Physiological characteristics of diabetic neuropathy in sucrose-fed Otsuka Long-Evans Tokushima fatty rats

Diabetic neuropathy is a very common complication of diabetes mellitus, and animal studies have contributed tremendously to its understanding. The aim of this study was to estimate the neuropathic alterations in the Otsuka Long-Evans Tokushima fatty (OLETF) rats, an animal model of human type 2 diabetes mellitus. For this purpose, four groups of animals were used: untreated OLETF rats, sucrose-fed for 2 months OLETF rats, untreated Long-Evans Tokushima Otsuka (LETO) nondiabetic rats as genetic controls of OLETF, and sucrose-fed LETO rats. All were examined at baseline, at the end of the sucrose treatment, and during a washout period. The following parameters were evaluated: motor nerve conduction velocity (MNCV), sensitivity to noxious thermal and mechanical stimuli using the tail-flick (TF) and tail-pressure (TP) tests, and blood glucose (BG) and HbA1c levels. Our results showed that BG and HbA1c were significantly higher in OLETF rats when compared with those in control LETO rats. Sucrose caused remarkable increase of BG and HbA1c in the OLETF rats, but not in the sucrose-fed LETO rats. MNCV and thermal nociception significantly decreased in OLETF rats in their 10th month, while the values of the TP test did not differ compared with those from LETO rats. Sucrose administration significantly decreased the MNCV, and increased the pain threshold evaluated by the TF and TP tests, compared with those in the control OLETF rats. The studied parameters were not significantly altered in sucrose-fed LETO rats. In conclusion, our findings show that signs of diabetic neuropathy appear late in the individual development of the OLETF rats, and MNCV and thermal nociception are selectively affected in this strain. Sucrose deteriorated the diabetic state, decreased MNCV, and caused thermal and mechanical hypoalgesia.     

Hydronephrosis in rats associated with postnatal exposure to a fatty acid oxidation inhibitor

Methyl palmoxirate, an inhibitor of long-chain fatty acid oxidation, was administered by gavage (0, 1, 5, or 20 mg/kg/day) to female rats over the last third of gestation and throughout lactation. Weight gain (mid- and high-dosage group) and survivability (high-dosage group) were significantly (p less than or equal to 0.05) reduced in offspring of methyl palmoxirate-treated dams as compared to control offspring. Mid- and high-dosage male offspring found dead after Day 4 of lactation exhibited grossly distended bladders and renal pelves. A dosage-related increased incidence of dilated renal pelves was observed in both sexes at necropsy of 21-day-old mid- and high-dosage group pups. Microscopic examination of the urinary tracts of a number of affected pups revealed renal parenchymal atrophy and urethral obstruction. Drug disposition studies indicated lactating pups were exposed to significant amounts of methyl palmoxirate via mammary secretions. Cross-fostering experimentation suggested that some of the adverse effects observed in offspring were due to lactational, rather than in utero, exposure.     

Induction of triglyceride accumulation in the liver of rats by perfluorinated fatty acids with different carbon chain lengths: comparison with induction of peroxisomal beta-oxidation

The potency to accumulate triglyceride (TG) was compared between perfluorinated fatty acids (PFCAs) with different carbon chain lengths in the liver of male and female rats and induction of peroxisomal beta-oxidation. In male rats, either perfluoroheptanoic acid (C7) or perfluorooctanoic acid (C8) had no effect, although perfluorononanonic acid (C9) and perfluorodecanoic acid (C10) markedly accumulated TG. In female rats, C7, C8, and C9 did not cause TG accumulation, whereas C10 caused TG accumulation at the same level as in male rats. TG accumulation induced by C9 was regulated by the level of testosterone in male rats. In contrast with TG accumulation, peroxisomal beta-oxidation was induced by C8, C9, and C10 in male rats and by C9 and C10 in female rats. Only a slight difference was observed in the induction by C9 between male and female rats. The induction of TG accumulation by these PFCAs occurred in a dose-dependent manner and significantly correlated with hepatic concentrations of PFCA regardless of their carbon chain length, as was observed with induction of peroxisomal beta-oxidation. There is, however, a striking difference in the hepatic concentration of PFCA required to cause induction of TG accumulation and that of peroxisomal beta-oxidation. The concentration of PFCA required to induce TG accumulation is much higher than that to induce peroxisomal beta-oxidation. These results strongly suggest that TG accumulation induced by PFCAs, as well as the induction of peroxisomal beta-oxidation, is dependent only on the number of PFCA molecules in hepatocytes, but is not due to the difference in their chemical structures, and that there is a marked difference in the PFCA threshold to cause distinct biological effects.     

Effects of silymarin and its combination with succinic acid on brain bioenergetics in rats with experimental inhibition of beta-oxidation of fatty acids

In the tests on rats with a model of encephalopathy caused by 4-pentenoic acid (inhibitor of the beta-oxidation of fatty acids), the hepatoprotective agent silymarin increases the respiratory activity in brain mitochondria, improves oxidative phosphorylation coupling and energization, and inhibits lipid peroxidation. Succinic acid (a regulator of bioenergetics) improves the damaged Krebs cycle metabolic pathways and produces an antioxidant effect. The combined use of silymarin and succinic demonstrated more expressed cerebroprotective action as compared to that of each agent administered separately.     

DDT increases hepatic testosterone metabolism in rats

DDT and its metabolites are considered as endocrine disruptors able to promote hormone-dependent pathologies. We studied the effects of technical-grade DDT on hepatic testosterone metabolism and testosterone hydroxylase activity ratios in the rat. Male and female Wistar rats were treated by gavage with a single dose of technical-grade DDT (0, 0.1, 1, 10, and 100 mg/kg body weight) and killed 24 h later. Hepatic microsomes were incubated with [4-¹⁴C]-testosterone and the metabolites were separated by thin-layer chromatography and quantified by radio scanning. DDT increased testosterone biotransformation and modified the profile of metabolites produced in a sex-dependent manner. Males treated with a representative dose (10 mg/kg) produced relatively less androstenedione (AD), 2-hydroxytestosterone (OHT), and 16-OHT but higher 6-OHT whereas treated females produced less 7-OHT and AD but higher 6-OHT and 6-OHT than their respective controls. In both sexes DDT decreased the relative proportion of AD and increased that of 6-OHT suggesting that the androgen-saving pathway was affected. The testosterone 6-/15-OHT ratio, a proposed indicator of demasculinization, was increased in treated males. This effect was in agreement with the demasculinizing ability proposed for DDT. The effects on 6-/16-OHT and 6-dehydrotestosterone/16-OHT ratios followed a similar tendency, with the ratio 6-/16-OHT being the most sensitive marker. Interestingly, these ratios were reduced in treated females suggesting that technical-grade DDT shifted testosterone hydroxylations toward a more masculine pattern. Thus, technical-grade DDT altered the hepatic sexual dimorphism in testosterone metabolism and decreased the metabolic differences between male and female rats.     

Reduced activity of hepatic microsomal fatty acid chain elongation synthesis in clofibrate-fed rats.

Na-clofibrate dissolved in drinking water was administered at a dose of 12 mg per day per animal to adult male Wistar rats. After different periods of drug administration, measurement was made of the activity of hepatic acetyl-CoA carboxylase, fatty acid synthetase and both microsomal and mitochondrial fatty acid chain elongation systems. Data obtained indicate a significant reduction in all synthetic activities, with the exception of the mitochondrial, even after dialysis of the investigated subcellular fractions. This reduction was found to increase with increased drug-administration periods, reaching the maximum after 7–8 days. A similar effect was previously shown by clofibrate in vitro [7]. The present results indicate an increase of 66.4% in hepatic cyclic AMP level as well after seven days of drug feeding to rats. Similarly, both serum aspartate aminotransferase and alanine amino-transferase activities were found to increase by about 30% after 13 days. The hypothesis is advanced that in vivo clofibrate probably reduces the synthetic activities under investigation by firmly binding to active enzyme-protein sites. In addition, the possibility that also the altered cyclic AMP level induced by this drug is responsible for both the above reductions and other metabolic variations reported elsewhere should not be excluded.     

Enhancement of peroxisomal beta-oxidation in the liver of rats and mice treated with valproic acid

The effects of valproic acid on peroxisomal beta-oxidation and on lipid levels of liver and serum in the rat and mouse were studied. When the animals were fed diet containing 1% valproic acid for 2 weeks, the activity of peroxisomal beta-oxidation increased 4-fold in the rat liver and 2-fold in the mouse liver. Other peroxisomal enzymes such as catalase and urate oxidase also increased by the treatment though to a lesser extent than beta-oxidation. The contents of triglyceride and cholesterol in the serum decreased significantly in the rat but not in the mouse. The time course curves of the activities of cyanide-insensitive palmitoyl-CoA oxidation and carnitine-dependent palmitoyltransferase indicated that peroxisomal beta-oxidation was enhanced more rapidly than that of mitochondrial. The distributions of these enzymes were not changed by the treatment with valproic acid, though increases in liver weight and protein content were observed. These results indicate that the action of valproic acid in enhancing hepatic beta-oxidation is similar to that of clofibrate and other hypolipidemic drugs.     

Technetium-99m-labeled long chain fatty acid analogues metabolized by beta-oxidation in the heart

The development of 99mTc-labeled fatty acid analogues metabolized by beta-oxidation in the myocardium constitutes an unsolved challenge. On the basis of our recent findings that [188Re]tricarbonyl(cyclopentadienylcarbonate)rhenium ([188Re]CpTR-COOH) was recognized as an aromatic compound and was metabolized as such in the body, [99mTc]cyclopentadienyltricarbonyltechnetium ([99mTc]CpTT) was conjugated at the omega-position of pentadecanoic acid to prepare [99mTc]CpTT-PA. When injected into rats, [99mTc]CpTT-PA exhibited the maximum myocardial accumulation and heart-to-blood ratio of 3.85 %ID/g at 1 min and 4.60 at 10 min postinjection, respectively. The metabolic study using isolated Langendorff perfused rat hearts demonstrated that approximately 67% of perfused [99mTc]CpTT-PA was incorporated and [99mTc]CpTT-propionic acid, the metabolite after six cycles of beta-oxidation of [99mTc]CpTT-PA, was detected as the major radiometabolite in the perfusate and myocardium. These findings indicate that [99mTc]CpTT-PA was recognized, transported, and metabolized as a long chain fatty acid analogue for energy production in the myocardium.     

Enhanced peroxisomal beta-oxidation of fatty acids and glutathione metabolism in rats exposed to phenoxyacetic acids

Peroxisomal beta-oxidation of fatty acids and the activities of glutathione-metabolizing enzymes in rat liver were measured after administration of 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), clofibrate [ethyl], 2-(p-chlorophenoxy)-2-methylpropionate], glyphosate (N-phosphonomethyl glycine, a herbicide not structurally related to phenoxy acids) or saline for 14 days. beta-Oxidation increased by 6-fold in the group given clofibrate, 3-fold in the 2,4-D-treated group, and 2-fold in the MCPA-treated group over the level in the controls (saline-treated). Glyphosate did not increase beta-oxidation. No significant change in reduced glutathione content from that in controls was found in any of the treated groups. Glutathione reductase activity increased by about 40% after administration of either 2,4-D or MCPA, and glutathione peroxidase activity increased by 30% in animals given MCPA. A slight decrease in glutathione S-transferase activity was found in the group treated with clofibrate. The marked increases in peroxisomal beta-oxidation of fatty acids were accompanied by only minor changes in the activities of enzymes involved in glutathione-dependent inactivation of organic hydroperoxides and other oxygen-centred reactive agents.     

Dietary lead: Effects on hepatic fatty acid composition in chicks

Arbor Acre broiler chicks were fed diets containing 0, 500, 750, 1000, 2000, or 4000 ppm lead (as Pb acetate · 3 H₂O) from day-old through 21 days of age. There were 8 groups of 10 male chicks per lead level. Eight chicks from each dietary lead level were killed at 21 days, and hepatic fatty acid composition was determined for each chick by gas-liquid chromatography. Increasing dietary lead levels decreased the concentrations of 16:1 and 18:1 fatty acids (first No. = No. carbons; second No. = No. double bonds) and increased the concentrations of 18:0 and 20:4. The concentration of 18:2 fatty acids did not differ significantly from control values for any level of lead. However, the ratio $\text{18:2}\text{20:4}$ declined from a control value of 3.3 to approximately 2 for all lead treatments. The ratio of saturated/monoenoic fatty acids increased with dietary lead levels above 1000 ppm. In a second experiment 10 male broiler chicks per treatment were fed either a control diet or the control diet plus 2000 ppm lead, 60 ppm cadmium, 500 ppm mercury, or 10 ppm selenium (as Pb acetate · 3 H₂O, CdSO₄, HgCl₂, or Na₂SeO₃, respectively) for 21 days. Six chicks from each group were killed at 21 days, and hepatic fatty acid composition was determined for each chick. In comparison to control, the ratio $\text{18:2}\text{20:4}$ was lowered by lead but unaffected by cadmium, mercury, and selenium. The data suggest that lead may increase tissue peroxidation (as noted by other workers) via a relative increase of 20:4 fatty acid and that a decrease of hepatic ratio $\text{18:2}\text{20:4}$ may be a specific sign of lead toxicity.     

Panipenum, a carbapenem antibiotic, increases the level of hepatic UDP-glucuronic acid in rats.

To investigate the mechanism for the enhanced glucuronidation of valproic acid (VPA) by panipenem (PAPM), a carbapenem antibiotic, in rat liver, we carried out studies to investigate whether PAPM increases the activity of UDP-glucuronosyltransferase or the level of hepatic UDP-glucuronic acid (UDPGA) in rats. PAPM had no effect on the UDP-glucuronosyltransferase activity toward VPA both in vivo and in vitro. On the other hand, in vivo treatment with PAPM significantly increased the hepatic UDPGA level by about 1.7-fold (control: 434.5 +/- 65.5 nmol/g of liver; PAPM-treated: 755.2 +/- 92.3 nmol/g of liver). The in vitro formation of VPA glucuronide increased proportionally as a function of the UDPGA concentration up to 0.8 mM. Therefore, the increase in the level of hepatic UDPGA by PAPM is likely to be one of the causal factors for enhancing VPA glucuronidation in vivo.     

Exposure of perfluorononanoic acid suppresses the hepatic insulin signal pathway and increases serum glucose in rats

Exposure to perfluorononanoic acid (PFNA), an increasingly persistent organic pollutant that has been detected in abiotic and biotic matrices, has been demonstrated to cause hepatotoxicity in animals. However, the effects of PFNA on hepatic glucose metabolism have not been fully characterized. In this study, male rats were exposed to 0, 0.2, 1 or 5mg/kg/d PFNA for 14 days to explore the specific effect of PFNA on hepatic glycometabolism and its underlying mechanisms. The results showed that administration of 5mg/kg/d PFNA significantly increased serum glucose and hepatic glycogen in rats. Quantitative real-time PCR analysis showed that PFNA exposure changed the expression levels of several genes related to hepatic glucose metabolism, such as the glucose-6-phosphatase (G6PC) gene and the glucose transporter 2 (GLUT2) gene, which were upregulated, and the glucokinase (GCK) gene and the phosphoinositide-3-kinase, catalytic, alpha polypeptide (PI3Kca) gene, which were decreased. The protein expression levels of phospho-insulin receptor 1(IRS1), phospho-PI3K, phospho-AKT and phospho-phosphoinositide-dependent kinase 1 (PDK1) were decreased in the livers of rats that received 5mg/kg/d PFNA. The expression of phospho-glycogen synthase kinase-3 beta (GSK3β, Ser 9) was increased, which explains the augment of hepatic glycogen. Significant increases in hydrogen peroxide (H(2)O(2)) and malondialdehyde (MDA) were found in the livers of 5mg/kg/d PFNA-treated rats. Thus, exposure to PFNA disordered glucose metabolism via inhibiting hepatic insulin signal pathway, accelerating the output of glucose and increasing glycogen synthesis in the rat liver. Furthermore, the oxidative stress induced by PFNA may be involved in this process.     

Nateglinide suppresses postprandial hypertriglyceridemia in Zucker fatty rats and Goto-Kakizaki rats: comparison with voglibose and glibenclamide

Postprandial hypertriglyceridemia, as well as postprandial hyperglycemia, are important factors contributing to the development of cardiovascular disease in patients with type 2 diabetes. Nateglinide is a recently approved antidiabetic that suppresses postprandial hyperglycemia by stimulating the early phase of insulin secretion. In the present study, we investigated the effects of nateglinide on postprandial hypertriglyceridemia in obese Zucker fatty (ZF) rats and non-obese diabetic Goto-Kakizaki (GK) rats. Administration of an oral fat load caused marked hypertriglyceridemia with a peak at 2 h in ZF and GK rats. Nateglinide (50 mg/kg) significantly suppressed the increase of plasma triglycerides after fat loading in both types of rat (delta AUC [0-4 h]: 15+/-69 mg.h/dl for nateglinide vs. 838+/-100 mg.h/dl for vehicle in ZF rats; p<0.01, 81+/-22 mg x h/dl for nateglinide vs. 164+/-17 mg.h/dl for vehicle in GK rats; p<0.01). In contrast, other antidiabetic agents (voglibose and glibenclamide) did not show a significant effect on the increase of triglycerides after fat loading. The triglyceride components suppressed by nateglinide were mainly at the origin and in the pre beta subfraction on agarose gel electrophoresis, suggesting that chylomicrons and very low density lipoproteins were decreased. Plasma insulin levels were significantly increased at 30 min in nateglinide-treated rats, but not in voglibose- or glibenclamide-treated rats. These results suggest that nateglinide not only suppresses postprandial hyperglycemia, but also suppresses postprandial hypertriglyceridemia, by promoting rapid and pulsatile insulin secretion in patients with type 2 diabetes.     

Soraphen A, an inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation

Acetyl CoA carboxylase (ACC1 and ACC2) generates malonyl CoA, a substrate for de novo lipogenesis (DNL) and an inhibitor of mitochondrial fatty acid β-oxidation (FAO). Malonyl CoA is also a substrate for microsomal fatty acid elongation, an important pathway for saturated (SFA), mono- (MUFA) and polyunsaturated fatty acid (PUFA) synthesis. Despite the interest in ACC as a target for obesity and cancer therapy, little attention has been given to the role ACC plays in long chain fatty acid synthesis. This report examines the effect of pharmacological inhibition of ACC on DNL and palmitate (16:0) and linoleate (18:2, n − 6) metabolism in HepG2 and LnCap cells. The ACC inhibitor, soraphen A, lowers cellular malonyl CoA, attenuates DNL and the formation of fatty acid elongation products derived from exogenous fatty acids, i.e., 16:0 and 18:2, n − 6; IC₅₀ ∼ 5 nM. Elevated expression of fatty acid elongases (Elovl5, Elovl6) or desaturases (FADS1, FADS2) failed to override the soraphen A effect on SFA, MUFA or PUFA synthesis. Inhibition of fatty acid elongation leads to the accumulation of 16- and 18-carbon unsaturated fatty acids derived from 16:0 and 18:2, n − 6, respectively. Pharmacological inhibition of ACC activity will not only attenuate DNL and induce FAO, but will also attenuate the synthesis of very long chain saturated, mono- and polyunsaturated fatty acids.     

心肌脂肪酸氧化抑制剂的研究进展

随着人们对缺血心肌能量代谢改变的深入研究,发现抑制脂肪酸的氧化可改善组织氧利用的有效性,为缺血性心脏病的改善和预后提供了新的治疗措施。本文综述了心肌脂肪酸氧化抑制剂的药理效应、作用机制以及此类药物的研究进展。     

Early hepatic changes in rats induced by permethrin in comparison with DDT

In this study permethrin [(3-phenoxyphenyl)-methyl-3-(2,2-dichloroethenyl)-2,2-dim ethylcyclopropanecarboxylate] and DDT [1,1-(2,2,2 trichloroethylidene)-bis-(4-chlorobenzene)] were compared in rats for their effects on early hepatic changes, proposed in the literature to be useful endpoints in screening for non-genotoxic hepatocarcinogenesis and/or liver tumour promotion. We compared the effects of both insecticides on the following endpoints: hepatomegaly, mitogenesis (DNA synthesis, mitotic activity, percentage of binuclear cells) and liver pathology. Male Wistar rats received permethrin (PERM) or DDT in one, three, five and 14 daily oral doses (at 24-h intervals) equivalent to 1/10 LD50. Distinct differences in early liver response between PERM and DDT were observed. DDT stimulated the early effect consisting of hepatomegaly accompanied by an increase in hepatocellular proliferation with signs of cell necrosis. Thus, it might be concluded, that the mitogenic effect of DDT was at least partly related to a regenerative liver response. Although PERM significantly affected DNA synthesis and increased binuclear hepatocytes, this compound did not increase the number of mitotic figures. These results suggest that PERM may inhibit of phase G2 in the cell cycle and consequently it may suppress the cell entering into the stage of mitosis (M-phase). In addition, the present findings provide evidence for the occurrence of abnormal mitoses in the hepatocytes of rats treated with DDT.     

基于UPLC-MS/MS的新型脂肪酸合成酶抑制剂4k在大鼠体内的药动学研究

目的 建立超高效液相色谱-串联质谱法(UPLC-MS/MS)测定大鼠血浆中新型脂肪酸合成酶抑制剂4k的浓度,并研究其在大鼠体内的药动学特征。方法 12只SD大鼠随机分为2组,分别单次尾静脉注射和灌胃给予4k。以三氯生为内标,建立并验证UPLC-MS/MS测定不同时间点大鼠血浆中4k的浓度,用DAS 2.0软件计算药动学参数。结果SD大鼠单次静脉注射后主要药动学参数为T_1/2 (0.54±0.39)h,T_max 0.033 h,C_max (2 730.72±803.13)ng·mL^-1,AUC_0-∞ (577.72± 174.58)ng·mL^-1·h,Vd (1 241.17±657.98)mL·kg^-1,CL (1 882.67±610.03)mL·kg^-1·h^-1,MRT_0-∞ (0.42±0.19)h。由于灌胃给药后,大鼠体内血药浓度低于定量下限,因此无法进行药动学参数计算。结论 本方法简单准确、快速灵敏,适用于大鼠血浆中4k浓度的测定及其药动学研究。