all diabetes cases and is associated with obesity and hyperinsulinemia. Various complications are associated with diabetes, including retinopathy, nephropathy, neuropathy, and cardiovascular disease , and the impact of diabetes on the central nervous system is gaining attention. It is also believed by some that diabetes accelerates brain aging [2, 3]. Aging and age-related diseases may involve abnormalities in the endosomal-lysosomal system, which is an early manifestation of neurodegeneration . Furthermore, lysosomal dysfunction contributes to the accumulation of protein aggregates, a common occurrence in neurodegenerative disorders [5, 6].
Lysosomes are involved in numerous functions, including cell death, exocytosis, endocytosis/phagocytosis, and autophagy. Many of these functions are dependent upon the action of acid hydrolase enzymes within the lysosome that can degrade lipids, carbohydrates, proteins, nucleic acids, or cellular debris. Hexosaminidase A is a lysosomal enzyme that converts GM2 ganglioside to G3M by removing an N-acetyl-glucosamine residue, thereby playing a role in the regulation of insulin sensitivity [7, 8]. In addition, cathepsin D is one of the major lysosomal proteases that contributes to the conversion of proinsulin to insulin in Langerhans cells and rat hepatocytes , and it is also involved in the degradation of insulin [10, 11].
Thiazolidinediones (TZDs), a class of drugs known to improve insulin sensitivity, are commonly used for the treatment of diabetes. TZDs are ligands for peroxisome proliferator-activated receptors (PPAR-γ) and improve insulin sensitivity by lowering serum glucose and insulin levels, increasing peripheral glucose uptake, and decreasing triglyceride levels [12, 13]. In fact, studies have demonstrated the beneficial effects of TZDs in neurodegenerative diseases associated with the abnormal accumulation of protein aggregates [14, 15, 16, 17]. One such TZD is pioglitazone (Actos), which improves hyperglycemia, reduces hyperinsulinemia, and ameliorates hypertriglyceridemia in a variety of animal models of obesity and insulin resistance [18, 19, 20, 21, 22, 23]. Pioglitazone does not improve cholesterol levels in mice (personal communication, ).
Hypercholesterolemia is present in 70 % of adults diagnosed with diabetes . In T2D, serum cholesterol is elevated secondary to altered cholesterol synthesis and absorption [26, 27]. When in excess, cholesterol is oxidized by enzymatic or reactive oxygen species (ROS)-mediated pathways. Oxidized cholesterol is increased in T2D [28, 29] and disrupt cellular membranes , especially lysosomal membranes . Disrupted lysosomes are incapable of effectively removing ROS-damaged macromolecules . This leads to a feed-forward cycle of damage, where ROS promote the oxidation of cholesterol, resulting in lysosomal injury.
Alterations in lysosomal function in diabetes have been documented in the liver, kidney, heart, saliva, whole brain, and plasma [33, 34, 35, 36]. Our previous work in the hippocampi of a well-characterized mouse model of T2D, the db/db mouse, revealed differential expression in genes related to the lysosome . Previous studies have reported alterations in the central nervous system including cognitive impairment and evidence of neurodegeneration in the db/db mouse [38, 39, 40, 41, 42, 43, 44]. Therefore, in this study, we characterized lysosomal function in the cortex and hippocampus of the db/db mouse. To determine a potential mechanism underlying lysosomal dysfunction in diabetes, we treated the db/db mice with pioglitazone to ameliorate diabetes and performed subsequent in vitro studies to confirm.
Materials and Methods
Control db+ and T2D db/db mice (BKS.Cg-m +/+ Leprdb/J, JAX mice stock no. 000642) were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were fed a standard rodent chow from Lab Diet (#5053) ad libitum. For pioglitazone studies, db+ and db/db mice were fed a standard diet (5LOD; Research Diets, New Brunswick, NJ) supplemented with or without 112.5 mg of pioglitazone per kg of chow for a final dosage of 15 mg/kg to the mouse beginning at 5 weeks of age. To document the persistence of diabetes, fasting blood glucose levels were measured every 4 weeks by analyzing one drop of tail blood after a 6-h fast using a standard Glucometer (One Touch Ultra, Milpitas, CA). Mice were euthanized at either ∼8 or ∼20 weeks of age.
The mice were euthanized according to our published protocols with an overdose of sodium pentobarbital, and the tissue was processed as follows per our previously published protocols . For western immunoblotting analyses and enzyme activity assays, the hippocampus and cortex from the dissected brains were prepared as previously described by homogenizing the tissues in tissue protein extraction reagent (Pierce, Rockford, IL) containing a protease inhibitor cocktail (Calbiochem, San Diego, CA). For immunohistochemistry (IHC), mice were perfused with 30 ml of 2 % paraformaldehyde-lysine-periodate, the whole brains were removed and immersed in the same fixative overnight, and the brains were then cryoprotected in PBS (0.1 M, pH 7.2) with 30 % sucrose prior to embedding in OCT compound (Sakura Finetek, Torrance, CA). The brains were then sectioned (20 μm) using a CM1850 cryostat (Leica Microsystems Inc., Bannockburn, IL), mounted onto SuperFrost glass slides (Fisher Scientific, Pittsburgh, PA), and stored at −20 °C until use. For cell fractionation and flow cytometry, the mice were perfused with 15-ml PBS, and the cortex and hippocampus were removed and immediately processed as described below.
Western immunoblotting was performed as previously described [45, 46]. Briefly, the tissue lysates were either separated by SDS-PAGE and transferred to a nitrocellulose membrane or used to determine enzyme activity as described below. Tris-buffered saline with Tween-20 supplemented with 5 % milk was used to block the membrane and to dilute the antibodies. Polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), β-hexosaminidase (ProteinTech Group, Inc., Chicago, IL), and actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), as well as appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.), were used for western immunoblotting. The signal was visualized using LumiGLO-enhanced chemiluminescence reagent (Cell Signaling Technology, Danvers, MA). Images were captured using the Chemidoc XRS system and analyzed by Quantity One software (Bio-Rad Laboratory, Hercules, CA).
The activity of β-hexosaminidase A and cathepsin D was measured in 4–5 μg of protein cell lysate from the cortex and hippocampus at 8 and 20 weeks of age (n ≥ 5 for each group) in a 96-well plate. To measure β-hexosaminidase A activity, 3.2 mM 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide (MUGS) potassium salt (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the cell lysate and incubated for 1 h at 37 °C. Next, 2-amino-2-methyl-1-propanol (0.1 M) was added, and fluorescence was read immediately with a 355-nm excitation filter and 460-nm emission filter using a Fluoroskan Ascent II plate reader (LabSystems, Helsinki). Cathepsin D activity was assessed using the cathepsin D activity assay kit (BioVision, Mountain View, CA) according to the manufacturer’s instructions. Fluorescence was read with a 320-nm excitation filter and 460-nm emission filter.
Dissected cortex and hippocampus were homogenized in homogenization medium (HM: 0.32 M sucrose, 1 mM Na2EDTA, 10 mM HEPES; pH 7.0) and centrifuged at 800g for 10 min at 4 °C. The supernatant was kept on ice, and the pellet was resuspended in HM and centrifuged again. The supernatants from both centrifugation steps were combined and centrifuged at 20,000g for 15 min at 4 °C. The supernatant was centrifuged at 300,000g for 2 h at 4 °C to obtain the cytosol, and the pellet was resuspended in HM and layered over a 27 % Percoll solution (Sigma) diluted with Percoll diluent (2.5 M sucrose, 10 mM Na2EDTA, 100 mM HEPES; pH 7.0). The sample was centrifuged for 95 min at 20,000g. Lysosomes were collected from the layer near the bottom of the gradient and centrifuged for 50 min at 100,000g.
Measurement of Intralysosomal pH
The measurement of intralysosomal pH was performed using flow cytometry with minced cortex and hippocampus (separately) that were trypsinized at 37 °C for 6 min. The tissue was triturated with Leibovitz (L15) media, filtered (70 μm), and centrifuged at 155g for 5 min at 4 °C. The pellet was resuspended in PBS. A standard curve was generated in LysoSensor Yellow/Blue DND-160 (2 mM; Invitrogen Molecular Probes) dye-loaded cell suspension using a series of phosphate-citrate buffers containing various mixtures of 300 mM KH2PO4 and 300 mM citric acid ranging in pH 2–6, supplemented with the inophores nigericin and monensin (Sigma Aldrich; 10 μmol/L) to facilitate the equilibration of intralysosomal pH with the buffer. The cell suspension was allowed to equilibrate for 10 min. The standard curve was generated by exciting at 355 nm and plotting the emission fluorescence ratio (550/21 nm) of DND-160-loaded cells as a function of the actual pH, which was assessed on a standard pH meter. The intralysosomal pH of the lysosomes in the cortex or hippocampus from db+ and db/db mice was calculated by extrapolation from the standard curve.
Brain sections were heated on a 55 °C slide warmer for 10 min, hydrated in PBS for 5 min, and permeabilized with PBS containing 0.3 % Triton X-100 and 3 % milk. Sections were incubated in primary antibodies diluted in PBS containing 0.3 % Triton X-100 and 1 % BSA in a humidified chamber overnight at 22 °C. A polyclonal antibody against lysosomal associated membrane protein-1 (LAMP-1; Abcam, Cambridge, MA) was used for IHC. After rinsing with PBS, sections were incubated with the appropriate secondary antibody conjugated with AlexaFluor 594 (Molecular Probes, Eugene, OR) for 1 h at room temperature. Following three rinses with PBS, the sections were incubated for 3 h at room temperature in the dark with 10 μg/ml filipin complex (Sigma, St. Louis, MO). After rinsing with PBS, coverslips were mounted with Prolong anti-fade mounting medium (Molecular Probes, Eugene, OR). Images were captured using a Spot-RT camera (Diagnostic Instruments Inc., Sterling Heights, MI) attached to a Nikon Microphot-FXA microscope.
Primary Cortical Neuron Experiments
Primary cortical neurons (CN) were prepared as previously described . Briefly, the cortex from E13 B6C3F1/J mice were dissected, dissociated with trypsin, and plated on poly-L-lysine (PLL)-coated tissue culture plates or coverslips. CN were maintained in neurobasal media (Invitrogen, Grand Island, NY) containing 5 mM glucose, supplemented with 1× B27 (without antioxidant; Invitrogen), antibiotics (penicillin, streptomycin, and neomycin; Sigma), 2.5 μg/ml albumin, 10 μg/ml apo-transferrin, 0.1 μg/ml biotin, 15 μg/ml D-galactose, 7 ng/ml progesterone, 16 μg/ml putrescine, 4 ng/ml selenium, 3 ng/ml β-estradiol, 4 ng/ml hydrocortisone, 3 μg/ml catalase, and 2.5 μg/ml superoxide dismutase. CN were cultured for 6 days prior to use, with an addition of fresh media on day 3. CN treatment media (neurobasal media without B27 and antibiotics) was used to carry out the experiments indicated below.
In vitro experiments involved treating primary CN with 25 mM of glucose  with or without 5 μM oxidized cholesterol (27-hydroxycholesterol; Medical Isotopes, Inc., Pelham, NH; Prasanthi et al. 2009) for 72 h. Lysosomal destabilization was measured using acridine orange (AO), a lysosomotropic weak base, and metachromatic fluorochrome. Briefly, 5 μg/ml AO in neurobasal medium was incubated with CN on coverslips for 15 min at 37 °C. After the cells were washed and treated as outlined above, microscopic assessment of lysosomes was performed using an Olympus IC-71 inverted confocal microscope and FluoView v4.3 software. Quantitative analysis was performed after staining and treatment of CN in a 96-well clear bottom/black walled plate using a Fluoroskan Ascent FL instrument. When excited by a blue light (450 nm), AO fluoresces red (612-nm wavelength emission) at high lysosomal concentrations and green (520-nm wavelength emission) at low nuclear and cytosolic concentrations. Rupture of AO-loaded lysosomes shows an increase in cytoplasmic diffuse green fluorescence and a decrease in granular red fluorescence [48, 49].
Data analyses were performed using Prism v6 (GraphPad Software, Inc.). Assumptions about Gaussian distribution of data were made using the D’Agostino and Pearson omnibus normality test. Data not corresponding to a Gaussian distribution were analyzed using an appropriate mathematical transformation, log (x). At least 10 mice per group at 8 and 20 weeks of age were used for metabolic studies. For all other measures, at least 5 mice per group were used. t test was used in experiments where T2D was compared to the nondiabetic control. For all experiments, *p < 0.05, **p < 0.01, #p < 0.001, and Φp < 0.0001 and bar graphs illustrate the mean ± standard error of the mean (SEM).
Cortical and Hippocampal Changes in Hexosaminidase A Protein Expression and Activity in T2D
Cortical and Hippocampal Changes in Cathepsin D Protein Expression and Activity in T2D
The processing of cathepsin D in the Golgi, endosomes, and lysosomes correspond to the 3 major forms—immature, intermediate, and mature, respectively. Alterations in the activity of cathepsin D are associated with the mature form of the protein. Since the protein expression of cathepsin D is significantly increased in the cortex of T2D but the activity is decreased, we assessed the 3 major forms of cathepsin D in the cortex to evaluate whether or not T2D alters the trafficking pattern of cathepsin D. We observed a significant increase in both the intermediate and mature forms of cathepsin D in T2D compared to the nondiabetic control (Fig. 2e, f).
Lysosomal Membrane Integrity, But Not pH, Is Compromised in T2D
Elevated Levels of Glucose Can Cause Destabilization of the Lysosomal Membrane
Pioglitazone Improves the Hyperglycemic Phenotype But Not Alterations in Lysosomal Membrane Integrity Associated with T2D
Elevated Levels of Cholesterol Within Lysosomes in T2D
Cholesterol Can Cause Destabilization of the Lysosomal Membrane
In the current study, we investigated the mechanisms underlying alterations in lysosomal function induced by diabetes. Hyperglycemia is a key player in many complications associated with diabetes. Thus, according to the American Diabetes Association, tight glycemic control with either drugs or diet and exercise is the most effective method for preventing diabetic complications. We show for the first time that cholesterol, and not hyperglycemia, may mediate the changes observed in lysosomal function during diabetes in the hippocampus/cortex. Abnormalities in the lysosomal system are early manifestations of neurodegeneration ; thus, altered cholesterol metabolism may play a role in diabetes-induced lysosomal changes and neurodegeneration.
The expression of both hexosaminidase A and cathepsin D is upregulated in neurodegenerative diseases [50, 51, 52, 53]. Hexosaminidase A removes N-acetyl-glucosamine residue from GM2 ganglioside, converting it to GM3. Obesity leads to a dramatic increase in the protein levels of GM2 in adipose tissue in a mouse model of T2D . Hence, the increase in protein expression observed in the hippocampus of the db/db mouse may be due to obesity. Consistent with our observed increase in hexosaminidase A activity in the db/db mouse hippocampus, T2D patients have increased activity of hexosaminidase A in plasma and serum . Thus, increased activity of hexosaminidase A in plasma and peripheral blood mononuclear cells may have diagnostic value for the detection of the early stages of dementia in Alzheimer’s disease (AD) patients with and without T2D .
Cathepsin D is the major lysosomal protease in neurons, contributing to nearly 90 % of protease degradation in the brain . The increase in protein expression we observed in both the cortex and hippocampus is consistent with previous studies in T2D patients reporting that the expression of cathepsin D is increased in serum and leucocytes [56, 9]. On the other hand, the decrease in the activity of cathepsin D we observed is unique to our studies in T2D and may be indicative of abnormalities in the trafficking of cathepsin D to lysosomes. The trafficking of cathepsin D was assessed by evaluating the 3 different isoforms—immature, intermediate, and mature—which correspond to trafficking from the Golgi, endosomes, and lysosomes, respectively . The acidic environment of lysosomes provides a platform for proteolytic cleavage of the intermediate form of cathepsin D to the mature form . Thus, any alterations in the pH of lysosomes may impact the levels of the mature form of the enzyme. Our data indicate that the pH within lysosomes is not altered during T2D. Alternatively, the elevated levels of cathepsin D in the cytosolic fraction in db/db mice suggests that the lysosomal membrane may be compromised. This would explain the elevated protein levels of cathepsin D and the decrease in the activity due to lack of acidic pH in the cytosol. The mechanism underlying this damage to the membrane of lysosomes is not known; however, oxidative stress is associated with leakage of cathepsin D into the cytosol from the lysosome in both in vivo and in vitro models [58, 59].
Hyperglycemia is the key player leading to oxidative stress during T2D. To investigate hyperglycemia as the mechanism underlying lysosomal membrane damage, we exposed CN to hyperglycemic conditions, demonstrating that hyperglycemia causes destabilization of the lysosomal membrane. A previous study demonstrated that hyperglycemia inhibited lysosomal function in macrophages and may contribute to diabetes-associated atherosclerosis . Thus, reversing hyperglycemia may have a profound impact on lysosomal membrane stability.
Pioglitazone is known to improve hyperglycemia, reduce hyperinsulinemia, and ameliorate hypertriglyceridemia in a variety of animal models of obesity and insulin resistance [22, 21, 20, 19, 18]; however, pioglitazone did not reverse the alterations in lysosomal enzymes and in fact lead to a further increase in the levels of cathepsin D in the cytosol. We previously demonstrated that pioglitazone does not improve hypercholesterolemia in db/db mice . In addition, in patients with T2D, serum cholesterol is elevated secondary to altered cholesterol synthesis and absorption [27, 26]. When in excess, cholesterol is oxidized by enzymatic or reactive oxygen species-mediated pathways to generate cholesterol oxides. Cholesterol oxide derivatives, known as oxysterols, are common components of oxidized LDLs, are increased in T2D [29, 28], and disrupt lysosomal membranes . We demonstrate that oxysterols are capable of disrupting lysosomal membranes in primary CN. Thus, it is possible that pioglitazone did not improve the T2D-induced effects on lysosomes due to its inability to reverse hypercholesterolemia in db/db mice.
In summary, it is likely that multiple mechanisms contribute to the alterations in lysosomal enzymes during diabetes. We demonstrate that improving hyperglycemia, insulin resistance, and hypertriglyceridemia alone is not sufficient to reverse diabetes-induced changes in lysosomal membrane stability, although our studies further demonstrate that hypercholesterolemia plays a role in lysosomal membrane destabilization. Future studies will focus on cholesterol as a potential therapeutic target to reverse or prevent the impact of T2D on lysosomal enzymes.
The authors wish to acknowledge Drs. Stacey A. Sakowski and Anne Heacock for expert editorial advice, Carey Backus and Janet Boggs for technical assistance, and the University of Michigan Flow Cytometry Core.
This work was supported by the National Institutes of Health (NIA Training Grant T32 AG000114, to C. S-R.; NINDS Neurology Training Grant T32 NS007222, to C. S-R.; and the Animal Models of Diabetic Complications Consortium NIH U01-DK076160), the A. Alfred Taubman Medical Research Institute, and the Program for Neurology Research and Discovery. This work also utilized the Chemistry Core of the Michigan Diabetes Research and Training Center (funded by NIDDK DK020572).
Conflict of Interest
The authors declare that they have no conflict of interest.
Compliance with Ethical Standards
All protocols and procedures were approved by the University of Michigan Committee on the Use and Care of Animals (UCUCA; approval number PRO00003694), and are in compliance with the University guidelines, State and Federal regulations, and the standards of the “Guide for the Care and Use of Laboratory Animals.” The University’s Animal Welfare Assurance Number on file with NIH Office of Laboratory Animal Welfare (OLAW) is A3114-01, and the University is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC, Intl.).
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