Hypoxia is a critical event for higher organisms, and cells and tissues react by increasing the oxygen supply by vasodilatation, angiogenesis, and erythropoiesis and maintaining cellular energy by increasing glycolysis and inhibiting anabolic pathways. Stimulation of glycolysis has been regarded as the main response that increases energy production during hypoxia; however, there is an obvious conflict during ischemia, because both the oxygen and glucose supply are insufficient. In this study, we found that exposure of HepG2 cells and normal fibroblasts to hypoxia induces cellular tolerance to glucose starvation. The tolerance induced by hypoxia is dependent on several amino acids, indicating a switch from glucose to amino acids as the energy source. When antisense RNA expression vector for 5-AMP-activated protein kinase or protein kinase B/Akt was transfected into HepG2 cells, the induction of tolerance to glucose was greatly inhibited, indicating that the tolerance was dependent on 5-AMP-activated protein kinase and protein kinase B/Akt. Similar tolerance was induced by nitric oxide exposure. The tolerance induced was observed in various cells and may represent a previously unknown physiological response related to hypoxia-preconditioning and tumor progression:austerity.

To clarify the properties of an inducible type of nitric oxide synthase (i‐NOS) in the brain, we examined whether lipopolysaccharide (LPS) induces NOS in glial cells cultured from neonatal rats. NOS activities (NO2− accumulation and L‐[14C]citrulline formation) were detected by treatment with LPS at 10 μg/ml for 6–72 hr. L‐[14C]citrulline formation by LPS‐induced i‐NOS was inhibited by NG‐monomethyl‐L‐arginine (a NOS inhibitor) and diphenyleneiodonium (a flavo‐protein inhibitor). The activity was not markedly changed in the presence or absence of Ca2+. The induction of i‐NOS by LPS was abolished by cycloheximide, actinomycin D, or dexamethasone. In addition, the induction was inhibited by herbimycin A (a tyrosine kinase inhibitor), but was not by staurosporine, W‐7, or FK‐506. After LPS stimulation, 130 kDa proteins were reacted with anti‐rat liver i‐NOS antibody 5–72 hr. i‐NOS induced from glial cells coupled tightly with endogenous calmodulin (CaM) even in the absence of Ca2+. These results suggest that LPS induces expression of 130‐kDa i‐NOS through an activation of tyrosine kinase, after which i‐NOS couples with CaM, and that NO is formed for 6–72 hr in glial cells. © 1996 Wiley‐Liss, Inc.

1. To elucidate the determining factors for elimination pathways of sulfate and glucuronide metabolites of xenobiotics, a single-pass perfusion of 4-methylumbelliferone (4MU) or p-nitrophenol (pNP) was performed with an isolated rat liver preparation. 2. Without bovine serum albumin in the perfusion system, clearance calculated based on the unbound concentration in the liver clearly showed that the net efflux clearances (CLeff) of sulfates from the sinusoidal membrane were much higher than those of glucuronides and that the biliary excretion clearances (CLb) of glucuronides were approximately two times larger than those of sulfates. 3. The ratios of CLeff to CLb were much higher for sulfates than those for glucuronides. The bile-oriented elimination of glucuronides or sinusoidal efflux-oriented elimination of sulfates was observed even using the perfusate including 3% bovine serum albumin, but the sinusoidal efflux of sulfates was extensively enhanced by bovine serum albumin in the perfusate. The mechanisms behind this stimulatory effect remain to be elucidated. 4. For both compounds, CLb of glucuronide was comparable with CLb of sulfate, meaning that CLb is not responsible for the biliary excretion of glucuronides at extensively higher rate than sulfates. 5. Higher concentration of glucuronides in the liver, partly caused by much lower CLeff of glucuronides than that of sulfates, is likely responsible for the bile-oriented excretion of glucuronides. The extensive sinusoidal efflux of sulfates, leading to the urine-oriented excretion, is attributed to the substantially higher CLeff than CLb. 6. In conclusion, the sinusoidal efflux is an important factor for determining elimination pathways of both sulfates and glucuronides, although further studies are needed to clarify the mechanisms of the sinusoidal efflux.

1. The hepatic and renal handling of glucuronides and sulphates of three phenolic compounds, 4-methylumbelliferone (4-MU), p-nitrophenol (pNP) and acetaminophen (APAP), were evaluated pharmacokinetically by in vivo constant infusion experiments in rat. It was shown that the urinary excretion rate at steady-state was larger than the biliary excretion rate for both glucuronides and sulfates, and sulfates, in particular, were extensively excreted into the urine. 2. For each glucuronide, however, biliary excretion clearances (CL(b)) calculated based on the total concentration and unbound concentration in the liver were much larger than the corresponding renal excretion clearances (CL(r)). Even in the case of sulfates, there was not any large difference between CL(r) and CL(b) based on the total and unbound concentration in tissues, which could not explain their extensive urinary excretion. From these results, these excretion clearances were recognized not to reflect necessarily the actual excretion rate obtained. 3. On the other hand, the tissue-to-plasma concentration ratio (K(p)) of both glucuronides and sulfates for every phenolic compound was much higher in the kidney than that in the liver. The results suggested that one of the most important determinants for the preferential excretion of these conjugates into the bile or urine is the extent of disposition of each compound to the liver or kidney. 4. In addition, K(p) of both glucuronides and sulfates in the liver, where these conjugates are mainly formed, was small. The K(p) of sulfates was quite low, suggesting that sulfates generated in the liver were subject to extensive sinusoidal efflux.

We have demonstrated that changes in the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) mRNA in streptozotocin-diabetic rats vary in different organs. The expression of different splice variants of PFKFB-3 mRNA is organ specific and changes in diabetes. We have identified a new splice variant of PFKFB-3 mRNA which has catalytic domains identical to those of the main isoform and other alternative splice variants of PFKFB-3 but differs by the length of C-terminus. The results of this investigation support a possible role of PFKFB-3 isozymes in adaptation of the cells to disordered carbohydrate metabolism resulting from the insulin deficiency.