Macroautophagy is an evolutionarily conserved vacuolar, self-digesting mechanism for cellular components, which end up in the lysosomal compartment. In mammalian cells, macroautophagy is cytoprotective, and protects the cells against the accumulation of damaged organelles or protein aggregates, the loss of interaction with the extracellular matrix, and the toxicity of cancer therapies. During periods of nutrient starvation, stimulating macroautophagy provides the fuel required to maintain an active metabolism and the production of ATP. Macroautophagy can inhibit the induction of several forms of cell death, such as apoptosis and necrosis. However, it can also be part of the cascades of events that lead to cell death, either by collaborating with other cell death mechanisms or by causing cell death on its own. Loss of the regulation of bulk macroautophagy can prime selfdestruction by cells, and some forms of selective autophagy and non-canonical forms of macroautophagy have been shown to be associated with cell demise. There is now mounting evidence that autophagy and apoptosis share several common regulatory elements that are crucial in any attempt to understand the dual role of autophagy in cell survival and cell death. The term 'autophagy' embraces several different mechanisms: microautophagy, macroautophagy and chaperone-mediated autophagy, 1 all of which are involved in the lysosomal degradation of cellular components. In this review, we will focus essentially on macroautophagy, because of the large body of data available about its cross talk with cell death. Macroautophagy (hereafter called 'autophagy') is a mechanism conserved among eukaryotic cells that starts with the formation of a multi-membrane-bound vacuole, known as an autophagosome, which ultimately fuses with the lysosomal compartment and the degradation of the sequestered material. 2 The seminal discovery of autophagy-related (ATG) genes initially in yeast and then in multicellular organisms 3,4 has led to an important breakthrough in the understanding of how autophagosomes are formed and of the part autophagy plays in cell physiology and human diseases. 1,5 To clarify how macroautophagy is modulated in response to stress situations, it is also absolutely essential to elucidate how the macroautophagy is regulated upstream of the Atg machinery. 6 Macroautophagy occurs at a basal rate in most cells, where it acts as a cytoplasmic quality-control mechanism to eliminate protein aggregates and damaged organelles. 1,5 The physiological importance of basal autophagy in maintaining tissue homeostasis has recently been demonstrated in conditional brain and liver Atg knockout mouse models. [7][8][9] These studies have demonstrated the role of autophagy in preventing the cytotoxic deposition of aggregate-prone proteins in the cytoplasm, and the contribution of autophagy to the elimination of ubiquitinated proteins that are efficient substrates for the proteasome.On the other hand, when the supply of nutrients is limited, starvation-induced autophagy contrib...
Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in plasma glucose and urinary paracetamol-glucuronide after infusion of [U-13 C]glucose, [2-13 C]glycerol, [1-2 H]galactose, and paracetamol. In hepatocytes, glucose-6-phosphate (Glc-6-P) content, net glycogen synthesis, and lactate production from glucose and dihydroxyacetone increased strongly in the presence of S4048 (10 M). In livers of S4048-treated rats (0.5 mg kg ؊1 min ؊1 ; 8 h) Glc-6-P content increased strongly (؉440%), and massive glycogen accumulation (؉1260%) was observed in periportal areas. Total glucose production was diminished by 50%. The gluconeogenic flux to Glc-6-P was unaffected (i.e. 33.3 ؎ 2.0 versus 33.2 ؎ 2.9 mol kg ؊1 min ؊1 in control and S4048-treated rats, respectively). Newly synthesized Glc-6-P was redistributed from glucose production (62 ؎ 1 versus 38 ؎ 1%; p < 0.001) to glycogen synthesis (35 ؎ 5% versus 65 ؎ 5%; p < 0.005) by S4048. This was associated with a strong inhibition (؊82%) of the flux through glucokinase and an increase (؉83%) of the flux through glycogen synthase, while the flux through glycogen phosphorylase remained unaffected. In livers from S4048-treated rats, mRNA levels of genes encoding Glc-6-P hydrolase (ϳ9-fold), Glc-6-P translocase (ϳ4-fold), glycogen synthase (ϳ7-fold) and L-type pyruvate kinase (ϳ 4-fold) were increased, whereas glucokinase expression was almost abolished. In accordance with unaltered gluconeogenic flux, expression of the gene encoding phosphoenolpyruvate carboxykinase was unaffected in the S4048-treated rats. Thus, acute inhibition of glucose-6-phosphatase activity by S4048 elicited 1) a repartitioning of newly synthesized Glc-6-P from glucose production into glycogen synthesis without affecting the gluconeogenic flux to Glc-6-P and 2) a cellular response aimed at maintaining cellular Glc-6-P homeostasis.
SUMMARYWe developed a quantitative histochemical assay for measurement of local glutamate concentrations in cryostat sections of rat liver. Deamination of glutamate by glutamate dehydrogenase (GDH) was coupled to the production of formazan and formazan precipitation was used for colorimetric visualization. The method was tested and validated with gelatin model sections with known glutamate concentrations. Calibration graphs showed linear relationships with high correlation coefficients ( Ͼ 96%) between glutamate concentrations or section thickness and absorbance values. The method was reproducible, with a constant percentage of 60 Ϯ 5% of glutamate being converted in gelatin model sections containing glutamate concentrations of 2 mM and higher. Glutamate concentrations were estimated in periportal, intermediate, and pericentral zones of liver lobules that contain low, intermediate, and high GDH activity, respectively. In fed adult male rat livers, periportal zones contained the highest concentrations of glutamate ( ف 14 mM) and intermediate and pericentral zones ف 13 and 9 mM, respectively. On starvation, glutamate concentrations increased only in the small rim of pericentral cells that express glutamine synthetase, to ف 15 mM. In livers of fetal and newborn rats, glutamate was homogeneously distributed, with a concentration of ف 5 mM. In suckling rat liver, distribution of glutamate was still homogeneous but the concentration was increased to ف 8 mM. These glutamate distribution patterns were in agreement with those detected immunohistochemically. (J Histochem Cytochem 45:1217-1229, 1997) I n the past decades, several methods have been developed to determine distribution patterns of enzymes in tissues and cells and to quantify their local activity (for review see Stoward and Pearse 1991). In these assays, enzyme activity is usually measured by incubating sections at saturating substrate concentrations for a particular enzyme. Nevertheless, metabolic fluxes through pathways depend not only on cellular concentrations of enzymes but also on concentrations of substrates, co-factors, and other regulatory factors. Several approaches were developed to measure local substrate and/or co-factor concentrations using either microbiochemistry (Teutsch 1988), electrochemistry (Thurman andKaufman 1985), positron emission tomography (Schelbert 1991;Messa et al. 1992), nuclear magnetic resonance spectroscopy (Schrader et al. 1993), or bioluminescence techniques (Paschen et al. 1981;Mueller-Klieser et al. 1990;Mueller-Klieser and Walenta 1993;Kim et al. 1993). An accurate insight into the relative contribution of enzyme and substrate concentrations is particularly relevant for the problem of metabolic zonation in the liver, because it remains to be established to what extent this zonation is determined by regional differences in the concentration of enzymes, their kinetic parameters, and/or local available substrates (for reviews see Jungermann and Katz 1989;Meijer et al. 1990; Van Noorden and Jonges 1995a,b).The ai...
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