Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Introduction

GLUT1 deficiency syndrome (GLUT1DS) (OMIM 606777) is an autosomal dominant genetic trait due to mutations in the glucose transporter SLC2A1 gene. GLUT1DS was first recognized in two children with seizures and persistently low glucose in the cerebrospinal fluid (CSF) (De Vivo et al. 1991; Seidner et al. 1998).

Since GLUT1 is the glucose transporter in the blood–brain barrier, the key hallmark is a low CSF glucose concentration in the presence of normoglycemia, with a CSF/blood glucose ratio commonly less than 0.4.

The clinical phenotype is variable; the most severe presentation is characterized by infantile epilepsy, developmental delay, cognitive impairment, spasticity, ataxia, and dystonia; some patients may present with paroxysmal head and eye movements, without epilepsy. A wide spectrum of mutations has been identified, but no obvious genotype–phenotype correlation has been detected (Pearson et al. 2013). The ketogenic diet is the standard of care, providing ketones in partial lieu of glucose as the alternative fuel for brain energy metabolism (Alter et al. 2015).

Recently, Yubero and colleagues reported a 15-year-old girl with GLUT1 deficiency and reduction of CoQ10 levels in muscle, plasma, and skin fibroblasts (Yubero et al. 2014).

CoQ supplementation in the patient leads to a striking improvement of neurological phenotype, suggesting that adjunctive treatment with CoQ10 may be wise when treating this disorder.

To determine if low levels of CoQ10 are a common biochemical finding in GLUT1 deficiency, we measured CoQ levels in fibroblasts, plasma, and white blood cells of several patients with GLUT1 deficiency and in affected and unaffected tissues of a model mice with GLUT1 haploinsufficiency (GLUT1+/−), as previously described (Wang et al. 2006).

Patients and Methods

Sixteen patients were diagnosed according to the following clinical and laboratory criteria: seizures, microcephaly, developmental delay, and low CSF/blood glucose ratio (~0.33 ± 0.01). Diagnosis was confirmed by molecular genetic analysis in 11. Informed consent was obtained from the parents of children who participated in this study. Plasma was collected from 14 patients; white blood cells were collected from eight patients, separated from plasma using Ficoll®-Paque, according to manufacturer instructions. Fibroblasts from five patients and six age-matched controls were cultured for CoQ10 assessment as previously described (Lopez et al. 2006). Briefly, cells were grown in 15-cm-diameter culture plates until confluent. CoQ10 was extracted in a hexane–ethanol mixture. The lipid component of the extract was separated by high-performance liquid chromatography (HPLC) on a reverse Symmetry® C18 3.5-μm, 4.6 × 150-mm column (Waters), using a mobile phase consisting of methanol, ethanol, 2-propanol, acetic acid (500:470:15:15), and 50 mM sodium acetate at a flow rate of 0.8 mL/min. The electrochemical detector consisted of an ESA Coulochem II with the following setting: guard cell (upstream of the injector) at +900 mV and conditioning cell at −650 mV (downstream of the column), followed by the analytical cell at +450 mV. CoQ10 concentration was estimated by comparison of the peak area with those of standard solutions of known concentration. Four GLUT1+/− heterozygous mice and four wild-type mice, aged 4 months, were euthanized; the brain, cerebellum, liver, kidney, muscle, and plasma were collected for biochemical analyses. CoQ9 and CoQ10 were extracted by mixing tissue extracts with 1-propanol, and measurement was performed as above. To measure activities of mitochondrial respiratory complexes, 40–70 mg tissue was homogenized in CPT medium (0.5 M Tris–HCl, 0.15 M KCl, pH 7.5) and centrifuged at 2,500g for 20 min at 4°C to obtain 10% homogenates. The supernatant was used for protein determination and enzymatic assays. Complex I and III (CI + III) activity was measured by observation of the reduction of cytochrome c (cyt c) at 550 nm. In brief, samples were incubated at 30°C in a medium containing 100 mM KH2PO4 (pH 7.5), 10 mM KCN, 2 mM NADH, and 1 mM cyt c. The increase of absorbance was observed for 1 min and for an additional 2 min after addition of rotenone (10 μg/mL). The residual activity in the presence of rotenone was subtracted from total activity. The results were expressed in nmol of reduced cyt c/min/g tissue. Complex II and III activity was measured by observation of the reduction of cytochrome c at 550 nm. Samples were incubated at 30°C in medium containing 100 mM KH2PO4 (pH 7.5), 30 mM succinate, 10 mM KCN, 2.5 μM rotenone KCN, and 1 mM cyt c, and increase of absorbance was observed for 2 min. The results were expressed in nmol of reduced cyt c/min/g tissue. Citrate synthase (CS) activity was measured following the reduction of 1 mM 5,5′-dithiobis(2-nitrobenzoic acid) in 1 M Tris–HCl (pH 7.5) at 412 nm (30°C) in the presence of sample, 10 mM acetyl-CoA, and 10 mM oxalacetic acid. The results of CI + III, CII + III, and CIV were normalized to CS activity and to protein.

To determine the level of oxidative damage in the cerebellum, we performed Western blot analysis of carbonyl group content using the OxyBlot kit (Chemicon, Millipore Corp, Billerica, MA, USA) following the manufacturer’s instructions. Briefly, 15 μg of proteins were incubated with 2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazine (DNP) derivates. 2,4-Dinitrophenyl-derivatized proteins were separated on a 12% polyacrylamide gel and transferred to polyvinyl prolidone membranes for 30 min at 50 V in Mini Trans-Blot cell apparatus (Bio-Rad, Hercules, CA, USA). The blots were hybridized with rabbit anti-DNP antibody and goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Chemicon, Millipore Corp, Billerica, MA, USA) and visualized by autoradiography using ECL substrate (Pierce, Rockford, IL, USA).

All animal studies were performed in accordance with protocols approved by the Columbia University Institutional Animal Care and Use Committee.

Mann–Whitney test was used to measure statistical significance (p < 0.05). Results are expressed as mean ± standard deviation.

Results

At the time of sample collection, clinically all patients (4 males and 12 females, mean age 15.4 ± 7.2 years) were symptomatic, and all but two were on a ketogenic diet (Table 1).

Table 1 Clinical features of GLUT1DS patients
Full size table

CoQ10 levels measured in plasma and white blood cells of seven patients showed no difference compared with controls (0.49 ± 0.06 vs. 0.41 ± 0.05 μg/mL; and 62.53 ± 20.97 vs. 68.05 ± 22.3 μg/mg prot, respectively) (Fig. 1a, b). We also assessed CoQ10 levels in cultured skin fibroblasts from an additional six patients with GLUT1 deficiency and found no differences compared to 6 age-matched controls (54.05 ± 11.9 vs. 46.6 ± 9.7 μg/mg prot) (Fig. 1c).

Fig. 1
figure 1

CoQ10 measured in (a) plasma of controls (ctrl; n = 9) and patients (GLUT1; n = 8), (b) white blood cells of controls (ctrl; n = 6) and patients (GLUT1; n = 6), and (c) skin fibroblasts of controls (ctrl; n = 6) and patients (GLUT1; n = 6). Data are expressed as mean; bars represent standard deviation

Full size image

CoQ10 deficiency has been shown to be tissue specific (Emmanuele et al. 2012). Thus, we measured CoQ levels in the brain, cerebellum, and skeletal muscle of GLUT1 mice. The liver and kidney were analyzed as non-affected tissues. CoQ9, the most common ubiquinone in rodents, as well as CoQ10 levels, was measured in wild-type and heterozygous animals. CoQ9 was normal in the brain (22.4 ± 2.6 vs. 22 ± 2.8 ng/mg tissue) (Fig. 2b), muscle (14.7 ± 4 vs. 17 ± 7 ng/mg tissue) (Fig. 2c), kidney (136.3 ± 3 vs. 136 ± 3 ng/mg tissue), and liver (85.7 ± 27.7 vs. 84 ± 26.5 ng/mg tissue). A very mild although statistically significant reduction of CoQ9 and CoQ10 was present in the cerebellum (24.45 ± 0.6 vs. 23.65 ± 0.3 ng/mg tissue; p < 0.05) (Fig. 2a).

Fig. 2
figure 2

CoQ9 (solid black) and CoQ10 (solid gray) in mutant (GLUT+/−; n = 4) and control (GLUT+/+; n = 4) mice: (a) cerebellum, (b) brain, (c) muscle, (d) plasma. Data are expressed as mean; error bars represent standard deviation. *p < 0.05

Full size image

We did not observe any difference between GLUT1+/− and wild-type animals when CoQ levels were normalized to CS, an index of mitochondrial mass (data not shown).

CoQ transports electrons from NADH dehydrogenase (complex I) and succinate dehydrogenase (complex II) to cytochrome c reductase (complex III). Therefore, to assess the functional impact of CoQ levels on the mitochondrial electron transport, we measured the activity of complexes I + III and II + III in the cerebellum, normalized to proteins and CS, and we did not find any difference between GLUT1+/− and wild-type animals (data not shown). Quantitative analysis of the blots carried out using the ImageJ software did not show any difference in protein oxidation between mutant and control mouse cerebellums (77.09 ± 19.05 vs. 68.22 ± 15.53).

Discussion

GLUT1DS is a rare neurological disorder with about 400 patients now reported in the literature worldwide (Pearson et al. 2013). The treatment with a ketogenic diet is based on the fact that brain metabolic fuels are limited to glucose and ketones. Recently, Yubero and colleagues reported a patient with GLUT1 deficiency, who showed consistently increased levels of blood lactate and alanine (Yubero et al. 2014). Mitochondrial function was investigated, and CoQ10 was reduced in skeletal muscle, fibroblasts, and plasma. Supplementation therapy with CoQ10 dramatically ameliorated some of the neurologic symptoms, especially cerebellar ataxia. Interestingly, CoQ10 deficiency has been observed in different conditions associated with cerebellar ataxia suggesting a specific susceptibility of this brain region to oxidative stress and/or mitochondrial bioenergetics impairment due to CoQ10 reduction (Emmanuele et al. 2012). GLUT1DS also is characterized clinically as an infantile-onset cerebellar phenotype, and the cerebellar region is unduly vulnerable as documented by brain imaging (Akman et al. 2015).

In order to assess if CoQ10 reduction is a common feature in GLUT1DS, we collected tissue samples from 16 patients with GLUT1 deficiency. Plasma CoQ10 levels, in seven patients, were comparable to controls. However, since plasma COQ10 levels can be influenced by diet (Emmanuele et al. 2012), we extended the studies to white blood cells (WBC) collected from peripheral blood. Again, the CoQ10 levels in WBC were not different from controls, congruent with the plasma data. We also studied 6 skin fibroblast cell lines derived from GLUT1 patients and found normal levels of CoQ10.

As is often the case in mitochondrial diseases, including CoQ deficiency, plasma and skin fibroblast CoQ levels do not necessarily reflect the biochemical phenotype (Emmanuele et al. 2012). Therefore, we decided to study affected and unaffected organs in model mice with GLUT1 deficiency. In GLUT1+/− mice, CoQ levels were mildly reduced only in the cerebellum (~7%), while CoQ levels elsewhere in the brain, muscle, kidney, and liver were normal. The cerebellum, and other specific brain regions, is noticeably vulnerable to GLUT1 deficiency (Akman et al. 2015). Therefore, we think the slightly reduced CoQ levels in this brain region reflect the tissue pathology as an epiphenomenon. Reduced brain lipid synthesis has been shown in a different GLUT1 mouse model (Marin-Valencia et al. 2012) and also may account for reduced CoQ levels in the cerebellum. However, such mild deficiency can hardly alter mitochondrial energy metabolism, as confirmed by our observations of normal transport of electrons from complexes I and II to complex III.

Since CoQ10 is the predominant antioxidant of the inner mitochondrial membrane, we studied oxidative stress as a possible consequence of CoQ10 deficiency, and we did not find any evidence of oxidative stress in GLUT1+/− mouse cerebellum, compared with controls.

In conclusion, our aggregate data from patients and model mice fail to support CoQ10 deficiency as a common finding in GLUT1 deficiency, and CoQ deficiency cannot be viewed as a direct biochemical consequence of defective glucose transport associated with a disease-causing mutation in the SLC2A1 gene. It is possible that the patient described by Yubero et al. had the double misfortune of inheriting GLUT1 deficiency and a second (yet undefined) genetic disorder causing CoQ10 deficiency.