Amarylis C.B.A. Wanschel

Chronic use of pravastatin reduces insulin exocytosis and increases β-cell death in hypercholesterolemic mice.

We have previously demonstrated that hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice secrete less insulin than wild-type mice. Removing cholesterol from isolated islets using methyl-beta-cyclodextrin reversed this defect. In this study, we hypothesized that in vivo treatment of LDLr(-/-) mice with the HMGCoA reductase inhibitor pravastatin would improve glucose-stimulated insulin secretion. Female LDLr(-/-) mice were treated with pravastatin (400mg/L) for 1-3 months. Isolated pancreatic islets were assayed for insulin secretion rates, intracellular calcium oscillations, cholesterol levels, NAD(P)H and SNARE protein levels, apoptosis indicators and lipidomic profile. Two months pravastatin treatment reduced cholesterol levels in plasma, liver and islets by 35%, 25% and 50%, respectively. Contrary to our hypothesis, pravastatin treatment increased fasting and fed plasma levels of glucose and decreased markedly (40%) fed plasma levels of insulin. In addition, ex vivo glucose stimulated insulin secretion was significantly reduced after two and three months (36-48%, p<0.05) of pravastatin treatment. Although reducing insulin secretion and insulinemia, two months pravastatin treatment did not affect glucose tolerance because it improved global insulin sensitivity. Pravastatin induced islet dysfunction was associated with marked reductions of exocytosis-related SNARE proteins (SNAP25, Syntaxin 1A, VAMP2) and increased apoptosis markers (Bax/Bcl2 protein ratio, cleaved caspase-3 and lower NAD(P)H production rates) observed in pancreatic islets from treated mice. In addition, several oxidized phospholipids, tri- and diacylglycerols and the proapoptotic lipid molecule ceramide were identified as markers of pravastatin-treated islets. Cell death and oxidative stress (H2O2 production) were confirmed in insulin secreting INS-1E cells treated with pravastatin. These results indicate that chronic treatment with pravastatin impairs the insulin exocytosis machinery and increases β-cell death. These findings suggest that prolonged use of statins may have a diabetogenic effect.

Lacking of estradiol reduces insulin exocytosis from pancreatic β-cells and increases hepatic insulin degradation

Low levels of plasma estrogens are associated with weight-gain, android fat distribution, and a high prevalence of obesity-related comorbidities such as glucose intolerance and type II diabetes. The mechanisms underlying the association between low levels of estrogens and impaired glucose homeostasis are not completely understood. To begin to test this, we used three-month-old female C57BL/6J mice that either underwent ovariectomy (OVX) or received a sham surgery (Sham), and we characterized glucose homeostasis. In a subsequent series of experiments, OVX mice received estradiol treatment (OVX+E2) or vehicle (OVX) for 6 consecutive days. As has been previously reported, lack of ovarian hormones resulted in dysregulated glucose homeostasis. To begin to explore the mechanisms by which this occurs, we characterized the impact of estrogens on insulin secretion and degradation in these mice. Insulin secretion and plasma insulin levels were lower in OVX mice. OVX mice had lower levels of pancreatic Syntaxin 1-A (Synt-1A) protein, which is involved in insulin extrusion from the pancreas. In the liver, OVX mice had higher levels of insulin-degrading enzyme (IDE) and this was associated with higher insulin clearance. Estradiol treatment improved glucose intolerance in OVX mice and restored insulin secretion, as well as normalized the protein content of pancreatic Synt-1A. The addition of estrogens to OVX mice reduced IDE protein to that of Sham mice. Our data suggest loss of ovarian estradiol following OVX led to impaired glucose homeostasis due to pancreatic β-cell dysfunction in the exocytosis of insulin, and upregulation of hepatic IDE protein content resulting in lower insulinemia, which was normalized by estradiol replacement.

Apolipoprotein CIII Overexpression-Induced Hypertriglyceridemia Increases Nonalcoholic Fatty Liver Disease in Association with Inflammation and Cell Death

Nonalcoholic fatty liver disease (NAFLD) is the principal manifestation of liver disease in obesity and metabolic syndrome. By comparing hypertriglyceridemic transgenic mice expressing apolipoprotein (apo) CIII with control nontransgenic (NTg) littermates, we demonstrated that overexpression of apoCIII, independent of a high-fat diet (HFD), produces NAFLD-like features, including increased liver lipid content; decreased antioxidant power; increased expression of TNFα, TNFα receptor, cleaved caspase-1, and interleukin-1β; decreased expression of adiponectin receptor-2; and increased cell death. This phenotype is aggravated and additional NAFLD features are differentially induced in apoCIII mice fed a HFD. HFD induced glucose intolerance together with increased gluconeogenesis, indicating hepatic insulin resistance. Additionally, the HFD led to marked increases in plasma TNFα (8-fold) and IL-6 (60%) in apoCIII mice. Cell death signaling (Bax/Bcl2), effector (caspase-3), and apoptosis were augmented in apoCIII mice regardless of whether a HFD or a low-fat diet was provided. Fenofibrate treatment reversed several of the effects associated with diet and apoCIII expression but did not normalize inflammatory traits even when liver lipid content was fully corrected. These results indicate that apoCIII and/or hypertriglyceridemia plays a major role in liver inflammation and cell death, which in turn increases susceptibility to and the severity of diet-induced NAFLD.

Monitoring of florfenicol residues in fish muscle by HPLC-UV with confirmation of suspect results by LC-MS/MS.

Florfenicol, a derivative of thiamphenicol in which the hydroxyl group at C-3 has been replaced with fluorine, is listed by the World Health Organization as an antibacterial agent for human medicine that is critically important in risk management strategies for non-human use. AOAC International has also identified it as an important molecule for the development of effective methods for the seafood sector. Following inspection missions from the European Union and United States of America, it was introduced in the Brazilian Residues Control Program to fulfill export and internal national requirements with a Maximum Residue Limit of 800 µg/kg. A high performance liquid chromatography method with ultraviolet detection at a wavelength of 230 nm (λ = 230 nm) for the detection of florfenicol in fish muscle was developed and validated according to the Brazilian Regulation 24/2009 (equivalent to European Union Decision 2002/657/EC). Fish samples were extracted with ethyl acetate and hexane followed by C18 solid phase clean-up and chromatographic separation on a reversed-phase C18 LC column with acetonitrile:water as mobile phase. The method results were also was compared with those obtained using liquid chromatography-tandem quadrupole mass spectrometry. The method meets the Brazilian regulatory requirements with a decision limit (CCα) of 840 µg/kg and detection capability (CCβ) of 879 µg/kg. This method is easy to use and has been implemented into Brazils residue control program, with liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation of any suspect samples using the same method.

Laboratory validation of an LC-MS/MS method for the detection of ractopamine, clenbuterol and salbutamol in bovine and swine muscle at sub-μg kg–1 regulatory limits

ABSTRACT Ractopamine (RAC), is a β-adrenergic agonist increasingly used in the swine and cattle industry. This compound redirects nutrients to favour leanness rather than fat deposition, improves growth and carcass traits gaining higher economic benefit to producers. Countries around the world are split over whether to allow the use of RAC in meat production. Clenbuterol (CLB) and salbutamol (SLB) are anillinic and phenolic β-agonists, respectively, with the same capacity of producing economic benefits for the meat sector. However, they are prohibited because of the potentially adverse reactions they can cause in consumers. The three β-agonist compounds have been included in the Brazilian National Regulatory Survey and consequentially there is an eminent need for reliable methods capable of detecting those substances at the same time and reduce analytical costs. Therefore, an LC-MS/MS method for the simultaneous determination of residual RAC, CLB and SAL in swine and cattle muscle was developed and validated with quantification levels respecting the action levels established for Brazil which are 0.1, 0.2 and 5 µg kg–1 for RAC, CLB and SAL, respectively. Samples were quantified using RAC-d5, CLB-d9 and SLB-d6 as internal standards. The validation was performed according to European Union Decision 2002/657, which includes criteria (CCα, CCβ, recovery, repeatability, reproducibility and calibration curve). The method meets the Brazilian regulatory requirement that establishes criteria and procedures for the determination of parameters such as CCα, CCβ, precision and recovery. CCα values were 0.02, 0.21 and 5.42 µg kg–1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples; CCβ values were 0.03, 0.22 and 5.8 µg kg–1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples. Average recoveries fortified with 0.05–7.5 µg kg–1 of the studied β-agonist leads around 95%. The method was demonstrated to be suitable for the determination of RAC, CLB and SLB in swine and cattle muscle samples.