Objectives: Our aim was to evaluate the structure and function of the upper gastrointestinal tract in a group of patients with autism who had gastrointestinal symptoms.
Study design: Thirty-six ...children (age: 5.7 ± 2 years, mean ± SD) with autistic disorder underwent upper gastrointestinal endoscopy with biopsies, intestinal and pancreatic enzyme analyses, and bacterial and fungal cultures. The most frequent gastrointestinal complaints were chronic diarrhea, gaseousness, and abdominal discomfort and distension.
Results: Histologic examination in these 36 children revealed grade I or II reflux esophagitis in 25 (69.4%), chronic gastritis in 15, and chronic duodenitis in 24. The number of Paneth’s cells in the duodenal crypts was significantly elevated in autistic children compared with non-autistic control subjects. Low intestinal carbohydrate digestive enzyme activity was reported in 21 children (58.3%), although there was no abnormality found in pancreatic function. Seventy-five percent of the autistic children (27/36) had an increased pancreatico-biliary fluid output after intravenous secretin administration. Nineteen of the 21 patients with diarrhea had significantly higher fluid output than those without diarrhea.
Conclusions: Unrecognized gastrointestinal disorders, especially reflux esophagitis and disaccharide malabsorption, may contribute to the behavioral problems of the non-verbal autistic patients. The observed increase in pancreatico-biliary secretion after secretin infusion suggests an upregulation of secretin receptors in the pancreas and liver. Further studies are required to determine the possible association between the brain and gastrointestinal dysfunctions in children with autistic disorder. (J Pediatr 1999;135:559-63)
Several recent studies have demonstrated that the metabolism of energy substrates takes place in multiple compartments in both astrocytes and synaptic terminals from brain. There are a number of ...differences in the metabolism of astrocytes and synaptic terminals primarily due to the localization of key enzymes such as pyruvate carboxylase and glutamine synthetase in astrocytes. The present study determined the rates of 14CO2 production from several energy substrates by primary cultures of astrocytes and cortical synaptic terminals from rat brain. The rates of 14CO2 production from labelled substrates by astrocytes were 0.96 +/- 0.13, 11.13 +/- 0.67, 10.51 +/- 0.35, 24.92 +/- 1.66 and 4.80 +/- 0.50 for D-6-14Cglucose, L-U-14Clactate, D-3-hydroxy3-14Cbutyrate, L-U-14Cglutamine and L-U-14Cma-late, respectively. The rates of 14CO2 production were also measured in the presence of 5 mM aminooxyacetate (AOAA) to determine the effect of inhibiting the malate-aspartate shuttle and other transaminase reactions on the oxidation of energy substrates. In astrocytes the addition of AOAA decreased the rate of glutamine oxidation 5-fold, consistent with other studies showing that glutamine enters the TCA cycle via transamination. AOAA increased the rate of 14CO2 production from labelled glucose 4-fold, suggesting that inhibition of alanine biosynthesis profoundly alters the utilization of glucose by astrocytes. AOAA also increased the oxidation of lactate and 3-hydroxybutyrate 36 and 58%, respectively. The rates of 14CO2 production from labelled substrates by synaptic terminals were 13.12 +/- 1.05, 35.29 +/- 3.58, 17.66 +/- 1.95, 30.18 +/- 1.10 and 9.95 +/- 1.29, respectively, for glucose, lactate, 3-hydroxybutyrate, glutamine and malate, demonstrating that all substrates were oxidized at a higher rate by synaptic terminals than by astrocytes. The addition of AOAA decreased the rate of 14CO2 production from labelled lactate by 57% suggesting that the use of lactate for energy in synaptic terminals is tightly coupled to the activity of the malate-aspartate shuttle. AOAA had no effect on the rate of 14CO2 production from labelled glutamine, demonstrating that exogenous glutamine enters the TCA cycle in synaptic terminals via glutamate dehydrogenase, not via transamination as is the case with astrocytes. AOAA had no significant effect on the rates of oxidation of glucose, 3-hydroxybutyrate and malate by synaptic terminals. These findings demonstrate that inhibiting transamination with AOAA had very different effects on the oxidation of energy substrates in the two preparations, suggesting that the regulation of metabolism is quite different in astrocytes and synaptic terminals.
Several reports indicate that lactate can serve as an energy substrate for the brain. The rate of oxidation of this substrate by cultured rat brain astrocytes was 3-fold higher than the rate with ...glucose, suggesting that lactate can serve as an energy source for these cells. Since transport into the astrocytes may play an important role in regulating nutrient use by individuals types of brain cells, we investigated the uptake of L-U-14Clactate by primary cultures of rat brain astrocytes. Measurement of the net uptake suggested two carrier-mediated mechanisms and an Eadie-Hofstee type plot of the data supported this conclusion revealing 2 Km values of 0.49 and 11.38 mM and Vmax values of 16.55 and 173.84 nmol/min/mg protein, respectively. The rate of uptake was temperature dependent and was 3-fold higher at pH 6.2 than at 7.4, but was 50% less at pH 8.2. Although the lactate uptake carrier systems in astrocytes appeared to be labile when incubated in phosphate buffered saline for 20 minutes, the uptake process exhibited an accelerative exchange mechanism. In addition, lactate uptake was altered by several metabolic inhibitors and effectors. Potassium cyanide and alpha-cyano-4-hydroxycinnamate inhibited lactate uptake, but mersalyl had little or no effect. Phenylpyruvate, alpha-ketoisocaproate, and 3-hydroxybutyrate at 5 and 10 mM greatly attenuated the rate of lactate uptake. These results suggest that the availability of lactate as an energy source is regulated in part by a biphasic transport system in primary astrocytes.
Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such ...as glutamine to provide pyruvate to maintain TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial fractions had very high mitochondrial malic enzyme (mME) activity with a
K
m and a
V
max of 0.37 mM and 32.6 nmol/min/mg protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The
K
m and
V
max for malic enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced glutathione in synaptic terminals.
: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the ...enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free‐moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min‐fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C‐amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for 14Cglutamate and 14Cglutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for U‐14Cglutamate and U‐14Cglutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of 14Cglutamine in the interstitial space when 14Cglutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.
Since lactate released by glial cells may be a key substrate for energy in neurons, the kinetics for the uptake of L-U-14Clactate by cortical synaptic terminals from 7- to 8-week-old rat brain were ...determined. Lactate uptake was temperature-dependent, and increased by 64.9% at pH 6.2, and decreased by 43.4% at pH 8.2 relative to uptake at pH 7.3. Uptake of monocarboxylic acids was saturable with increasing substrate concentration. Eadie-Hofstee plots of the data gave evidence of two carrier-mediated uptake mechanisms with a high-affinity Km of 0.66 mM and Vmax of 3.66 mM for pyruvate, and a low-affinity system with a Km of 9.9 mM for both lactate and pyruvate and Vmax values of 16.6 and 23.1 nmol/30 s/mg protein for lactate and pyruvate, respectively. Saturable uptake was seen in the presence of 10 mM alpha-cyano-4-hydroxycinnamate. Lactate transport by synaptic terminals was much more sensitive to inhibition by sulfhydryl reagents than transport in astrocytes. Addition of 0.5 and 2 mM mersalyl decreased the uptake of 1 mM lactate by synaptic terminals by 59.3 and 66.37%, respectively. Pyruvate moderately decreased lactate transport, whereas 3-hydroxybutyrate had little effect. Quercetin, an inhibitor of lactate release, had little effect on the content of 14C lactate in synaptic terminals, supporting the concept that the majority of lactate produced within brain is from glial cells. Oxidation of L-U-14Clactate by synaptosomes was saturable, and yielded a Km of 1.23 mM and a Vmax of 116 nmol/h/mg protein. Overall the studies show that synaptic terminals from adult brain have a high capacity for transport and oxidation of lactate, consistent with the proposed role for this compound in metabolic trafficking in brain. Furthermore, the data provide kinetic evidence of two carrier-mediated mechanisms for monocarboxylic acid transport by synaptosomes and demonstrate that uptake of lactate by synaptic terminals is regulated differently than transport by astrocytes. Uptake of lactate by synaptic terminals also has differences from the systems described for neurons.
Malate has a number of key roles in the brain, including its function as a tricarboxylic acid (TCA) cycle intermediate, and as a participant in the malate-aspartate shuttle. In addition, malate is ...converted to pyruvate and CO2 via malic enzyme and may participate in metabolic trafficking between astrocytes and neurons. We have previously demonstrated that malate is metabolized in at least two compartments of TCA cycle activity in astrocytes. Since malic enzyme contributes to the overall regulation of malate metabolism, we determined the activity and kinetics of the mitochondrial and cytosolic forms of this enzyme from cultured astrocytes. Malic enzyme activity measured at 37 degrees C in the presence of 0.5 mM malate was 4.15 +/- 0.47 and 11.61 +/- 0.98 nmol/min/mg protein, in mitochondria and cytosol, respectively (mean +/- SEM, n = 18-19). Malic enzyme activity was also measured in the presence of several endogenous compounds, which have been shown to alter intracellular malate metabolism in astrocytes, to determine if these compounds affected malic enzyme activity. Lactate inhibited cytosolic malic enzyme by a noncompetitive mechanism, but had no effect on the mitochondrial enzyme. alpha-Ketoglutarate inhibited both cytosolic and mitochondrial malic enzymes by a partial noncompetitive mechanism. Citrate inhibited cytosolic malic enzyme competitively and inhibited mitochondrial malic enzyme noncompetitively at low concentrations of malate, but competitively at high concentrations of malate. Both glutamate and aspartate decreased the activity of mitochondrial malic enzyme, but also increased the affinity of the enzyme for malate. The results demonstrate that mitochondrial and cytosolic malic enzymes have different kinetic parameters and are regulated differently by endogenous compounds previously shown to alter malate metabolism in astrocytes. We propose that malic enzyme in brain has an important role in the complete oxidation of anaplerotic compounds for energy.
Leucine and alpha-ketoisocaproate (alpha-KIC) were perfused at increasing concentrations into rat brain hippocampus by microdialysis to mimic the conditions of maple syrup urine disease. The effects ...of elevated leucine or alpha-KIC on the oxidation of L-U-14Cglutamate and L-U-14Cglutamine in the brain were determined in the non-anesthetized rat. 14CO2 generated by the metabolic oxidation of 14Cglutamate and 14Cglutamine in brain was measured following its diffusion into the eluant during the microdialysis. Leucine and alpha-KIC exhibited differential effects on 14CO2 generation from radioactive glutamate on glutamine. Infusion of 0.5 mM alpha-KIC increased 14Cglutamate oxidation approximately 2-fold; higher concentrations of alpha-KIC did not further stimulate 14Cglutamate oxidation. The enhanced oxidation of 14Cglutamate may be attributed to the function of alpha-KIC as a nitrogen acceptor from 14Cglutamate yielding 14Calpha-ketoglutarate, an intermediate of the tricarboxylic acid cycle. 14Cglutamine oxidation was not stimulated as much as 14Cglutamate oxidation and only increased at 10 mM alpha-KIC reflecting the extra metabolic step required for its oxidative metabolism. In contrast, leucine had no effect on the oxidation of either 14Cglutamate or 14Cglutamine. In maple syrup urine disease elevated alpha-KIC may play a significant role in altered energy metabolism in brain while leucine may contribute to clinical manifestations of this disease in other ways.
Studies from several groups have provided evidence that glutamate and glutamine are metabolized in different compartments in astrocytes. In the present study we measured the rates of 14CO2 production ...from U-14Cglutamate and U-14Cglutamine, and utilized both substrate competition experiments and the transaminase inhibitor aminooxyacetic acid (AOAA) to obtain more information about the compartmentation of these substrates in cultured rat brain astrocytes. The rates of oxidation of 1 mM glutamine and glutamate were 26.4 +/- 1.4 and 63.0 +/- 7.4 nmol/h/mg protein, respectively. The addition of 1 mM glutamate decreased the rate of oxidation of glutamine to 26.3% of the control rate, demonstrating that glutamate can effectively compete with the oxidation of glutamine by astrocytes. In contrast, the addition of 1 mM glutamine had little or no effect on the rate of oxidation of glutamate by astrocytes, demonstrating that the glutamate produced intracellularly from exogenous glutamine does not dilute the glutamate taken up from the media. The addition of 5 mM AOAA decreased the rate of 14CO2 production from glutamine to 29.2% of the control rate, consistent with earlier studies by our group. The addition of 5 mM AOAA decreased the rate of oxidation of concentrations of glutamate < or = 0.1 mM by approximately 50%, but decreased the oxidation of 0.5-1 mM glutamate by only approximately 20%, demonstrating that a substantial portion of glutamate enters the tricarboxylic acid (TCA) cycle via glutamate dehydrogenase (GDH) rather than transamination, and that as the concentration of glutamate increases the relative proportion entering the TCA cycle via GDH also increases. To determine if the presence of an amino group acceptor (i.e. a ketoacid) would increase the rate of metabolism of glutamate, pyruvate was added in some experiments. Addition of 1 mM pyruvate increased the rate of oxidation of glutamate, and the increase was inhibited by AOAA, consistent with enhanced entry of glutamate into the TCA cycle via transamination in the presence of pyruvate. Enzymatic studies showed that pyruvate increased the activity of mitochondrial aspartate aminotransferase (AAT). Overall, the data demonstrate that glutamate formed intracellularly from glutamine enters the TCA cycle primarily via transamination, but does not enter the same TCA cycle compartment as glutamate taken up from the extracellular milieu. In contrast, extracellular glutamate enters the TCA cycle in astrocytes via both transamination and GDH, and can compete with, or dilute, the oxidation of glutamate produced intracellularly from glutamine.
Glucose has been thought to be the primary substrate for energy metabolism in the developing lung; however, alternate substrates are used for energy metabolism in other organs. To examine the role of ...alternate substrates in the lung, we measured rates of oxidation of glutamine, glucose, lactate, and 3-hydroxybutyrate in type II pneumocytes isolated from d 19 fetal rat lungs by measuring the production of 14CO2 from labeled substrates. Glutamine had a rate of 24.36 +/- 4.51 nmol 14CO2 produced/ h/mg of protein (mean +/- SEM), whereas lactate had a significantly higher rate, 40.29 +/- 4.42. 3-Hydroxybutyrate had a rate of 14.91 +/- 1.93. The rate of glucose oxidation was 2.13 +/- 0.36, significantly lower than that of glutamine. To examine the interactions of substrates normally found in the intracellular milieu, we measured the effect of unlabeled substrates as competitors on labeled substrate. This identifies multiple metabolic compartments of energy metabolism. Glucose, but not lactate, inhibited the oxidation of glutamine, suggesting a compartmentation of tricarboxylic acid cycle activity, rather than simple dilution by glucose. Glucose and lactate had reciprocal inhibition. Our data suggest at least two separate compartments in the type II cells for substrate oxidation, one for glutamine metabolism and a second for glucose metabolism. In summary, we have documented that glutamine and other alternate substrates are oxidized preferentially over glucose for energy metabolism in the d 19 fetal rat lung type II pneumocyte. In addition, we have delineated some of the compartmentation that occurs within the developing type II cell, which may determine how these substrates are used.
![Compartmentation of [14C]Gl...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0022-3042/small "Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis")
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