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*
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil; and
Centro de Ciências da Saúde, Universidade do Vale do Rio dos Sinos, São Leopoldo, RS, Brazil
2To whom correspondence should be addressed. E-mail: casg{at}ufrgs.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • ketogenic diet • protein phosphorylation • ß-hydroxybutyrate • epilepsy • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Recent evidence showed clearly that the efficacy of these diets can be evaluated in rats by studying seizures induced with kainic acid (2
) or pentylenetetrazole (3
). However, we know little about the underlying biochemical bases of this treatment (4
) or about the mechanism(s) of epileptogenesis and the cellular and molecular actions of the current antiepileptic drugs (5
).
There are many hypotheses in the literature about action of KD [see (4
) for a review]. The hypothesis that has received most attention focuses on alterations in brain metabolism induced by ketone bodies as a result of changing from a glucose-based to a ketone-based energy substrate (1
). One of the first studies of metabolic changes induced by a KD demonstrated an increase in the ATP/ADP ratio in rat brains (6
). ATP levels fall rapidly during seizures and it has been suggested that a putative protective effect of ATP is involved in controlling this chronic disorder. However, it is not clear how increasing the energy reserve in the brain could reduce brain excitability by increasing the seizure threshold (3
).
Protein phosphorylation is the major regulatory mechanism of signal transduction and has been implicated in modulating neuronal excitability (7
). Therefore, changes in protein phosphorylation may be involved in seizure discharge (8
). General and specific alterations in protein phosphorylation have been reported in brain biopsy samples from epileptic patients (9
) and in rat models of seizures (10
–12
). In this study, we investigated basal protein phosphorylation in slices from different brain regions of young rats fed a KD. We also evaluated the effect of this diet on body weight during development and on the health of these rats assessed by observations on blood biochemistry.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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[32P]Na2HPO4 was purchased from CNEN (São Paulo, Brazil). Acrylamide, bis-acrylamide, tris(hydroxymethyl)-aminomethane (Tris), sodium dodecyl sulfate (SDS) and other chemicals used for electrophoresis were purchased from Sigma (St. Louis, MO). NaCl, KCl, MgCl2, MgSO4, CaCl2, HEPES and glucose were purchased from MERCK (Rio de Janeiro, Brazil).
Animals.
Male 30-d-old Wistar rats came from the local breeding colony (Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul). They were maintained on a 12-h light/dark cycle in a ventilated room at 21°C with free access to food and water. All animal procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the local authorities.
Diets.
Rats were weight-matched and divided into two groups: C, control rats that received regular laboratory ration (Nuvilab-CR1; Nuvital, Curitiba, Brazil) and K, rats that received the KD (Table 1
). All rats consumed food and water ad libitum for 6–8 wk. Rats were weighed at weekly intervals.
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Blood samples were incubated at 37°C for 10 min and centrifuged at 800 x g for 10 min (Eppendorf 5402; Hamburg, Germany). Serum was stored at 8°C for 24 h. Biochemical analysis was carried out in a Multi-test Analyzer (Mega; Merck, Darmstadt, Germany), using specific kits supplied by Merck as follows: total protein (protein-SMT, 1.1970 3.0001,biuret method); albumin (albumin-SMT, 1.1972 2.0001,bromocresol method); glucose (GLUC-DH 1.07116.0001); urea (urea-SMT 1.1970 2.0001,UV test); creatinine (creatinine-SMT 1.1972 6.0001,UV test); uric acid (uric acid-SMT, 1.1973 9.0001,color test); triglycerides (SMT-triglyceride, 1.19706.0001,GPO-PAP method); cholesterol (cholesterol-SMT, 1.1973 8.0001,CHOD-PAP method); and LDL cholesterol (1.14992.0001, CHOD-PAP method). HDL cholesterol was determined using a kit (HDL cholesterol direct FS) from DiaSys (Diagnostic Systems International, Holzheim, Germany). Ketonemia was determined by a semiquantitative kit (Keto-Diastix) from Bayer Diagnostics (Buenos Aires, Argentina).
Preparation of microslices and labeling of phosphoproteins.
This procedure was carried out as described (13
). Briefly, rats were killed by decapitation and the hippocampi, cerebral cortex and cerebellum dissected on ice. Transverse slices (0.4 mm) were prepared with a Mcllwain chopper (The Mickle Laboratory Engineering Co., UK) and microslices (1 mm diameter) were then punched out. One microslice from each brain region was preincubated at 30°C in 0.1 mL of medium containing: 0.124 mol/L NaCl, 0.004 mol/L KCl, 0.0012 mol/L MgSO4, 0.025 mol/L HEPES (pH 7.4), 0.012 mol/L glucose, and 0.001 mol/L CaCl2. In some experiments, 0.0025 mol/L sodium DL-hydroxybutirate and 0.0025 mol/L sodium acetoacetate were added to this medium (see Results in Fig. 3
). After 30 min, the medium was replaced by 0.1 mL of the same medium containing 40 µCi of [32P]phosphate. The labeling reaction was allowed to proceed for 1 h at 30°C and stopped with 1 mL of 0.60 mol/L tricholoroacetic acid. The microslices were washed twice by decantation with 0.24 mol/L of tricholoroacetic acid to remove excess radioactivity, briefly with water to remove acid and then immediately dissolved in 0.1 mL sodium dodecyl sulfate-poliacrilamide gel electrophoresis (SDS-PAGE) buffer (0.138 mol/L SDS, 0.002 mol/L EDTA, 0.05 mol Tris-HCl, pH 6.8 and 0.64 mol/L mercaptoethanol) or in 0.06 mL two-dimensional electrophoresis buffer (9.2 mol/L urea, 0.012 mol/L lysine, 0.007 mol/L SDS, 4% (v/v) Nonidet-P40, and 0.25 mol/L 2-mercaptoethanol).
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For SDS-PAGE, 25 µL of glycerol medium [40% (v/v) glycerol, 0.64 mol/L mercaptoethanol, Tris-HCl, pH 6.8 and bromophenol blue] were added to each sample. The samples (30 µL) were applied to10% gels (14
). The gels were dried, exposed to X-ray films at -70°C with intensifying screens. For two-dimensional electrophoresis, samples were analyzed by nonequilibrium pH gradient electrophoresis followed by SDS-PAGE in 10% slab gels as described previously (13
,15
). Assessment of the extent of protein phosphorylation was made by densitometric scanning of the autoradiographs. Densitometric values from hippocampal slices of normal fed rats were normalized to 100%.
Statistical methods.
Significant differences among the treatment groups were analyzed by paired or unpaired Student’s t test or ANOVA with posthoc Duncan’s or least significant difference test. Differences were considered significant at P < 0.05. All analyses were done using SPSS program, Version 7.5 (SPSS, Chicago, IL). Values are means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). However, at 7–8 wk, weight gain of the KD-fed rats was significantly less than that of controls (P < 0.05).
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). The ketonemia was similar to ß-hydroxybutyrate concentrations in Sprague-Dawley rats that had started a KD at postnatal d 28 (3
). Other variables, including lipemia, indicated normal health. Interestingly, serum urea was lower (P < 0.05) in ketonemic rats than in controls, but the meaning of this is unclear. Preliminary experiments using a KD with 20% protein (as used in children) instead 25% protein caused undernutrition of the rats as shown by a significant loss of weight and hair (data not shown). This result may be related to the Wistar strain of rats used in this study, because Sprague-Dawley rats did not show signs of undernutrition when fed a KD containing 10% protein, even when the diet was started at 22 d postnatal (3
).
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40% greater total protein phosphorylation in all brain regions compared with control rats (Fig. 2
, A and B). Furthermore, when slices of cerebral cortex from ketonemic rats were incubated with [32P]phosphate in a medium containing glucose or glucose plus ketone bodies, there were no differences in protein phosphorylation (Fig. 3
). Interestingly, protein phosphorylation was higher in slices from rats fed the control diet when incubated in the presence of ketone bodies (Fig. 3)
. Similar results were obtained with slices from hippocampus and cerebellum (data not shown).
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). Five phosphoproteins were identified by immunoblotting: glial fibrillary acidic protein (GFAP) (14
,15
), synapsin I (16
) and growth-associated protein of 43 kDa (GAP-43) (17
) or by electrophoretic mobility: myristoylated alanine-rich C kinase substrate of 87 kDa (MARCKS) (18
) and pp42C (19
). Autoradiography showed a general increase in protein phosphorylation in brain slices from ketogenic rats (426%). A large increment of phosphorylation was observed in synapsin I, GAP-43 and MARCKS (685%, 646% and 501%, respectively) and an increment still higher (950%) was observed in two nonidentified phosphoproteins (spots 6 and 7; Fig. 4
). GFAP and ppC42 phosphorylation increased 171% and 269%, respectively. These proteins had an increment significantly lower than the overall mean increment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The first experimental evidence for a beneficial effect of a KD on seizures appeared in the 1970s (20
,21
). More recently, KD were used successfully in adult rats to increase the seizure threshold (2
,3
).
Young Wistar rats fed with ketogenic and normal diets gained weight during treatment, but a lower gain at 7–8 wk was observed in the ketogenic group. This difference disappeared after 16 wk of treatment (data not shown). In other studies using adult rats, ketogenic and control groups did not differ in weight gain (22
). Possibly weight gain as well as changes in seizure threshold in KD-fed rats depends on age (3
). Besides weight, other variables were used to evaluate the health of ketogenic rats, particularly lipemia and proteinemia that are severely altered in some children comsuming this diet (23
). These variables and others (see Table 2
) indicated that our rats remained healthy when consuming a KD for 8 wk.
Brain slices from KD-fed rats had more protein phosphorylation in all regions analyzed compared with control rats. Our results may suggest a lasting effect (independent of the presence of ketone bodies) of the KD that could involve some metabolic change induced by high circulating levels of ketone bodies or perhaps by a lipid component of this diet.
We can offer possible explanations for the general increment in protein phosphorylation. For example, an elevated ratio of ATP to ADP, as suggested previously (6
), could be accompanied by an elevation in general protein phosphorylation.
Other possibilities such as different phosphate uptake, different ATP synthesis or differences in the activity of protein phosphatases that have a broad specificity when compared to protein kinases cannot be excluded. This issue deserves further investigation. In fact, phosphate uptake was 2.5-fold greater in the presence of ketone bodies in hippocampal slices from both normal and ketogenic rats (unpublished data). Ketone bodies by themselves seem to directly increase protein phosphorylation by changing phosphate uptake. This result maybe explain why phosphorylation is increased in brain slices from normal rats incubated in the presence of ketone bodies, but it does not explain why phosphorylation was greater in slices from ketogenic rats without ketone bodies in the medium.
Other in vitro metabolic alterations directly induced by ketone bodies have been described such as a diminution of available oxaloacetate in synaptosomes (24
) and an increase in citrate in astrocytes (25
), which could be associated to changes in the neurotransmitters glutamate and 6 
-aminobutyric acid. However, electrophysiological observations indicate that ketone bodies per se did not reduce neuronal excitability (26
).
Although a general increase in protein phosphorylation was observed in brain slices from rats fed the KD, two-dimensional electrophoresis showed different increments for specific proteins. Neuronal phosphoproteins synapsin I and GAP-43, involved in neurotransmitter release and synaptic plasticity, respectively, showed high increments in phosphorylation compared with GFAP, a glial protein involved in the intermediate filament plasticity (27
). Two proteins associated with neural development, MARCKS (18
) and ppC42 (19
), presented a different profile of increase in the phosphorylation. All these proteins have different cell localizations and brain region distribution, as well as being substrates for different protein kinases. However, despite different increments in the extent of protein phosphorylation, currently it is not possible to conclude any functional meaning from our observations or even to determine whether this reflects specific changes on cell types or on phosphorylating systems.
Our results support the observation that a KD induces metabolic changes affecting the basal status of protein phosphorylation. This change could affect the mechanisms of signal transduction in neural cells involved in the increased seizure threshold. However, a relationship between them has not been established.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: GAP-43, growth-associated protein of 43 kDa; GFAP, glial fibrillary acidic protein; KD, ketogenic diet; MARCKS, myristoylated alanine-rich C kinase substrate; SDS-PAGE, sodium dodecyl sulfate-poliacrilamide gel electrophoresis.
Manuscript received 10 August 2001. Initial review completed 3 October 2001. Revision accepted 10 December 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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