Hans J. Seitz

The problem of tissue sampling from experimental animals with respect to freezing technique, anoxia, stress and narcosis: A new method for sampling rat liver tissue and the physiological values of glycolytic intermediates and related compounds

Abstract The problems of the measurement of physiological concentrations of glycolytic intermediates and related compounds in rat liver were investigated. Five hundred milligrams, tissue samples, immersed in liquid nitrogen, remained at a temperature of more than 30 ° for about 15 sec. Not till then temperature dropped rapidly. When samples up to 24 g were cooled only a disproportionately small prolongation of the initial phase was observed. The dependency of freezing time on tissue weight was investigated for liquid nitrogen, CClF3 and isopentane. Using the latter two the freezing time could be shortened significantly. Yet, even for isopentane, it remained still longer than 12 sec for a 500 mg tissue sample. A mathematical approach proved that—even under ideal conditions—500 mg of tissue immersed should need about 7 sec to freeze through. A new method of liver sampling from unanaesthetized rats was developed and the physiological substrate concentrations were determined. AMP values were found to be as low as 15 nmoles/g liver. The time course of the alterations of substrate concentrations during anoxia was investigated and found to be nearly linear during the first 13.6 sec. Nembutal was shown to influence substrate concentrations severely, independent on narcosis-induced anoxia. AMP was found to be a highly sensitive parameter to stress.

Regulation of mitochondrial biogenesis by thyroid hormone.

Thyroid hormone (T3) has a profound effect on mitochondrial biogenesis. T3‐regulated gene expression is mediated by thyroid hormone receptor (TR) binding to thyroid hormone response elements (TREs). In concert with the action of various coactivators and corepressors this interaction leads to a modulation of the chromatin structure and subsequently to a modulation of gene expression of adjacent target genes. However, as numerous genes are endogenous regulated by T3 whereas a TRE appears to be absent in their regulatory elements, a TR‐independent pathway of T3‐mediated gene regulation is likely. In this review, we discuss the direct mechanisms of TR‐dependent regulation of gene expression on the nuclear and mitochondrial genome by T3. We also summarise recent observations on an indirect mechanism of T3 action via intermediate factor(s). We discuss the regulation of nuclear respiratory factor 1 (NRF‐1) and peroxisome proliferator‐activated receptor gamma coactivator 1 alpha (PGC‐1α) by T3, suggesting NRF‐1 and PGC‐1α as attractive candidate factors for an intermediate factor of T3 action in vivo.

Coordinate control of intermediary metabolism in rat liver by the insulin/glucagon ratio during starvation and after glucose refeeding: Regulatory significance of long-chain acyl-CoA and cyclic AMP

Abstract The levels of serum insulin, glucagon, and free fatty acids (FFA) and the tissue concentrations of hepatic cyclic AMP, long-chain acyl-CoA (LCA), adenine nucleotides, inorganic phosphate, the intermediates of the Embden-Meyerhof pathway, the citric acid cycle (including acetyl-CoA and free CoA), and the cytoplasmic and mitochondrial redox couples were determined in the rat 12, 24, and 48 h after food withdrawal and 5, 10, 20, 40, 60, and 120 min after the refeeding of glucose. Using the measured metabolite contents in the liver, the alterations in the concentration of malate, oxaloacetate, citrate, and α-ketoglutarate and the changes in the energy state of the adenine nucleotide system and the redox state of the NAD system were attributed to the cytoplasmic and mitochondrial compartments by applying established calculation methods. Glucose refeeding provoked significant alterations in all parameters investigated. These changes occurred within minutes, reversing the hormone and metabolite pattern which had developed within 24 h in response to food withdrawal. Particularly, glucose refeeding resulted in a drastic increase in the insulin/glucagon ratio. Simultaneously, the level of serum FFA and the concentration of LCA in the liver declined. The latter alteration was accompanied by an increase in the cytoplasmic and a decrease in the mitochondrial ATP ADP × P ratios. Moreover, the redox state of the cytoplasmic NAD system was shifted toward the oxidized state. When the appropriate data were plotted against each other, highly significant correlations were obtained (i) between the insulin/glucagon ratio and the serum FFA concentration, (ii) between the serum FFA concentration and the concentration of hepatic LCA, (iii) between the hepatic LCA concentration and the cytoplasmic energy state, and (iv) between the cytoplasmic energy state and the redox state of the cytoplasmic NAD system. These findings are interpreted to support the hypothesis derived from experiments carried out in vitro that the insulin/glucagon ratio via the FFA-dependent concentration of hepatic LCA might affect the translocation of adenine nucleotides between the cytoplasmic and the mitochondrial compartment, thereby regulating the cytoplasmic energy state and the redox state of the cytoplasmic NAD system, consequently. Glucose refeeding provoked rapid coordinate changes in the concentration of the intermediates of both the citric acid cycle and the Embden-Meyerhof chain, indicating the altered substrate flow through these pathways. Those metabolites, known to modulate the activity of key regulatory enzymes in vitro, were analyzed with respect to their suggested regulatory function. As to the established shift from pyruvate carboxylation to pyruvate decarboxylation after glucose refeeding, the data revealed that the decrease in pyruvate carboxylase activity can be attributed to the decrease in the intramitochondrial ATP ADP ratio and the simultaneous fall in acetyl-CoA concentration, while the coordinate increase in pyruvate dehydrogenase activity can be ascribed to the decline in the concentration of LCA and, consequently, in the ratios of ATP ADP , NADH NAD , and acetyl- CoA CoA within the mitochondria. As for the citric acid cycle, increased citrate synthesis from acetyl-CoA and oxaloacetate was supported by the rapid drop in the concentration of the established inhibitor of citrate synthesis, LCA. In contrast, the concentration of succinyl-CoA, an inhibitor of the enzyme in vitro, remained practically constant, questioning its regulatory function under the present experimental conditions. In addition to the activation of citrate synthase, the coordinate activation of isocitrate dehydrogenase was indicated by the LCA-mediated decline in both the mitochondrial ATP ADP and the NADH NAD ratios. Glucose refeeding immediately reduced urea excretion to basal values. This alteration was preceded by a drastic fall in the tissue concentration of cyclic AMP, supporting the physiological role of the nucleotide in the control of hepatic gluconeogenesis. In contrast, the observed changes in the concentration of the effectory acting metabolites (ATP, AMP, fructose 1,6-diphosphate, citrate, and alanine) were incompatible with the suggested function of these intermediates in switching over the substrate flow through the Embden-Meyerhof pathway from gluconeogenesis to glycolysis. The results are discussed in reference to the known rapid stimulation of fatty acid biosynthesis in the liver and to the transfer of reducing equivalents by the different shuttles of the inner mitochondrial membrane. In summary, it can be concluded that the insulin/glucagon ratio in a moment-to-moment fashion controls the glucose balance across the liver by regulating hepatic intermediary metabolism via the concentration of both LCA and cyclic AMP.

Rapid rise in plasma glucagon induced by acute cold exposure in man and rat

The effect of acute cold exposure on the concentration of glucagon in the blood was investigated in man and in intact and adrenalectomized rats.In man fasted overnight acute cold exposure, which caused a twofold increase in O2-consumption resulted in a rapid rise in plasma glucagon. The levels of insulin and blood glucose remained unaltered, while the concentration of serum free fatty acids and β-hydroxybutyrate increased.In fasted intact rats acute cold exposure lead to similar effects. A close parallelism between the rise in plasma glucagon and the concentration of hepatic cycloAMP was observed. Adrenalectomy did not impair the cold induced rise in plasma glucagon and hepatic cycloAMP.It is concluded that acute cold exposure caused a rapid rise in the concentration of plasma glucagon leading to an increase in the concentration of hepatic cycloAMP, thus enhancing the rate of hepatic gluconeogenesis and ketogenesis. As these alterations were similar in the absence of glucocorticoids and medulla-derived catecholamines, it is suggested that glucagon may play a role in the metabolic adaptation to acute cold exposure.

Long-term perfusion of the isolated rat liver maintenance of its functional state by use of a fluorocarbon emulsion

Abstract In order to establish a long-term perfusion system a fluorocarbon emulsion was developed and employed for the perfusion of isolated rat liver up to 20 h. Its suitability for maintaining some specific organ functions was compared with that of a commonly used red cell-containing medium. All livers perfused with the fluorocarbon medium released phosphoglucose isomerase, glutamate-oxaloacetate transaminase and glutamate dehydrogenase almost linearly at a low basal rate, glutamate dehydrogenase release beginning after 5 h perfusion. In contrast to that, a certain percentage of the livers perfused with the red cell-containing medium showed an exponential enzyme release which was over two standard deviations above the mean of the livers perfused with fluorocarbon medium, the values being 25% for phosphoglucose isomerase, 38% for glutamate-oxaloacetate transmiinase and 87% for glutamate dehydrogenase after 10 h of perfusion. In each case the exponential release of phosphoglucose isomerase signaled the functional impairment of the preparation. Thus, defining those livers as “intact” only if their phosphoglucose isomerase release was within two standard deviations of the means of the fluorocarbon-perfused livers, the following liver functions were examined in fluorocarbon-perfused and, for comparison, in “intact” cell-perfused livers during a 10-h period: Metabolite state, galactose elimination from the perfusate, induction of tyrosine aminotransferase by dexamethasone, and gluconeogenesis from lactate and bile production. It was found that the fluorocarbon medium provided at least the same or an even better hepatic function than did the red cell-containing medium. However, while in red cell-perfused livers functional impairment always occurred at various percentages under the conditions mentioned above, this was never observed with the fluorocarbon medium. Electron microscopic examination of the livers perfused with the fluorocarbon medium showed no disturbance of the mitochondrial matrix and cristae after a 10 h perfusion. While within a large number of liver cells the ergastoplasm was seen in normal appearance, in other liver cells the cisternae of rough endoplasmic reticulum were vacuolated. Some important physicochemical data of the fluorocarbon medium such as O 2 capacity, viscosity and particle size are reported, and the technique and the problems of its preparation are described. The advantages of the fluorocarbon medium for long as well as short term perfusion experiments are discussed.

In vivo glucose turnover in hypo- and hyperthyroid starved rat.

The effect of hypo- and hyperthyroidism on glucose turnover in vivo was determined in unanesthetized rats starved for 48 h. Glucose pool and decay rate of specific radioactivity of blood glucose was measured after bolus injection of a mixture of3H-(2)- and14C-(U)-glucose under steady state conditions. Compared with euthyroid controls (=100%), hypothyroidism resulted in a decrease of blood glucose concentration (81%), glucose pool (52%), glucose disappearance rate (39%), and total glucose recycling (12%). In contrast, hyperthyroidism led to an increase of blood glucose concentration (148%), glucose pool (121%), glucose disappearance rate (185%), and and total glucose recycling (163%).T1/2 for glucose was calculated to be 46 min in the hypo-, 34 min in the eu-, and 22 min in the hyperthyroid state.The concentration of circulating glucoregulatory hormones, corticosterone and glucagon were elevated in hyperthyroid rats, while glucagon was diminished in hypothyroid animals. No difference in the level of insulin was found.These data demonstrate that glucose turnover in vivo is a function of the thyroid state being reduced in hypo- and considerably increased in hyperthyroidism.

Testis-specific expression of rat mitochondrial glycerol-3-phosphate dehydrogenase in haploid male germ cells.

Abstract Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is regulated by multiple promoters in a tissue-specific manner. We characterized the testis-specific promoter C of the mGPDH gene and investigated the cellular localization of mGPDH within the testis. Electrophoretic mobility shift experiments identified a cAMP-response element (CRE) site at −57 that was active in the testis. An in vitro-translated CRE modulator (CREM) protein was able to bind this CRE site, and an anti-CREM antibody interfered with this complex. Ectopic expression of the testis-specific transcriptional activator CREMτ and protein kinase A in human hepatocarcinoma HepG2 cells activated a promoter C-driven luciferase construct in transient transfection experiments. Furthermore, mGPDH expression was undetectable in testis of CREM-deficient mice. The cellular localization of mGPDH expression and translation in adult rat testis was determined by in situ hybridization and immunohistochemistry techniques. The mGPDH transcripts were detected solely in postmeiotic germ cells. Expression of mGPDH was restricted from round spermatids to early elongating spermatids. The mGPDH protein was delayed in postmeiotic germ cells, restricted from late elongating spermatids to mature spermatids. Our results indicate that rat mGPDH is expressed by a testis-specific promoter from haploid male germ cells in a stage-specific manner.

Multiple promoters direct the tissue-specific expression of rat mitochondrial glycerol-3-phosphate dehydrogenase.

Abstract The mitochondrial FADdependent glycerol-3-phosphate dehydrogenase (mGPDH) is an essential component of the glycerol phosphate shuttle which transfers reduction equivalents from the cytoplasm into the mitochondria. We analyzed the distribution of different exon 1-containing transcripts by RTPCR in various tissues in vivo. Exon 1a was predominantly expressed in brain, brown adipose tissue and pancreas, exon 1b was ubiquitously expressed, and exon 1c was exclusively expressed in testis. In transient transfection assays the ubiquitous promoter B showed a detectable activity, whereas promoters A and C were completely silent. A deletion mutational analysis located the basal promoter B activity to a 316 bp core sequence upstream of the transcription start site.

Effect of thyroid state on ketogenic capacity of the isolated perfused liver of starved rats

The effect of different thyroid states on the oxidation of free fatty acids was investigated in the isolated perfused liver of 24-h-starved rats. 1. Compared with the euthyroid control the oxidation of oleate to ketone bodies as well as to CO2 was increased in hyper-, while it was unchanged in hypothyroid livers. 2. The addition of carnitine stimulated oleate oxidation in livers from eu- and hyperthyroid rats, but was without effect in hypothyroid livers. 3. Glucose did not affect the thyroid hormone-mediated effect of oleate conversion to ketone bodies. 4. Hepatic oxidation of octanoate was similar in all thyroid states. 5. Re-esterification of oleate was enhanced in hypo-, but reduced in hyperthyroidism. 6. The concentration of hepatic malonyl-CoA was decreased in hypo- and unchanged in hyperthyroid livers. 7. The concentration of cyclic AMP was elevated in the liver of hyperthyroid rats, no differences were observed between eu- and hypothyroid livers. However, increasing the hepatic cyclic AMP content by the addition of glucagon did not stimulate ketogenesis in eu- and hypothyroid livers. 8. The results indicate that thyroid hormones stimulate oleate oxidation by an accelerated transport of its CoA derivative into the mitochondrial compartment.

Physiologic significance of glucocorticoids and insulin in the regulation of hepatic gluconeogenesis during starvation in rats.

The physiologic significance of glucocorticoids and insulin in the regulation of hepatic gluconeogenesis was investigated during a 48-hr starvation period by studying the factors presumed to control the rate of glucose synthesis in the final gluconeogenetic pathway. Rats were used, in which glucorticoids were removed by adrenalectomy before starvation, and in which serum insulin was kept constant before and after food withdrawal by pre-feeding with a proteinfree diet. It was found that adrenalectomized rats at constantly low serum insulin levels responded to starvation as rapidly, and to the same degree, as intact control subjects (1) by a significant increase in plasma glucagon and, consequently, in hepatic cAMP concentration; (2) by a coordinate elevation of the activities of hepatic pyruvate carboxylase, P-enolpyruvate carboxykinase, and fructose-1,6-diphosphatase; (3) by systematic alterations in the concentration of effectors of gluconeogenetic key enzymes; (4) by a shifting of the cytoplasmic NAD system towards the reduced state; (5) by a decrease in the intrahepatic concentration of glycogenic precursor substrates. These results suggest that the hepatic gluconeogenic response to starvation with respect to the regulatory factors 1-5 occurs independently from changes in the concentration of plasma glucocorticoids and insulin. The crossing over of the gluconeogenetic intermediates between pyruvate and P-enolpyruvate (PEP), which was observed in intact but not in adrenalectomized rats, supports the assumption that during starvation, glucocorticoids enhance the rate of glucose production by the liver predominantly by permitting hepatic cAMP to stimulate the yet undefined mechanism, which has been demonstrated in the isolated perfused rat liver to control the substrate flow between pyruvate and PEP.