Acute non-insulin-like stimulation of rat muscle glucose metabolism by troglitazone in vitro

1. The direct short-term effects of troglitazone on parameters of glucose metabolism were investigated in rat soleus muscle strips. 2. In muscle strips from Sprague-Dawley rats, troglitazone (3.25 micromol l(-1)) increased basal and insulin-stimulated glucose transport by 24% and 41%, respectively (P<0.01 each). 3. In the presence of 5 nmol l(-1) insulin, stimulation of glucose transport by 3.25 micromol l(-1) troglitazone was accompanied by a 36% decrease in glycogen synthesis, while glycolysis was increased (112% increase in lactate production) suggesting a catabolic response of intracellular glucose handling. 4. Whereas insulin retained its stimulant effect on [3H]-2-deoxy-glucose transport in hypoxia-stimulated muscle (by 44%; c.p.m. mg(-1) h(-1): 852+/-77 vs 1229+/-75, P<0.01), 3.25 micromol l(-1) troglitazone failed to increase glucose transport under hypoxic conditions (789+/-40 vs 815+/-28, NS) suggesting that hypoxia and troglitazone address a similar, non-insulin-like mechanism. 5. No differences between troglitazone and hypoxia were identified in respective interactions with insulin. 6. Troglitazone acutely stimulated muscle glucose metabolism in a hypoxia/contraction-like manner, but it remains to be elucidated whether this contributes to the long-term antidiabetic and insulin enhancing potential in vivo or is to be regarded as an independent pharmacological effect.     

Comparative metabolism of bis(2-methoxyethyl)ether in isolated rat hepatocytes and in the intact rat: effects of ethanol on in vitro metabolism

The metabolism of the reproductive and developmental toxicant bis(2-methoxyethyl)ether (diglyme) was studied in isolated rat hepatocytes and in the intact rat. Male Sprague-Dawley rats (190-220 g) were used in both studies. Hepatocytes, isolated by a two-step in situ collagenase perfusion of the liver, were cultured as monolayers and incubated with [14C]diglyme at 1, 10, 30, and 50 microM for up to 48 h. For the in vivo study, rats were given single oral doses of [14C]diglyme at 5.1 mmol/kg body wt, and urine was collected for up to 96 h. Radioactive compounds in the culture medium or in the urine were separated by high performance liquid chromatography and quantified with an in-line radioactivity monitor. Metabolites were identified by comparison of their chromatographic retention times and their mass spectra with those of authentic compounds. The principal metabolite from hepatocytes and in the urine was (2-methoxyethoxy)acetic acid (MEAA). This metabolite accounted for approximately 36% of the radioactivity in the 48-h culture medium and about 67% of the administered dose in the 48-h urine. Other prominent metabolites common to both systems included 2-(2-methoxyethoxy)ethanol, methoxyacetic acid (MAA), 2-methoxyethanol, and diglycolic acid. The diglyme metabolite profiles from urine and from hepatocytes were qualitatively similar, demonstrating that, in the rat, hepatocytes serve as a good model system for predicting the urinary metabolites of diglyme. Moreover, MEAA was shown to be the metabolite best suited for use as a short-term biological marker of exposure to diglyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of aniline on ethanol oxidation and carbohydrate metabolism in isolated hepatocytes

The addition of aniline to isolated hepatocytes derived from fasted rats and incubated with ethanol, caused a 30-60% decrease in the rate of ethanol oxidation. The degree of inhibition was dependent on aniline concentration, 5 mM causing near-maximal inhibition. Aniline reduced the activity of alcohol dehydrogenase in a noncompetitive manner, but had no effect on aldehyde dehydrogenase activity nor on reducing-equivalent transfer between the cytoplasm and mitochondria. The inhibition of alcohol dehydrogenase by aniline was associated with a decrease in the inhibitory effects of ethanol on glycolysis. Aniline, added to hepatocytes in the presence or absence of ethanol, inhibited gluconeogenesis from lactate and pyruvate, but not from sorbitol or fructose.     

Oxidative metabolism in a rat hepatoma (N13) and isolated rat hepatocytes: a flow cytometric comparative study

Recently, we have developed a new and fast kinetic method for assessing mitochondrial membrane potential by flow cytometry, based on the quantitation of the initial rate of rhodamine 123 (Rh123) uptake by living cells. This test has proved suitable to detect metabolic and toxic effects on mitochondria. To characterize energy metabolism in a rat hepatoma cell line (N13), we applied this method to assess several metabolic pathways that eventually generate mitochondrial membrane potential. Using this approach, we found that N13 hepatoma cells retain an oxidative capacity comparable with that observed in isolated hepatocytes under the same conditions. These results show that this cell line may represent an adequate biological model to perform metabolic and toxicological studies in vitro.     

Effect of hypothyroidism on glucose transport and metabolism in rat small intestine

A cell line (SMKT-R3) established from human renal cell carcinoma was characterized for the presence of sulfolipids and glycolipid sulfotransferases. Sulfolipids were found to constitute a large part of the acidic glycolipid fraction in SMKT-R3 cells. These findings were confirmed by metabolic labelling with 35S-sulfate. These sulfolipids were expressed at the surface of SMKT-R3 cells as ascertained by cytofluorometry using a monoclonal antibody directed to sulfolipids. Furthermore, markedly high activity levels of glycolipid sulfotransferases were observed in SMKT-R3 cells compared with other cell lines. These results suggest that the increased synthesis of sulfolipids in renal cell carcinoma tissue (Sakakibara et al., 1989. Cancer Res., 49, 335-339) is due to the elevation of the sulfotransferase activities of renal carcinoma cells themselves.     

Stereochemical aspects of 1,3-butadiene metabolism and toxicity in rat and mouse liver microsomes and freshly isolated rat hepatocytes

In the last decade, silver staining of nucleolar organizer region-associated proteins (AgNORs) has been widely used in tumour pathology both for diagnostic and for prognostic purposes. However, a reliable and reproducible assessment of these proteins on routinely processed archival tissues has only become possible since the recent introduction of standardized staining method and computer-aided morphometric analysis. In the present study, the AgNOR content at the invasive front of 80 squamous cell carcinomas of the floor of the mouth/tongue was investigated using this novel approach, with regard to prognosis and a variety of clinico-pathological parameters. All standardized AgNOR parameters [mean of AgNOR number, mean of AgNOR area, coefficients of variation (CV) of both AgNOR number and area] were statistically significantly associated with the clinical course. The strongest correlation was found for the AgNOR-area univariate analysis (P = 0.006). In multivariate analysis, the mean of AgNOR number could independently predict both overall (P = 0.01) and disease-free survival (P = 0.001). It is concluded that standardized staining and computer-aided analysis of AgNORs are prerequisites for an objective and reproducible AgNOR assessment, which has potential as a supplementary diagnostic and prognostic tool in oral cancer.     

Effects of troglitazone and metformin on glucose and lipid metabolism: alterations of two distinct molecular pathways

Using an experimental model of rat colon adenocarcinoma, we have recently shown that the presence of H blood-group antigen on variants of the CD44 adhesion molecule carrying amino acids encoded by exon v6 (CD44v6), increased the cells' tumorigenicity. In the present study, colon adenocarcinomas were induced by 1,2-dimethylhydrazine treatment in rats. Using immunohistochemistry, biopsies of normal, precancerous and carcinomatous colon mucosa were evaluated for expression A and H blood group antigens and CD44s and CD44v6 antigens. Normal rat colon showed strong and homogeneous expression of blood-group antigen A, but weak expression of H antigen. Several weeks before the appearance of tumours, dysplastic glands were strongly stained with anti-H reagents, while their A antigen was lost. Expression of CD44v6 was weak and restricted to some cells at the bottom of normal crypts. No obvious change was observed before appearance of severe dysplasia. In carcinomas, a strong but irregular expression of A, H and CD44v6 antigens was observed. In moderately differentiated carcinomas, A and H antigens were present at the apical surface of cells, whereas CD44v6 was found at the basolateral side. Only carcinomatous cells with loss of polarity, found in poorly differentiated cancers or infiltrated in the muscularis mucosae, were found to coexpress blood-group H or A and CD44v6 antigens at their surface.     

Effects of fatty acid oxidation on glucose utilization by isolated hepatocytes

We have studied the inhibitory action of long- and short-chain fatty acids on hepatic glucose utilization in hepatocytes isolated from fasted rats. The rates of hepatic glucose phosphorylation and glycolysis were determined from the tritiated products of [2-3H] and [6-3H]glucose metabolism, respectively. The difference between these was taken as an estimate of the 'cycling' between glucose and glucose-6-phosphate. In the presence of 40 mM glucose this cycling was estimated at 0.68 mumol/min/g wet wt. Glucose phosphorylation was unaffected during palmitate and hexanoate oxidation to ketone bodies but glycolysis was inhibited. The rate of glucose cycling was increased during this phase to 1.25 mumol/min/g. Following the complete metabolism of the fatty acids, glycolysis was reinstated and cycling rates returned to control levels. Hepatic glucose cycling appears to be an important component of the glucose/fatty acid cycle.     

Oxidative metabolism in rat hepatocytes and mitochondria during sepsis

We hypothesized that cellular oxygen consumption is abnormal during sepsis as a result of increased oxidative stress and selective mitochondrial damage. In a rat model of sepsis (cecal ligation and puncture), we studied the respiratory characteristics of isolated hepatocytes and liver mitochondria 16 h after onset of septic injury. Endogenous respiration by isolated cells was decreased during sepsis, while cyanide-resistant (nonmitochondrial) respiration was unaffected. Maximal oxygen consumption in ADP-supplemented, permeabilized hepatocytes was decreased with succinate as the substrate, but not with malate + glutamate or TMPD + ascorbate. In contrast, maximum oxygen consumption (State 3) by isolated liver mitochondria increased up to 35% during sepsis using either succinate or malate + glutamate as substrate. The electrophoretic features and mobility of nondenatured mitochondrial respiratory complexes were similar in control and septic hepatocytes, with the exception of decreased Complex V protein in sepsis. Structural evaluation of mitochondria in fixed liver slices by electron microscopy showed mitochondrial swelling in most of the septic animals. Measurements of oxidative stress during sepsis suggested an increase in hydroxylation of salicylate by isolated hepatocytes, and mitochondrial protein carbonyl content was increased significantly. Induction of iNOS in hepatocytes after 16 h of sepsis was variable, and little release of the oxidation products of NO. was detected. These findings are interpreted to mean that hepatocytes contain a mixed population of injured and hyperfunctional mitochondria during sepsis.     

The effect of stevioside on glucose metabolism in rat

This study was conducted to determine the effect of stevioside (SVS) on glucose metabolism. The experiments were performed in male Wistar rats treated with SVS either by intravenous infusion or feeding. SVS infusion (150 mg/mL) was carried out in doses of 0.67, 1.00, and 1.33 mL.kg-1 body weight.h-1. The plasma glucose level significantly increased both during and after SVS infusion, whereas it was not affected by SVS feeding (13.3 mL.kg-1 body weight). The glucose turnover rate (GTR) of [14C(U)]glucose and [3(-3)H]glucose was not significantly different between control and SVS infusion animals. Percent glucose carbon recycling and glucose clearance were reduced from 28.7 +/- 1.3 to 23.0 +/- 1.6% (p < 0.05) and from 6.46 +/- 0.34 to 4.99 +/- 0.20 mL.min-1.kg-1 body weight (p < 0.01), respectively. The plasma insulin level did not change, whereas the plasma glucose level significantly increased from 120.3 +/- 5.9 to 176.8 +/- 10.8 mg% (p < 0.01) during SVS infusion. Animals pretreated with angiotensin II and arginine vasopressin showed no significant effect, while animals pretreated with prazosin had an attenuated hyperglycemic effect of SVS infusion. Pretreatment with indomethacin or N omega-nitro-L-arginine methyl ester (L-NAME) alleviated the plasma glucose level during the second period of SVS infusion. Pretreatment with the combination infusion of indomethacin and L-NAME reduced the plasma glucose level from 117.0 +/- 1.8 to 109.0 +/- 1.7 mg% (p < 0.001), and normalized the plasma glucose level in the second period of SVS infusion. Insulin infusion inhibited the hyperglycemic effect of SVS infusion. The present results show that the elevation of the plasma glucose level during SVS infusion is not due to the reduction of the insulin level. It is probably the effect of SVS on glucose transport across the cell. Insulin response to a high plasma glucose level is suppressed during SVS infusion. Several interactions among norepinephrine, prostaglandin, and nitric oxide are involved in modulating the hyperglycemia during SVS infusion.     

Effect of bodyweight on the rate of glucose uptake by isolated rat muscles

The uptake of glucose by isolated extensor digitorum longus muscles was measured in rats of 78-350 g bodyweight. The rate of uptake per unit weight of muscle fell as the weight of the animal increased. It is concluded that in metabolic studies with isolated rat skelatal muscles, only muscles from weight-matched rats should be compared.     

The actions of avenaciolide and ethanol on glucose metabolism and on related enzyme activities in the isolated perfused rat liver

Six hundred and seventy five cases of attempted suicide observed in a resuscitation department were confronted with a certain number of biometeorological factors recorded daily: atmospheric pressure, air temperature, degree of insolation, precipitation, relative humidity, water vapour pressure, wind (speed and direction), hydrometeores, index of solar eruption, density of F2 layer. The confrontation is made for the two days before the intoxication and for the day when suicide is attempted. Parameters are then studied by statistical calculation (calculation of X2 and of the number of degrees of freedom). There does not seem to be any significant relationship, in spite of disconcerting series, between most meteorological factors and the number of attempted suicides observed. However, it is noted that no suicides were recorded during periods of solar eruption, and that there seems to be a marked correlation between suicide and winds, particularly according to their direction. Thus, winds charged with ionised particles seem to coincide with a high rate of self-destruction.     

Adenosine-mediated inhibition of glutathione synthesis in rat isolated hepatocytes

The modulatory effect of adenosine on hepatic glutathione (GSH) synthesis has been investigated in an experimental model in which GSH synthesis from methionine was monitored in rat isolated hepatocytes previously depleted of GSH. Adenosine inhibited GSH synthesis in this system, with complete inhibition occurring at 1 mM. Studies with adenosine receptor agonists and antagonists and adenosine analogues devoid of agonist activity have indicated that this effect of adenosine is not receptor-mediated nor is it mediated through changes in ATP level or redox balance. Rather, the data are consistent with an inhibitory effect of adenosine on the methionine-->cysteine transsulphuration pathway, probably at the level of the enzyme S-adenosylhomocysteine hydrolase. Submaximal inhibitory concentrations of adenosine produced an effect on GSH synthesis additive to that obtained with a maximal inhibitory concentration of adrenaline, which inhibits GSH synthesis at the level of gamma-glutamylcysteinyl synthetase. Furthermore, exposure of hepatocytes to adenosine subsequent to brief treatment with the toxicant menadione precipitated cytotoxicity. These results suggest that adenosine-mediated inhibition of hepatic GSH synthesis may have a role in various pathological or toxicological states.     

Piroxicam modifies the effects of ethanol on isolated rat hepatocytes

It has been reported that piroxicam prevents the hepatic increase of triacylglycerides and thiobarbituric acid-reactive substances observed after acute ethanol intoxication in rats and also causes a decrease in blood ethanol concentration. The aim of this study was to assess the effect of piroxicam on these 3 metabolic indicators, using isolated rat hepatocytes incubated with ethanol or lactate, supplemented or not with epinephrine. Epinephrine stimulated the consumption of lactate, but not of ethanol. In the isolated hepatocytes, and in a dose-dependent fashion, piroxicam alone raised the consumption of lactate and ethanol, increased the triacylglyceride pool in cells incubated with lactate or ethanol, and decreased the content of thiobarbituric acid-reactive substances in cells incubated with ethanol, but not with lactate. Epinephrine blocked these actions of piroxicam, except the lowering of the content of thiobarbituric acid-reactive substances. Thus, piroxicam helps to control the oxidative stress produced in isolated hepatocytes by ethanol.