Reduced hepatic insulin clearance in rats with dietary-induced obesity

Insulin uptake in the in situ perfused liver from rats that were moderately obese after overfeeding was diminished in comparison with controls. The obese rats had higher levels of portal free fatty acids (FFA) and liver triglyceride contents but not of insulin concentration in the portal vein. There were strong negative correlations between hepatic triglyceride and insulin clearance (r approximately 0.8-0.9). The perfusions were performed with lower FFA concentrations than those in vivo in the portal vein. It is suggested that the inhibited insulin uptake in the obese rats was due to exposure of these livers in vivo to elevated FFA concentrations, and that this inhibition remained during the experiment and was associated with the triglyceride contents of the livers. It is also suggested that this mechanism was responsible for the moderate peripheral hyperinsulinemia seen in these rats. A mechanism of regulation of insulin uptake in the liver via FFA and liver triglyceride might be of importance in several conditions with hyperinsulinemia and known elevation of portal FFA, and liver triglyceride contents.     

Intrasplenic hepatocellular transplantation corrects hepatic encephalopathy in portacaval-shunted rats.

The aim of this work was to evaluate the effect of intrasplenic hepatocellular transplantation on hepatic encephalopathy in an experimental model of chronic liver failure induced by end-to-side portacaval shunt in the rat. Inbred male Wistar Furth rats were divided into three groups: rats subjected to portacaval shunt (n = 10), rats subjected to portacaval shunt and intrasplenic hepatocellular transplantation of 10(7) hepatocytes isolated from livers of syngeneic rats (n = 10) and sham-operated rats (n = 10). Behavior tests were performed in a blind fashion at 3 wk, at 2 mo and at 3 mo after surgery. Spontaneous activity and nose-poke exploration by individual rats were studied in automated open field boxes equipped with infrared cells. Each cell beam interruption was automatically recorded on a microcomputer and transformed into a score index (counts/hour). Plasma levels of amino acids, ammonia and total biliary acids were measured. Portacaval shunt rats showed reduced spontaneous activity and nose-poke exploration scores. Intrasplenic hepatocellular transplantation significantly increased spontaneous activity after 2 mo and improved nose-poke exploration after 3 wk. At 3 mo, spontaneous activity and nose-poke exploration in portacaval shunt/intrasplenic hepatocellular transplantation rats were not significantly different from those of sham rats. Increases in plasma ammonia levels after portacaval shunt were not corrected. Amino acid imbalance and bile acid concentration in plasma were partially corrected by intrasplenic hepatocellular transplantation. These data show that intrasplenic hepatocellular transplantation can correct the neurological symptoms of hepatic encephalopathy in an experimental model of chronic liver failure and suggest that intrasplenic hepatocellular transplantation might be of therapeutic interest in chronic liver failure.     

Hepatic oxidant and antioxidant systems in portacaval-shunted rats.

The purpose of the present study was to determine the effects of chronic portal diversion on antioxidant levels in the rat liver. Male Sprague-Dawley rats (n = 32) were used for these studies. An end-to-side portacaval anastomosis was constructed in 17 of the rats. Sham-operated rats (n = 15) served as controls. Two weeks later, hepatic blood flow was measured by the radioactive microsphere technique and the liver was harvested for biochemical measurement of catalase, manganese superoxide dismutase, copper-zinc superoxide dismutase, selenium glutathione peroxidase, xanthine oxidase, xanthine dehydrogenase and reduced glutathione (acid soluble sulfhydryls). Total hepatic blood flow was approx. 40% lower in portacaval-shunted rats when compared to sham-operated control rats. Total superoxide dismutase (SOD) and xanthine dehydrogenase (XD) levels were significantly reduced in the liver of shunted rats when compared to controls. Xanthine oxidase activity was unaltered. The decreased superoxide dismutase levels were exclusively due to reductions in the cytosolic Ca/Zn SOD; Mn SOD levels were unaltered. These data are consistent with oxidant stress and suggest that the liver of subjects with conditions characterized by decreased portal blood flow may be more susceptible to oxidant-induced liver injury.     

Prevention of degradation of human polymorphonuclear leukocyte proteins by diisopropylfluorophosphate

Proteases can complicate the characterization of proteins from cells, especially human polymorphonuclear leukocytes (PMN), which contain abundant neutral proteases. We tested the ability of agents to inhibit proteolysis, with special reference to the subunit polypeptides of the contractile proteins actin, myosin, and actin-binding protein (ABP). Phenylmethylsulfonyl fluoride (PMSF), O-phenanthroline, EGTA, EDTA, N- ethylmaleimide, alone or in combinations, failed to prevent extensive proteolysis of the PMN proteins during solubilization of cells with dodecyl sulfate. These inhibitors and also alpha-1-antitrypsin and soybean trypsin inhibitor similarly could not prevent proteolysis during homogenization of cells in cold isosomolar sucrose. Treatment of PMN with greater than or equal to mM diisopropylfluorophosphate (DFP) prior to solubilization or homogenization markedly inhibited proteolysis. PMSF and DFP were equally effective in inhibiting proteolysis in PMN extracts, suggesting that the efficacy of DFP may result from its permeation of intact cells and granules before barriers are disrupted by detergents or homogenization. Treatment of PMN with DFP under conditions inhibiting proteolysis did not affect their rate of phagocytosis. We recommend the use of DFP in future studies correlating functions and protein structure of PMN.     

Long-term lung clearance in humans studied with Teflon particles labeled with chromium-51

Six healthy nonsmoking males inhaled 4-micron diameter Teflon particles labeled with chromium-51. Lung retention was measured for approximately 300 days. Three subjects inhaled "high-leaching" particles and three "low-leaching" particles. The high-leaching leaching particles leached 0.26%/day in water at 37 degrees C for the first 100 days and the low-leaching particles 0.065%/day. On an average, urine sampled for 24 h contained 0.13% of the lung burden for the high-leaching particles and 0.02% for the low-leaching particles. Thirty percent of the long-term clearance occurred with a fast phase, with half-times ranging from 4.5-45 days, and 70% with a slow phase, with half-times ranging from 200-2500 days. The fast phase was similar for the high-leaching and low-leaching groups. The slow phase varied widely among the subjects. There was no clear-cut relationship between half-time and type of particle. However, it seems probable that the slow phase was dependent on the degree of leaching, i.e., the half-times for elimination of intact particles from the lung are even slower. The large differences in long-term clearance among subjects indicate that long-term exposure to highly insoluble particles will result in large differences in lung burden. Subjects with a low capacity to eliminate alveolarly deposited particles are thus expected to be more susceptible to diseases in the alveolar part of the lung, i.e., diseases caused by certain insoluble particles.     

Reduced quinidine clearance in elderly persons.

The influence of age on quinidine pharmacokinetics was assessed in 22 healthy male and female volunteers; 14 of the subjects were young (aged 23 to 34 years) and 8 elderly (aged 60 to 69 years). All subjects received 180 to 300 mg of quinidine base by constant rate intravenous infusion over 10 to 15 minutes. The concentration of total and unbound quinidine in multiple serum samples and in urine collected within 48 hours after the administration of quinidine qas determined with spectrophotofluorometric assay. Mean kinetic values for total quinidine in the young subjects were: elimination half-life (t 1/2 beta), 7.3 hours; total volume of distribution (Vd), 2.39 liters/kg; total clearance, 4.04 ml/min per kg; renal clearance 1.43 ml/min per kg; and percent unbound, 24.6 In the elderly subjects, the values for Vd (2.18 liters/kg) and percent unbound (28.2) did not differ significantly from these values in the young subjects. However, in the elderly subjects t 1/2 beta was significantly longer (9.7 hours, P less than 0.05) and total quinidine clearance significantly less (2.64 ml/min per kg, P less than 0.005) than in the young subjects. Renal clearance of quinidine in the elderly was also significantly less (0.99 ml/min per kg, P less than 0.05) than in the young and was associated with lower rates of creatinine clearance in the elderly (r = 0.66). Reduced clearance of quinidine and prolongation of its elimination half-life could predispose to toxicity in the elderly unless the dose were appropriately adjusted.     

Effect of orally administered L-carnitine on blood ammonia and L-carnitine concentrations in portacaval-shunted rats

L-Carnitine (16 mmoles per kg, injected intraperitoneally) is reported to protect mice against subsequent injection of ammonium acetate given at the unprotected LD100. The present studies in rats show a variable protective effect of L-carnitine (16 mmoles per kg) administered 1 hr prior to an LD100 dose of ammonium acetate. Survival ranged from 100% to 35%. In two experiments, protection was highly significant; in a third experiment, L-carnitine did not protect against death but did significantly prolong time to death. Although the cause of this variability is not known, the data establish the protective effect in rats of L-carnitine given 1 hr before ammonium acetate. D-Carnitine and deoxycarnitine, chemically related analogs unable to substitute for L-carnitine metabolically, are without protective effect. The protective effect of L-carnitine is short-lived and is, for example, completely lost if ammonium acetate is given 24 hr after L-carnitine administration. In contrast, the free carnitine content of brain rises slowly but continuously for at least 24 hr following a single dose of L-carnitine. The observation that protection from ammonia toxicity is not correlated with brain carnitine levels strongly suggests a major peripheral component to the protective effect. Chronically hyperammonemic (portacaval-shunted) rats were found to have significantly depressed total and free carnitine levels in blood compared to normal and sham-operated controls. The hypocarnitinemia, but not the hyperammonemia, was completely reversed in portacaval-shunted rats given drinking water containing 10 mM L-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity labeled somatomedin-C-binding proteins in rat sera

We have developed an affinity labeling technique that uses disuccinimidyl suberate to covalently cross-link [125I]somatomedin-C (Sm-C) to specific binding proteins in rat serum. Normal rat serum contains four major classes of intensely labeled [125I]Sm-C-binding protein complexes which are sensitive to competition with unlabeled Sm-C with relative molecular masses of 95, 49, 36-33, and 26-23 K. In addition, less intensely labeled complexes are observed migrating between 175 and 115 K. Of the Sm-C binding complexes observed in normal serum, hypophysectomized (hypox) serum contains only an intensely labeled 36-33-K complex and a faint 49-K complex. Chronic administration of ovine (100 micrograms, ip, daily) to hypox rats induces the 95-K complex and possibly complexes between 175-115 K. With increasing duration of treatment, these complexes as well as the 49-K complex appear to increase in intensity. Binding proteins in both hypox and normal sera do not appear to distinguish between Sms, since both unlabeled Sm-C and multiplication-stimulating activity were equally potent in competing with [125I]Sm-C for binding. This affinity labeling technique appears to be a useful investigative tool to study the physiology and structure of Sm-binding proteins.     

Reduced insulin clearance in normal subjects due to extreme hyperinsulinemia

Insulin clearance was assessed in five normal subjects infused with insulin at a rate of 10 mU.kg-1.min-1 for 12-16 hours, which produced insulin levels of 1500-2000 microU/ml (approximately 10(-8) M). This level approximates the Kd of low affinity insulin binding sites, whereas previous clearance studies had been performed at insulin concentrations of 10(-9) M or less, approximating the Kd of the high affinity insulin receptor. The metabolic clearance rate for insulin during the infusion averaged 214 +/- 29 ml.min-1.m-2, which is half of that reported previously when lower insulin levels were achieved. Upon termination of the insulin infusion, the disappearance of insulin was markedly prolonged with an average "half-life" of 62 minutes. The rapidity with which hyperinsulinemia altered clearance suggested that down-regulation of insulin receptors was probably not the explanation for the reduced clearance. To elucidate the cause for the observed decrease in insulin clearance, five additional subjects were studied. If insulin was infused for 3.0-4.5 hours, the half-life of insulin disappearance was intermediate between that for an insulin bolus dose and that for a 12-16 hour insulin infusion. Administration of an insulin bolus dose at the end of a 12-hour infusion, while the insulin concentration was still approximately 10(-8) M, or 140 min later, when the insulin concentration was 10(-9) M, was followed by rapid disappearance with half-lives of 1.5 and 6-8 minutes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced erythrocyte survival following clearance of malarial parasitaemia in Thai patients

Erythrocyte survival times were measured in healthy Thai controls and in patients following clearance of asexual P. falciparum or P. vivax parasitaemia. In five controls the mean cell life (MCL) of compatible donor erythrocytes was 89.6 d (mean range 73-101 d) compared with a mean MCL of 56.8 d (range 30-66 d) for autologous erythrocytes in 12 falciparum patients. In one of these patients the survival curve was biphasic with a rapid loss of some labelled cells. The survival of compatible donor erythrocytes was also studied in 10 patients and two types of survival curve could be distinguished. In five patients the cells had a mean MCL of 64.4 d (range 42-90 d). In the others survival curves were curvilinear, suggesting a complex mechanism of cell clearance or the presence of more than one cell population. There was initially a more rapid rate of destruction. In P. vivax malaria the MCL of autologous erythrocytes in seven patients was a mean of 67.2 d (range 34-74 d) and that of compatible donor cells in six patients was 66.8 d (range 54-76 d). In all except one of these patients both autologous and donor cell survival curves could be fitted to straight lines. No increase in cell-bound IgG or C3 was evident in 12 patients tested. The differences between the mean MCL in all the groups of patients and the controls were statistically significant at the 5% level. This indicates an increased rate of erythrocyte destruction following clearance of P. falciparum or P. vivax parasites which is not antibody or complement mediated. The mechanism is unknown, but appears to be extrinsic to the erythrocytes themselves and may result from nonspecific activation of the reticuloendothelial function associated with the parasitic infection.     

Reduced adipose glucocorticoid reactivation and increased hepatic glucocorticoid clearance as an early adaptation to high-fat feeding in Wistar rats

Altered peripheral glucocorticoid metabolism may be important in the pathogenesis of obesity in humans and animal models. Genetically obese Zucker rats, Lep/ob mice, and obese humans exhibit increased regeneration of active glucocorticoids selectively in adipose tissue by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) and increased glucocorticoid clearance by hepatic A-ring reductases. We have examined whether dietary obesity in rats induces the same changes in glucocorticoid metabolism. Male Wistar rats were weaned onto high-fat (HF; 45% kcal from fat) or control (10% fat) diets. After 3 wk, HF rats showed no differences in weight but were glucose intolerant, had lower 11beta-HSD-1 activity in liver (3.8 +/- 0.2 vs. 4.9 +/- 0.2 pmol product/min.mg protein; P <0.01), sc fat (0.03 +/- 0.01 vs. 0.09 +/- 0.01 pmol product/min.mg protein; P <0.01), and omental fat (0.02 +/- 0.001 vs. 0.03 +/- 0.003 pmol/ product/min.mg protein; P <0.05) and higher hepatic 5beta-reductase activity (0.26 +/- 0.05 vs. 0.10 +/- 0.007 pmol product/min.mg protein; P <0.05). After 20 wk, HF rats were obese, hyperglycemic, and hyperinsulinemic, but differences in 11beta-HSD-1 and 5beta-reductase activities were no longer apparent. Mature male rats given HF diets for 24 or 72 h showed increased hepatic 5beta-reductase activity and a trend for decreased sc adipose 11beta-HSD-1 activity. Dietary obesity is not accompanied by the changes in 11beta-HSD-1 and 5beta-reductase expression and activity observed in genetically obese rodents. Acute exposure to HF diet alters glucocorticoid metabolism, predicting lower hepatic and adipose intracellular glucocorticoid concentrations, which may be a key mechanism protecting against the metabolic complications of obesity.     

Stimulation of Brucella clearance in rats after treatment with BCG suspensions

Intraperitoneal application of BCG induced a significantly increased clearance of Brucella in rats. The effect starts three days after application of 7 mg BCG to the rat and continued for eight weeks. This effect could be used for estimation of efficiency of BCG for nonspecific resistance stimulation. Comparative investigation of seven different batches reveals that three BCG preparations induce an unspecific resistance more than eight weeks, one for two weeks and three batches exerted no effect. Addition of Gelafusal as a stabilizer prolonged durability for about two weeks.     

Reduced arsenic clearance and increased toxicity in aquaglyceroporin-9-null mice

Expressed in liver, aquaglyceroporin-9 (AQP9) is permeated by glycerol, arsenite, and other small, neutral solutes. To evaluate a possible protective role, AQP9-null mice were evaluated for in vivo arsenic toxicity. After injection with NaAsO₂, AQP9-null mice suffer reduced survival rates (LD₅₀, 12 mg/kg) compared with WT mice (LD₅₀, 15 mg/kg). The highest tissue level of arsenic is in heart, with AQP9-null mice accumulating 10–20 times more arsenic than WT mice. Within hours after NaAsO₂ injection, AQP9-null mice sustain profound bradycardia, despite normal serum electrolytes. Increased arsenic levels are also present in liver, lung, spleen, and testis of AQP9-null mice. Arsenic levels in the feces and urine of AQP9-null mice are only ≈10% of the WT levels, and reduced clearance of multiple arsenic species by the AQP9-null mice suggests that AQP9 is involved in the export of multiple forms of arsenic. Immunohistochemical staining of liver sections revealed that AQP9 is most abundant in basolateral membrane of hepatocytes adjacent to the sinusoids. AQP9 is not detected in heart or kidney by PCR or immunohistochemistry. We propose that AQP9 provides a route for excretion of arsenic by the liver, thereby providing partial protection of the whole animal from arsenic toxicity.Evolutionary exposure to environmental arsenic has led to selection of organisms ranging from microbes to mammals with mechanisms for coping with arsenic toxicity. Humans may be inadvertently exposed to arsenic from water contaminated from geological sources or industrial pollution. Up to 57 million people in Bangladesh presently drink groundwater with arsenic concentrations above the World Health Organization acceptable standard (1). In clinical medicine, arsenic trioxide is used in tandem with all-trans-retinoic acid to treat acute promyelocytic leukemia (2, 3), and drugs containing arsenic and antimony are used to treat parasitic infections, African sleeping sickness, and leishmaniasis (4).Transport proteins are critical in the response to arsenic toxicity, because uptake and export are two key features in the cellular drug response. Members of the ATP-binding cassette transporter family export certain drugs, and some cell lines resistant to arsenic have increased expression of multidrug-resistance proteins (MRPs) (5–7). MRP1-null mice exhibit increased sensitivity to sodium arsenite (8). Multidrug-resistance gene MDR1a/1b double-null mice are even more sensitive and accumulate arsenic in their organs after exposure (9).Several lines of evidence indicate that aquaglyceroporins are involved in arsenite uptake by mammalian cells. A member of the aquaporin gene family, yeast glycerol transporter (Fps1p), was shown to facilitate arsenite [As(III)] and antimonite [Sb(III)] uptake by eukaryotic cells (10). The mammalian aquaglyceroporins AQP3, AQP7, and AQP9 were shown to transport arsenite and antimonite (7, 11). Studies of multiple cultured cell lines revealed increased AQP3 or AQP9 expression, resulting in increased arsenic accumulation and toxicity (7, 12–15). A human lung adenocarcinoma cell line resistant to arsenic was found to have decreased levels of AQP3 (7). When AQP3 expression was reduced further, arsenite uptake declined still further, and cellular resistance to arsenic toxicity increased (7).Known to transport a range of small, neutral solutes (16), including arsenite, antimonite, and methylarsonous acid [MAs(III)] (11, 17), AQP9 is expressed in liver, testes, brain, and leukocytes (16, 18, 19), some tissues being sensitive to arsenite. In vivo studies may be complicated by conversion of arsenite to other forms by oxidation, reduction (20), methylation (21, 22), and glutathionylation (20, 23). Despite plentiful evidence that aquaglyceroporins are involved in arsenite transport, arsenic toxicity has not been reported in AQP9-null mice.Here, we describe arsenic toxicity studies of AQP9-null mice compared with WT mice. We determined that AQP9-null mice suffer reduced survival after injection with sodium arsenite (NaAsO₂). Increased accumulation of arsenic was observed in multiple organs of AQP9-null mice, with highest levels of arsenic in heart, accompanied by profound bradycardia. Excretion of arsenic in urine and feces of AQP9-null mice is greatly reduced. Although AQP9 is known to facilitate uptake of arsenite by single cells in culture (12–15), our studies cause us to propose that AQP9 also facilitates in vivo arsenic excretion by the liver, thereby providing partial protection against arsenic toxicity.     

Endogenous catecholamine stimulates alveolar fluid clearance in rats with acute pancreatitis

Background and objective: Acute pancreatitis causes pulmonary oedema with the accumulation of fluid in the alveolar spaces, possibly due to reduced clearance. This study tested the hypothesis that acute pancreatitis decreases alveolar fluid clearance in a rat model of pulmonary oedema during acute pancreatitis. Methods: Acute pancreatitis was induced by a retrograde injection of 5% taurocholate sodium (0.2 mL) into the common bile duct. The lungs were isolated 4, 24 and 48 h after the induction of acute pancreatitis and alveolar fluid clearance was measured in the absence of pulmonary perfusion. Results: Alveolar fluid clearance increased to 31.0 ± 3.5% of instilled volume/h in rats with acute pancreatitis for 4 h compared with 17.3 ± 1.0% of instilled volume/h in sham rats (P < 0.01), then returned to the control level 48 h after acute pancreatitis (16.0 ± 4.1% of instilled volume/h). In contrast, the lung water to dry lung weight ratio decreased maximally 24 h after acute pancreatitis (P < 0.01), then returned to the control level 48 h after acute pancreatitis. The plasma epinephrine levels increased to 25‐fold higher in rats with acute pancreatitis for 4 h than in sham rats without acute pancreatitis. Prazosin (an α₁‐adrenergic antagonist, 10^?4 mol/L), yohimbine (an α₂‐adrenergic antagonist, 10^?4 mol/L) or a bilateral adrenalectomy inhibited the increase in part, a combination of prazosin (10^?4 mol/L) and yohimbine (10^?4 mol/L) completely inhibited the increase in alveolar fluid clearance in rats after acute pancreatitis for 4 h, whereas propranolol (a β‐adrenergic antagonist, 10^?4 mol/L) had no effect. Conclusions: Endogenous catecholamine stimulates α‐adrenoceptors and increases alveolar fluid clearance in rats with acute pancreatitis.     

Metabolic clearance rates of catechol estrogens in rats

MCRs of the catechol estrogens 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) and of the parent estrogen 17 beta-estradiol (E2) were determined in rats. Long term ovariectomized Wistar rats were infused with the steroids at a constant rate for 3 days via a catheter placed in the abdominal aorta. Blood samples were drawn discontinually by retroorbital puncture, and the serum concentrations of E2, 4-OHE2, and 2-OHE2 were measured by RIA. Steady state was reached within 24 h of infusion. Mean serum MCRs were calculated to be 740 +/- 117 ml/h for E2, 2700 +/- 1000 ml/h for 4-OHE2, and 8300 +/- 1700 ml/h for 2-OHE2. Thus, the MCRs of the catechol estrogens were definitely higher than the MCR of E2 resulting in an apparent ratio of 1:4:11 (E2:4-OHE2:2-OHE2).     

Reduced noradrenaline turnover in streptozotocin-induced diabetic rats

Summary To clarify whether activity of the sympathetic nervous system is decreased in streptozotocin-induced diabetic rats, noradrenaline turnover, which is a reliable indicator of sympathetic nervous system activity, was measured in the interscapular brown adipose tissue, heart and pancreas of streptozotocin diabetic rats. Results from studies using inhibition of noradrenaline biosynthesis with -methyl-p-tyrosine demonstrated significant reductions (p<0.05-0.001) in sympathetic nervous system activity in the interscapular brown adipose tissue, heart and pancreas of streptozotocin (65 mg/kg) diabetic rats, compared with measurements in streptozotocin (35 mg/kg) diabetic and saline-control rats. The daily injections of neutral protamine Hagedorn insulin to streptoz/otocin (65 mg/kg) diabetic rats prevented the decrease of noradrenaline turnover in the interscapular brown adipose tissue and heart significantly (p<0.02), but this was less marked in pancreas, compared with non-treated streptozotocin (65 mg/kg) diabetic rats. Furthermore reduced noradrenaline turnover was also observed in the control rats which showed comparable changes in body weight to the rats injected with streptozotocin (65 mg/kg). These results suggest that poorly controlled streptozotocin diabetic rats may have reduced sympathetic nervous function, and that insulin therapy might prevent this.