Rafaela Fadoni Alponti

Aspartyl, arginyl and alanyl aminopeptidase activities in the hippocampus and hypothalamus of streptozotocin-induced diabetic rats

Acid (aspartyl), basic (arginyl) and neutral (alanyl) aminopeptidases degrade angiotensins, vasopressin, oxytocin, bradykinin and enkephalins. These peptides regulate memory, energy homeostasis, water-salt balance and blood pressure, functions that are mainly exerted in the hippocampus and hypothalamus, and that can be affected by diabetes mellitus. To evaluate the relationship between the diabetes mellitus and processing and inactivation roles of these representative aminopeptidases, we measured their activities in both brain structures of control and streptozotocin-diabetic rats. Hypothalamic soluble aspartyl and arginyl aminopeptidases presented significant decreased activity levels in diabetic rats, which were mitigated by insulin therapy. In addition to membrane-bound puromycin sensitive and insensitive alanyl aminopeptidases, its soluble puromycin sensitive form did not differ between diabetic and control rats in both brain structures. Glucose and/or insulin did not seem to alter in vitro the hypothalamic activities of soluble aspartyl and arginyl aminopeptidases. The implied hypothalamic control of regulatory peptide activity by aspartyl and arginyl aminopeptidases supports the hypothesis that the hydrolytic ability of these enzyme types could be a common link for the disruptions of water-salt balance, blood pressure and energy homeostasis in diabetes mellitus.

Prospective evaluation of aminopeptidase activities in plasma and peripheral organs of streptozotocin-induced diabetic rats

The cleavage of peptides by aminopeptidase enzyme types could be among the mechanisms related to certain disruptions on mediator and modulatory functions in diabetes mellitus. In order to examine this hypothesis, we measured representative aminopeptidase activities in tissues of peripheral organs of control and streptozotocin-diabetic rats. None of the examined aminopeptidase activities differed between diabetics and controls in plasma, ileum, stomach or lung. Soluble and membrane-associated alanyl, and membrane-associated cystyl aminopeptidase activities were higher in the kidney of diabetics. Decreased activity was observed in soluble and membrane-associated aspartyl and soluble dipeptidyl-peptidase IV, while increased activity was observed in soluble alanyl, arginyl, and cystyl aminopeptidases in the pancreas of diabetics. In the jejunum, soluble cystyl aminopeptidase increased in diabetics. Soluble arginyl and type-1-pyroglutamyl aminopeptidase and membrane-associated dipeptidyl-peptidase IV activities increased in the liver of diabetics. Membrane-associated dipeptidyl-peptidase IV and alanyl aminopeptidase activities in the spleen were higher in diabetics than in controls. Membrane-associated alanyl aminopeptidase activity also increased in the heart of diabetics. All these changes in streptozotocin-treated rats were avoided by the administration of insulin. Our comparative analysis of a diverse array of aminopeptidase activities supported the proposal that the regulation of peptide cleavage by these enzyme types is associated with the effects of strep-tozotocin-diabetes mellitus on peripheral organs.

Differential efficiency of simvastatin and lipoic acid treatments on Bothrops jararaca envenomation-induced acute kidney injury in mice

Snake bite accidents by Bothrops genus is an important public health issue in Brazil and one of its most serious complications is the acute kidney injury (AKI). Here we evaluated the effects of Bothrops jararaca venom (vBj) and the treatments with lipoic acid (LA) and simvastatin (SA) on renal function, aminopeptidase (AP) activities and renal redox status. Primordial events for establishment of AKI by vBj were hyperuricemia, hypercreatinemia, urinary hyperosmolality, renal oxidative stress and reduction of hematocrit and protein content in the membrane of renal cortex and medulla and in the plasma. In the renal cortex and medulla the changes caused by vBj in soluble and membrane-bound AP activities had a similar pattern. The beneficial effects of LA and SA on envenomed mice were similar on the hyperuricemia, renal oxidative stress and reduction of hematocrit. LA mitigated the hypercreatinemia, but exacerbated the urinary urea and creatinine, whereas SA mitigated the decrease of plasma urea, urinary hyperosmolality and hypercreatinuria induced by vBj. The beneficial effects of LA and especially of SA on renal effects of vBj open a new perspective for clinical investigations of these drugs as coadjuvant agents in the serotherapy of Bothrops envenomation.

Basic aminopeptidase activity is an emerging biomarker in collagen-induced rheumatoid arthritis

The objective of this study was to investigate the catalytic activity of basic aminopeptidase (APB) and its association with periarticular edema and circulating tumor necrosis factor (TNF)-alpha and type II collagen (CII) antibodies (AACII) in a rat model of rheumatoid arthritis (RA) induced by CII (CIA). Edema does not occur in part of CII-treated, even when AACII is higher than in control. TNF-alpha is detectable only in edematous CII-treated. APB in synovial membrane is predominantly a membrane-bound activity also present in soluble form and with higher activity in edematous than in non-edematous CII-treated or control. Synovial fluid and blood plasma have lower APB in non-edematous than in edematous CII-treated or control. In peripheral blood mononuclear cells (PBMCs) the highest levels of APB are found in soluble form in control and in membrane-bound form in non-edematous CII-treated. CII treatment distinguishes two categories of rats: one with arthritic edema, high AACII, detectable TNF-alpha, high soluble and membrane-bound APB in synovial membrane and low APB in the soluble fraction of PBMCs, and another without edema and with high AACII, undetectable TNF-alpha, low APB in the synovial fluid and blood plasma and high APB in the membrane-bound fraction of PBMCs. Data suggest that APB and CIA are strongly related.

Novel adipocyte aminopeptidases are selectively upregulated by insulin in healthy and obese rats

The lack of a complete assembly of the sensitivity of subcellular aminopeptidase (AP) activities to insulin in different pathophysiological conditions has hampered the complete view of the adipocyte metabolic pathways and its implications in these conditions. Here we investigated the influence of insulin on basic AP (APB), neutral puromycin-sensitive AP (PSA), and neutral puromycin-insensitive AP (APM) in high and low density microsomal and plasma membrane fractions from adipocytes of healthy and obese rats. Catalytic activities of these enzymes were fluorometrically monitoring in these fractions with or without insulin stimulus. Canonical traffic such as insulin-regulated AP was not detected for these novel adipocyte APs in healthy and obese rats. However, insulin increased APM in low density microsomal and plasma membrane fractions from healthy rats, APB in high density microsomal fraction from obese rats and PSA in plasma membrane fraction from healthy rats. A new concept of intracellular compartment-dependent upregulation of AP enzyme activities by insulin emerges from these data. This relatively selective regulation has pathophysiological significance, since these enzymes are well known to act as catalysts and receptor of peptides directly related to energy metabolism. Overall, the regulation of each one of these enzyme activities reflects certain dysfunction in obese individuals.

Adipocyte aminopeptidases in obesity and fasting.

This study checked the existence of a diverse array of aminopeptidase (AP) enzymes in high (HDM) and low (LDM) density microsomal and plasma membrane (MF) fractions from adipocytes of control, monosodium glutamate obese and food deprived rats. Gene expression was detected for ArgAP, AspAP, MetAP, and two AlaAP (APM and PSA). APM and PSA had the highest catalytic efficiency, whereas AspAP the highest affinity. Subcellular distribution of AP activities depended on metabolic status. Comparing catalytic levels, AspAP in HDM, LDM and MF was absent in obese and control under food deprivation; PSA in LDM was 3.5-times higher in obese than in normally fed control and control and obese under food deprivation; MetAP in MF was 4.5-times higher in obese than in food deprived obese. Data show new AP enzymes genetically expressed in subcellular compartments of adipocytes, three of them with altered catalytic levels that respond to whole-body energetic demands.

Insulin-regulated aminopeptidase in adipocyte is Cys-specific and affected by obesity

Insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) in adipocytes is well known to traffic between high (HDM) and low (LDM) density microsomal fractions toward the plasma membrane (MF) under stimulation by insulin. However, its catalytic preference for aminoacyl substrates with N-terminal Leu or Cys is controversial. Furthermore, possible changes in its traffic under metabolic challenges are unknown. The present study investigated the catalytic activity attributable to EC 3.4.11.3 in HDM, LDM and MF from isolated adipocytes of healthy (C), food deprived (FD) and monosodium glutamate (MSG) obese rats on aminoacyl substrates with N-terminal Cys or Leu, in absence or presence of insulin. Efficacy and reproducibility of subcellular adipocyte fractionation procedure were demonstrated. Comparison among HDM vs LDM vs MF intragroup revealed that hydrolytic activity trafficking from LDM to MF under influence of insulin in C, MSG and FD is only on N-terminal Cys. In MSG the same pattern of anterograde traffic and aminoacyl preference occurred independently of insulin stimulation. The pathophysiological significance of IRAP in adipocytes seems to be linked to comprehensive energy metabolism related roles of endogenous substrates with N-terminal cysteine pair such as vasopressin and oxytocin.

Hypothalamic activity during altered salt and water balance in the snake Bothrops jararaca

The effects of water and salt overload on the activities of the supraoptic and paraventricular nuclei and the adjacent periventricular zone of the hypothalamus of the snake Bothrops jararaca were investigated by measurements of Fos-like immunoreactivity (Fos-ir). Both water and salt overload resulted in changes in body mass, plasma osmolality, and plasma concentrations of sodium, potassium, and chloride. Hyper-osmolality increased Fos immunoreactivity in the rostral supraoptic nucleus (SON), the paraventricular nucleus (PVN), and adjacent periventricular areas. Both hyper- and hypo-osmolality increased Fos immunoreactivity in the intermediate SON, but not in other areas of the hypothalamus. Immunostaining was abundant in cerebrospinal fluid (CSF)-contacting tanycyte-like cells in the ependymal layer of the third ventricle. These data highlight some features of regional distribution of Fos immunoreactivity that are consistent with vasotocin functioning as a hormone, and support the role of hypothalamic structures in the response to disruption of salt and water balance in this snake.

Efficacy of parenteral administration of bee venom in experimental arthritis in the rat: A comparison with methotrexate

The use of bee venom (BV) to treat inflammation and pain in arthritis has become increasingly common. This study aimed to compare the effects of BV and methotrexate (MTX), the most used disease-modifying anti-rheumatic drug, in arthritic rats. Edema, erythema, cyanosis, hyperalgesia, reduction of the body mass gain, high circulating tumor necrosis factor alpha (TNF-α) and anti-type II collagen antibodies (AACII), and altered activity of basic (APB) and neutral (APN) aminopeptidases and dipeptidyl peptidase IV (DPPIV) are present in arthritic rats. MTX and/or BV do not affect AACII in healthy or arthritic individuals, but restores TNF-α to normal levels in arthritic rats. BV restores body mass gain to normal levels and MTX ameliorates body mass gain. BV contains DPPIV. BV restores APN in synovial fluid (SF) and in soluble fraction (S) from synovial tissue (ST), and DPPIV in solubilized membrane-bound fraction (M) from peripheral blood mononuclear cells (PBMCs). MTX restores APN of SF, as well as ameliorates APB of S-PBMCs, APN of S-ST and DPPIV of M-PBMCs. The combination therapy does not overcome the effects of BV or MTX alone on the peptidase activities. Edema is ameliorated by MTX or BV alone. MTX, but not BV, is effective in reducing hyperalgesia. Data show that anti-arthritic effects of BV at non-acupoints are not negligible when compared with MTX.

Anti-obesogenic and hypolipidemic effects of a glucagon-like peptide-1 receptor agonist derived from the saliva of the Gila monster

Introduction: Glucagon‐like peptide‐1 (GLP‐1) receptor (R) agonists are a class of incretin mimetic drugs that have been used for the treatment of type 2 diabetes mellitus and also considered strong candidates for the treatment of obesity. The original prototypical drug in this class is the exenatide, a synthetic peptide with the same structure as the native molecule, exendin‐4, found in the saliva of the Gila monster (Heloderma suspectum suspectum lizard). Objectives: To identify and compare the anti‐obesogenic, antidyslipidemic and antidiabetogenic effects of agonism in GLP‐1R by exenatide on two distinct models of obesity: induced by hypothalamic injury (MSG) or high‐calorie diet (DIO). Methods: To obtain MSG, neonatal rats were daily subcutaneously injected with 4 g monosodium glutamate/kg, for 10 consecutive days. To obtain DIO, 72–75 days old rats received hyperlipid food and 30% sucrose for drinking up to 142–145 days old. Untreated healthy rats with the same age were used as control. General biometric and metabolic parameters were measured. Results: MSG was characterized by decreased naso‐anal length, food and fluid intake, plasma protein and glucose decay rate per minute after insulin administration (KITT), as well as increased Lee index (body mass0.33/naso‐anal length), mass of retroperitoneal and periepididymal fat pads, glycemia, triglycerides (TG), LDL and VLDL. Exenatide ameliorated KITT and food and fluid intake, and it also restored glycemia in MSG. DIO was characterized by glucose intolerance, increased body mass, Lee index, fluid intake, mass of retroperitoneal and periepididymal fat pads, glycemia, glycated hemoglobin (HbA1c), TG, VLDL and total cholesterol, as well as decreased food intake and KITT. Exenatide restored glycemia, HbA1c, TG, VLDL, total cholesterol and body mass, and it also ameliorated food and fluid intake, KITT and mass of retroperitoneal fat pad in DIO. Conclusions: The hypothalamic injury and the high‐calorie diet induce dyslipidemia and glycemic dysregulation in addition to obesity in rats. The usual therapeutic dose of exenatide in humans is antidiabetogenic in both these obesity models, but is anti‐obesogenic and hypolipidemic only in diet‐induced obesity. Agonists of GLP‐1R are promising anti‐obesogenic and antidyslipidemic drugs in the early stages of the obesity, in which the integrity of the nervous system was unaffected. HighlightsCentral or diet‐induced obesities promote dyslipidemia and glycemic dysregulation.GLP‐1R agonist exenatide (EXE) is antidiabetogenic in these obesity models.EXE is anti‐obesogenic and hypolipidemic only in diet‐induced obesity.Neuronal integrity is related to anti‐obesogenic and lipid‐lowering effects of EXE.GLP‐1R agonists are promising drugs in the early stages of obesity and dyslipidemia.