Molecular and Cellular Endocrinology 394 (2014) 29–36

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Endurance training increases brain lactate uptake during hypoglycemia by up regulation of brain lactate transporters Malihe Aveseh a,b, Rohollah Nikooie b,⇑, Vahid Sheibani a, Saeed Esmaeili-Mahani c a

Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran Department of Exercise Physiology, Faculty of Physical Education and Sport Science, Shahid Bahonar University of Kerman, Kerman, Iran c Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran b

a r t i c l e

i n f o

Article history: Received 2 March 2014 Received in revised form 26 June 2014 Accepted 30 June 2014 Available online 5 July 2014 Keywords: Monocarboxylate transporters, Hypoglycemia Endurance training Lactate

a b s t r a c t The capacity of the brain to metabolize non-glucose substrates under hypoglycemic state maintains its energy requirements. We hypothesized that exercise-induced increase in capacity for brain utilization of lactate by up regulation of the monocarboxylate transporters (MCTs) may contribute metabolic substrates during hypoglycemia in diabetic rats induced by streptozotocin. The induced diabetes increased MCT1 and MCT2 expression in the cortex and the hippocampus in the sedentary diabetic animals. There were exercise-induced increases in MCT1 in the cortex and the hippocampus and MCT2 expression in the cortex in trained diabetic animals; whereas, no changes were found in the healthy trained animals. Both diabetic and healthy trained animals showed higher values for brain lactate uptake during insulin-induced hypoglycemia when animals were intraperitoneally injected by L(+)-lactic acid. However, the response of counterregulatory hormones during hypoglycemia were blunted in the diabetic trained animals which indicates to carefully monitoring of glycemic targets both during and following prolonged exercise. Ó 2014 Published by Elsevier Ireland Ltd.

1. Introduction In normal individuals, the level of plasma glucose remains within a fairly tight range of 4–7 mM despite periods of feeding and fasting. However, the body’s intrinsic insulin-sensitive ability to utilize glucose is disrupted in a group of heterogeneous metabolic disorders known as diabetes mellitus (Bloomgarden, 2005). Patients with type 1 diabetes mellitus experience hypoglycemia in this disease. Due to the brain dependency almost exclusively on glucose, recurrent hypoglycemia may be a threat for cognitive dysfunction and cerebral damage (Languren et al., 2013). Metabolic strategies to cope with hypoglycemia and to protect the brain include counter-regulatory endocrine responses to increase hepatic glucose production (Sandoval et al., 2004) as well as selection of alternative fuels by the brain (Maran et al., 2000). When compared with the control-group, the diabetic patient’s brain has more capacity to use other blood-derived energy substrates, such as acetate and lactate, to maintain its energy requirements during a hypoglycemic state (Mason et al., 2006; Maran et al., 2000). Subsequently, in diabetic patient with recurring hypoglycemia the brain suppresses some of the counterregulatory responses to this state ⇑ Corresponding author. Tel.: +98 341 2811001/2; fax: +98 341 2812777. E-mail address: [email protected] (R. Nikooie). http://dx.doi.org/10.1016/j.mce.2014.06.019 0303-7207/Ó 2014 Published by Elsevier Ireland Ltd.

(Maran et al., 2000), hypoglycemia unawareness has been documented in these patients (Cryer, 1992). Glucagon response to hypoglycemia in type 1 diabetes is almost absent (Gerich et al., 1973). This leaves the patients dependent on epinephrine to counter the falling levels of glucose. Previous studies in human have demonstrated that blood lactate plays an important role in the brain’s metabolism (Boumezbeur et al., 2010; Overgaard et al., 2012; Rasmussen et al., 2011; Van et al., 2009). Studies using 13C-magnetic resonance spectroscopy (MRS) have demonstrated that lactate has the potential to provide up to 10% of the brain’s energy needs under euglycemic conditions in healthy subjects (Boumezbeur et al., 2010; Van et al., 2009). In animals, lactate is the important contributor to sustain tricarboxylic acid cycle (TCA) during hypoglycemia and has the potential to provide over 30% of oxidative energy (Herzog et al., 2013). However, lactate utilization by the brain for energy production is dependent to uptake of lactate from the bloodstream and its exchange among various portions of the brain. Transport of lactate across the blood–brain barrier is performed in a pH – dependent manner and mediated by a family of proton-coupled monocarboxylate transporters (MCTs) (Pierre and Pellerin, 2005; Pellerin et al., 2005). MCT1, MCT2, and MCT4 are the three most commonly isoforms that are found in the cortex, the hippocampus, and the cerebellum of the rat brain and each of these isoforms

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M. Aveseh et al. / Molecular and Cellular Endocrinology 394 (2014) 29–36

exhibits a distinct regional and cellular distribution (Pierre and Pellerin, 2005; Pierre et al., 2007). MCT1 is commonly expressed in astrocytes and in microvessel endothelial cells (Gerhart et al., 1997, 1998) whilst MCT2 expressions were reported in astrocytes (Gerhart et al., 1997, 1998), and in the synapses and Purkinje cell spines (Bergersen et al., 2001). MCT4 is preferentially expressed in astrocyte and is involved in the astrocytic processes (Bergersen et al., 2001; Pierre and Pellerin, 2005). The plasma membrane abundances of MCTs changes significantly in diabetes which suggests that these transporters may be involved in the metabolism regulation (Enoki et al., 2003; Nikooie et al., 2013). In the diabetic animals, MCTs expression in heart and skeletal muscle is extensively altered in response to chronic exercise (Enoki et al., 2003; Metz et al., 2005; Nikooie et al., 2013). The brain also has the ability to adjust its supply of monocarboxylates to meet specific energy requirements, under specific physiological condition such as exercise in human (Overgaard et al., 2012) and rats (Yamada et al., 2010). As the arterial blood lactate concentrations rise during exercise, therefore, exercise is often accompanied by shifts in the supply of fuel to the brain (Overgaard et al., 2012). Although increased lactate uptake by the human brain during exercise was reported in some studies (Overgaard et al., 2012; Van et al., 2009), on the contrary, no study has investigated the MCTs expression in the brain post-endurance training. Counterregulatory responses to hypoglycemia are also affected by endurance training. Previous studies have reported a higher adrenaline response to exercise in the endurance-trained as compared with untrained subjects in response to the intense exercise at the same relative intensity, however, they have an attenuated response of epinephrine during moderate exercise (Zouhal et al., 2008). Despite the numerous therapeutic benefits of exercise, hypoglycemia is often associated with physical activity and it has been shown that antecedent prolonged moderate exercise blunts neuroendocrine and metabolic counterregulatory responses to subsequent hypoglycemia in normal (Galassetti et al., 2001) and diabetic patients (Galassetti et al., 2003; Sandoval et al., 2004). However, in all these studies the counterregulatory responses to hypoglycemia have been investigated 2–24 h after a single session exercise. Therefore, it is difficult to define whether the observed responses were due to the body’s adaptation or a temporary response to the acute exercise. In this study, we hypothesized that endurance training can augment the brains lactate uptake during the hypoglycemic state, by the up regulation of the brains MCTs expressions. The primary aim of this study was to investigate and examine the long term effect of endurance training on the expression of MCT1 and MCT2 in the cortex of the brain. The hippocampus and the cerebellum of the brain in the healthy and diabetic rats and their brains capacity and ability for utilization of lactate under the hypoglycemic condition were also examined. Secondly, the study proposed to investigate the long term effect of endurance training on counterregulatory responses to hypoglycemia.

2. Material and methods All procedures in the present study were approved by the Ethical Committees on Animal Care at the Neuroscience Research Center of Kerman, University of Medical Sciences, Kerman, Iran. Healthy and diabetic male wistar rats were inducted into a training program or remained sedentary throughout the experiment. Fortyeight hours following the last exercise session, some of the animals were selected for a short-term insulin-induced hypoglycemia (IIH) in which counterregulatory hormones were examined and finally autopsied for the brains MCT1 and MCT2 expression. The remaining animals experienced hypoglycemia in which the effects of the

intraperitoneal injection of L(+)-lactic acid on the brain lactate uptake were investigated. 2.1. Animals Male wistar rats (6 weeks old) were purchased from Pastor Institute (Tehran) and housed in an air conditioned room (temperature, 22 ± 3 °C) on a 12-h light–12-h dark cycle. All rats were fed rat chow for 2 weeks ad libitum, and their body weights were measured daily. After an acclimation period of 2 weeks, the animals were assigned randomly into four groups, according to their body weight: control (C; n = 25), trained (T; n = 25), control diabetic (CD; n = 25), and trained diabetic (TD; n = 25). Seven animals – four and three from CD and TD, respectively – were died during the experiment. All deaths occurred after induction of diabetes. 2.2. Diabetes induction procedure At the onset of 8 weeks of age, the diabetes was induced by intraperitoneally injection of streptozotocin (STZ; 50 mg/kg, ip) in a 0.1-M citrate buffer (pH 4.5) (Enoki et al., 2003); 48 h after the STZ injection, nonfasting glucose concentrations were determined via blood sample that were taken from the orbital sinus. Blood collected via heparinized tube and plasma separation was performed by centrifugation at 3000g for 10 min at 4 °C. Plasma glucose concentration was determined by glucose oxidase method (Wincey and Marks, 1961), and the animal showing FBG of P180 mg/dl were considered as diabetic. 2.3. Training intervention Treadmill exercise was performed for 60 min/day, 5 days/week, for 8 week. Initially, the TD and T groups were familiarized with a motor-driven treadmill running at low speeds (12–15 m/min) for 15–20 min/day for the first 5 days. Thereafter, the speed and duration were increased progressively over the 8-week period until the animals were running at 30 m/min for 60 min for the last 2 weeks. The control and sedentary diabetic animals remained sedentary in their cages for the duration of the 8-week training program. 2.4. Short-term insulin-induced hypoglycemia (IIH) Forty-eight hours following the last exercise session, six fed rats from each group were selected for a short-term insulin-induced hypoglycemia (IIH). Hypoglycemia was induced by an intraperitoneal injection of 10 U/kg of regular insulin (Oliveira-Yamashita et al., 2009). Blood samples were taken 30, 60 and 120 min following insulin injection to calculate epinephrine and glucagon level. Serum glucagon was measured with Glucagon Quantikine ELISA Kit (Catalog No: DGCG0, R & D Sestems). Plasma epinephrine was measured with Chemiluminescence immunoassay kit (Catalog No: E-CL-R0018, Elabscience). These animals were autopsied 48 h after the IIH test; the cortex, the cerebellum, and the hippocampus were excised immediately and frozen in liquid nitrogen, and stored at 80 °C for subsequent analysis. 2.5. Western blotting Approximately 20–150 mg tissue from the cortex, the hippocampus, and the cerebellum were powdered with cold mortar and pestle in liquid nitrogen. Tissue were homogenized in ice-cold RIPA buffer (50 mM Tris–HCl, 1 mM EDTA, 150 mM NaCl, NP40 1%, Na deoxycholate 1%, SDS 1%, Protease and phosphatase inhibitor 0.01 M, pH 7.4). The homogenate was centrifuged at 14,000 RPM for 15 min at 4 °C. Supernatant was discovered and total protein was determined with Bradford protein assay using bovine serum

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albumin (BSA) as a standard; 20 lg total protein of each sample was loaded and separated on 12.5% SDS–PAGE and transferred by electroblotting onto polyvinylidenedifluoride membranes (Amersham). Thereafter, PVDF membranes were stained in 0.1% Ponceau S in 1% acetic acid for 3 min with shaking. Membranes were destained by rinsing in water for 2 min. Ponceau S staining total protein loading was recorded to monitor transfer efficiency and quantification of whole protein loading. Then, after washing in TBST for 5 min membranes were incubated for 1.5 h at room temperature on an orbital shaker in blocking buffer (150 mM NaCl, 5% skimmed milk, 0.1% Tween 20, and 50 mM Tris, pH 7.5) and incubated in primary antibody overnight at 4 °C in blocking buffer. Membranes were washed and then incubated for 90 min at room temperature with secondary antibody in TBST. Membranes were washed and protein expression was then detected by enhanced chemiluminescence according to manufacture instructions. Autoradiographic film were exposed to membranes and developed. Molecular weight standards were used to identify appropriate antibody binding. Band densities were determined with ImageJ densitometer software. Primary antibodies used were rabbit anti-MCT1 (AB3540P, Millipore) and rabbit anti-MCT2 (AB3542, Millipore) and Goat Anti-Rabbit IgG Antibody, Peroxidase Conjugated (AP132P, Millipore) was used as the secondary antibody. Rat erythrocyte ghost was used as a positive control and to fix an arbitrary unit to allow comparison between experiments (1 equals the MCT1 signal generated by 5 lg of rat erythrocyte ghost).

2.6. Rat erythrocyte ghost preparation Fresh blood from a rat was mixed with 7 vol. of acid citrate– dextrose buffer (75 mM sodium citrate, 38 mM citric acid, and 138 mM D-glucose) and centrifuged at 16,000g. The supernatant and buffy coat are removed and pellet washed thrice in 66 mM NACL. The sedimented cell was diluted in 66 mM NaCl for 25% cell suspension. Cell suspension mixed with hemolyzed buffer (1 mM EDTA, 9.64 mM NaCl, 3.61 mM Na2HPO4, and 1.2 mM KH2PO4; pH 7.2) in 1:7 volume ratio and placed on ice for 20 min. The solution was centrifuged at 20,000g for 15 min at 4 °C, and pellet diluted with ten times volume of buffer containing 9.6 mM Tris– HCl, 20 mM NaCl; pH 7.2) and washed once in this buffer and again in buffer containing 4.8 mM Tris–HCl and 10 mM NaCl. Pellet washed once in 100 mM KCl and two times in water and diluted in CO2-free water (Schwoch and Pasoow, 1984).

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2.8. Plasma and brain preparations Blood (0.2 ml) was collected from the neck vessels at the time of sacrifice and immediately mixed with 20 volumes of 0.5 M perchloric acid, centrifuged at 1500g for 10 min at 4 °C and the supernatant was recovered. Forebrain lactate content was determined from perchloric acid extracts. Approximately 50 mg of forebrain was powdered with cold mortar and pestle in liquid nitrogen and incubated for 10 min in 8 vol. of ice-cold 6% perchloric and centrifuged at 1500g for 10 min at 4 °C (Gutman and Wahlefeld, 1974). The supernatant was removed and then lactate concentration was measured by lactate assay kit (cat. No. K607-100, Biovision) according to manufacturer’s instructions for both tissue and plasma samples. 2.9. Statistical analysis Values between- and within groups were tested using analysis of variance (ANOVA). Tukey’s post hoc test were used to determine wherever significant differences occurred. Before proceeding with the statistical analysis, the residuals in the ANOVA were examined for a normal distribution through examination of a histogram and a normal plot. If residuals were considered not to be normally distributed, data were log transformed. This was the case for the data on the lactate injection test and thus for these measurements log transformed data were used in the subsequent statistical analysis. Therefore, all the data on the lactate injection test are reported as geometric means with a 95% confidence interval (C.I.). The remaining data (MCTs and hormones) are represented as means ± SD. The P-values are based on the analyses of transformed data where transformations were used. In all comparisons, the significant level was set at a = 0.05. 3. Results 3.1. Diabetes induction and endurance training increased MCT1 and MCT2 expressions in the cortex and the hippocampus The induced diabetes significantly increased MCT1 expression in the cortex (2 ± 0.2-fold, P < 0.01) and in the hippocampus (1.7 ± 0.2-fold, P < 0.01) in CD in comparison to C group. MCT1 expression in the cerebellum was not significantly different between CD and C groups (Fig. 1). After 8 weeks of endurance training MCT1 expression in the cortex (P < 0.05) and hippocampus

2.7. Lactate injection in insulin-induced hypoglycemic rats A 0.33 M L(+)-lactic acid solution was prepared from a 30% solution (Sigma Chemical Co., USA) under sterilized condition. The pH of the solution was adjusted to 7.2 by the addition of 10 N NaOH. Forty-eight hours following the last exercise session, twelve animals from each group were selected for IIH. When the animals exhibited signs of hypoglycemic stupor or coma, lactate (4 mmol/ kg) were intraperitoneally injected into nine animals from each group. The animals were autopsied by decapitation, at intervals of 10, 20, and 30 min (three animals for each time) following lactate injection and the head was directly inserted in liquid nitrogen and stored at 80 °C for subsequent lactate and glucose measurements. Blood samples were collected from the neck vessels in heparinized tubes and used for determination of plasma lactate and glucose concentration. Brain/plasma lactate ratio was used for determination of permeability of the blood–brain barrier to lactate in the healthy and diabetic rats (Thurston et al., 1983).

Fig. 1. MCT1 expression relative to standard (St; MCT1 signal generated by 5 lg of rat erythrocyte ghost) in the cortex, the hippocampus, and the cerebellum. Values are means ± SD, C (control, n = 6), T (trained, n = 6), CD (control diabetic, n = 6), TD (trained diabetic, n = 6). *Significant difference between groups (P < 0.05), ** (P < 0.01).

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(P < 0.05) were significantly higher in TD in comparison to CD group (Fig. 1). The induced diabetes also significantly increased MCT2 expression in the cortex (2.7 ± 0.4-fold, P < 0.01) and in the hippocampus (1.6 ± 0.2-fold, P < 0.01) in CD compared with C group. There was no significantly difference for MCT2 expression in the cerebellum between these groups (Fig. 2). At the end of the study higher values for MCT1 expression was only found in the cortex (P < 0.05) of TD group in comparison to CD animals (Fig. 2). 3.2. Trained animals showed the higher values of brain lactate and Permeability of the blood–brain barrier to lactate compared with control during hypoglycemia The resting values of plasma lactate concentrations were (C: 2.09 ± 0.15 mmol/L; T: 1.95 ± 0.3 mmol/L; CD: 2.93 ± 0.28 mmol/ L; TD: 2.84 ± 0.37 mmol/L) before the intraperitoneal injection of insulin. One-hour and a half following the insulin injection, plasma lactate concentration were reduced in the healthy (57.6 ± 1.1%) and diabetic animals (70.4 ± 1.7%) compared to the resting values of plasma lactate concentration. However, the animals of four groups have shown almost the same values for plasma lactate concentrations before the intraperitoneal injection of insulin (Table 1). The plasma and brain lactate concentration and brain/plasma lactate ratio of hypoglycemic animals before and at intervals following the intraperitoneal injection of L(+)-lactic acid are shown in Table 1. The intraperitoneal injection of lactate raised the plasma lactate concentration toward 4.4–4.9 (mmol/l) values in all groups (Table 1). No significant difference was established for plasma lactate concentration among four groups 10 min following the lactate injection, but the difference became significant 20 (P < 0.05) and 30 min (P < 0.05) following the injection between C and T groups. The difference between CD and TD was only significant at 30 min (P < 0.05) following the intraperitoneal injection of L(+)-lactic acid. Ten minutes following the lactate injection, the brain lactate concentration increased in the C group (2.3 ± 0.3-fold), T (2.4 ± 0.2-fold), CD (2.5 ± 0.2-fold), and TD (3 ± 0.2-fold) furthermore there was no significant difference among four groups. Thirty minutes following the injection, a significant difference was established between C and T (P < 0.05), CD and TD (P < 0.05). There was a sharp drop in the brain/plasma lactate concentration ratio following the lactate injection in all groups. Significant difference were established between C and T (P < 0.05), CD and TD (P < 0.05) groups 30 min following the injection.

3.3. Increased lactate utilization by brain could not return the lowered brain glucose concentration to resting values during hypoglycemia The resting values of plasma glucose concentrations were (C: 5.34 ± 0.21 mmol/L; T: 5.33 ± 0.27 mmol/L; CD: 12.5 ± 0.99 mmol/ L; TD: 11.28 ± 0.95 mmol/L) before the intraperitoneal injection of insulin. One-hour and a half following the insulin injection, plasma glucose concentration were reduced in the healthy (61.7 ± 1.6%) and diabetic animals (80.5 ± 2.2%). Table 2 illustrates the plasma and brain glucose concentrations of the hypoglycemic animals before and after the lactate injections. The plasma glucose concentration values did not differ significantly from the pre-injection values in all groups at any time. However, minimal non-significant reductions in plasma glucose concentration were established in C and T groups 20 min following the lactate injection. The intraperitoneal injection of L(+)-lactic acid resulted in an elevation in brain glucose concentration in all groups. In both C and T groups, brain glucose concentration values at intervals 20 (P < 0.01) and 30 (P < 0.01) were significantly higher in comparison to their pre-injection values. There was a significant difference between the values corresponding to pre-injection time and 10 min following the lactate injection for T group (P < 0.05). In both CD and TD groups, brain glucose concentration values at 30 (P < 0.01) were significantly higher in comparison to their preinjection values. However, the absolute values of brain glucose concentration for 30 min following the lactate injection were still very low (14.3 ± 0.33% for healthy animals and 10.3 ± 0.37% for diabetic groups) in comparison to the resting values in all groups (C: 1.09 ± 0.07 mmol/kg; T: 1.12 ± 0.05 mmol/kg; CD: 1.14 ± 0.06 mmol/kg; TD: 1.11 ± 0.07 mmol/kg). 3.4. Response of counterregulatory hormones were blunted in trained diabetic animals compared with control In both the diabetic groups, basal plasma concentrations of glucagon were significantly higher (P < 0.01) and concentrations of epinephrine were significantly lower (P < 0.01) compared with the healthy groups (Table 3). Plasma glucagon increased in response to hypoglycemia with significant difference (P < 0.01) between normal and diabetic animals (Fig. 3a). The increase was not significant in TD animals and their plasma concentrations of glucagon were non-significantly lower at any time than CD group. Fig. 3b shows the plasma D [glucagon] during hypoglycemia period in all groups. Significant difference was found between CD and TD groups (P < 0.05) and there was no significant difference between C and T. The plasma concentrations of epinephrine were significantly increased in response to hypoglycemia in all groups (P < 0.01). At any time during hypoglycemia period, the plasma concentrations of epinephrine were lower in diabetic animals in comparison to healthy groups (Fig. 3c). The peak plasma epinephrine concentrations during the hypoglycemia were lower in the diabetic groups than in the healthy animals (P < 0.01). Plasma concentrations of epinephrine at 30, 60 and 120 min were not significant between CD and TD. Fig. 3d shows the plasma D [epinephrine] during the hypoglycemia period in all groups. There was no significant difference neither between T and C nor CD and TD. 4. Discussion

Fig. 2. MCT2 expression relative to standard (St; MCT1 signal generated by 5 lg of rat erythrocyte ghost) in the cortex, the hippocampus, and the cerebellum. Values are means ± SD, C (control, n = 6), T (trained, n = 6), CD (control diabetic, n = 6), TD (trained diabetic, n = 6). *Significant difference between groups (P < 0.05), ** (P < 0.01).

This study, examined the long term effects of the endurance training sessions on the monocarboxylate transporters (MCT1 and MCT2) expressions and their role in the brains lactate permeability and the brains utilization of the lactate during a hypoglycemic state in the diabetic/healthy animals. The unique results

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M. Aveseh et al. / Molecular and Cellular Endocrinology 394 (2014) 29–36 Table 1 Effect of lactate injection on plasma and brain lactate concentration and brain/plasma lactate ratio of hypoglycemic animals from all groups. Variable

Groups

Before injection

After injection (min)

Plasma lactate (mmol/l)

C (n = 3) T (n = 3) CD (n = 3) TD (n = 3)

0.85 0.80 0.87 0.83

(0.91, (0.95, (0.97, (0.97,

0.80) 0.67) 0.77) 0.71)

4.41 4.52 4.84 4.88

(4.50, (4.67, (5.03, (5.27,

4.32) 4.37) 4.65) 4.52)

3.95 3.41 4.18 4.02

(4.11, (3.62, (4.43, (4.22,

3.80) 3.21)a 3.94) 3.83)

3.19 2.57 3.64 3.14

Brain lactate (mmol/kg)

C (n = 3) T (n = 3) CD (n = 3) TD (n = 3)

0.59 0.59 0.64 0.57

(0.66, (0.68, (0.69, (0.62,

0.53) 0.51) 0.6) 0.52)

1.41 1.48 1.68 1.74

(1.58, (1.59, (1.80, (1.79,

1.25) 1.37) 1.56) 1.69)

1.50 1.54 1.67 1.79

(1.59, (1.95, (1.79, (1.82,

1.41) 1.44) 1.57) 1.75)

1.36 (1.42, 1.30) 1.48 (1.57, 1.41)a 1.5 (1.62, 1.39) 1.75 (1.84, 1.67)b

Brain/plasma lactate ratio (L/kg)

C (n = 3) T (n = 3) CD (n = 3) TD (n = 3)

0.69 0.74 0.74 0.68

(0.79, 0.61) (1.01, 0.53) (0.078, 0.70) (0.74, 0.63)

0.31 0.32 0.34 0.35

(0.36, (0.34, (0.38, (0.38,

0.27) 0.31) 0.31) 0.33)

0.38 0.45 0.40 0.44

(0.41, 0.35) (0.48, 0.42)a (0.44, 0.35) (045, 0.43)

10

20

30

0.42 0.57 0.41 0.55

(3.43, (2.95, (3.70, (3.19,

(0.47, (0.64, (0.43, (0.57,

2.97) 2.23)a 3.59) 3.10)b

0.38) 0.52)a 0.38) 0.54)b

Animals received i.p. injections of 0.33 M L(+)-lactic acid (4 mmol/kg), 1.5 h after insulin injection and measurements were performed at intervals of 10, 20, and 30 min. For details, see Section 2. Data are back log-transformed log(10) means. The values in parentheses represent the 95% confidence interval. a Significant difference with C. b Significant difference with CD.

Table 2 Effect of lactate injection on plasma and brain glucose concentration of hypoglycemic animals from all groups. Variable

Groups

Before injection

After injection (min) 10

20

Plasma glucose (mmol/l)

C (n = 3) T (n = 3) CD (n = 3) TD (n = 3)

2.1 (2.49, 1.77) 2.06 (2.32, 1.82) 2.16 (2.53, 1.84) 2.09 (2.34, 1.87)

2.27 2.22 2.35 2.30

(2.62, 1.97) (2.54, 1.94) (2.69, 2.05) (259, 2.03)

Brain glucose (mmol/kg)

C (n = 3) T (n = 3) CD (n = 3) TD (n = 3)

0.069 (0.075, 0.064) 0.07 (0.08, 0.061) 0.071 (0.082, 0.063) 0.07 (0.086, .057)

0.102 0.119 0.097 0.102

(0.12, 0.087) (0.141, 0.1)# (0.106, 0.089) (0.120, 0.087)

1.96 2.02 2.30 2.33

30 (2.23, (2.30, (2.60, (2.53,

1.73) 1.76) 2.03) 2.15)

0.122 (0.140, 0.107)# 0.154 (0.192, 0.124#,a 0.111 (0.143, 0.086) 0.0114 (0.147, .0089)

2.02 1.98 2.33 2.21

(2.26, (2.25, (2.65, (2.63,

0.139 0.189 0.115 0.121

1.81) 1.74) 2.05) 1.86)

(.163, .118)# (0.201, .0178)#,a (0.140, 0.094)# (0.153, 0.096)#

Effects of i.p. injections of 0.33 M L(+)-lactic acid (4 mmol/kg), on plasma and brain glucose concentration in hypoglycemic animals. Measurements were performed at intervals of 10, 20, and 30 min. For details, see Section 2. Data are back log-transformed log(10) means. The values in parentheses represent the 95% confidence interval. a Significant difference with C. # Significant difference with pre-injection values.

Table 3 Resting values of serum glucagon and epinephrine concentration in the groups of study.

Glucagon (Pg/ml) Epinephrine (Pg/ml)

Control

Trained

Control diabetic

Trained diabetic

41.6 ± 3.9 65.7 ± 7.4

42.6 ± 4.4 68.3 ± 9.8

61.3 ± 13.2a 45 ± 4.6a

60.7 ± 9.1a 47.8 ± 10.2a

Values are means ± SD, each value is the average for six animals. a Significant difference with control (P < 0.01).

ascertained in these trials were as follows: (1) diabetes markedly increases MCT1 and MCT2 expression in the cortex and in the hippocampus portions of the brain (2) the endurance training sessions increases MCT1 expression in the cortex and the hippocampus of the brain and the MCT2 expression in the cortex of the diabetic rats and (3) increased expression of MCT1 and MCT2 enhances the brain lactate uptake during hypoglycemia and preserves the brain metabolism in the hypoglycemia state. Induced diabetes markedly increases MCT1 and MCT2 expression in the cortex and in the hippocampus as previously described (Pierre et al., 2007). A feature commonly observed in STZ-induced diabetic rats is an elevation of circulating levels of the lactate (Enoki et al., 2003; Nikooie et al., 2013). It is purported that the enhanced expression of MCT1 and MCT2 is an adaptive process to allow the brain to use the circulating lactate and other monocarboxylic acids. Consistent with this hypothesis are the findings that

MCT2 expression in the brain is most abundant in the first 3 postnatal weeks and supports brain lactate utilization and metabolism (Rafiki et al., 2003) and in adulthood, cerebral MCT1 expression was shown to be up-regulated after 3 weeks consumption of a ketogenic diet (Leino et al., 2001). In addition, we observed that adaptation of MCT1 and MCT2 to diabetes do not occur uniformly throughout the brain as it only takes place in certain areas of the brain (e.g. hippocampus or cortex) but not others (e.g. cerebellum). Although the specific reasons for such regional heterogeneities are unknown, they are likely to be related to differential activation of brain regions to hypoglycemia (Leino et al., 2001). It has been shown that some regions of the brain (e.g. cortex, hypothalamus, and brainstem) are more active in response to hypoglycemia in comparison to others (e.g. cerebellum) in type 1 diabetes (Leino et al., 2001). Therefore, the differential activation of brain regions to hypoglycemia can partially explain the observed

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Fig. 3. Time course changes of serum glucagon (a) and epinephrine (c) concentration during hypoglycemia periods. In order to calculation of plasma D [glucagon] and plasma D [epinephrine] the peak (the highest value that obtained throughout the hypoglycemia period) minus resting value was calculated for each animal, then the average of each groups was calculated (b and d). Values are means ± SD, control (n = 6), trained (n = 6), control diabetic (n = 6), trained diabetic (n = 6). *Significant difference between groups (P < 0.05).

regional heterogeneities in MCTs expression, however, other possible factors such as differential distribution of effector mechanisms, e.g. receptors and/or transduction mechanisms, are also involved. The data collected in this study are the first to describe gradual and extensive changes in the expression of MCT1 and MCT2 in the brain in healthy and diabetic adult animals exposed to endurance training. In the diabetic animals, endurance training increased the brains MCTs expression in a regional-specific manner and had no effect on the healthy animals. Increased MCT expression in the cardiac and skeletal muscle has been reported in previous studies (Enoki et al., 2003; Nikooie et al., 2013), however, the effects of exercise are not limited to skeletal muscle, and the non-exercised tissues can also be affected by exercise. Exercise has long been thought to primarily modify the brains molecular physiology by increasing the amount of the brain-derived neurotrophic factor (Stranahan et al., 2009), nevertheless, other factors such as circulating lactate may also mediate the non-muscle effects of exercise (Lezi et al., 2013). It has been demonstrated that lactate could mediate some of the effects that exercise has on the brain, and that lactate itself can act as a partial exercise mimetic (Lezi et al., 2013). Another study also found that exercise increases brain peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1a) mRNA levels (Steiner et al., 2011). In number of tissues, PGC-1a acts as a master regulator of MCTs regulation and cell energy metabolism. Taken together, increased blood lactate concentration during exercise and up-regulation of PGC-1a mRNA can explains the exercise-induced changes in the brains MCT expression in the trained diabetic animals. In order to determine the role of exercise and the enhanced expression of MCT1 and MCT2 in the brains lactate uptake, six animals from each group were selected for lactate injections (4 mmol/kg)

during hypoglycemic condition. This value of lactate was selected in order to elevate blood lactate concentration to an average value of [lactate] that animal experienced during exercise. Although the basal values of plasma lactate concentration were significantly higher in the diabetic groups, conversely, the animals of four groups have shown almost the same values of plasma lactate concentration before the lactate injection. The pre-injection values of plasma lactate concentration (0.83) were almost similar to km value of MCT2 (0.1–0.74 mM, the lowest km for MCTs) for L-lactate (Halestrap, 2012). Therefore, it can be hypothesized that the plasma lactate was almost completely metabolized by lactate consumers such as the liver, brain and heart during hypoglycemic state. Thirty minutes following the injection of lactate, the decrease in plasma lactate levels (La clearing) were greater in trained groups vs. respective sedentary controls. These higher values of lactate clearance were concomitant with their higher values of the brain lactate concentration and the brain/plasma lactate ratio. Taken together, these results imply significant effect of endurance training in the brains lactate uptake in T and TD groups. The finding could be attributed to the increased expression of the brain MCT1 and MCT2 in trained diabetic which facilities lactate uptake from circulation, but since we did not observe enhanced expression levels of MCTs in the T group, their higher values were most likely due to an increase in the activity of existing transporters stimulated by the endurance exercise. Metz et al. (2005) have demonstrated that endurance training improves impairment of lactate transport capacity in skeletal muscle of Zucker falfa rats and Pilegaard et al. (1993) have reported a higher affinity of lactate transporters in rat skeletal muscle after 7 weeks of moderateintensity treadmill training. In addition, two important notes must be considered. First, the brain lactate levels in trained diabetic

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animals did not change appreciably between 10 and 30 min following the lactate injection despite the increase in the brain/ plasma lactate ratio. This means that any lactate taken up during this interval must have been metabolized in the brain. In favor of this assertion are the observation that lactate can be rapidly metabolized in the brain during an extreme hypoglycemic state (Herzog et al., 2013; Maran et al., 2000; Thurston et al., 1983). Moreover, the brain/plasma lactate ratio in the CD group did not change between 10 and 30 min following the lactate injection, but the animals showed the decrease in the brain lactate concentration at 30 min following the lactate injection which implies significant effect of diabetes on the brain lactate usage. Second, since blood lactate levels were lower at 20 in comparison to 10 min, the lack of decrease in brain lactate levels from 10 to 20 min means that lactate movement into brain (by both carrier-mediated and diffusional processes) has a defined capacity and almost certainly becomes saturated at a specific level of blood lactate concentration (above 3.6 mM in the present study). On the other hand, when blood lactate concentration reduced to below of this value at 30 min following the lactate injection, T and TD groups demonstrated the higher values for the brain lactate concentrations and the brain/plasma lactate ratio in comparison to the C and CD groups, respectively. These findings are reported for the first time by this study and means that the brain lactate uptake is a trainable factor but the exercise-induced changes in the brains lactate uptake are more predominant at the lower levels of blood lactate concentration. Also, the significant difference between CD and TD groups for the brain lactate concentrations and the brain/plasma lactate ratio at 30 min following the lactate injection seems to suggest the higher impact of training over that of diabetes. Aside from the attained results, it should be noted that we did not use labeled lactate and this may be a clear limitation of the present study, since the brain lactate concentration and the brain/plasma lactate concentration ratio may also be affected by lactate utilization, anaerobic production of lactate, or both. In the healthy animals the plasma glucose levels were reduced 20 min following the lactate injection. Because the diabetic animals did not show similar reduction and their basal levels of plasma insulin were much lower than healthy groups, this finding suggests the possible stimulation of insulin secretion by high levels of blood lactate (Ribes et al., 1979). The large increase (twofold) of brain glucose concentration following the lactate injection was possibly the result of a decreased brain glucose oxidation. Unfortunately, we have no evidence to confirm this assertion, but other studies have shown that lactate can partly replace glucose as fuel for brain undergoing a hypoglycemic state (Herzog et al., 2013; Miller et al., 1984; Thurston et al., 1983). Nevertheless, lactate oxidation in the brain could not return the lowered brain glucose concentration to resting values. In order to determine whether exercise-induced increased lactate transport at the blood–brain barrier contributes to hypoglycemia unawareness, glucagon and epinephrine responses to hypoglycemia were also investigated. Exercise had no effect on counterregulatory responses in the healthy trained animals as previously reported (Koyama et al., 2002; Rattarasarn et al., 1994). On the other hand, glucagon responses to hypoglycemia were almost absent and epinephrine responses were blunted in both CD as previously reported (Cryer, 1992; Maran et al., 2000) and TD. The absent of glucagon response leaves diabetic animals dependent on epinephrine to counter falling glucose levels during hypoglycemia. Because TD animals also showed the slower response of epinephrine in comparison to CD, it be concluded that despite performing endurance training for a long period, hypoglycemia unawareness still remains. The blunted response of epinephrine in TD animals may probably relate to a diminished sympathetic drive to the adrenal medulla resulted in lower epinephrine release

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after the endurance training (Deuster et al., 1989; Kjær et al., 1988). Duo to the frequent, prolonged exercise bouts the rats were hypoglycemic not only at the end of each exercise bout, but also between training sessions and may have adapted to hypoglycemia by a reduced sympathetic nervous output to the adrenal medulla. An increased sensitivity of subcutaneous adipose tissue to the lipolytic action of epinephrine due to an enhanced response of the beta-adrenergic pathways (Gerich et al., 1973) and exerciseinduced reduction in the antilipolytic action of catecholamine’s mediated by a2-adrenergic receptors (Richterova et al., 2004) are others possible mechanisms that interpret the diminished epinephrine response to hypoglycemia in TD animals. In conclusion, the results of this study determined that diabetes and endurance training significantly increased the monocarboxylate transporters (MCTs) expression in the brain, and thus facilitated brain uptake of lactate to preserve brain metabolism during hypoglycemia, despite blunting the response of glucagon and epinephrine to hypoglycemia in diabetic animals. Acknowledgments This study was supported by a Centre grant from the Neuroscience Research Center of Kerman. We also acknowledge all our collaborators. References Bergersen, L., Waerhaug, O., Helm, J., Thomas, M., Laake, P., Davies, A.J., Wilson, M.C., Halestrap, A.P., Ottersen, O.P., 2001. A novel postsynaptic density protein: the monocarboxylate transporter MCT2 is colocalized with delta-glutamate receptors in postsynaptic densities of parallel fibre-Purkinje cell synapses. Exp. Brain Res. 136, 523–534. Bloomgarden, Z.T., 2005. Concepts of insulin resistance. Metab. Syndr. Relat. Disord. 3, 284–293. Boumezbeur, F., Petersen, K.F., Cline, G.W., Mason, G.F., Behar, K.L., Shulman, G.I., Rothman, D.L., 2010. The contribution of blood lactate to brain energy metabolism in humans measured by dynamic 13c nuclear magnetic resonance spectroscopy. J. Neurosci. 30, 13983–13991. Cryer, P.E., 1992. Iatrogenic hypoglycemia as a cause of hypoglycemia-associated autonomic failure in IDDM. Diabetes 41, 255–260. Deuster, P.A., Chrousos, G.P., Luger, A., DeBolt, J.E., Trostmann, U.H., Kyle, S.B., Montgomery, L.C., Loriaux, D.L., 1989. Hormonal and metabolic responses of untrained, moderately trained, and highly trained men to three exercise intensities. Metabolism 38, 141–148. Enoki, T., Yoshida, Y., Hatta, H., Bonen, A., 2003. Exercise training alleviates MCT1 and MCT4 reductions in heart and skeletal muscles of STZ-induced diabetic rats. J. Appl. Physiol. 94, 2433–2438. Galassetti, P., Mann, S., Tate, D., Neill, R.A., Costa, F., Wasserman, D.H., Davis, S.N., 2001. Effects of antecedent prolonged exercise on subsequent counterregulatory responses to hypoglycemia. Am. J. Physiol. Endocrinol. Metab. 280, E908–E917. Galassetti, P., Tate, D., Neill, R.A., Morrey, Sachiko., Wasserman, D.H., Davis, S.N., 2003. Effect of antecedent hypoglycemia on counterregulatory responses to subsequent euglycemic exercise in type 1 diabetes. Diabetes 52, 1761–1769. Gerhart, D.Z., Enerson, B.E., Zhdankina, O.Y., Leino, R.L., Drewes, L.R., 1997. Expression of monocarboxylate transporter MCT1 by brain endothelium and glia in adult and suckling rats. Am. J. Physiol. 273, E207–E213. Gerhart, D.Z., Enerson, B.E., Zhdankina, O.Y., Leino, R.L., Drewes, L.R., 1998. Expression of the monocarboxylate transporter MCT2 by rat brain glia. Glia 22, 272–281. Gerich, J.E., Langlois, M., Noacco, C., Karam, J., Forsham, P.H., 1973. Lack of a glucagon response to hypoglycemia in diabetes: evidence for an intrinsic pancreatic alpha-cell defect. Science 182, 171–173. Gutman, I., Wahlefeld, A.W., 1974. L-lactate determination with lactate dehydrogenase and NAD. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, second ed. Academic Press, New York, pp. 1464–1468. Halestrap, A.P., 2012. The monocarboxylate transporter family-structure and functional characterization. IUBMB Life 64, 1–9. Herzog, R.I., Jiang, L., Herman, P., Zhao, C., Sanganahalli, B.G., Mason, G.F., Hyder, F., Rothman, D.L., Sherwin, R.S., Behar, K.L., 2013. Lactate preserves neuronal metabolism and function following antecedent recurrent hypoglycemia. J. Clin. Invest. 123, 1988–1998. Kjær, M., Bangsbo, J., Lortie, G., Galbo, H., 1988. Hormonal response to exercise in man: influence of hypoxia and physical training. Am. J. Physiol. 254, R197– R203. Koyama, Y., Galassetti, P., Coker, R.H., Pencek, R.R., Lacy, D.B., Davis, S.N., Wasserman, D.H., 2002. Prior exercise and the response to insulin-induced hypoglycemia in the dog. Am. J. Physiol. Endocrinol. Metab. 282, E1128–E1138.

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