THE EFFECTS OF STREPTOZOTOCIN DIABETES AND DIETARY IRON INTAKE ON CATALASE, GLUTATHIONE PEROXIDASE, SUPEROXIDE DISMUTASE AND LIPID PEROXIDATION IN CARDIAC AND SKELETAL MUSCLES OF RATS

Catalase, glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) prevent oxygen free radical mediated tissue damage. Diabetes increases and a low dietary intake of iron decreases catalase activity in muscle. Therefore, the combined effects of diabetes and iron deficiency on the free radical scavenging enzyme system and lipid peroxidation were studied. Male, weanling rats were injected with streptozotocin (65 mg/kg, IV) and fed diets containing either 35 ppm iron (Db + Fe) or 8 ppm iron (Db $-$ Fe). Sham injected animals served as iron adequate (C + Fe) or iron deficient (C $-$ Fe) controls. Heart, gastrocnemius (GT), soleus and tibialis anterior (TA) muscles were dissected, weighted and analyzed for catalase, GSH-Px and SOD activities after 3, 6 or 9 weeks on the respective diets. The TBA assay was used to assess lipid peroxidation in the GT muscle. Diabetes elevated catalase activity in all muscles while it had a slight lowering effect on SOD and GSH-Px activities in the GT and TA muscles. In the C $-$ Fe rats, catalase activity declined and remained depressed in all muscles except the heart. There was an elevation in GSH-Px and SOD in the GT muscles of these animals after 6 weeks but not after 9 weeks of consuming the low iron diet. The Db $-$ Fe animals were unable to respond to the diabetic state with catalase activity as high as observed in the Db + Fe rats. Treatment with insulin or iron returned catalase to control levels. The C $-$ Fe animals had significantly lower levels of lipid peroxidation than the other groups at 6 and 9 weeks. Refeeding an iron adequate diet resulted in an increase in lipid peroxidation levels. These studies indicate that skeletal muscle free radical scavenging enzymes are sensitive to metabolic states and that dietary iron influences lipid peroxidation in this tissue. ^

The Effect of Parathyroid Hormone on Osseointegration in Insulin-Treated Diabetic Rats.

OBJECTIVES Uncontrolled diabetes mellitus is associated with impaired osseointegration. Diabetic individuals might benefit from bone anabolic therapies. Intermittent administration of 1-34 parathyroid hormone (PTH) stimulates bone formation in rodent models. However, this anabolic effect fails in diabetic rats. Whether the anabolic effect of PTH can be achieved in insulin-controlled diabetic rats has not been investigated yet. MATERIALS AND METHODS After diabetes induction with streptozotocin in 40 female Wistar rats, the animals were randomly divided into 4 groups: diabetes, diabetes plus PTH, insulin-treated diabetes, and insulin-treated diabetes plus PTH. After 1 week, miniscrews were inserted in the tibiae. Osmotic pumps with insulin or saline solution were implanted. Animals received 60 mg/kg PTH or saline solution. Histomorphometric analysis was performed. RESULTS In diabetic rats, no changes of medullary periimplant bone area or bone-to-implant contacts (BICs) were achieved with or without treatment with PTH. However, also animals treated with insulin failed to response significantly to PTH regarding bone area (7.4 ± 4.1% and 8.1 ± 4.1%) and BICs (33.7 ± 16.9% and 49.9 ± 11.9%). CONCLUSION These results demonstrate that the metabolic characteristics of the diabetic rats produced a condition unable to respond to PTH treatment, even when hyperglycemia was controlled with insulin.

Impact of Diabetic Status on Outcomes After Revascularization With Drug-Eluting Stents in Relation to Coronary Artery Disease Complexity: Patient-Level Pooled Analysis of 6081 Patients.

BACKGROUND Diabetes mellitus and angiographic coronary artery disease complexity are intertwined and unfavorably affect prognosis after percutaneous coronary interventions, but their relative impact on long-term outcomes after percutaneous coronary intervention with drug-eluting stents remains controversial. This study determined drug-eluting stents outcomes in relation to diabetic status and coronary artery disease complexity as assessed by the Synergy Between PCI With Taxus and Cardiac Surgery (SYNTAX) score. METHODS AND RESULTS In a patient-level pooled analysis from 4 all-comers trials, 6081 patients were stratified according to diabetic status and according to the median SYNTAX score ≤11 or >11. The primary end point was major adverse cardiac events, a composite of cardiac death, myocardial infarction, and clinically indicated target lesion revascularization within 2 years. Diabetes mellitus was present in 1310 patients (22%), and new-generation drug-eluting stents were used in 4554 patients (75%). Major adverse cardiac events occurred in 173 diabetics (14.5%) and 436 nondiabetic patients (9.9%; P<0.001). In adjusted Cox regression analyses, SYNTAX score and diabetes mellitus were both associated with the primary end point (P<0.001 and P=0.028, respectively; P for interaction, 0.07). In multivariable analyses, diabetic versus nondiabetic patients had higher risks of major adverse cardiac events (hazard ratio, 1.25; 95% confidence interval, 1.03-1.53; P=0.026) and target lesion revascularization (hazard ratio, 1.54; 95% confidence interval, 1.18-2.01; P=0.002) but similar risks of cardiac death (hazard ratio, 1.41; 95% confidence interval, 0.96-2.07; P=0.08) and myocardial infarction (hazard ratio, 0.89; 95% confidence interval, 0.64-1.22; P=0.45), without significant interaction with SYNTAX score ≤11 or >11 for any of the end points. CONCLUSIONS In this population treated with predominantly new-generation drug-eluting stents, diabetic patients were at increased risk for repeat target-lesion revascularization consistently across the spectrum of disease complexity. The SYNTAX score was an independent predictor of 2-year outcomes but did not modify the respective effect of diabetes mellitus. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00297661, NCT00389220, NCT00617084, and NCT01443104.

Identification of a renal-specific oxido-reductase in newborn diabetic mice

Aldose reductase (ALR2), a NADPH-dependent aldo-keto reductase (AKR), is widely distributed in mammalian tissues and has been implicated in complications of diabetes, including diabetic nephropathy. To identify a renal-specific reductase belonging to the AKR family, representational difference analyses of cDNA from diabetic mouse kidney were performed. A full-length cDNA with an ORF of 855 nt and yielding a ≈1.5-kb mRNA transcript was isolated from a mouse kidney library. Human and rat homologues also were isolated, and they had ≈91% and ≈97% amino acid identity with mouse protein. In vitro translation of the cDNA yielded a protein product of ≈33 kDa. Northern and Western blot analyses, using the cDNA and antirecombinant protein antibody, revealed its expression exclusively confined to the kidney. Like ALR2, the expression was up-regulated in diabetic kidneys. Its mRNA and protein expression was restricted to renal proximal tubules. The gene neither codistributed with Tamm–Horsfall protein nor aquaporin-2. The deduced protein sequence revealed an AKR-3 motif located near the N terminus, unlike the other AKR family members where it is confined to the C terminus. Fluorescence quenching and reactive blue agarose chromatography studies revealed that it binds to NADPH with high affinity (KdNADPH = 66.9 ± 2.3 nM). This binding domain is a tetrapeptide (Met-Ala-Lys-Ser) located within the AKR-3 motif that is similar to the other AKR members. The identified protein is designated as RSOR because it is renal-specific with properties of an oxido-reductase, and like ALR2 it may be relevant in the renal complications of diabetes mellitus.

Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin response element (IRE) that resembles IREs of other insulin-repressed genes

Recent data indicate that sustained elevations in plasma insulin suppress the mRNA for IRS-2, a component of the insulin signaling pathway in liver, and that this deficiency contributes to hepatic insulin resistance and inappropriate gluconeogenesis. Here, we use nuclear run-on assays to show that insulin inhibits transcription of the IRS-2 gene in the livers of intact rats. Insulin also inhibited transcription of a reporter gene driven by the human IRS-2 promoter that was transfected into freshly isolated rat hepatocytes. The human promoter contains a heptanucleotide sequence, TGTTTTG, that is identical to the insulin response element (IRE) identified previously in the promoters of insulin-repressed genes. Single base pair substitutions in this IRE decreased transcription of the IRS-2-driven reporter in the absence of insulin and abolished insulin-mediated repression. We conclude that insulin represses transcription of the IRS-2 gene by blocking the action of a positive factor that binds to the IRE. Sustained repression of IRS-2, as occurs in chronic hyperinsulinemia, contributes to hepatic insulin resistance and accelerates the development of the diabetic state.

Correction of diabetic alterations by glucokinase.

Hyperglycemia is a common feature of diabetes mellitus. It results from a decrease in glucose utilization by the liver and peripheral tissues and an increase in hepatic glucose production. Glucose phosphorylation by glucokinase is an initial event in glucose metabolism by the liver. However, glucokinase gene expression is very low in diabetic animals. Transgenic mice expressing the P-enolpyruvate carboxykinase/glucokinase chimeric gene were generated to study whether the return of the expression of glucokinase in the liver of diabetic mice might prevent metabolic alterations. In contrast to nontransgenic mice treated with streptozotocin, mice with the transgene previously treated with streptozotocin showed high levels of both glucokinase mRNA and its enzyme activity in the liver, which were associated with an increase in intracellular levels of glucose 6-phosphate and glycogen. The liver of these mice also showed an increase in pyruvate kinase activity and lactate production. Furthermore, normalization of both the expression of genes involved in gluconeogenesis and ketogenesis in the liver and the production of glucose and ketone body by hepatocytes in primary culture were observed in streptozotocin-treated transgenic mice. Thus, glycolysis was induced while gluconeogenesis and ketogenesis were blocked in the liver of diabetic mice expressing glucokinase. This was associated with normalization of blood glucose, ketone bodies, triglycerides, and free fatty acids even in the absence of insulin. These results suggest that the expression of glucokinase during diabetes might be a new approach to the normalization of hyperglycemia.

Prevention of diabetic alterations in transgenic mice overexpressing Myc in the liver.

Recent studies have demonstrated that the overexpression of the c-myc gene in the liver of transgenic mice leads to an increase in both utilization and accumulation of glucose in the liver, suggesting that c-Myc transcription factor is involved in the control of liver carbohydrate metabolism in vivo. To determine whether the increase in c-Myc might control glucose homeostasis, an intraperitoneal glucose tolerance test was performed. Transgenic mice showed lower levels of blood glucose than control animals, indicating that the overexpression of c-Myc led to an increase of blood glucose disposal by the liver. Thus, the increase in c-Myc might counteract diabetic hyperglycemia. In contrast to control mice, transgenic mice treated with streptozotocin showed normalization of concentrations of blood glucose, ketone bodies, triacylglycerols and free fatty acids in the absence of insulin. These findings resulted from the normalization of liver metabolism in these animals. While low glucokinase activity was detected in the liver of diabetic control mice, high levels of both glucokinase mRNA and enzyme activity were noted in the liver of streptozotocin-treated transgenic mice, which led to an increase in intracellular levels of glucose 6-phosphate and glycogen. The liver of these mice also showed an increase in pyruvate kinase activity and lactate production. Furthermore, normalization of both the expression of genes involved in the control of gluconeogenesis and ketogenesis and the production of glucose and ketone bodies was observed in streptozotocin-treated transgenic mice. Thus, these results suggested that c-Myc counteracted diabetic alterations through its ability to induce hepatic glucose uptake and utilization and to block the activation of gluconeogenesis and ketogenesis.

Implications physiopathologiques de la Nestine lors du remodelage pulmonaire et cardiaque à la suite de l’infarctus du myocarde, du diabète et de l’hypertension pulmonaire

Il est reconnu que la protéine filamenteuse intermédiaire Nestine est exprimée lors du processus de cicatrisation et du remodelage fibrotique. De plus, nous avons identifié l’expression de la Nestine au sein de deux populations distinctes qui sont directement impliquées dans les réponses de fibroses réparative et réactive. Ainsi, une population de cellules souches neurales progénitrices résidentes du coeur de rat adulte exprime la Nestine et a été identifiée à titre de substrat de l’angiogenèse et de la neurogenèse cardiaque. Également, la Nestine est exprimée par les myofibroblastes cicatriciels cardiaques et il a été établi que la protéine filamenteuse intermédiaire joue un rôle dans la prolifération de ces cellules. Ainsi, l’objectif général de cette thèse était de mieux comprendre les évènements cellulaires impliqués dans la réponse neurogénique des cellules souches neurales progénitrices résidentes cardiaques Nestine(+) (CSNPRCN(+)) lors de la fibrose réparative cardiaque et d’explorer si l’apparition de fibroblastes Nestine(+) est associée avec la réponse de fibrose réactive secondaire du remodelage pulmonaire. Une première publication nous a permis d’établir qu’il existe une régulation à la hausse de l’expression de la GAP43 (growth associated protein 43) et que cet événement transitoire précède l’acquisition d’un phénotype neuronal par les CSNPRCN(+) lors du processus de cicatrisation cardiaque chez le rat ayant subi un infarctus du myocarde. De plus, la surimposition de la condition diabétique de type 1, via l’injection unique de Streptozotocine chez le rat, abolit la réponse neurogénique des CSNPRCN(+), qui est normalement induite à la suite de l’ischémie cardiaque ou de l’administration de 6-hydroxydopamine. Le second article a démontré que le développement aigu de la fibrose pulmonaire secondaire de l’infarctus du myocarde chez le rat est associé avec une augmentation de l’expression protéique de la Nestine et de l’apparition de myofibroblastes pulmonaires Nestine(+). Également, le traitement de fibroblastes pulmonaires avec des facteurs de croissances peptidiques pro-fibrotiques a augmenté l’expression de la Nestine par ces cellules. Enfin, le développement initial de la condition diabétique de type 1 chez le rat est associé avec une absence de fibrose réactive pulmonaire et à une réduction significative des niveaux protéiques et d’ARN messager de la Nestine pulmonaire. Finalement, la troisième étude représentait quant à elle un prolongement de la deuxième étude et a alors examiné le remodelage pulmonaire chronique chez un modèle établi d’hypertension pulmonaire. Ainsi, les poumons de rats adultes mâles soumis à l’hypoxie hypobarique durant 3 semaines présentent un remodelage vasculaire, une fibrose réactive et une augmentation des niveaux d’ARN messager et de la protéine Nestine. De plus, nos résultats ont démontré que la Nestine, plutôt que l’alpha-actine du muscle lisse, est un marqueur plus approprié des diverses populations de fibroblastes pulmonaires activés. Également, nos données suggèrent que les fibroblastes pulmonaires activés proviendraient en partie de fibroblastes résidents, ainsi que des processus de transition épithélio-mésenchymateuse et de transition endothélio-mésenchymateuse. Collectivement, ces études ont démontré que des populations distinctes de cellules Nestine(+) jouent un rôle majeur dans la fibrose réparative cardiaque et la fibrose réactive pulmonaire.

Determinants of subclinical diabetic heart disease

Aims/hypothesis: Subclinical left ventricular (LV) dysfunction has been shown by tissue Doppler and strain imaging in diabetic patients in the absence of coronary disease or LV hypertrophy, but the prevalence and aetiology of this finding remain unclear. This study sought to identify the prevalence and the determinants of subclinical diabetic heart disease. Methods: A group of 219 unselected patients with type 2 diabetes without known cardiac disease underwent resting and stress echocardiography. After exclusion of coronary artery disease or LV hypertrophy, the remaining 120 patients ( age 57 +/- 10 years, 73 male) were studied with tissue Doppler imaging. Peak systolic strain of each wall and systolic (Sm) and diastolic ( Em) velocity of each basal segment were measured from the three apical views and averaged for each patient. Significant subclinical LV dysfunction was identified according to Sm and Em normal ranges adjusted by age and sex. Strain and Em were correlated with clinical, therapeutic, echocardiographic and biochemical variables, and significant independent associations were sought using a multiple linear regressionmodel. Results: Significant subclinical LV dysfunction was present in 27% diabetic patients. Myocardial systolic dysfunction by peak strain was independently associated with glycosylated haemoglobin level ( p< 0.001) and lack of angiotensin- converting enzyme inhibitor treatment ( p= 0.003). Myocardial diastolic function ( Em) was independently predicted by age ( p= 0.013), hypertension ( p= 0.001), insulin ( p= 0.008) and metformin ( p= 0.01) treatment. Conclusions/ interpretation: In patients with diabetes mellitus, subclinical LV dysfunction is common and associated with poor diabetic control, advancing age, hypertension and metformin treatment; ACE inhibitor and insulin therapies appear to be protective.

Do changes in transglutaminase activity alter latent transforming growth factor beta activation in experimental diabetic nephropathy?

Background. Diabetic nephropathy is the leading cause of end-stage kidney failure worldwide. It is characterized by excessive extracellular matrix accumulation. Transforming growth factor beta 1 (TGF-ß1) is a fibrogenic cytokine playing a major role in the healing process and scarring by regulating extracellular matrix turnover, cell proliferation and epithelial mesanchymal transdifferentiation. Newly synthesized TGF-ß is released as a latent, biologically inactive complex. The cross-linking of the large latent TGF-ß to the extracellular matrix by transglutaminase 2 (TG2) is one of the key mechanisms of recruitment and activation of this cytokine. TG2 is an enzyme catalyzing an acyl transfer reaction leading to the formation of a stable e(?-glutamyl)-lysine cross-link between peptides.Methods. To investigate if changes in TG activity can modulate TGF-ß1 activation, we used the mink lung cell bioassay to assess TGF-ß activity in the streptozotocin model of diabetic nephropathy treated with TG inhibitor NTU281 and in TG2 overexpressing opossum kidney (OK) proximal tubular epithelial cells.Results. Application of the site-directed TG inhibitor NTU281 caused a 25% reduction in kidney levels of active TGF-ß1. Specific upregulation of TG2 in OK proximal tubular epithelial cells increased latent TGF-ß recruitment and activation by 20.7% and 19.7%, respectively, in co-cultures with latent TGF-ß binding protein producing fibroblasts.Conclusions. Regulation of TG2 directly influences the level of active TGF-ß1, and thus, TG inhibition may exert a renoprotective effect by targeting not only a direct extracellular matrix deposition but also TGF-ß1 activation and recruitment.

Elevated ε-(γ-glutamyl)lysine in human diabetic nephropathy results from increased expression and cellular release of tissue transglutaminase

Introduction: Diabetic nephropathy (DN) is the leading cause of chronic kidney failure, however the mechanisms underlying the characteristic expansion of the extracellular matrix (ECM) in diabetic kidneys remain controversial and unclear. In non-diabetic kidney scarring the protein crosslinking enzyme tissue transglutaminase (tTg) has been implicated in this process by the formation of increased ε-(γ-glutamyl)lysine bonds between ECM components in both experimental and human disease. Studies in db/db diabetic mice and in streptozotocin-treated rats have suggested a similar mechanism, although the relevance of this to human disease has not been addressed. Methods: We have undertaken a retrospective analysis of renal biopsies from 16 DN patients with type 2 diabetes mellitus using an immunohistochemical and immunofl uorescence approach, with tTg and ε-(γ-glutamyl)lysine crosslink quantified by confocal microscopy. Results: Immunofl uorescent analysis of human biopsies (confocal microscopy) showed increases in levels of tTg (+1,266%, p <0.001) and ε-(γ-glutamyl)lysine (+486%, p <0.001) in kidneys with DN compared to normal. Changes were predominantly in the extracellular periglomerular and peritubular areas. tTg staining correlated with e-(?-glutamyl)lysine (r = 0.615, p <0.01) and renal scarring (Masson's trichrome, r = 0.728, p <0.001). Significant changes in e-(?-glutamyl)lysine were also noted intracellularly in some (=5%) tubular epithelial cells. This is consistent with cells undergoing a novel transglutaminase-mediated cell death process in response to Ca influx and subsequent activation of intracellular tTg. Conclusion: Changes in tTg and ε-(γ- glutamyl)lysine occur in human DN. Cellular export of tTg may therefore be a factor in the perpetuation of DN by crosslinking and stabilisation of the ECM, while intracellular activation may lead to cell death contributing towards tubular atrophy. Copyright © 2004 S. Karger AG, Basel.

Cardiomyopathy in Offspring of Pregestational Diabetic Mouse Pregnancy

PURPOSE: To investigate cardiomyopathy in offspring in a mouse model of pregestational type 1 diabetic pregnancy.

METHODS: Pregestational diabetes was induced with STZ administration in female C57BL6/J mice that were subsequently mated with healthy C57BL6/J males. Offspring were sacrificed at embryonic day 18.5 and 6-week adolescent and 12-week adult stages. The size and number of cardiomyocyte nuclei and also the extent of collagen deposition within the hearts of diabetic and control offspring were assessed following cardiac tissue staining with either haematoxylin and eosin or Picrosirius red and subsequently quantified using automated digital image analysis.

RESULTS: Offspring from diabetic mice at embryonic day 18.5 had a significantly higher number of cardiomyocyte nuclei present compared to controls. These nuclei were also significantly smaller than controls. Collagen deposition was shown to be significantly increased in the hearts of diabetic offspring at the same age. No significant differences were found between the groups at 6 and 12 weeks.

CONCLUSIONS: Our results from offspring of type 1 diabetic mice show increased myocardial collagen deposition in late gestation and have increased myocardial nuclear counts (hyperplasia) as opposed to increased myocardial nuclear size (hypertrophy) in late gestation. These changes normalize postpartum after removal from the maternal intrauterine environment.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target to prevent retinal vasopermeability during diabetes

Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood-retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents.

Alteration of the bone tissue material properties in type 1 diabetes mellitus: A Fourier transform infrared microspectroscopy study

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The Cratylia Mollis Seed Lectin Induces Membrane Permeability Transition In Isolated Rat Liver Mitochondria And A Cyclosporine A-insensitive Permeability Transition In Trypanosoma Cruzi Mitochondria.

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca(2+) was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca(2+) this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.