Thiamine Deprivation Produces a Liver ATP Deficit and Metabolic and Genomic Effects in Mice: Findings Are Parallel to Those of Biotin Deficiency and Have Implications for Energy Disorders.

Thiamine is one of several essential cofactors for ATP generation. Its deficiency, like in beriberi and in the Wernicke-Korsakoff syndrome, has been studied for many decades. However, its mechanism of action is still not completely understood at the cellular and molecular levels. Since it acts as a coenzyme for dehydrogenases of pyruvate, branched-chain keto acids, and ketoglutarate, its nutritional privation is partly a phenocopy of inborn errors of metabolism, among them maple syrup urine disease. In the present paper, we report metabolic and genomic findings in mice deprived of thiamine. They are similar to the ones we have previously found in biotin deficiency, another ATP generation cofactor. Here we show that thiamine deficiency substantially reduced the energy state in the liver and activated the energy sensor AMP-activated kinase. With this vitamin deficiency, several metabolic parameters changed: blood glucose was diminished and serum lactate was increased, but insulin, triglycerides, and cholesterol, as well as liver glycogen, were reduced. These results indicate a severe change in the energy status of the whole organism. Our findings were associated with modified hepatic levels of the mRNAs of several carbon metabolism genes: a reduction of transcripts for liver glucokinase and fatty acid synthase and augmentation of those for carnitine palmitoyl transferase 1 and phosphoenolpyruvate carboxykinase as markers for glycolysis, fatty acid synthesis, beta-oxidation, and gluconeogenesis, respectively. Glucose tolerance was initially increased, suggesting augmented insulin sensitivity, as we had found in biotin deficiency; however, in the case of thiamine, it was diminished from the 3rd week on, when the deficient animals became undernourished, and paralleled the changes in AKT and mTOR, 2 main proteins in the insulin signaling pathway. Since many of the metabolic and gene expression effects on mice deprived of thiamine are similar to those in biotin deficiency, it may be that they result from a more general impairment of oxidative phosphorylation due to a shortage of ATP generation cofactors. These findings may be relevant to energy-related disorders, among them several inborn errors of metabolism, as well as common energy disorders like obesity, diabetes, and neurodegenerative illnesses.

The role of KATP channels on propofol preconditioning in a cellular model of renal ischemia-reperfusion.

BACKGROUND: Propofol (2,6-diisopropylphenol) has been shown to protect several organs, including the kidneys, from ischemia-reperfusion (I-R)-induced injury. Although propofol affects adenosine triphosphate-sensitive potassium (K(ATP)) channels in nonrenal tissues, it is still not clear by which mechanisms propofol protects renal cells from such damage. In this study, we investigated whether propofol induces renal preconditioning through renal K(ATP) channels. METHODS: A reversible ATP depletion (antimycin A) followed by restoration of substrate supply in LLC-PK1 cells was used as an in vitro model of renal I-R. Cell viability was assessed by dimethylthiazol-diphenyltetrazol bromide and trypan blue dye exclusion test assays. Apoptosis was evaluated by annexin V-fluorescein isothiocyanate staining by flow cytometry and immunofluorescence. Propofol treatments were initiated at various time intervals: 1 or 24 h before ischemia, only during ischemia, or only during reperfusion. To evaluate the mechanisms of propofol protection, specific K(ATP) channel inhibitors or activators were used in some experiments during propofol pretreatment. RESULTS: Propofol attenuated I-R injury on LLC-PK1 cells when present either 1 or 24 h before initiated I-R, and also during the recovery period, but not when added only during ischemia. Propofol pretreatment significantly protected LLC-PK1 from I-R-induced apoptosis. The protective effect of propofol was prevented by glibenclamide (a sarcolemmal ATP-dependent K(+) channel blocker) and decreased by 5-hydroxidecanoic acid (a mitochondrial ATP-dependent K(+) channel blocker), but it was not modified by diazoxide (a selective opener of ATP-sensitive K(+) channel). CONCLUSION: Propofol protected cells against apoptosis induced by I-R. This protection was probably due to a preconditioning effect of propofol and was, at least in part, mediated by K(ATP) channels.

Astrogliosis is temporally correlated with enhanced neurogenesis in adult rat hippocampus following a glucoprivic insult.

2-Deoxy-d-glucose (2-DG) administration causes transient depletion of glucose derivates and ATP. Hence, it can be used in a model system to study the effects of a mild glycoprivic brain insult mimicking transient hypoglycemia, which often occurs when insulin or oral hypoglycemic agents are administered for diabetes control. In the present study, the effect of a single 2-DG application (500mg/kg, a clinically applicable dose) on glial reactivity and neurogenesis in adult rat hippocampus was examined, as well as a possible temporal correlation between these two phenomena. Post-insult (PI) glial reactivity time course was assessed by immunoreaction against glial-fibrillary acidic protein (GFAP) during the following 5 consecutive days. A clear increase of GFAP immunoreactivity in hilus was observed from 48 to 96h PI. Moreover, enhanced labeling of long radial processes in the granule cell layer adjacent to hilus was evidenced. On the other hand, a transient increase of progenitor cell proliferation was detected in the subgranular zone, prominently at 48h PI, coinciding with the temporal peak of glial activation. This increase resulted in an augment of neuroblasts double labeled with 5-bromo-deoxyuridine (BrdU) and with double cortin (DCX) at day 7 PI. Around half of these cells survived 28 days showing matured neuronal phenotype double labeled by BrdU and a neuronal specific nuclear protein marker (NeuN). These findings suggest that a transient neuroglycoprivic state exerts a short-term effect on glial activation that possibly triggers a long-term effect on neurogenesis in hippocampus.

New vanadate-induced Ca2+ pathway in human red cells.

Vanadate is a commonly used Ca2+ pump blocker, exerting a substantial effect on Ca2+ extrusion at millimolar concentrations in human red cells. At such levels, vanadate also seems to open an L type-like Ca2+ channel in these cells (J Biol Chem 257 (1982) 7414; Gen Physiol Biophys 16 (1997) 359). Since neither a dose-dependence effect nor a metabolic requirement for the latter action could be found in the literature, we have addressed this matter in the present work. Accordingly, vanadate action on Ca2+ entry was systematically investigated in both young and old human red cells after metabolic depletion. Although vanadate enhanced Ca2+ entry indifferently in either cell type, a distinct over-all effect was paradoxically found depending on whether or not metabolic substrates that give rise to ATP were present. In ATP-depleted cells, unlike with ATP-containing cells, vanadate-stimulated Ca2+ entry was neither blocked by raising external K+ nor by adding voltage-dependent Ca2+ channel blockers (nifedipine, calciseptine, FTX3.3) or compounds affecting polyphosphoinositide metabolism (Li+, neomycin). Likewise, full substitution of external Na+ by other cations did not inhibit vanadate-enhanced Ca2+ entry. Regardless of the cell age, stimulation by vanadate depended strongly on internal Na+ (0-30 mM). Vanadate stimulation was significantly reduced (about 55%) by heparin (10 mg/ml) only in young cells and by ryanodine (about 35%, 250 microM) in old cells. The results suggest presence of a new vanadate-induced Ca2+ entry pathway in ATP-depleted cells.

Caffeine activates a mechanosensitive Ca(2+) channel in human red cells.

Caffeine is known to activate influx of both mono- and divalent cations in various cell types, suggesting that this xanthine opens non-selective cation channels at the plasma membrane. This possibility was investigated in human erythrocytes, studying the caffeine action on net Ca(2+), Na(+) and K(+) movements in ATP-depleted cells. Whole populations and subpopulations of young and old erythrocytes were employed. Caffeine was tested in the presence of known mechanosensitive channel blockers (Gd(3+), neomycin and amiloride) and ruthenium red as a possible inhibitor. Caffeine enhanced net cation fluxes in a concentration-dependent way. In whole populations, the Ca(2+) entry elicited by 20 mM caffeine was fully suppressed by Gd(3+) (5 microM), amiloride (250 microM) and ruthenium red (100 microM) and partially blocked by neomycin (100 microM). The above blockers also inhibited caffeine-dependent Na(+) entry whilst showing antagonistic effects on the corresponding K(+) efflux. These compounds fully suppressed hypotonically-induced (-35 mOsm/kg) Ca(2+) influx at nearly the same concentrations completely blocking caffeine-stimulated Ca(2+) entry. The effect of inhibitors on Ca(2+) influx in young cells exceeded that in old cells at similar concentrations. The results clearly show that caffeine stimulates a stretch-activated Ca(2+) channel in human red cells and that aged cells are less susceptible to mechanosensitive channel blockers.

Functional expression of Kir 6.1/SUR1-K(ATP) channels in frog retinal Müller glial cells.

The retinae and brains of larval and adult amphibians survive long-lasting anoxia; this finding suggests the presence of functional K(ATP) channels. We have previously shown with immunocytochemistry studies that retinal glial (Müller) cells in adult frogs express the K(ATP) channel and receptor proteins, Kir6.1 and SUR1, while retinal neurons display Kir6.2 and SUR2A/B (Skatchkov et al., 2001a: NeuroReport 12:1437-1441; Eaton et al., in press: NeuroReport). Using both immunocytochemistry and electrophysiology, we demonstrate the expression of Kir6.1/SUR1 (K(ATP)) channels in adult frog and tadpole Müller cells. Using conditions favoring the activation of K(ATP) channels (i.e., ATP- and spermine-free cytoplasm-dialyzing solution containing gluconate) in Müller cells isolated from both adult frogs and tadpoles, we demonstrate the following. First, using the patch-clamp technique in whole-cell recordings, tolbutamide, a blocker of K(ATP) channels, blocks nearly 100% of the transient and about 30% of the steady-state inward currents and depolarizes the cell membrane by 5-12 mV. Second, inside-out membrane patches display a single-channel inward current induced by gluconate (40 mM) and blocked by ATP (200 microM) at the cytoplasmic side. The channels apparently show two sublevels (each of approximately 27-32 pS) with a total of 85-pS maximal conductance at -80 mV; the open probability follows a two-exponential mechanism. Thus, functional K(ATP) channels, composed of Kir6.1/SUR1, are present in frog Müller cells and contribute a significant part to the whole-cell K+ inward currents in the absence of ATP. Other inwardly rectifying channels, such as Kir4.1 or Kir2.1, may mediate the remaining currents. K(ATP) channels may help maintain glial cell functions during ATP deficiency.

MgATP counteracts intracellular proton inhibition of the sodium-calcium exchanger in dialysed squid axons.

Intracellular Na(+) and H(+) inhibit Na(+)-Ca(2+) exchange. ATP regulates exchange activity by altering kinetic parameters for Ca(2+)(i), Na(+)(i) and Na(+)(o). The role of the Ca(2+)(i)regulatory site on Na(+)(i)-H(+)(i)-ATP interactions was explored by measuring the Na(+)(o)-dependent (45)Ca(2+) efflux (Na(+)(o)-Ca(2+)(i) exchange) and Ca(2+)(i)-dependent (22)Na(+) efflux (Na(+)(o)-Na(+)(i) exchange) in intracellular-dialysed squid axons. Our results show that: (1) without ATP, inhibition by Na(+)(i) is strongly dependent on H(+)(i). Lowering the pH(i) by 0.4 units from its physiological value of 7.3 causes 80 % inhibition of Na(+)(o)-Ca(2+)(i) exchange; (2) in the presence of MgATP, H(+)(i) and Na(+)(i) inhibition is markedly diminished; and (3) experiments on Na(+)(o)-Na(+)(i) exchange indicate that the drastic changes in the Na(+)(i)-H(+)(i)-ATP interactions take place at the Ca(2+)(i) regulatory site. The increase in Ca(2+)(i) affinity induced by ATP at acid pH (6.9) can be mimicked by a rise in pH(i) from 6.9 to 7.3 in the absence of the nucleotide. We conclude that ATP modulation of the Na(+)-Ca(2+) exchange occurs by protection from intracellular proton and sodium inhibition. These findings are predicted by a model where: (i) the binding of Ca(2+) to the regulatory site is essential for translocation but not for the binding of Na(+)(i) or Ca(2+)(i) to the transporting site; (ii) H(+)(i) competes with Ca(2+)(i) for the same form of the exchanger without an effect on the Ca(2+)(i) transporting site; (iii) protonation of the carrier increases the apparent affinity and changes the cooperativity for Na(+)(i) binding; and (iv) ATP prevents both H(+)(i) and Na(+)(i)-effects. The relief of H(+) and Na(+) inhibition induced by ATP could be important in cardiac ischaemia, in which a combination of acidosis and rise in [Na(+)](i) occurs.