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[Studies on the excitation-metabolism coupling mechanism in brown adipose tissues, 2; Effects of K+ and norepinephrine on the lipolysis in brown adipose tissues (author's transl)].

This investigation was undertaken to clarify the lipolytic response caused by K+ and norepinephrine in brown adipose tissues. 1. Lipolytic response was obviously observed at the concentration of 50 mM in the K+-induced stimulation, and at 1.0 microgram/ml in the norepinephrine-induced stimulation, respectively. 2. K+- and norepinephrine-stimulated lipolysis were inhibited in the Ca2+-deficient tissues, and were inhibited by the addition of Mg2+, Mn2+ and La3+. 3. In the K+-stimulated lipolysis Ca2+ could be substituted only by Sr2+, while in the norepinephrine-stimulated lipolysis the substitution was possible with both Sr2+ and Ba2+. 4. K+- and norepinephrine-stimulated lipolysis were inhibited by the addition of propranolol and procaine. Accordingly, these positive results suggest that K+- and norepinephrine-stimulated lipolysis are dependent on the presence of Ca2+, and are related to the movements of Ca2+ and cyclic AMP system in the cells.

Adipose Tissue, Brown

Cyclic nucleotide metabolism coupled to cytodifferentiation of Blastocladiella emersonii.

Cyclic GMP and cyclic AMP levels during growth and differentiation of B. emersonii were measured by use of chemical, biochemical, and immunologic assays. Of particular interest was the finding that net synthesis of cyclic GMP occurred during a single stage of its life cycle, sporulation, when intracellular levels increased 50- to 100-fold in a process requiring protein and RNA synthesis.

Blastocladiella

Parallel evolution of pairs of dehydrogenase isoenzymes.

Lactate dehydrogenase and glycerol 3-phosphate dehydrogenase are metabolically coupled by the anaerobic dismutation of glyceraldehyde 3-phosphate and by the NAD redox state. This causes the concentrations of lactate and glycerol 3-phosphate to accumulate proportionally during anaerobic muscle contraction; these concentrations are high relative to those in aerobic tissues such as liver. We show that the isoenzymes of lactate dehydrogenase and glycerol 3-phosphate dehydrogenase from chicken breast muscle have Km values for lactate and glycerol 3-phosphate, respectively, that are 10-fold higher than the Km values measured for the lactate dehydrogenase and glycerol 3-phosphate dehydrogenase isoenzymes from chicken liver. The association of proportionally higher Km values with the potential for proportionally higher accumulation of substrates suggests that the isoenzymes of lactate dehydrogenase and glycerol 3-phosphate dehydrogenase from chicken muscle have evolved in parallel as a coupled metabolic unit distinct from the coupled isoenzymes in liver. The parallelism observed for the reduced substrates extends to the oxidized substrates, and to the coenzymes, NAD+ and NADH.

Animals

Cell-to-cell communication and myogenesis.

Cell-to-cell communication was characterized in prefusion chick embryo myoblast cultures, and it was determined that the prefusion myoblasts can interact via gap junctions, ionic coupling, and metabolic coupling. The biological relevance of this communication was supported by the detection of gap junctions between myoblasts in embryonic muscle. Communication was also examined in fusion-arrested cultures to determine its potential relationship to fusion competency. In cultures that were fusion arrested by treatment with either 1.8 mM ethyleneglycolbis-(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), 3.3 X 10(-6) M 5-bromodeoxyuridine (BUdR), or 1 microgram/ml cycloheximide (CHX), both gap junctions and ionic coupling were present. Therefore, it is possible to conclude that cell communication is not a sufficient property by itself, to generate fusion between myob-asts. The potential role of communication in myogenesis is discusssed with respect to these observations.

Cell Communication

The thermodynamic degree of coupling between metabolism and sodium transport in frog skin.

The tightness of coupling between two processes is advantageously evaluated by the thermodynamic degree of coupling q, varying in absolute value from zero for uncoupled processes to unity for processes which are related stoichiometrically. Two methods for the determination of q in the active pathway in frog skin have been developed, employing amiloride to abolish active sodium transport. The values of q in 6 frog skins varied, but were always less than unity (mean 0.79 +/- 0.06 S.E. according to one method, 0.78 +/- 0.06 S.E. according to the other). This indicates that metabolism and sodium transport are incompletely coupled in this tissue even when passive transepithelial leakage pathways are taken into account.

Amiloride

Evolutionary diversification of invertase paralogs couples carbon metabolism and sexual reproduction in fission yeasts.

Dynamic patterns of gene gain and loss play a major role in the diversification of eukaryotes, reflecting adaptation to a broad range of ecological contexts. Reconstructing the evolutionary history of genes provides a powerful framework for understanding how functional innovation shapes life-history traits. Here we report a comprehensive analysis of gene gain and loss across the fission yeast clade, whose evolutionary trajectory remains elusive. Reductive evolution of metabolic genes is a major contributor to species diversification, as observed in other fungal taxa. Notably, we uncovered an evolutionary scenario in which an ancestral gene duplication was followed by lineage-specific loss of one or the other paralog, except in S. pombe, which retained both. We demonstrate that these paralogs encode catalytically-active invertases, named Inv1 and Inv2, with distinct enzymatic properties, localization, regulation, and physiological roles. Inv1 is a secreted enzyme subject to glucose catabolite repression and is the sole invertase required for sucrose assimilation, resembling canonical yeast invertases. In contrast, Inv2 is intracellular, constitutively expressed, and required for inducing sexual differentiation in response to nutrient availability. Overall, these findings reveal an unexpected role for carbon metabolism in modulating the haploid-diploid cycle of fission yeasts, suggesting that diversification of core metabolic functions may contribute to adaptation to environments with distinct sugar compositions.

Evolution

Coupling of metabolism and electrical activity in cortical astrocytes.

Cortical mouse astrocytes in culture were impaled with two-channel microelectrodes. These mouse astrocytes have the same responses to different K+ concentrations, ouabain, and glutamate as cultured rat astrocytes, with the exception that a large barium-sensitive K+ conductance clamps the membrane potential at the K+ equilibrium potential. Glycolytic and mitochondrial inhibitors have little effect on the mouse astrocytes. Total blockade of energy metabolism leads to an irreversible, calcium-dependent depolarization, but only if applied for longer than 45 min. Increasing the extracellular K+ concentration to 60 mM increases the intracellular K+ concentration by 43 mM and the bicarbonate concentration by 22 mM and leads to a concomitant fast swelling. Together with the 20 mM increase in Cl- concentration reported in the literature this is a good indication for a Boyle- and Conway-mediated K(+)-anion influx with water. This influx is accomplished by the depolarization-induced opening of Cl- channels as reported in the literature. In conclusion, ischemia-like conditions have little direct, immediate impact on astrocytes. In contrast, ischemia-induced release of substances from neurones, such as K+, produces an immediate and fast response.

Animals

Leucine transport in Escherichia coli. The resolution of multiple transport systems and their coupling to metabolic energy.

The multiple active transport systems mediating L-leucine accumulation in Escherichia coli strain 7 (K12) and ML 308-225 have been examined. In addition to the previously characterized osmotic shock-sensitive LS (L-leucine-specific) and LIV-I (L-leucine; L-isoleucine-, and L-valine-specific) activities, a third system (designated LIV-II) has been detected, confirming a report by Rahmanian et al. (RAHMANIAN, M., CLAUS, D.R., and OXENDER, D. L. (1973) J. Bacteriol. 116, 1258-1266). This third system transports L-leucine, L-isoleucine, and L-valine with a relatively low affinity (apparent transport Km equals 4 muM for L-leucine) and it is resistant to repression by cell growth on L-leucine. Exploitation of these properties and of the differential sensitivity of the three transport activities to inhibition by L-leucine analogues permits estimation of the contribution by each system to the total transport activity under varying conditions. Such experiments show that, unlike systems LS and LIV-I, system LIV-II is resistant to osmotic shock. The L-leucine, L-iosleucine, and L-valine transport activity in membrane vesicles from strain ML 308-225 has the properties of system LIV-II. Although the L-leucine transport activities in strains 7 and ML 308-225 are in all other respects similar, membrane vesicles from strain 7 do not transport L-leucine, L-isoleucine, or L-valine. L-leucine transport under various conditions of energy supply has been measured in strain ML 308-225 and the corresponding Mg-2+-ATP-ASE-DEFICIENT STRAIN, DL-54. These measurements support the view that the osmotic shock-sensitive LS and LIV-I activities depend on the synthesis of ATP, while the osmotic shock-resistant LIV-II activity depends on the energized membrane state generated by electron flow but not on ATP synthesis, per se. This conclusion is not supported by the inhibitory effects of the energy poisons arsenate and 2,4-dinitrophenol, but these compounds may have secondary chemical effects on the transport systems.

Arsenates

Mechanochemical coupling in flagella. IV. Absence of coupling between metabolism and motility in Chaetopterus spermatozoa.

The metabolic rate of suspensions of spermatozoa from the annelid worm, Chaetopterus, does not change significantly when the viscosity of the suspension is increased by the addition of methyl cellulose. At high viscosities, where the spermatozoa swim with about 1/3 the normal beat frequency and 1.5-1.8 times the normal bend angle, the calculated power output against external viscous resistances increases to 4-5 times its normal value. This large increase in power output can be accommodated without an increase in metabolic rate only if internal viscous resistances to flagellar bending are relatively low.

Animals

Metabolic models of microcirculatory regulation.

The functions and integrity of body tissues are critically dependent on an adequate oxygen supply. Because the transport of oxygen to the cells is intimately linked to the microcirculation, the concept of microcirculation-metabolism coupling has received much attention. In essence, the metabolic theory of intrinsic control of the microcirculation states that microvascular tone is locally modulated to maintain adequate oxygen levels in the parenchymal cells. We propose a two-component control system for the regulation of tissue O2 delivery in accordance with metabolic needs. A precapillary sphincter control mechanism maintains tissue PO2 by governing the number of perfused capillaries. Functional capillary density in turn determines surface area available for diffusion and capillary-to-cell diffusion distance. On the other hand, the arteriolar control system modulates local blood flow in accordance with parenchymal O2 utilization and thereby minimizes changes in capillary PO2 when the O2 availability/demand ratio is decreased. We propose that the precapillary sphincters are more sensitive to changes in tissue PO2 than are the flow-regulating arterioles. Consequently, for mild stresses, adequate tissue oxygenation is maintained mainly by precapillary sphincter control of diffusion parameters without the need for changes in blood flow. However, as metabolic stresses become greater, blood flow regulation becomes the dominant factor in the control of tissue O2 delivery. Thus, by working in concert, the local mechanisms regulating microvascular resistance and effective capillary density provide a wide margin of safety against the development of cellular hypoxia.

Capillaries

Identification and distribution of gap junctions in the mesoderm of the developing chick limb bud.

Sub-ridge, core, anterior and posterior borders of mesoderm were dissected from stages 22-24 chick wing buds to investigate whether structures for intercellular coupling develop between mesenchymal cells. Fine structure was examined using techniques of transmission electron microscopy, freeze-fracture and scanning electron microscopy. Gap (communicating) junctions which were observed between mesenchymal cells of all limb bud regions were distributed between apposed cell bodies, points of contact between cell processes and other cell bodies, and between contacting tips of slender cell projections. In addition particularly in the the subridge region, filopodia were observed to extend through the intercellular matrix to contact other cells several micrometers distant. The observations reported in this paper show that mesodermal cells throughout the limb have the structural capability for electrotonic and metabolic coupling during a critical period of morphogenesis in the avian limb. Whether intercellular signals which are thought to be transmitted through gap junctions are active in normal limb development remains to be investigated.

Animals

Lactylome Reprogramming Mediates Therapeutic Response and Adaptation to Neoadjuvant Chemotherapy in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) exhibits high prevalence in China and poor prognosis despite neoadjuvant chemotherapy (NACT), with significant chemoresistance development. Tumor-associated metabolic reprogramming and NACT-induced cellular stress promote lactate accumulation, which serves as a precursor for lysine lactylation (Kla), a post-translational modification potentially regulating cancer progression. We hypothesized that systematic characterization of the lactylome in response to NACT could reveal critical molecular mechanisms underlying treatment and identify new therapeutic vulnerabilities in ESCC. Herein, through comprehensive proteomic and lactylome profiling of tumor and adjacent normal adjacent tissues from 31 ESCC patients (with or without NACT treatment), we identified 8281 proteins and 1836 Kla sites across 62 samples. NACT induced substantial lactylome alterations with 307 differentially expressed Kla sites predominantly in nonhistone proteins involved in DNA damage response and metabolic pathways. Our data revealed that while NACT-induced suppression of energy metabolism, coupled with upregulated 3-hydroxy-3-methylglutaryl reductase degradation 1 complex expression, may exert potential proapoptotic effects, the activation of ribosome biogenesis and increased nucleoprotein lactylation triggered tumor-protective mechanisms. Mechanistically, we demonstrated that DNA damage and elevated lactate levels induced poly(ADP-ribose) polymerase 1 K654 lactylation, enhancing its enzymatic activity and augmenting poly(ADP-ribosyl)ation of downstream targets, potentially playing a pivotal role in chemotherapy resistance-associated pathways. This comprehensive tissue-level landscape of Kla dynamics in ESCC response to chemotherapy establishes Kla as a critical regulatory mechanism in treatment response, potentially offering novel therapeutic targets and predictive biomarkers for personalized treatment strategies.

Humans

Urinary acidification in turtle bladder is due to a reversible proton-translocating ATPase.

Adverse proton electrochemical gradients (delta muH) applied across the turtle urinary bladder decrease active H+ transport in this epithelium. A delta muH of 180 mV abolishes both transport and its tightly coupled metabolic reaction. Larger gradients should, in theory, reverse the direction of H+ transport and the metabolic reaction leading to synthesis of ATP if the pump is an ATPase, or cause an increase in the oxidized state of a redox pair if it is a redox pump. To distinguish between these two possibilities, we measured ATP levels in epithelial cells that were poisoned to inhibit cellular mechanisms of ATP synthesis. At delta muH of 120 mV or less no ATP synthesis was found. At delta muH of greater than 120 mV there was a linear increase in ATP synthesis. Dinitrophenol, a H+ carrier, prevented synthesis at delta muH of 310 mV. Dicyclohexylcarbodiimide, an inhibitor of H+ transport that works at the cell surface, prevented ATP synthesis at delta muH of 310 mV. These results demonstrate that a reversible proton-translocating ATPase in the mucosal border of the bladder is the H+ pump responsible for urinary acidification.

Adenosine Triphosphatases

Adenosine in rat cerebral cortex: its determination, normal values, and correlation to AMP and cyclic AMP during shortlasting ischemia.

It has recently been suggested that adenosine is a metabolic coupling factor responsible for an increased cerebral blood flow during hypoxia or increased functional activity. However, tissue adenosine concentrations have been reported to increase in situations previously shown to be unassociated with changes in tissue AMP concentrations. The present experiments were undertaken to assess cerebral cortex concentrations of adenosine under normal circumstances, and to relate changes in adenosine, AMP and cyclic AMP during shortlasting ischemia. Following freezing and extraction of tissue, adenosine was measured using high pressure liquid chromatography. In paralyzed and anaesthetized (70% N2O) rats, freezing of tissue through intact skull bone gave an adenosine concentration of 0.9 +/- 0.1 mumol-kg-1 (mean +/- S.E.M.). With freezing through the exposed dura the concentration was 3 times as high with a large scatter. When special precautions were taken to avoid tissue trauma during craniotomy, the adenosine concentration was 1.1 +/- 0.1 mumol-kg-1. It is concluded that previously reported values are erroneously high. During the first 60 s of total ischemia there was a linear correlation between increase in AMP and in adenosine concentration (as well as between adenosine and cyclic AMP concentrations). It is concluded that increases in tissue adenosine concentration only occur if AMP accumulates. However, since (relative) changes in adenosine concentrations are at least twice those of AMP, analyses of adenosine may provide sensitive measures of a change in phosphorylation state.

Adenosine