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Proton NMR spectrum of lactate and alanine at 9.4 Tesla. Regions A, B, C, and D were fit to the expected number of peaks, and processed as described in the text.
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Recent studies of isotope exchange across lactate dehydrogenase (LDH) and alanine aminotransferase (AAT) in hearts call into question whether both reactions are in equilibrium. To compare the oxidative and non-oxidative fates of glycolytic end products, isolated rabbit hearts were perfused with 5 mM [2-13C] glucose and 2.5 mM [3-13C] pyruvate: with...
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... the four regions are labeled A 1974). Glycogen levels were determined by hy- through D as in Figure 1, the resulting calculation drolysis of the glycogen to glucose and measure- for percentage [3-13 C] is: ment of the glucose by standard spectroscopic techniques. (Bergmeyer, 1974) Alanine levels were determined by high resolution proton and 13 C NMR %[3-13 C] alanine= 2 * A (B−D)+2 * A * 100% spectroscopy. ...
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In this study, we investigated how colonic epithelial cells maintained pyridine nucleotide (NADH/NAD⁺) redox homeostasis upon acute metabolic variation imposed by glucose deprivation or supplementation with mitochondrial substrates, succinate and malate/glutamate (M/G). Our results showed that low glucose caused cellular NADH/NAD⁺ redox imbalance t...
Ever since it was shown for the first time that lactate can support neuronal function in vitro as a sole oxidative energy substrate, investigators in the field of neuroenergetics have been debating the role, if any, of this glycolytic product in cerebral energy metabolism. Our experiments employed the rat hippocampal slice preparation with electrop...
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... extracts were obtained with the use of a 5-mm probe placed in a 14.1 T or 9.4 T Burker NMR magnet. 1 H spectra of lactate and alanine methyl group protons revealed the fractional 13 C enrichment of each metabolite. [39][40][41] The fractional contribution (Fc) of LCFAs to acetyl CoA entering the tricarboxylic acid (TCA) cycle was determined by glutamate isotopomer analysis from the 13 C NMR spectra as previously described. 32,42,43 Enrichment of glutamate from glucose and lactate was used to index the relative extent of carbohydrate oxidation and incorporation into the TCA cycle. ...
Brown adipose tissue (BAT) is correlated to cardiovascular health in rodents and humans, but the physiological role of BAT in the initial cardiac remodeling at the onset of stress is unknown. Activation of BAT via 48 h cold (16°C) in mice following transverse aortic constriction (TAC) reduced cardiac gene expression for LCFA uptake and oxidation in male mice and accelerated the onset of cardiac metabolic remodeling, with an early isoform shift of carnitine palmitoyltransferase 1 (CPT1) toward increased CPT1a, reduced entry of long chain fatty acid (LCFA) into oxidative metabolism (0.59 ± 0.02 vs. 0.72 ± 0.02 in RT TAC hearts, p < .05) and increased carbohydrate oxidation with altered glucose transporter content. BAT activation with TAC reduced early hypertrophic expression of β‐MHC by 61% versus RT‐TAC and reduced pro‐fibrotic TGF‐β1 and COL3α1 expression. While cardiac natriuretic peptide expression was yet to increase at only 3 days TAC, Nppa and Nppb expression were elevated in Cold TAC versus RT TAC hearts 2.7‐ and 2.4‐fold, respectively. Eliminating BAT thermogenic activation with UCP1 KO mice eliminated differences between Cold TAC and RT TAC hearts, confirming effects of BAT activation rather than autonomous cardiac responses to cold. Female responses to BAT activation were blunted, with limited UCP1 changes with cold, partly due to already activated BAT in females at RT compared to thermoneutrality. These data reveal a previously unknown physiological mechanism of UCP1‐dependent BAT activation in attenuating early cardiac hypertrophic and profibrotic signaling and accelerating remodeled metabolic activity in the heart at the onset of cardiac stress.
... Cell-tracking experiments performed over a period of 6 hr on LnCAP cells treated with MK2206 revealed a decrease in motility from 0.72 ± 0.04 to 0.46 ± 0.03 µm/s, p<0.05 as shown in Figure 2C. Additionally, cellular metabolism was also altered, with both Alanine enrichment from 13 C-labeled glucose can be attributed to alanine transaminase activity that converts glucose-derived pyruvate to alanine (21). A similar trend was observed with lactate (79.3 ± 13.0 vs. 37.6 ±10.0 %, DMSO vs. MK2206, p<0.05), as shown in Figure 2F. ...
The PI3K/AKT/mTOR (PAM) signaling pathway is frequently mutated in prostate cancer. Specific AKT inhibitors are now in advanced clinical trials, and this study investigates the effect of MK2206, a non-ATP-competitive inhibitor, on the cellular metabolism of prostate cancer cells. We observed a reduction in cell motility and aerobic glycolysis in prostate cancer cells with treatment. These changes were not accompanied by a reduction in the ratio of high-energy phosphates or a change in total protein levels of enzymes and transporters involved in glycolysis. However, a decreased ratio of NAD+/NADH was observed, motivating the use of hyperpolarized magnetic resonance spectroscopy (HP-MRS) to detect treatment response. Spectroscopic experiments were performed on tumor spheroids, 3D structures that self-organize in the presence of an extracellular matrix. Treated spheroids showed decreased lactate production with on-target inhibition confirmed using IHC, demonstrating that HP-MRS can be used to probe treatment response in prostate cancer spheroids and can provide a biomarker for treatment response. Mol Cancer Res; 1-8. ©2018 AACR.
... The heart is a highly metabolically active and energy demanding organ that is able to utilize a variety of energy substrates, dependent upon their relative abundance, prevailing hormonal conditions, workload and oxygen availability (1). A number of experimental methods are available for investigating cardiac metabolism, including the use of radioactive tracer approaches (2,3) and steady-state incorporation of 13 C labeled metabolites (4,5). It has recently been shown to be possible to probe cardiac metabolism in real time (both in vivo and ex. ...
Hyperpolarized (13) C MR measurements have the potential to display non-linear kinetics. We have developed an approach to describe possible non-first-order kinetics of hyperpolarized [1-(13) C] pyruvate employing a system of differential equations that agrees with the principle of conservation of mass of the hyperpolarized signal. Simultaneous fitting to a second-order model for conversion of [1-(13) C] pyruvate to bicarbonate, lactate and alanine was well described in the isolated rat heart perfused with Krebs buffer containing glucose as sole energy substrate, or glucose supplemented with pyruvate. Second-order modeling yielded significantly improved fits of pyruvate-bicarbonate kinetics compared with the more traditionally used first-order model and suggested time-dependent decreases in pyruvate-bicarbonate flux. Second-order modeling gave time-dependent changes in forward and reverse reaction kinetics of pyruvate-lactate exchange and pyruvate-alanine exchange in both groups of hearts during the infusion of pyruvate; however, the fits were not significantly improved with respect to a traditional first-order model. The mechanism giving rise to second-order pyruvate dehydrogenase (PDH) kinetics was explored experimentally using surface fluorescence measurements of nicotinamide adenine dinucleotide reduced form (NADH) performed under the same conditions, demonstrating a significant increase of NADH during pyruvate infusion. This suggests a simultaneous depletion of available mitochondrial NAD(+) (the cofactor for PDH), consistent with the non-linear nature of the kinetics. NADH levels returned to baseline following cessation of the pyruvate infusion, suggesting this to be a transient effect. Copyright © 2016 John Wiley & Sons, Ltd.
... Tissue metabolites were extracted from frozen ventricle with the use of 7% perchloric acid and assayed according to published methods. 36,37 Myocardial triglyceride content was extracted and quantified as described previously. 38 In vitro nuclear magnetic resonance (NMR) data were collected on a 9.4 T NMR system (Bruker Instruments). ...
... 38 In vitro nuclear magnetic resonance (NMR) data were collected on a 9.4 T NMR system (Bruker Instruments). 36,37 The fractional 13 C enrichment acetyl-CoA produced from [2,4,6,8,10,12,14,16-13 C 8 ] palmitate (Fc) was determined from the relative signal intensities and multiplet peaks of Preconditioning raises LCFA use and ischaemic function the glutamate 4-and 3-carbon resonance signals, using established methods. 37 -40 Malonyl-CoA and acetyl-CoA were assayed by highpressure liquid chromatography with UV detection, as previously described, and the tissue content expressed as per gram wet weight of tissue. ...
... Increased non-oxidative, glycolytic metabolism in the ischaemic myocardium is confirmed by elevated lactate and alanine content, both of which are metabolites of the glycolytic end product, pyruvate ( Table 2). 37 As expected, lactate was increased in ischaemic tissue in all groups. In control tissue, the alanine and lactate content was approximately equal, whereas lactate represented slightly more of this pool of glycolytic end products in stenosis (subepicardium: 60%; subendocardium: 63%) and SWOP + stenosis (subepicardium: 59%; subendocardium: 63%) tissue. ...
Although a major mechanism for cardioprotection is altered metabolism, little is known regarding metabolic changes in ischaemic preconditioning and subsequent ischaemia. Our objective was to examine the effects of the second window of preconditioning (SWOP), the delayed phase of preconditioning against infarction and stunning, on long-chain free fatty acid (LCFA) oxidation during ischaemia in chronically instrumented, conscious pigs.
We studied three groups: (i) normal baseline perfusion (n = 5); (ii) coronary artery stenosis (CAS; n = 5); (iii) CAS 24 h following 2 × 10 min coronary occlusions and 10 min reperfusion (n = 7). Ischaemia was induced by a left anterior descending (LAD) stenosis (40% flow reduction) for 90 min, dropping systolic wall thickening by 72%. LCFA oxidation was assessed following LAD infusion of (13)C palmitate, i.e. during control or stenosis, by in vitro nuclear magnetic resonance of the sampled myocardium. Stenosis reduced subendocardial blood flow subendocardially, but not subepicardial, yet induced transmural reductions in LCFA oxidation and increased non-oxidative glycolysis. During stenosis, preconditioned hearts showed normalized contributions of LCFA to oxidative ATP synthesis, despite increased lactate accumulation. SWOP induced a shift towards LCFA oxidation during stenosis, despite increased malonyl-CoA, and marked protection of contractile function with a significant improvement in systolic wall thickening.
Thus, the second window of preconditioning normalized oxidative metabolism of LCFA during subsequent ischaemia despite elevated non-oxidative glycolysis and malonyl-CoA and was linked to protection of regional contractile function resulting in improved mechanical performance. Interestingly, the metabolic responses occurred transmurally while ischaemia was restricted solely to the subendocardium.
... Tissue lactate content was assayed by UV spectrophotometric analysis [17]. Alanine content was determined by 1 H NMR relative to the signal from known lactate concentration from assays [18][19][20]. Glycogen was extracted and content measured following digestion with amyloglucosidase and detection of glycosyl units, as previously described [21]. P-AMPK/AMPK ratios were determined from total ventricular homogenates (20-30 mg) using previously described methods to isolated ventricular proteins [22,23]. ...
... Lyophilized tissue extracts were reconstituted in 0.5 mL D 2 O and proton ( 1 H) NMR spectra were collected using a 5 mm 1 H probe (Bruker Instruments, Billerica, MA) as described elsewhere [19,20]. 1 H spectra of the 3-carbon methyl group resonances of lactate and alanine revealed the fractional 13 C enrichment of each metabolite from the observed splitting of the proton resonances due to protoncarbon-13 coupling, distinguishing 12 CH 3 from 13 CH 3 groups, as previously described [18][19][20]. ...
... Lyophilized tissue extracts were reconstituted in 0.5 mL D 2 O and proton ( 1 H) NMR spectra were collected using a 5 mm 1 H probe (Bruker Instruments, Billerica, MA) as described elsewhere [19,20]. 1 H spectra of the 3-carbon methyl group resonances of lactate and alanine revealed the fractional 13 C enrichment of each metabolite from the observed splitting of the proton resonances due to protoncarbon-13 coupling, distinguishing 12 CH 3 from 13 CH 3 groups, as previously described [18][19][20]. ...
Changes in metabolic and myofilament phenotypes coincide in developing hearts. Posttranslational modification of sarcomere proteins influences contractility, affecting the energetic cost of contraction. However, metabolic adaptations to sarcomeric phenotypes are not well understood, particularly during pathophysiological stress. This study explored metabolic adaptations to expression of the fetal, slow skeletal muscle troponin I (ssTnI). Hearts expressing ssTnI exhibited no significant ATP loss during 5 min of global ischemia, while non-transgenic littermates (NTG) showed continual ATP loss. At 7 min ischemia TG-ssTnI hearts retained 80±12% of ATP versus 49±6% in NTG (P<0.05). Hearts expressing ssTnI also had increased AMPK phosphorylation. The mechanism of ATP preservation was augmented glycolysis. Glycolytic end products (lactate and alanine) were 38% higher in TG-ssTnI than NTG at 2 min and 27% higher at 5 min. This additional glycolysis was supported exclusively by exogenous glucose, and not glycogen. Thus, expression of a fetal myofilament protein in adult mouse hearts induced elevated anaerobic ATP production during ischemia via metabolic adaptations consistent with the resistance to hypoxia of fetal hearts. The general findings hold important relevance to both our current understanding of the association between metabolic and contractile phenotypes and the potential for invoking cardioprotective mechanisms against ischemic stress. This article is part of a Special Issue entitled "Possible Editorial".
... In Protocol 2, the four sham and hypertrophied groups, with and without DCA-treatment received 13 C glucose and unlabeled palmitate (Sham, n=4; DCA Sham, n=8; Hyp, n=6; DCA Hyp, n=7 ). 13 C and 1 H NMR spectra were collected from in vitro samples, reconstituted in deuterium oxide, using a 5 mm 13 C/ 1 H NMR probe in a 14.1 T NMR magnet (Bruker Instruments, Inc., Billerica, MA) (4-6,13). 13 C spectra were collected with WALTZ-16 proton decoupling. ...
... In Protocol 2, the four sham and hypertrophied groups, with and without DCA-treatment received 13 C glucose and unlabeled palmitate (Sham, n=4; DCA Sham, n=8; Hyp, n=6; DCA Hyp, n=7 ). 13 C and 1 H NMR spectra were collected from in vitro samples, reconstituted in deuterium oxide, using a 5 mm 13 C/ 1 H NMR probe in a 14.1 T NMR magnet (Bruker Instruments, Inc., Billerica, MA) (4-6,13). 13 C spectra were collected with WALTZ-16 proton decoupling. ...
... In Protocol 2, the four sham and hypertrophied groups, with and without DCA-treatment received 13 C glucose and unlabeled palmitate (Sham, n=4; DCA Sham, n=8; Hyp, n=6; DCA Hyp, n=7 ). 13 C and 1 H NMR spectra were collected from in vitro samples, reconstituted in deuterium oxide, using a 5 mm 13 C/ 1 H NMR probe in a 14.1 T NMR magnet (Bruker Instruments, Inc., Billerica, MA) (4-6,13). 13 C spectra were collected with WALTZ-16 proton decoupling. ...
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 (13)C 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 micromol/g dry weight) was lower than SHAM (13.5+/-1.0 micromol/g dry weight). Interestingly, reduced anaplerosis in HYP+DCA corresponded with normalized TAG (14.9+/-0.6 micromol/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.
... As shown in Figure 1, labeled glucose and endogenous glycogen contribute to the formation of both pyruvate and alanine via glycolysis. While intracellular pyruvate content is too low for NMR detection, isotopic equilibrium with the readily NMR detected alanine pool indicates enrichment of glycolytic end products (19,20). Thus, the labeled fraction of alanine (F A ) corresponds to exogenous 13 C-glucose utilization and the unlabeled fraction of alanine (1-F A ) corresponds to the endogenous glycogen utilization, which was further corrected for incorporation of 13 C glucose into glycogen (3.5 −3.8% enrichment) using 13 C NMR of glycogen extracts against a glucose concentration and enrichment standard [21]. ...
Intramyocardial lipid handling in pressure-overload-induced heart failure remains poorly understood, and the balance between endogenous and exogenous lipid utilization for mitochondrial ATP production is essentially unknown. In this study, we determined the contribution of endogenous triacylglycerols (TAG) to mitochondrial oxidation relative to that of exogenous palmitate, glucose, and endogenous glycogen in the failing, pressure-overloaded rat heart. TAG content and turnover were also assessed to determine if lipid availability and mobility were altered. Dynamic-mode (13)C NMR was performed in intact hearts from aortic banded and sham operated Spraque-Dawley rats perfused with (13)C-labeled palmitate or glucose to assess TAG turnover rate and palmitate oxidation rate. The fractional contributions from palmitate, glucose, glycogen, and TAG to mitochondrial ATP production were determined from NMR analysis of heart extracts. TAG oxidation was not evident in HF, whereas the contribution of TAG to oxidative ATP production was significant in shams. TAG content was 39% lower in HF compared to sham, and TAG turnover rate was 60% lower in HF. During adrenergic challenge, TAG sources were again not oxidized in the HF group. In early cardiac failure, endogenous TAG oxidation was reduced in parallel to increased carbohydrate oxidation, with no change in exogenous palmitate oxidation. This finding was consistent with reduced TAG storage and mobilization. These data further elucidate the role of intermediary and lipid metabolism in the progression of LVH to failure, and contribute to emerging evidence linking the disruption of myocardial substrate use to cardiomyopathies.
... 18,25 Alanine content was determined by in vitro 1 H-NMR. 26 The percent of labeled acetyl CoA entering the TCA cycle was determined from in vitro 13 C-NMR spectra. 27,28 Atrial natriuretic factor expression was determined after total RNA isolation by single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture. ...
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.
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-(13)C8] 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.
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.
... glycerol (TAG) storage and turnover rates in the intact, beating rat heart were determined for the first time using dynamic mode 13 C-NMR spectroscopy to elucidate profound differences between hearts from diabetic rats (DR, streptozotocin treatment) and normal rats (NR). The incorporation of [2,4,6,8,10,12,14, C8]palmitate into the TAG pool was monitored in isolated hearts perfused with physiological (0.5 mM palmitate, 5 mM glucose) and elevated substrate levels (1.2 mM palmitate, 11 mM glucose) characteristic of the diabetic condition. Surprisingly, although the normal hearts were enriched at a near-linear profile for Ն2 h before exponential characterization, exponential enrichment of TAG in diabetic hearts reached steady state after only 45 min. ...
... The hearts were first perfused for 30 min with medium containing unlabeled substrates. Then, one protocol provided normal hearts (n ϭ 6) and hearts from diabetics (n ϭ 6) with recirculated medium containing 0.5 mM [2,4,6,8,10,12,14, C8]palmitate plus 5 mM unlabeled glucose. The concentrations of substrates were selected to represent near-normal physiological levels. ...
... After 2 h, the hearts were freeze-clamped for additional analysis. The protocol was repeated for a second group of normal (n ϭ 8) and diabetic hearts (n ϭ 8) perfused with medium containing concentrations of substrates more characteristic of the in vivo diabetic state (1.2 mM [2,4,6,8,10,12,14, C8]palmitate plus 11 mM unlabeled glucose). ...
Triacylglycerol (TAG) storage and turnover rates in the intact, beating rat heart were determined for the first time using dynamic mode (13)C- NMR spectroscopy to elucidate profound differences between hearts from diabetic rats (DR, streptozotocin treatment) and normal rats (NR). The incorporation of [2,4,6,8,10,12,14,16-(13)C(8)]palmitate into the TAG pool was monitored in isolated hearts perfused with physiological (0.5 mM palmitate, 5 mM glucose) and elevated substrate levels (1.2 mM palmitate, 11 mM glucose) characteristic of the diabetic condition. Surprisingly, although the normal hearts were enriched at a near-linear profile for >or=2 h before exponential characterization, exponential enrichment of TAG in diabetic hearts reached steady state after only 45 min. Consequently, TAG turnover rate was determined by fitting an exponential model to enrichment data rather than conventional two-point linear analysis. In the high-substrate group, both turnover rate (DR 820+/- 330, NR 190 +/-150 nmol.min(-1).g(-1) dry wt; P< 0.001) and [TAG] content (DR 78 +/-10, NR 32+/- 4 micromol/g dry wt; P< 0.001) were greater in the diabetic group. At lower substrate concentrations, turnover was greater in diabetics (DR 530+/-300, NR 160+/- 30; P<0.05). However, this could not be explained by simple mass action, because [TAG] content was similar between groups [DR 34+/- 7, NR 39+/- 9 micromol/g dry wt; not significant (NS)]. Consistent with exponential enrichment data, (13)C fractional enrichment of TAG was lower in diabetics (low- substrate groups: DR 4+/-1%, NR 10+/- 4%, P<0.05; high-substrate groups: DR 8+/- 3%, NR 14+/- 9%, NS), thereby supporting earlier speculation that TAG is compartmentalized in the diabetic heart.
... Extensive evidence indicates a coupling of metabolic cascades to specific processes in different types of cells, and this concept is now emerging as a more general feature of cellular organization. Compartmentation of carbohydrate metabolism has been shown in a wide range of tissues (21)(22)(23), including reports on the compartmentation of glycolysis with the plasma membrane of cells (2,(24)(25)(26)(27). The physical basis for the binding of glycolytic enzymes to the plasma membrane has been characterized only in erythrocytes, mainly through the interaction of glycolytic enzymes with the transmembrane protein band 3 anion transporter (27)(28)(29). ...
... For many years, there have been reports of compartmentation of glycolytic metabolism in a wide range of tissues (21)(22)(23), including reports of one compartment of glycolysis somehow associated with the plasma membrane of cells (2,(24)(25)(26)(27). However, only in the erythrocyte (25,(27)(28)(29) has the physical basis for the plasma membrane-associated glycolytic pathway been established. ...
Compartmentation of carbohydrate metabolism has been shown in a wide range of tissues including reports of one compartment of glycolysis associated with the plasma membrane of cells. However, only in the erythrocyte has the physical basis for plasma membrane-associated glycolytic pathway been established. We have previously found that phosphofructokinase (PFK) appeared to colocalize with the fairly ubiquitous plasma membrane protein caveolin-1 (CAV-1), consistent with a role for CAV-1 as an anchor for glycolysis to the plasma membrane. To test the hypothesis that CAV-1 functions as a scaffolding protein for PFK, we transfected human lymphocytes (a cell without CAV-1 expression) with human CAV-1 cDNA. We demonstrate that expression of CAV-1 in lymphocytes results in the formation of caveolae at the plasma membrane and affects the subcellular localization of PFK by recruiting PFK to the plasma membrane. Targeting of PFK by CAV-1 also was validated by the significant colocalization between the proteins after transfection, which resulted in a correlation of 0.97 +/- 0.004 between the two fluorophores. This finding is significant in as much as it illustrates the CAV-1 feasibility of generating binding sites for glycolytic enzymes on the plasma membrane. We therefore conclude that CAV-1 functions as a scaffolding protein for PFK and that this may contribute to the elucidation of the basis for carbohydrate compartmentation to the plasma membrane in a wide variety of cell types.
