Kathryn F. LaNoue

Diabetic Retinopathy Seeing Beyond Glucose-Induced Microvascular Disease

Diabetic retinopathy remains a frightening prospect to patients and frustrates physicians. Destruction of damaged retina by photocoagulation remains the primary treatment nearly 50 years after its introduction. The diabetes pandemic requires new approaches to understand the pathophysiology and improve the detection, prevention, and treatment of retinopathy. This perspective considers how the unique anatomy and physiology of the retina may predispose it to the metabolic stresses of diabetes. The roles of neural retinal alterations and impaired retinal insulin action in the pathogenesis of early retinopathy and the mechanisms of vision loss are emphasized. Potential means to overcome limitations of current animal models and diagnostic testing are also presented with the goal of accelerating therapies to manage retinopathy in the face of ongoing diabetes.

Diabetic Retinopathy: More Than Meets the Eye

Retinal microvascular dysfunction in diabetes is a major component of diabetic retinopathy. This review highlights recent observations regarding the cellular anatomy that contributes to the blood-retinal barrier and its breakdown, the alterations of macroglial, neuronal, and microglial cells in diabetes, and how these changes lead to loss of vision. In addition, the effects of systemic pathophysiologic influences, including metabolic control, blood pressure, and fluid volume on the formation of diabetic macular edema are discussed. Finally, an overview of inflammatory mechanisms and responses in the retina in diabetes is provided. Together, these new observations provide a broader clinical and research perspective on diabetic retinal vascular dysfunction than previously considered, and provide new avenues for improved treatments to prevent loss of vision.

Neuroglial metabolism in the awake rat brain: CO2 fixation increases with brain activity

Glial cells are thought to supply energy for neurotransmission by increasing nonoxidative glycolysis; however, oxidative metabolism in glia may also contribute to increased brain activity. To study glial contribution to cerebral energy metabolism in the unanesthetized state, we measured neuronal and glial metabolic fluxes in the awake rat brain by using a double isotopic-labeling technique and a two-compartment mathematical model of neurotransmitter metabolism. Rats (n = 23) were infused simultaneously with 14C-bicarbonate and [1-13C]glucose for up to 1 hr. The 14C and 13C labeling of glutamate, glutamine, and aspartate was measured at five time points in tissue extracts using scintillation counting and 13C nuclear magnetic resonance of the chromatographically separated amino acids. The isotopic 13C enrichment of glutamate and glutamine was different, suggesting significant rates of glial metabolism compared with the glutamate-glutamine cycle. Modeling the 13C-labeling time courses alone and with 14C confirmed significant glial TCA cycle activity (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(V_{\mathrm{PDH}}^{(\mathrm{g})},{\sim}0.5\) \end{document} μmol · gm-1 · min-1) relative to the glutamate-glutamine cycle (VNT) (∼0.5-0.6 μmol · gm-1 · min-1). The glial TCA cycle rate was ∼30% of total TCA cycle activity. A high pyruvate carboxylase rate (VPC, ∼0.14-0.18 μmol · gm-1 · min-1) contributed to the glial TCA cycle flux. This anaplerotic rate in the awake rat brain was severalfold higher than under deep pentobarbital anesthesia, measured previously in our laboratory using the same 13C-labeling technique. We postulate that the high rate of anaplerosis in awake brain is linked to brain activity by maintaining glial glutamine concentrations during increased neurotransmission.

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 α‐ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the α‐amino group to glutamate. Measurements of synthesis of 14C‐glutamate and of 14C‐glutamine from H14CO3 have shown that 14C‐amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mm. The specific radioactivity of 14C‐glutamine indicated that ∼30% of glutamine was derived from 14CO2 fixation. Using gabapentin, an inhibitor of the cytosolic branched‐chain aminotransferase, synthesis of 14C‐glutamate and 14C‐glutamine from H14CO3− was inhibited by 31%. These results suggest that transamination of α‐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 H14CO3−. 14C‐metabolites in brain extracts were measured to determine net incorporation of 14CO2 and specific radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from 14CO2 fixation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for ∼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.

Recruitment of Compensatory Pathways to Sustain Oxidative Flux With Reduced Carnitine Palmitoyltransferase I Activity Characterizes Inefficiency in Energy Metabolism in Hypertrophied Hearts

Background— Transport rates of long-chain free fatty acids into mitochondria via carnitine palmitoyltransferase I relative to overall oxidative rates in hypertrophied hearts remain poorly understood. Furthermore, the extent of glucose oxidation, despite increased glycolysis in hypertrophy, remains controversial. The present study explores potential compensatory mechanisms to sustain tricarboxylic acid cycle flux that resolve the apparent discrepancy of reduced fatty acid oxidation without increased glucose oxidation through pyruvate dehydrogenase complex in the energy-poor, hypertrophied heart. Methods and Results— We studied flux through the oxidative metabolism of intact adult rat hearts subjected to 10 weeks of pressure overload (hypertrophied; n=9) or sham operation (sham; n=8) using dynamic 13C–nuclear magnetic resonance. Isolated hearts were perfused with [2,4,6,8,10,12,14,16-13C8] palmitate (0.4 mmol/L) plus glucose (5 mmol/L) in a 14.1-T nuclear magnetic resonance magnet. At similar tricarboxylic acid cycle rates, flux through carnitine palmitoyltransferase I was 23% lower in hypertrophied (P<0.04) compared with sham hearts and corresponded to a shift toward increased expression of the L–carnitine palmitoyltransferase I isoform. Glucose oxidation via pyruvate dehydrogenase complex did not compensate for reduced palmitate oxidation rates. However, hypertrophied rats displayed an 83% increase in anaplerotic flux into the tricarboxylic acid cycle (P<0.03) that was supported by glycolytic pyruvate, coincident with increased mRNA transcript levels for malic enzyme. Conclusions— In cardiac hypertrophy, fatty acid oxidation rates are reduced, whereas compensatory increases in anaplerosis maintain tricarboxylic acid cycle flux and account for a greater portion of glucose oxidation than previously recognized. The shift away from acetyl coenzyme A production toward carbon influx via anaplerosis bypasses energy, yielding reactions contributing to a less energy-efficient heart.

Cardiolipin Remodeling by ALCAT1 Links Oxidative Stress and Mitochondrial Dysfunction to Obesity

Oxidative stress causes mitochondrial dysfunction and metabolic complications through unknown mechanisms. Cardiolipin (CL) is a key mitochondrial phospholipid required for oxidative phosphorylation. Oxidative damage to CL from pathological remodeling is implicated in the etiology of mitochondrial dysfunction commonly associated with diabetes, obesity, and other metabolic diseases. Here, we show that ALCAT1, a lyso-CL acyltransferase upregulated by oxidative stress and diet-induced obesity (DIO), catalyzes the synthesis of CL species that are highly sensitive to oxidative damage, leading to mitochondrial dysfunction, ROS production, and insulin resistance. These metabolic disorders were reminiscent of those observed in type 2 diabetes and were reversed by rosiglitazone treatment. Consequently, ALCAT1 deficiency prevented the onset of DIO and significantly improved mitochondrial complex I activity, lipid oxidation, and insulin signaling in ALCAT1(-/-) mice. Collectively, these findings identify a key role of ALCAT1 in regulating CL remodeling, mitochondrial dysfunction, and susceptibility to DIO.

Interrelationships between malate-aspartate shuttle and citric acid cycle in rat heart mitochondria☆

Abstract The control of substrate utilization was investigated using rat heart mitochondria incubated under conditions of state 4, state 3, oligomycin-inhibited and uncoupled respiration. A comparison of the changes in metabolite levels after addition of pyruvate or acetylcarnitine showed that cycle flux was controlled primarily at citrate synthase, which appeared to be regulated by the intramitochondrial oxalacetate concentration. A secondary control site located between α-ketoglutarate and succinate was revealed by addition of oligomycin which caused an increased flux through α-ketoglutarate dehydrogenase and thereby a decreased efflux of α-ketoglutarate from the mitchondria. The increased α-ketoglutarate dehydrogenase activity was caused by the fall of the ATP/ADP ratio, which by increasing the availability of GDP for substrate level phosphorylation produced diminished product inhibition by succinyl CoA. Because transport of NADH into mitochondria by the malate-aspartate shuttle requires a stoichiometric influx of malate and glutamate and efflux of aspartate and α-ketoglutarate from the mitochondria, alterations in the rate of efflux of α-ketoglutarate can significantly alter flux through the shuttle and the rate of utilization of cytosolic NADH. Studies with mitochondria oxidizing glutamate and malate in the presence and absence of acetylcarnitine or octanoate showed that α-ketoglutarate efflux could be strongly effected by the intramitochondrial ATP/ADP ratio. Rates of α-ketoglutarate efflux were also increased by extramitochondrial malate (half maximal stimulation at 0.6 mM). Glutamate transamination was inhibited by uncoupling agents and subsequent studies, measuring intramitochondrial aspartate levels, showed that this was due to an inhibition of aspartate efflux from the mitochondria in the uncoupled state. Conclusions drawn regarding regulation of the transport of reducing equivalents were verified using mitochondria supplemented with the extramitochondrial components of the malate aspartate shuttle.

Kinetic analysis of dynamic 13C NMR spectra: Metabolic flux, regulation, and compartmentation in hearts

Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.

Substrate–Enzyme Competition Attenuates Upregulated Anaplerotic Flux Through Malic Enzyme in Hypertrophied Rat Heart and Restores Triacylglyceride Content. Attenuating Upregulated Anaplerosis in Hypertrophy

Recent work identifies the recruitment of alternate routes for carbohydrate oxidation, other than pyruvate dehydrogenase (PDH), in hypertrophied heart. Increased carboxylation of pyruvate via cytosolic malic enzyme (ME), producing malate, enables “anaplerotic” influx of carbon into the citric acid cycle. In addition to inefficient NADH production from pyruvate fueling this anaplerosis, ME also consumes NADPH necessary for lipogenesis. Thus, we tested the balance between PDH and ME fluxes in hypertrophied hearts and examined whether low triacylglyceride (TAG) was linked to ME-catalyzed anaplerosis. Sham-operated (SHAM) and aortic banded rat hearts (HYP) were perfused with buffer containing either 13C-palmitate plus glucose or 13C glucose plus palmitate for 30 minutes. Hearts remained untreated or received dichloroacetate (DCA) to activate PDH and increase substrate competition with ME. HYP showed a 13% to 26% reduction in rate pressure product (RPP) and impaired dP/dt versus SHAM (P<0.05). DCA did not affect RPP but normalized dP/dt in HYP. HYP had elevated ME expression with a 90% elevation in anaplerosis over SHAM. Increasing competition from PDH reduced anaplerosis in HYP+DCA by 18%. Correspondingly, malate was 2.2-fold greater in HYP than SHAM but was lowered with PDH activation: HYP=1419±220 nmol/g dry weight; HYP+DCA=343±56 nmol/g dry weight. TAG content in HYP (9.7±0.7 &mgr;mol/g dry weight) was lower than SHAM (13.5±1.0 &mgr;mol/g dry weight). Interestingly, reduced anaplerosis in HYP+DCA corresponded with normalized TAG (14.9±0.6 &mgr;mol/g dry weight) and improved contractility. Thus, we have determined partial reversibility of increased anaplerosis in HYP. The findings suggest anaplerosis through NADPH-dependent, cytosolic ME limits TAG formation in hypertrophied hearts.

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

Abstract: 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 →α‐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 α‐[14C]ketoglutarate to [14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α‐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 α‐ketoglutarate to glutamate may frequently be the rate‐limiting step in this process of glutamate synthesis.