Modulation of Autophagy Influences the Function and Survival of Human Pancreatic Beta Cells Under Endoplasmic Reticulum Stress Conditions and in Type 2 Diabetes.

Autophagy is the major mechanism involved in degradation and recycling of intracellular components, and its alterations have been proposed to cause beta cell dysfunction. In this study, we explored the effects of autophagy modulation in human islets under conditions associated to endoplasmic reticulum (ER) stress. Human pancreatic islets were isolated by enzymatic digestion and density gradient purification from pancreatic samples of non-diabetic (ND; n = 17; age 65 ± 21 years; gender: 5 M/12 F; BMI 23.4 ± 3.3 kg/m2) and T2D (n = 9; age 76 ± 6 years; 4 M/5 F; gender: BMI 25.4 ± 3.7 kg/m2) organ donors. Nine ND organ donors were treated for hypertension and 1 for both hypertension and hypercholesterolemia. T2D organ donors were treated with metformin (1), oral hypoglycemic agents (2), diet + oral hypoglycemic agents (3), insulin (3) or insulin plus metformin (3) as for antidiabetic therapy and, of these, 3 were treated also for hypertension and 6 for both hypertension and hypercholesterolemia. Two days after isolation, they were cultured for 1-5 days with 10 ng/ml rapamycin (autophagy inducer), 5 mM 3-methyladenine or 1.0 nM concanamycin-A (autophagy blockers), either in the presence or not of metabolic (0.5 mM palmitate) or chemical (0.1 ng/ml brefeldin A) ER stressors. In ND islets palmitate exposure induced a 4 to 5-fold increase of beta cell apoptosis, which was significantly prevented by rapamycin and exacerbated by 3-MA. Similar results were observed with brefeldin treatment. Glucose-stimulated insulin secretion from ND islets was reduced by palmitate (-40 to 50%) and brefeldin (-60 to 70%), and rapamycin counteracted palmitate, but not brefeldin, cytotoxic actions. Both palmitate and brefeldin induced PERK, CHOP and BiP gene expression, which was partially, but significantly prevented by rapamycin. With T2D islets, rapamycin alone reduced the amount of p62, an autophagy receptor that accumulates in cells when macroautophagy is inhibited. Compared to untreated T2D cells, rapamycin-exposed diabetic islets showed improved insulin secretion, reduced proportion of beta cells showing signs of apoptosis and better preserved insulin granules, mitochondria and ER ultrastructure; this was associated with significant reduction of PERK, CHOP and BiP gene expression. This study emphasizes the importance of autophagy modulation in human beta cell function and survival, particularly in situations of ER stress. Tuning autophagy could be a tool for beta cell protection.

Prevention by metformin of alterations induced by chronic exposure to high glucose in human islet beta cells is associated with preserved ATP/ADP ratio.

AIM: We have explored whether the insulin secretory defects induced by glucotoxicity in human pancreatic islets could be prevented by metformin and investigated some of the possible mechanisms involved. METHODS: Human pancreatic islets and INS-1E cells were cultured for 24h with or without high glucose (16.7mM) concentration in the presence or absence of therapeutical concentration of metformin and then glucose-stimulated insulin release, adenine nucleotide levels and mitochondrial complex I and II activities were measured. Islet ultrastructure was analyzed by electron microscopy. RESULTS: Compared to control islets, human islets cultured with high glucose showed a reduced glucose-stimulated insulin secretion that was associated with lower ATP levels and a lower ATP/ADP ratio. These functional and biochemical defects were significantly prevented by the presence of metformin in the culture medium, that was also able to significantly inhibit the activity of mitochondrial complex I especially in beta cells exposed to high glucose. Ultrastructural observations showed that mitochondrial volume density was significantly increased in high glucose cultured islets. The critical involvement of mitochondria was further supported by the observation of remarkably swollen organelles with dispersed matrix and fragmented cristae. Metformin was able to efficiently prevent the appearance of all these ultrastructural alterations in human islets exposed to high glucose. CONCLUSIONS: Our results show that the functional, biochemical and ultrastructural abnormalities observed in human islet cells exposed to glucotoxic condition can be significantly prevented by metformin, further highlighting a direct beneficial effect of this drug on the insulin secreting human pancreatic beta cells.

The aryl receptor inhibitor epigallocatechin-3-gallate protects INS-1E beta-cell line against acute dioxin toxicity.

The aim of this research was to investigate the mechanism(s) underlying the acute toxicity of dioxin in pancreatic beta cells and to evaluate the protective effects of epigallocatechin-3-gallate (EGCG), the most abundant of the green tea's catechins and a powerful inhibitor of the aryl hydrocarbon receptor (AhR). Using the insulin-secreting INS-1E cell line we have explored the effect of 1h exposure to different concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), alone or in the presence of EGCG, on: (a) cell survival; (b) cellular ultrastructure; (c) intracellular calcium levels; (d) mitochondrial membrane potential; (e) glucose-stimulated insulin secretion and (f) activation of MAP kinases. Our results demonstrate that TCDD is highly toxic for INS-1E cells, suggesting that pancreatic beta cells should be considered a relevant and sensitive target for dioxin acute toxicity. EGCG significantly protects INS-1E cells against TCDD-induced toxicity in terms of both cell survival and preservation of cellular ultrastructure. The mechanism of this protective effect seems to be related to: (a) the ability of EGCG to preserve the mitochondrial function and thus to prevent the TCDD-induced inhibition of glucose-stimulated insulin secretion and (b) the ability of EGCG to inhibit the TCDD-induced activation of selected kinases, such as e.g. ERK 1/2 and JNK. Our results clearly show that EGCG is able to protect pancreatic beta cells against dioxin acute toxicity and indicate the mitochondrion as the most likely target for this beneficial effect.

Persistent correction of hyperglycemia in streptozotocin-nicotinamide-induced diabetic mice by a non-conventional radical scavenger.

We previously reported that in a diabetes mouse model, characterised by moderate hyperglycaemia and reduced beta-cell mass, the radical scavenger bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate di-hydrochloride (IAC), a non-conventional cyclic hydroxylamine derivative, improves metabolic alterations by counteracting beta-cell dysfunction associated with oxidative stress. The aims of this study were to ascertain whether the beneficial effects of IAC treatment could be maintained after its discontinuation and further elucidate the underlying mechanisms. Diabetes was induced in C57Bl/6J mice by streptozotocin (STZ) and nicotinamide (NA) administration. Diabetic mice were treated for 7 weeks with various doses of IAC (7.5, 15, or 30 mg/kg b.w./die i.p.) and monitored for additional 8 weeks after suspension of IAC. Then, pancreatic tissue was used for determination of beta-cell mass by immunohistochemistry and beta-cell ultrastructural analysis. STZ-NA mice showed moderate hyperglycaemia, glucose intolerance and reduced beta-cell mass (25% of controls). IAC-treated STZ-NA mice (at both doses of 15 and 30 mg/kg b.w.) showed long-term reduction of hyperglycaemia even after discontinuation of treatment, attenuation of glucose intolerance and partial preservation of beta-cell mass. The lowest IAC dose was much less effective. Plasma nitrotyrosine levels (an oxidative stress index) significantly increased in untreated diabetic mice and were lowered upon IAC treatment. At ultrastructural level, beta cells of IAC-treated diabetic mice were protected against degranulation and mitochondrial alterations. In the STZ-NA diabetic mouse model, the radical scavenger IAC induces a prolonged reduction of hyperglycaemia associated with partial restoration of beta-cell mass and function, likely dependent on blockade of oxidative stress-induced damaging mechanisms.

Expression of gamma-glutamyltransferase in cancer cells and its significance in drug resistance.

The expression of gamma-glutamyltransferase (GGT), a cell surface enzyme involved in cellular glutathione homeostasis, is often significantly increased in human tumors, and its role in tumor progression, invasion and drug resistance has been repeatedly suggested. As GGT participates in the metabolism of cellular glutathione, its activity has been mostly regarded as a factor in reconsitution of cellular antioxidant/antitoxic defences. On this basis, an involvement of GGT expression in resistance of cancer cells to cytotoxic drugs (in particular, cisplatin and other electrophilic agents) has been envisaged. Mechanistic aspects of GGT involvement in antitumor pharmacology deserve however further investigations. Recent evidence points to a more complex role of GGT in modulation of redox equilibria, with effects acting both intracellularly and in the extracellular microenvironment. Indications exist that the protective effects of GGT may be independent of intracellular glutathione, and derive rather from processes taking place at extracellular level and involving reactions of electrophilic drugs with thiol metabolites originating from GGT-mediated cleavage of extracellular glutathione. Although expression of GGT cannot be regarded as a general mechanism of resistance, the involvement of this enzyme in modulation of redox metabolism is expected to have impact in cellular response to several cytotoxic agents. The present commentary is a survey of data concerning the role of GGT in tumor cell biology and the mechanisms of its potential involvement in tumor drug resistance.

Calorie restriction counteracts the impairment of adipocyte insulin-stimulated lipogenesis in aging rats, but not that induced by dexamethasone treatment.

In order to detect metabolic derangements that could be implicated in the pathogenesis of age-related insulin resistance, insulin-stimulated lipogenesis was investigated in isolated adipocytes from 24-month-old Sprague-Dawley rats, and the protective influence of caloric restriction was assessed. For comparison, the effects of glucocorticoid administration, used as a pharmacological tool to alter insulin sensitivity, were also studied. Caloric restriction consisted in a 40% reduction of the daily food intake of controls starting at 3 months of age. Dexamethasone (0.13 mg/kg/day) was administered for 14 days prior to sacrifice to both ad libitum-fed and dietary-restricted aging rats. Three-month-old animals, treated or untreated with dexamethasone, served as young controls. The results showed a significant age-related decrease of insulin-stimulated lipogenesis, which was fully prevented by a lifelong regimen of dietary restriction. Dexamethasone treatment markedly reduced insulin-stimulated lipogenesis in adipocytes isolated from all groups of rats, including those submitted to calorie restriction. In conclusion, our data indicate that the mechanism by which aging alters adipose tissue insulin-induced lipogenesis is reversed by dietary intervention and appears to be different from that triggered by dexamethasone. This particular defect might contribute to an imbalance of fat distribution among tissues that could induce or aggravate peripheral insulin resistance in old age.

Endogenous oxidative stress induces distinct redox forms of tumor necrosis factor receptor-1 in melanoma cells.

Receptors of the TNFR superfamily possess abundant thiols in their extracellular domains, which makes them susceptible to redox modulation by prooxidant agents and processes. Previous studies from our laboratory have documented that membrane gamma-glutamyltransferase (GGT) activity can originate reactive oxygen species in the extracellular milieu, during the GGT-mediated metabolism of extracellular glutathione. The present study was aimed thus to verify a possible redox-modulating effect of GGT activity on TNFR1 receptors. The thiol-specific probe maleimide-polyethylene glycol was used to selectively label the reduced thiol groups in proteins of cell lysates; fractions corresponding to TNFR1 were then identified by immunoblot. In human melanoma Me665/2 cells, expressing varying GGT levels, at least five distinct forms of TNFR1 have been thus identified. The more oxidized forms appear to be prevalent in the 2/60 clone, expressing higher GGT levels, as compared to clone 2/21. Stimulation of GGT activity in the latter induced an increase of the oxidized TNFR1 forms. It is conceivable that different redox states of TNFR1 may correspond to different binding affinity and/or changes in the transducing function of the receptor. As GGT is frequently expressed by malignant tumors, the described phenomena might concur to alter the sensitivity of cancer cells to agents targeted on activation of TNF-alpha-dependent signaling pathways.

Dexamethasone-induced insulin resistance and pancreatic adaptive response in aging rats are not modified by oral vanadyl sulfate treatment.

OBJECTIVE: To explore the adaptive response of the endocrine pancreas in vivo and in vitro and the possible beneficial effect of the insulino-mimetic agent vanadyl sulfate (VOSO(4)), using glucocorticoid treatment to increase insulin resistance, in aging rats. DESIGN AND METHODS: Dexamethasone (Dex) (0.13 mg/kg b.w.) was administered daily for 13 days to 3- and 18-month old Sprague-Dawley rats and oral VOSO(4) was given from the 5th day. Plasma glucose, insulin and free fatty acids (FFA) concentrations were measured during these treatments and the insulin secretory response of the isolated perfused pancreas was assessed at the end of the experiment. RESULTS AND CONCLUSIONS: In both young and aging rats, particularly in the latter, hyperinsulinemia and increased in vitro insulin responsiveness to glucose were observed in response to Dex treatment, concomitant with an increase in plasma FFA concentrations. Thus, in glucocorticoid-treated animals, the beta-cell adaptive response occurred in both age groups and could possibly be mediated by increased circulating FFA; however, it was insufficient to prevent hyperglycemia in 60% of aging animals. Oral VOSO(4) administration failed to correct Dex-induced alterations in glucose and lipid metabolism, although it influenced in vitro beta-cell responsiveness to stimuli in aging rats.

Age-related changes in the urinary excretion of aldehydes in ad libitum fed and food-restricted rats.

Age-related changes in the urinary excretion of aldehydes arising from lipid peroxidation have been investigated in male Sprague-Dawley rats aged 2, 4, 6, 12, 18, 24 and 27 months, fed ad libitum or subjected to two different regimens of calorie restriction (namely every-other-day ad libitum feeding--EOD--and 40% calorie restriction--40%DR). For only some age groups, results were compared with those obtained in ad libitum fed male Fisher 344 and Lewis rats. Results show that the urinary excretion of malondialdehyde (MDA) and formaldehyde (FA) significantly decreases, whereas that of propionaldehyde (PROP) progressively increases with age, and that urinary excretion of acetaldehyde (ACT) does not show any significant age-related variations. Dietary restriction significantly increases the urinary levels of MDA, FA and PROP without affecting their age-related modifications, and does not affect ACT urinary excretion. In conclusion, results indicate that the quantitative pattern of aldehyde production and urinary excretion may be altered by the process of aging.

Age-dependent reduction in GLUT-2 levels is correlated with the impairment of the insulin secretory response in isolated islets of Sprague-Dawley rats.

In this study we have investigated the insulin secretory response to glucose and other secretagogues (2-ketoisocaproate, 3-isobutyl-1-methyl-xanthine and arginine) of pancreatic islets isolated from Sprague-Dawley rats of various ages (from 2 to 28 months). Our results showed a significant decline in the glucose-stimulated insulin secretion, starting at 12 months of age. On the other hand, the response to non-glucose secretagogues (and mainly to 2-ketoisocaproate) was less impaired with advancing age than that to glucose. We also observed a progressive age-related decline of protein levels of the glucose transporter GLUT-2 in pancreatic islets, which was temporally concomitant and quantitatively comparable with the beta-cell alteration in glucose responsiveness (-40/50%). Finally, we observed a significant increase of the islets insulin content in older rats with respect to younger animals. We conclude that in the islet of older rats the impaired capability to respond to glucose could be dependent, at least in part, on the age-dependent reduction in GLUT-2 and could be compensated by mechanisms including a preserved responsiveness to non-glucose secretagogues and/or the development of islet hypertrophy.

Effects of low-dose VOSO(4) on age-related changes in glucose homeostasis in rats.

The effects of low doses of vanadyl sulfate (0.2 mg/ml in the drinking water) on the age-related impairment of glucose homeostasis in Sprague-Dawley rats were investigated. VOSO(4) administration was initiated in 5-month-old animals and lasted 3 months. Thus, in 8-month-old rats, we investigated glucose metabolism in vivo and insulin secretory function in vitro. Results showed that VOSO(4) allowed the disposal of an oral glucose load at lower insulin levels than in age-matched controls. No significant changes were found in muscle glucose transporter (GLUT-4) levels or in glycogen content upon VOSO(4) treatment. Islets isolated from VOSO(4)-treated rats released less insulin than control islets, but showed a better preserved sensitivity to secretagogues, in terms of incremental release over basal release, secretory efficiency, and maintenance of the priming effect of glucose. In conclusion, chronic low-dose VOSO(4) treatment facilitates insulin action by a mechanism independent of muscle GLUT-4 levels and helps preserve the appropriate sensitivity of beta cells to stimuli, thereby preventing age-dependent functional alterations.

Insufficient adaptive capability of pancreatic endocrine function in dexamethasone-treated ageing rats.

This study was aimed at exploring the capability of the pancreatic endocrine adaptive mechanisms of ageing Sprague-Dawley rats to counteract the metabolic challenge induced by the prolonged administration of dexamethasone (DEX) (0.13 mg/kg per day for 13 days). DEX treatment induced peripheral insulin resistance in 3-, 18- and 26-month-old rats, as indicated by the significant and persistent rise of plasma insulin levels in each age group (plasma insulin in 3-, 18- and 26-month-old rats from basal values of 4.3+/-0.8, 4.7+/-0.5 and 5.6+/-1.0 ng/ml (means+/-s.e.m.) respectively, rose to 11.9+/-1.7, 29.1+/-5.5 and 27.9+/-2.7 ng/ml respectively, after 9 days of administration). However, plasma glucose concentrations remained unchanged during the treatment in young rats, whereas they increased up to frankly diabetic levels in most 18-month-old and in all 26-month-old animals after a few days of DEX administration. Plasma free fatty acid concentrations increased 2-fold in 3- and 26-month-old rats and 4-fold in 18-month-old rats and could possibly be involved in the glucocorticoid-induced enhancement in insulin resistance, although they showed no significant correlation with glycaemic values. Incubation of pancreatic islets obtained from treated rats showed that DEX administration increased the insulin responsiveness of islets from not only younger but also older donors. However, in the islets of ageing rats, which already showed an age-dependent impairment of the sensitivity to glucose and other secretagogues, this enhancing effect was clearly attenuated with respect to the younger counterpart. Furthermore, DEX treatment depressed significantly the priming effect of glucose in islets isolated from all the three age groups. In conclusion, our results show that ageing rats are unable to counteract effectively a prolonged hyperglycaemic challenge as such induced by DEX administration. This homeostatic defect can be ascribed to the age-dependent failure of the endocrine pancreas to provide enough insulin to overcome the aggravation of an antecedent state of increased peripheral insulin resistance.

Glycated plasma proteins in experimentally induced acute toxic renal failure by dichromate injection: evidence for loss with urine and decreased plasma levels.

In the rat, a single subcutaneous injection of sodium dichromate (20 mg/kg) causes acute renal injury and significant polyuria, proteinuria, and glycosuria (peaking 2-3 days after treatment, and returning to normal by day 5) without any changes in the plasma levels of protein, glucose, and glycated haemoglobin. Surprisingly, the percentage levels of glycated plasma total proteins and albumin (assayed by boronate affinity chromatography) transiently and significantly decrease during recovery from proteinuria (days 4 and 10 after treatment) and were found in the normal range of values by day 18. These changes are concomitant with a significant increase in the percentage level of glycated albumin in urine. Constancy of total plasma protein and the temporal pattern of levels of glycation suggest that changes in the percentage values of glycated proteins are secondary to a transient selective loss of glycated plasma proteins in urine.

Determination of glycated hemoglobins in the rat: comparison between two different chromatographic methods and application in experimental diabetology.

Due to the unusual presence of several different hemoglobin components in the rat, determination of glycated hemoglobin (Hb) has been considered difficult and often unreliable in this animal species. In the present study, we compare a fully automated high-performance liquid chromatographic (HPLC) method of analysis of glycated hemoglobin that has been assessed for clinical use with an affinity chromatography technique using boronate micro-columns; we used blood samples taken from Sprague-Dawley rats of various ages and streptozotocin-diabetic rats. In nondiabetic rats, the sum of HbA1c and other minor glycated hemoglobins separated by the HPLC method is close to the total glycated hemoglobin obtained by affinity chromatography for each age group of animals. In diabetic rats, the glycated hemoglobins measured by whatever method show a linear increase during the first 3 weeks following streptozotocin administration, with the difference that glycated hemoglobin values obtained by affinity chromatography are markedly higher than those obtained by HPLC technique. Interestingly, a comparative determination of glycated hemoglobin in diabetic patients gives the same results with both methods. Therefore, it appears that in the rat, unlike man, at high glucose concentrations glycation occurs preferentially at the amino groups of hemoglobin components, which are not separated by the HPLC method. Our results indicate that while affinity chromatography should be used to detect the total extent of hemoglobin glycosylation in diabetic rats, the utilization of rapid and automatized HPLC procedures can be a very convenient alternative for the determination of glycated hemoglobin in both euglycemic and hyperglycemic rats.

The induction of mitochondrial myopathy in the rat by feeding beta-guanidinopropionic acid and the reversibility of the induced mitochondrial lesions: a biochemical and ultrastructural investigation.

The long-lasting depletions of creatine phosphate induced by feeding rats with a beta-guanidinopropionic acid (GPA)-supplemented diet induces specific mitochondrial alterations in skeletal muscles very similar to those observed in human mitochondrial myopathies. The slow-twitch soleus muscle appears to be affected primarily, while the fast-twitch extensor digitorum longus is affected less severely and only after a longer period of treatment (6 months). Changes in the enzyme activities of glucose metabolism appear to be secondary and differ between the two muscles. Withdrawal of GPA from the diet after 2 months of treatment shows that both mitochondrial alterations and biochemical modification are reversible.

Beneficial effects of the oral administration of vanadyl sulphate on glucose metabolism in senescent rats.

We investigated the effects of the oral administration of vanadyl sulphate (0.5 mg/ml in the drinking water) on glucose homeostasis of 3-month- and 24-month-old rats. Results show that aging is associated with alteration of the oral glucose tolerance test and impairment of the postprandial accumulation of glycogen in skeletal muscles and that the oral administration of vanadyl sulphate rapidly normalizes the inbalance of glucose metabolism in senescent rats. It is suggested that vanadate administration may restore the ability of skeletal muscles of senescent rats to respond to circulating insulin efficiently.

Effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on proliferation of young and senescent WI-38 human diploid fibroblasts.

The effects of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on the proliferation, protein kinase C activity (PKC), and c-fos gene expression were examined in cultures of young and senescent (90-95% lifespan completed) WI-38 human diploid fibroblasts. We observed that, following stimulation with medium containing 10% fetal bovine serum (FBS), the translocation of PKC from the cytosol to the particulate compartment was less efficient in senescent WI-38 cells than in young cells. However, when PMA was added to the medium, the intracellular distribution of PKC activity in old cells became nearly identical to that observed in young cells. The inducibility of c-fos mRNA by serum addition, which is a protein kinase C-dependent event, was significantly amplified in the presence of PMA. Moreover, the duration of peak c-fos expression, after stimulation by FBS and PMA, increased in senescent cells as compared to young cells. Our results reveal that the normal signal transduction pathway is altered in senescent, slowly proliferating human fibroblasts and that it can be partially restored in the presence of the tumor promoter PMA.

Effects of the administration of thyroid hormone on the plasma levels of atrial natriuretic peptides and on atrial myoendocrine cells in the rat: an immunochemical, ultrastructural, and stereological study.

The effects of an altered thyroidal status on the levels of immunoreactive (ir-) atrial natriuretic peptide (ANP) in serum and in the right and left atria, as well as on the subcellular structures of atrial myoendocrine cells were explored in groups of male Sprague Dawley rats given the vehicle or triiodothyronine in the toxicological dose-range (50 micrograms/100 g bw/day) for 0,5, 1,2,4,7 or 14 days. Plasma levels of ir-ANP were 30% higher in T3-treated rats compared with controls at 0,5 and 1 day after hormone administration and then decreased to levels 30-40% lower than controls at days 2 and 4 to rise again above control values on day 7 and 14. Atrial ir-ANP levels decreased at first both in the right and in the left atria with different latencies (1 and 2 days, respectively) and rose back towards control levels by day 4. Changes in the numerical density of specific granules followed a parallel temporal pattern. An increased in the individual volume of the granules followed was also observed. Investigation into the circulatory effects of T3 administration showed that the heart rate was increased by hour 12 after hormone administration (simultaneously with the early rise in plasma ir-ANP levels) and that blood pressure was increased by day 2.(ABSTRACT TRUNCATED AT 250 WORDS)

[The enzymatic activity of adenosine metabolism in the rat heart. The effects of cardiac hypertrophy]. / Attività degli enzimi del metabolismo dell'adenosina nel cuore di ratto. Effetti dell'ipertrofia cardiaca.

The transmural distribution of the adenosine metabolizing enzyme activities 5'nucleotidase (5'N) and adenosine deaminase (ADA) across the left ventricular wall was explored in rats with heart hypertrophy induced by aortocaval shunt (FAC), or coarctation of the abdominal aorta (S) or by tireotoxicosis (T). FAC caused largest heart hypertrophy with a very short latency (1 day: + 0%, 3.5 days: + 30%, 7 days: + 41%, 21 days: + 49%). A 30% increase in the left ventricle free wall was observed after 3.5, 14 or 28 days of treatment with FAC, T or S respectively. Different changes in enzyme levels and alteration of transmural distribution profiles were observed with the different types of heart hypertrophy. Level of 5'N was decreased with FAC, did not change with T and increased with S; levels of ADA were always increased. The pattern of the transmural distribution of 5'N was affected by S but not by T and FAC, whereas distribution of ADA was not affected by S and T and was altered by FAC transiently.