Biomedical subjects

K F LaNoue

Publications and source records attributed to K F LaNoue.

At least 19 recordsLinked to original sources

PubMed2004

Whole-brain glutamate metabolism evaluated by steady-state kinetics using a double-isotope procedure: effects of gabapentin.

Cerebral rates of anaplerosis are known to be significant, yet the rates measured in vivo have been debated. In order to track glutamate metabolism in brain glutamatergic neurons and brain glia, for the first time unrestrained awake rats were continuously infused with a combination of H14CO3- and [1 - 13C]glucose in over 50 infusions ranging from 5 to 60 min. In whole-brain extracts from these animals, the appearance of 14C in brain glutamate and glutamine and appearance of 13C in the C-4 position of glutamate and glutamine were measured as a function of time. The rate of total neuronal glutamate turnover, the anaplerotic rate of synthesis of glutamine and glutamate from H14CO3-, flux through the glutamate/glutamine cycle, and a minimum estimate of whole-brain anaplerosis was obtained. The rate of synthesis of 14C-glutamate from H14CO3- was 1.29 +/- 0.11 nmoles/min/mg protein, whereas the rate of synthesis of 14C-glutamine was 1.48 +/- 0.10 nmoles/min/mg protein compared to total glutamate turnover of 9.39 +/- 0.73 nmoles/min/mg protein. From the turnover rate of glutamine, an upper limit for flux through the glutamate/glutamine cycle was estimated at 4.6 nmoles/min/mg protein. Synthesis of glutamine from H14CO3- was substantial, amounting to 32% of the glutamate/glutamine cycle. These rates were not significantly affected by a single injection of 100 mg/kg of the antiepileptic drug gabapentin. In contrast, acute administration of gabapentin significantly lowered incorporation of H14CO3- into glutamate and glutamine in excised rat retinas, suggesting metabolic effects of gabapentin may require chronic treatment and/or are restricted to brain areas enriched in target enzymes such as the cytosolic branched chain aminotransferase. We conclude that the brain has a high anaplerotic activity and that the combination of two tracers with different precursors affords unique insights into the compartmentation of cerebral metabolism.

Acetates↗

PubMed2001

Role of specific aminotransferases in de novo glutamate synthesis and redox shuttling in the retina.

In this study aminotransferase inhibitors were used to determine the relative importance of different aminotransferases in providing nitrogen for de novo glutamate synthesis in the retina. Aminooxyacetate, which inhibits all aminotransferases, blocked de novo glutamate synthesis from H(14)CO(3)(-) by more than 60%. Inhibition of neuronal cytosolic branched chain amino acid transamination by gabapentin or branched chain amino acid transport by the L-system substrate analog, 2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid, lowered total de novo synthesis of glutamate by 30%, suggesting that branched chain amino acids may account for half of the glutamate nitrogen contributed by transamination reactions. L-cycloserine, an inhibitor of alanine aminotransferase, inhibited glutamate synthesis less than 15% when added in the presence of 5 mM pyruvate but 47% in the presence of 0.2 mM pyruvate. Although high levels of pyruvate blunted the inhibitory effectiveness of L-cycloserine, the results indicate that, under physiological conditions, alanine as well as branched chain amino acids are probably the predominant sources of glutamate nitrogen in ex vivo retinas. The L-cycloserine results were also used to evaluate activity of the malate/aspartate shuttle. In this shuttle, cytosolic aspartate (synthesized in mitochondria) generates cytosolic oxaloacetate that oxidizes cytosolic NADH via malate dehydrogenase. Because L-cycloserine inhibits cytosolic but not mitochondrial aspartate aminotransferase, L-cycloserine should prevent the utilization of aspartate but not its generation, thereby increasing levels of (14)C-aspartate. Instead, L-cycloserine caused a significant decline in (14)C-aspartate. The results suggest the possibility that shuttle activity is low in retinal Müller cells. Low malate/aspartate shuttle activity may be the molecular basis for the high rate of aerobic glycolysis in retinal Müller cells.

Acetates↗

PubMed2001

Excessive hexosamines block the neuroprotective effect of insulin and induce apoptosis in retinal neurons.

In addition to microvascular abnormalities, neuronal apoptosis occurs early in diabetic retinopathy, but the mechanism is unknown. Insulin may act as a neurotrophic factor in the retina via the phosphoinositide 3-kinase/Akt pathway. Excessive glucose flux through the hexosamine biosynthetic pathway (HBP) is implicated in the development of insulin resistance in peripheral tissues and diabetic complications such as nephropathy. We tested whether increased glucose flux through the HBP perturbs insulin action and induces apoptosis in retinal neuronal cells. Exposure of R28 cells, a model of retinal neurons, to 20 mm glucose for 24 h attenuated the ability of 10 nm insulin to rescue them from serum deprivation-induced apoptosis and to phosphorylate Akt compared with 5 mm glucose. Glucosamine not only impaired the neuroprotective effect of insulin but also induced apoptosis in R28 cells in a dose-dependent fashion. UDP-N-acetylhexosamines (UDP-HexNAc), end products of the HBP, were increased approximately 2- and 15-fold after a 24-h incubation in 20 mm glucose and 1.5 mm glucosamine, respectively. Azaserine, a glutamine:fructose-6-phosphate amidotransferase inhibitor, reversed the effect of 20 mm glucose, but not that of 1.5 mm glucosamine, on attenuation of the ability of insulin to promote cell survival and phosphorylate Akt as well as accumulation of UDP-HexNAc. Glucosamine also impaired insulin receptor processing in a dose-dependent manner but did not decrease ATP content. By contrast, in L6 muscle cells, glucosamine impaired insulin receptor processing but did not induce apoptosis. These results suggest that the excessive glucose flux through the HBP may direct retinal neurons to undergo apoptosis in a bimodal fashion; i.e. via perturbation of the neuroprotective effect of insulin mediated by Akt and via induction of apoptosis possibly by altered glycosylation of proteins. The HBP may be involved in retinal neurodegeneration in diabetes.

Adenosine Triphosphate↗

PubMed2001

Effects of adenosine receptor antagonism on protein tyrosine phosphatase in rat skeletal muscle.

Earlier studies have shown that whole body adenosine receptor antagonism increases skeletal muscle insulin sensitivity in insulin-resistant Zucker rats. To find which steps in the insulin signaling pathway are influenced by adenosine receptors, muscle from lean and obese Zucker rats, treated for 1 week with the adenosine receptor antagonist, 1,3-dipropyl-8-(4-acrylate)-phenylxanthine (BWA1433), were analyzed. All rats were first anesthetized and injected intravenously (i.v.) with 1 IU of insulin. About 3 min later the gastrocnemius was freeze clamped. Insulin receptors were partially purified on wheat germ agglutinin (WGA) columns and insulin receptor kinase activity measured in control and BWA1433-treated lean and obese Zucker rats. Protein tyrosine phosphatase (PTPase) activity was also analyzed in subcellular fractions, including the cytosolic fraction, a high-speed particulate fraction and the insulin receptor fraction eluted from WGA columns. Administration of BWA1433 increased insulin receptor kinase activity in obese but not lean Zucker rats. PTPase activities were higher in the untreated obese rat muscle particulate fractions than in the lean rat particulate fractions. The BWA1433 administration lowered the PTPase activity of the obese rats but not the lean rats. Although the PTPase activity in WGA eluate fractions containing crude insulin receptors were similar in lean and obese animals, BWA1433 administration was found to lower the PTPase activities in the fractions obtained from obese but not from the lean rats. PTPases may be upregulated in muscles from obese rats due to activated adenosine receptors. Adenosine receptor blockade, by reducing PTPase activity, may thereby increase insulin signaling.

Animals↗

PubMed2001

Nitrogen shuttling between neurons and glial cells during glutamate synthesis.

The relationship between neuronal glutamate turnover, the glutamate/glutamine cycle and de novo glutamate synthesis was examined using two different model systems, freshly dissected rat retinas ex vivo and in vivo perfused rat brains. In the ex vivo rat retina, dual kinetic control of de novo glutamate synthesis by pyruvate carboxylation and transamination of alpha-ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the alpha-amino group to glutamate. Measurements of synthesis of (14)C-glutamate and of (14)C-glutamine from H(14)CO(3) have shown that (14)C-amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mM. The specific radioactivity of (14)C-glutamine indicated that approximately 30% of glutamine was derived from (14)CO(2) fixation. Using gabapentin, an inhibitor of the cytosolic branched-chain aminotransferase, synthesis of (14)C-glutamate and (14)C-glutamine from H(14)CO(3)(-) was inhibited by 31%. These results suggest that transamination of alpha-ketoglutarate to glutamate in Müller cells is slow, the supply of branched-chain amino acids may limit flux, and that branched-chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysis suggests that the glutamate/glutamine cycle accounts for 15% of total neuronal glutamate turnover in the ex vivo retina. To examine the contribution of the glutamate/glutamine cycle to glutamate turnover in the whole brain in vivo, rats were infused intravenously with H(14)CO(3)(-). (14)C-metabolites in brain extracts were measured to determine net incorporation of (14)CO(2) and specific radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from (14)CO(2) fixation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for approximately 60% of total neuronal turnover. Finally, differences between glutamine/glutamate cycle rates in these two model systems suggest that the cycle is closely linked to neuronal activity.

Animals↗

PubMed2001

Function of leucine in excitatory neurotransmitter metabolism in the central nervous system.

A novel hypothesis for the role of branched-chain amino acids (BCAA) in regulating levels of the major excitatory neurotransmitter glutamate in the central nervous system is described. It is postulated that the branched-chain aminotransferase (BCAT) isoenzymes (mitochondrial BCATm and cytosolic BCATc) are localized in different cell types and operate in series to provide nitrogen for optimal rates of de novo glutamate synthesis. BCAA enter the astrocyte where transamination is catalyzed by BCATm, producing glutamate and branched-chain alpha-keto acids (BCKA). BCKA, which are poorly oxidized in astrocytes, exit and are taken up by neurons. Neuronal BCATc catalyzes transamination of the BCKA with glutamate. The products, BCAA, exit the neuron and return to the astrocyte. The alpha-ketoglutarate product in the neurons may undergo reductive amination to glutamate via neuronal glutamate dehydrogenase. Operation of the shuttle in the proposed direction provides a mechanism for efficient nitrogen transfer between astrocytes and neurons and synthesis of glutamate from astrocyte alpha-ketoglutarate. Evidence in favor of the hypothesis is: 1) The two BCAT isoenzymes appear to be localized separately in the neurons (BCATc) or in the astroglia (BCATm). 2) Inhibition of the shuttle in the direction of glutamate synthesis can be achieved by inhibiting BCATc using the neuroactive drug gabapentin. Although gabapentin does not inhibit BCATm, it does block de novo glutamate synthesis from alpha-ketoglutarate. 3) Conversely, gabapentin stimulates oxidation of glutamate. Inhibition of BCATc may allow BCKA to accumulate in the astroglia, thus facilitating conversion of glutamate to alpha-ketoglutarate.

Acetates↗

PubMed2000

Diabetes reduces glutamate oxidation and glutamine synthesis in the retina. The Penn State Retina Research Group.

Retinas of diabetic individuals develop early functional changes measurable by electrophysiological and psychometric testing. Using a rat model of diabetes, we previously identified diabetes-induced alterations in metabolism of the neurotransmitter glutamate which may ultimately lead to accumulation of glutamate in the retina (Diabetes, 47: 815, 1998). We therefore investigated the function of enzymes that mediate the synthesis and breakdown of glutamate in retinas from rats made diabetic by injection of streptozotocin. De novo synthesis of nitrogen-containing amino acids including glutamate, glutamine and aspartate was assessed by measuring the rate of carbon fixation in freshly dissected retinas, and was unchanged by diabetes. In contrast, the oxidation of glutamate was significantly reduced in retinas from diabetic rats (62%, P < 0.05). Furthermore, diabetic retinas were less susceptible to inhibition of glutamate oxidation by the transaminase inhibitor aminoxyacetate (80%, N.S.), compared to the significant decrease seen in control rats (61%, P < 0.001). The activity and content of glutamine synthetase were also significantly reduced in retinas from rats diabetic for 2-6 months [range of 48% (P < 0.005) to 83% (P < 0.05) compared to control]. The activity of glutamine synthetase was normalized by acute injections of insulin, but not by reducing blood sugar levels with injections of phlorizin. These results indicate two enzymatic abnormalities in the glutamate metabolism pathway in the retina during diabetes: transamination to alpha-ketoglutarate and amination to glutamine. The reduced flux through these pathways may be associated with the accumulation of glutamate. These results are also consistent with the possibility that some of the glial changes in the retina during diabetes may be caused by hypoinsulinemia rather than hyperglycemia.

Animals↗

PubMed2000

New insights into the pathophysiology of diabetic retinopathy: potential cell-specific therapeutic targets.

Diabetic retinopathy, a leading cause of vision impairment, is classically defined by its vascular lesions. This review examines how diabetes affects vascular cells, as well as neurons, macroglia, and microglia. The cellular and clinical elements of diabetic retinopathy have many features of chronic inflammation. Understanding the individual cell-specific and global inflammatory changes in the retina may lead to novel therapeutic approaches to prevent vision loss.

Diabetes Mellitus↗

PubMed1999

Potassium-channel opener cardioplegia is superior to St. Thomas' solution in the intact animal.

BACKGROUND: In isolated hearts, the potassium-channel opener pinacidil is an effective cardioplegic agent. This study tested the hypothesis that pinacidil is superior to St. Thomas' solution in the more clinically relevant intact animal. METHODS: Sixteen pigs were placed on full cardiopulmonary bypass. Hearts underwent 2 hours of global ischemia (10 degrees to 15 degrees C). Either St. Thomas' or 100 micromol/L pinacidil was administered every 20 minutes (10 mL/kg). Preischemic and postreperfusion slopes of the preload-recruitable stroke work relationship were determined. Changes in myocardial adenine nucleotide levels and cellular ultrastructure were analyzed. RESULTS: Pinacidil cardioplegia resulted in an insignificant change in the slope of the preload-recruitable stroke work relationship (40.6+/-2.1 mm Hg/mm before ischemia and 36.5+/-3.7 mm Hg/mm after ischemia; p = 0.466). In contrast, St. Thomas' solution resulted in a significant decrease in the slope after reperfusion (34.3+/-5.5 mm Hg/mm and 13.5+/-2.3 mm Hg/mm; p = 0.003). Adenine nucleotide levels, myocardial tissue water, and ultrastructural changes were similar between groups. CONCLUSIONS: Pinacidil ameliorated myocardial stunning associated with traditional hyperkalemic cardioplegia without causing significant differences in cellular metabolism.

Adenine Nucleotides↗

PubMed1998

Role of branched-chain aminotransferase isoenzymes and gabapentin in neurotransmitter metabolism.

Because it is well known that excess branched-chain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with alpha-ketoglutarate to form the branched-chain alpha-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BCAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic (BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-14C]glutamate we found that BCKAs, but not BCAAs, stimulate glutamate transamination to alpha-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an alpha-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAA/BCKA shuttle, where BCATs and BCAAs provide the amino group for glutamate synthesis from alpha-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1998

A1 adenosine receptor antagonism improves glucose tolerance in Zucker rats.

The A1 adenosine receptor (A1ar) antagonist 1,3-dipropyl-8-(p-acrylic)-phenylxanthine (BW-1433) was administered to lean and obese Zucker rats to probe the influence of endogenously activated A1ars on whole body energy metabolism. The drug induced a transient increase in lipolysis as indicated by a rise in serum glycerol in obese rats. The disappearance of the response by day 7 of chronic studies was accompanied by an increase in A1ar numbers. Glucose tolerance tests were administered to rats treated with BW-1433. Peak serum insulin levels and areas under glucose curves (AUGs) were 34 and 41% lower in treated obese animals than in controls, respectively, and 19 and 39% lower in lean animals. With chronic administration (6 wk), AUGs decreased 47 and 33% in obese and lean animals, respectively. There was no effect of BW-1433 in either lean or obese rats on weight gain or percent body fat. Thus the major sustained influence of whole body A1ar antagonism in both lean and obese animals was an increase in whole body glucose tolerance at lower levels of insulin.

Adipocytes↗

PubMed1998

Dehydrogenase regulation of metabolite oxidation and efflux from mitochondria in intact hearts.

To test how alpha-ketoglutarate dehydrogenase (alpha-KGDH) activity influences the balance between oxidative flux and transmitochondrial metabolite exchange, we monitored these rates in isolated mitochondria and in perfused rabbit hearts at an altered kinetics (Km) of alpha-KGDH for alpha-ketoglutarate (alpha-KG). In isolated mitochondria, relative Km dropped from 0.23 mM at pH = 7.2 to 0.10 mM at pH 6.8 (P < 0.05), and alpha-KG efflux decreased from 126 to 95 nmol.min-1.mg-1. In intact hearts, Km was reduced with low intracellular pH, while matching control workload and respiratory rate with increased Ca2+ (pHi = 7.20, perfusate CaCl2 = 1.5 mM; pHi = 6.89, perfusate CaCl2 = 3 +/- 1 mM). Sequential 13C nuclear magnetic resonance spectra from hearts oxidizing [2-13C]acetate provided tricarboxylic acid cycle flux and the exchange rate between alpha-KG and cytosolic glutamate (F1). Tricarboxylic acid cycle flux was 10 mumol.min-1.g-1 in both groups, but F1 fell from a control of 9.3 +/- 0.6 to 2.8 +/- 0.4 mumol.min-1.g-1 at low Km. The results indicate that increased activity of alpha-KGDH occurs at the expense of alpha-KG efflux during support of normal workloads.

Acetates↗

PubMed1998

Effects of hindlimb contraction on pressor and muscle interstitial metabolite responses in the cat.

We used the microdialysis technique to measure the interstitial concentration of several putative metabolic stimulants of the exercise pressor reflex during 3- and 5-Hz twitch contractions in the decerebrate cat. The peak increases in heart rate and mean arterial pressure during contraction were 20 +/- 5 beats/min and 21 +/- 8 mmHg and 27 +/- 9 beats/min and 37 +/- 12 mmHg for the 3- and 5-Hz stimulation protocols, respectively. All variables returned to baseline after 10 min of recovery. Interstitial lactate rose (P < 0. 05) by 0.41 +/- 0.15 and 0.56 +/- 0.16 mM for the 3- and 5-Hz stimulation protocols, respectively, and were not statistically different from one another. Interstitial lactate levels remained above (P < 0.05) baseline during recovery in the 5-Hz group. Dialysate phosphate concentrations (corrected for shifts in probe recovery) rose with stimulation (P < 0.05) by 0.19 +/- 0.08 and 0.11 +/- 0.03 mM for the 3- and 5-Hz protocols. There were no differences between groups. The resting dialysate K+ concentrations for the 3- and 5-Hz conditions were 4.0 +/- 0.1 and 3.9 +/- 0.1 meq/l, respectively. During stimulation the dialysate K+ concentrations rose steadily for both conditions, and the increase from rest to stimulation (P < 0.05) was 0.57 +/- 0.19 and 0.81 +/- 0.06 meq/l for the 3- and 5-Hz conditions, respectively, with no differences between groups. Resting dialysate pH was 6.915 +/- 0.055 and 6.981 +/- 0.032 and rose to 7.013 (P < 0.05) and 7.053 (P < 0.05) for the 3- and 5-Hz conditions, respectively, and then became acidotic (6. 905, P < 0.05) during recovery (5 Hz only). This study represents the first time simultaneous measurements of multiple skeletal muscle interstitial metabolites and pressor responses to twitch contractions have been made in the cat. These data suggest that interstitial K+ and phosphate, but not lactate and H+, may contribute to the stimulation of thin fiber muscle afferents during contraction.

Animals↗

PubMed1997

Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes.

CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C-labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C] glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate --> alpha-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14C incorporation). Low rates of conversion of alpha-[14C]ketoglutarate to [14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of alpha-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of alpha-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis.

Ammonia↗

PubMed1997

Role of the matrixin MMP-2 in multicellular organization of adipocytes cultured in basement membrane components.

Primary rat adipocytes cultured in basement membrane component gels migrated and organized into large, three-dimensional, multicellular clusters. Gross morphological changes seen during this reorganization are described. The rate of cluster formation decreased with age of the rats and was stimulated by insulin in older, but not in younger rats. Echistatin, a disintegrin, partially inhibited the formation of multicellular clusters in a concentration-dependent fashion (50% inhibitory concentration approximately 10 nM). The original extracellular matrix was initially remodeled and eventually destroyed by the time large multicellular clusters were observed. This implied that one or more matrix-degrading protease(s) were being secreted. Adipocyte-conditioned medium was found to contain a divalent cation-sensitive gelatinase activity at approximately 72 and/or approximately 62 kDa. The elution profile of this activity from gelatin-Sepharose 4B was similar to matrix metalloproteinase 2 (MMP-2, a 72-kDa matrixin with a 62-kDa mature form), and the dimethyl sulfoxide eluant from these columns contained MMP-2 immunoreactivity. MMP-2 concentration and activity were greater in conditioned medium from young than from older animals; however, insulin did not affect the amount of MMP-2 in adipocyte-conditioned media. The matrixin inhibitor 1,10-phenanthroline not only blocked gelatinase activity in zymograms but also prevented extracellular matrix remodeling and destruction, as well as adipocyte migration and the formation of cell-cell contacts in adipocyte cultures. These observations are consistent with the hypothesis that the matrixin MMP-2 is secreted by adipocytes. Whereas matrixin activity alone may not be sufficient for the formation of multicellular clusters, the data indicate that it may have a requisite role in this process.

Adipocytes↗

PubMed1997

Effect of carbonic anhydrase inhibition and acetoacetate on anaplerotic pyruvate carboxylase activity in cultured rat astrocytes.

In peripheral tissues, carbonic anhydrase (CA) inhibition secondarily decreases the anaplerotic activity of pyruvate carboxylase activity leading to a decline in citric acid cycle intermediates and glutamate. In view of the important role of pyruvate carboxylase in the brain, we examined the effects of CA inhibition on pyruvate-carboxylase-mediated [14C]CO2 fixation in cultured astrocytes from postnatal rat brains. Incubation with H[14C]O3 led to radiolabeling of metabolites found both in the cells and in the medium. These were separated by ion exchange chromatography for identification. Ethoxyzolamide (ETZ), a sulfonamide CA inhibitor (SCAI) with a heterocyclic side group, caused a 43-73% decrease in cell lysate [alpha-ketoglutarate] and 14C incorporation into major products of pyruvate carboxylation in the cell lysates and cell medium (i.e., released products). Half-maximal inhibition of [14C]CO2 fixation was observed between 1 and 3 x 10(7) M. This is similar to the IC50 value for ETZ inhibition of events in other cells that are thought to be mediated by CA. Inhibition was also observed with trifluormethanesulfonamide, an aliphatic SCAI, providing further evidence that this effect is mediated by CA. Western blot analysis using isozyme-specific antisera indicated that astrocytes contain CA II, a cytosolic isozyme, but CA III, CA IV and CA V could not be detected. This finding is unusual since the effects of SCAIs on pyruvate carboxylation in other tissues have been attributed to inhibition of the intramitochondrial isozyme. CA V. [14C]CO2 fixation was also decreased by lowering media [pyruvate] or by addition of 5 mM acetoacetate. It is hypothesized that SCAIs may inhibit pyruvate carboxylation in astrocytes by limiting the supply of bicarbonate to this enzyme while ketone bodies, by inhibiting glucose oxidation, may limit the supply of pyruvate. Interestingly, both SCAIs and ketogenic diets are used to treat adolescent forms of epilepsy. The possibility that these treatments might ultimately work by affecting anaplerotic pyruvate carboxylase activity in the brain is discussed.

Acetoacetates↗

PubMed1997

Altered metabolite exchange between subcellular compartments in intact postischemic rabbit hearts.

To examine metabolic regulation in postischemic hearts, we examined oxidative recycling of 13C within the glutamate pool (GLU) of intact rabbit hearts. Isolated hearts oxidized 2.5 mmol/L [2-13C]acetate during normal conditions (n = 6) or during reperfusion after 10 minutes of ischemia (n = 5). 13C-Nuclear magnetic resonance spectra were acquired every 1 minute. Kinetic analysis of 13C incorporation into GLU provided both tricarboxylic acid (TCA) cycle flux and the interconversion rate (F1) between the TCA cycle intermediate, alpha-ketoglutarate (alpha-KG), and the largely cytosolic GLU. The rate-pressure product in postischemic hearts was 46% of normal (P < .05). No difference in substrate utilization occurred between groups, with acetate accounting for 92% of the carbon units entering the TCA cycle at the citrate synthase step. TCA cycle flux in postischemic hearts was normal (normal hearts, 10.7 mumol.min-1.g-1; postischemic hearts, 9.4 mumol.min-1.g-1), whereas F1 was 72% lower at 2.9 +/- 0.4 versus 10.2 +/- 2.5 mumol.min-1.g-1 (mean +/- SE) in normal hearts (P < .05). From additional hearts perfused with 2.5 mmol/L [2-13C]acetate plus supplemental 5 mmol/L glucose, any potential differences in endogenous carbohydrate availability were proved not to account for the reduced rate alpha-KG and GLU exchange, which remained depressed in postischemic hearts. However, specific activities of the transaminase enzyme, catalyzing chemical exchange of alpha-KG and GLU, were the same, and transaminase flux was 100 mumol.min-1.g-1 in postischemic hearts versus 68 mumol.min-1.g-1 in normal hearts. Normal transaminase activity and the increased flux in postischemic hearts are contrary to the reduced F1. The findings indicate reduced metabolite transport rates across the mitochondrial membranes of stunned myocardium, particularly through the reversible alpha-KG-malate carrier.

Animals↗

PubMed1996

Multiplet structure of 13C NMR signal from glutamate and direct detection of tricarboxylic acid (TCA) cycle intermediates.

For the first time, 13C NMR signals are shown from 13C-enriched, low-level tricarboxylic acid (TCA) cycle intermediates from extracts of normal cardiac tissue. As the low tissue content of the key intermediates alpha-ketoglutarate (alpha-KG) and succinate (SUC) in normal, well perfused tissues has until now precluded direct NMR detection from intact tissues and tissue extracts, 13C NMR signal from glutamate has generally been used to infer the isotopomer patterns of intermediates that are in chemical exchange with glutamate. However, the required assumptions regarding intracellular compartmentation for such indirect analysis have not been previously tested, as glutamate is largely cytosolic while the TCA cycle enzymes are located in the mitochondria. Chromatographic isolation of alpha-KG and SUC from heart tissue extracts allowed isotopomer analysis to be performed for comparison with that of glutamate. At steady state, a direct relationship between glutamate and alpha-ketoglutarate isotopomers was found, but succinate isotopomers matched those of glutamate only in hearts that displayed negligible contributions from the oxidation of unlabeled endogenous carbon sources.

Animals↗