Ana Vasilijević

Beneficial effects of L‐arginine–nitric oxide‐producing pathway in rats treated with alloxan

In an attempt to elucidate molecular mechanisms and factors involved in β cell regeneration, we evaluated a possible role of the l‐arginine–nitric oxide (NO)‐producing pathway in alloxan‐induced diabetes mellitus. Diabetes was induced in male Mill Hill rats with a single alloxan dose (120 mg kg−1). Both non‐diabetic and diabetic groups were additionally separated into three subgroups: (i) receiving l‐arginine · HCl (2.25%), (ii) receiving l‐NAME · HCl (0.01%) for 12 days as drinking liquids, and (iii) control. Treatment of diabetic animals started after diabetes induction (glucose level ≥ 12 mmol l−1). We found that disturbed glucose homeostasis, i.e. blood insulin and glucose levels in diabetic rats was restored after l‐arginine treatment. Immunohistochemical findings revealed that l‐arginine had a favourable effect on β cell neogenesis, i.e. it increased the area of insulin‐immunopositive cells. Moreover, confocal microscopy showed colocalization of insulin and pancreas duodenum homeobox‐1 (PDX‐1) in both endocrine and exocrine pancreas. This increase in insulin‐expressing cells was accompanied by increased cell proliferation (observed by proliferating cell nuclear antigen‐PCNA immunopositivity) which occurred in a regulated manner since it was associated with increased apoptosis (detected by the TUNEL method). Furthermore, l‐arginine enhanced both nuclear factor‐kB (NF‐kB) and neuronal nitric oxide synthase (nNOS) immunopositivities. The effect of l‐arginine on antioxidative defence was observed especially in restoring to control level the diabetes‐induced increase in glutathione peroxidase activity. In contrast to l‐arginine, diabetic pancreas was not affected by l‐NAME supplementation. In conclusion, the results suggest beneficial l‐arginine effects on alloxan‐induced diabetes resulting from the stimulation of β cell neogenesis, including complex mechanisms of transcriptional and redox regulation.

Antioxidative defence alterations in skeletal muscle during prolonged acclimation to cold: role of l-arginine/NO-producing pathway

SUMMARY Early in cold acclimation (1–7 days), heat is produced by shivering, while late in cold acclimation (12–45 days), skeletal muscle contributes to thermogenesis by tissue metabolism other than contractions. Given that both thermogenic phases augment skeletal muscle aerobic power and reactive species production, we aimed in this study to examine possible changes in skeletal muscle antioxidative defence (AD) during early and late cold acclimation with special emphasis on the influence of the l-arginine/nitric oxide (NO)-producing pathway on the modulation of AD in this tissue. Adult Mill Hill hybrid hooded rat males were divided into two main groups: a control group, which was kept at room temperature (22±1°C), and a group maintained at 4±1°C for 45 days. The cold-acclimated group was divided into three subgroups: untreated, l-arginine treated and Nω-nitro-l-arginine methyl ester (l-NAME) treated. The AD parameters were determined in the gastrocnemius muscle on day 1, 3, 7, 12, 21 and 45 of cold acclimation. The results showed an improvement of skeletal muscle AD in both early and late cold acclimation. Clear phase-dependent changes were seen only in copper, zinc superoxide dismutase activity, which was increased in early cold acclimation but returned to the control level in late acclimation. In contrast, there were no phase-dependent changes in manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase, the activities of which were increased during the whole cold exposure, indicating their engagement in both thermogenic phases. l-Arginine in early cold acclimation accelerated the cold-induced AD response, while in the late phase it sustained increases achieved in the early period. l-NAME affected both early and late acclimation through attenuation and a decrease in the AD response. These data strongly suggest the involvement of the l-arginine/NO pathway in the modulation of skeletal muscle AD.

Nitric oxide regulates mitochondrial re-modelling in interscapular brown adipose tissue: ultrastructural and morphometric-stereologic studies

As a complex, cell‐specific process that includes both division and clear functional differentiation of mitochondria, mitochondriogenesis is regulated by numerous endocrine and autocrine factors. In the present ultrastructural study, in vivo effects of l‐arginine‐nitric oxide (NO)‐producing pathway on mitochondriogenesis in interscapular brown adipose tissue (IBAT) were examined. For that purpose, adult Mill Hill hybrid hooded rats were receiving l‐arginine, a substrate of NO synthases (NOSs), or Nω‐nitro‐l‐arginine methyl ester (l‐NAME), an inhibitor of NOSs, as drinking liquids for 45 days. All experimental groups were divided into two sub‐groups – acclimated to room temperature and cold. IBAT mitochondria were analyzed by transmission electron microscopy and stereology. l‐Arginine treatment acted increasing the number of mitochondrial profiles per cell profile, as well as volume fraction of mitochondria per cell volume in animals maintained at room temperature. Cold‐induced enhancement of number of mitochondrial profiles per cell profile was additionally increased in l‐arginine‐treated rats. Ultrastructural examinations of l‐arginine‐treated cold‐acclimated animals clearly demonstrated thermogenically active mitochondria (larger size, lamellar, more numerous and well‐ordered cristae in their profiles), which however were inactive in l‐arginine‐receiving animals kept at room temperature (small mitochondria, tubular cristae). By contrast, l‐NAME treatment of rats acclimated to room temperature induced mitochondrial alterations characterized by irregular shape, short disorganized cristae and megamitochondria formation. These results showed that NO is a necessary factor for mitochondrial biogenesis and that it acts intensifying this process, but NO alone is not a sufficient stimulus for in vivo induction of mitochondriogenesis in brown adipocytes.

Expression pattern of thermogenesis-related factors in interscapular brown adipose tissue of alloxan-treated rats: Beneficial effect of l-arginine

Metabolic abnormalities underlying diabetes can be abrogated by L-arginine. Here we examined the molecular basis of disturbed interscapular brown adipose tissue (IBAT) thermogenesis and the possible role of nitric oxide (NO) in the IBAT of diabetic rats. To induce diabetes, adult Mill Hill hybrid hooded male rats were given a single alloxan dose (120 mg/kg). Both non-diabetic and diabetic groups were further divided into three subgroups receiving: (i) L-arginine.HCl (2.25%) or (ii) N(omega)-nitro-L-arginine methyl ester (L-NAME.HCl, 0.01%) for 12 days in drinking water and (iii) untreated controls. Treatment of the diabetic animals started after diabetes induction (glucose level 12 mmol/L). Diabetes led to a decrease in the mRNA levels of uncoupling protein 1 (UCP1), peroxisomal proliferator activator receptor gamma (PPARgamma) and endothelial NO synthase (eNOS) as revealed by RT-PCR. The diabetic rats had reduced eNOS and inducible NOS (iNOS) protein contents accompanied by low tissue vascularization, a parameter directly related to tissue thermogenic state. Downregulation of glutathione peroxidase (GSH-Px) and catalase (CAT) transcripts were also observed in diabetes. In contrast, the expression level of PPARgamma coactivator-1alpha (PGC-1alpha) mRNA was elevated. Supplementation with L-arginine not only restored diabetes-induced changes in the expressions of these molecules important for IBAT regulation, but also increased the vascularity. Interestingly, L-NAME induced similar patterns of changes in vascularity and PGC-1alpha mRNA level as did l-arginine. In summary, our results provide insight into the molecular basis underlying diabetes-induced metabolic and functional disturbances in the IBAT and suggest a beneficial role for the L-arginine-NO production pathway.

NO modulates the molecular basis of rat interscapular brown adipose tissue thermogenesis.

Molecular mechanisms underlying interscapular brown adipose tissue (IBAT) thermogenesis were elucidated. Namely, gene and/or protein expression of uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor gamma (PPARgamma), PPARgamma-coactivator-1alpha (PGC-1alpha), vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) - key molecules that regulate thermogenesis-related processes - mitochondriogenesis, angiogenesis and IBAT hyperplasia, in rats subjected to cold (4+/-1 degrees C) for 1, 3, 7, 12, 21 and 45days were investigated. Particularly, to examine influence of nitric oxide (NO) on IBAT thermogenic-program, cold-exposed animals were treated by l-arginine or N(omega)-nitro-l-arginine-methyl ester (L-NAME). Related to control (22+/-1 degrees C), cold induced time-coordinated UCP1, PPARgamma and PGC-1alpha transcriptional activation accompanied by PCNA activation and increased VEGF immunolabeling that correlate with endothelial NO synthase (eNOS) transcriptional activation suggesting NO involvement in these thermogenic-factors activation. Observed molecular changes were translated into increased mitochondrial-remodeling, angiogenesis, and IBAT hyperplasia. l-Arginine augmented and prolonged cold-induced increase of eNOS, inducible NOS and thermogenic-molecules expression, IBAT nerve supply, vascularity, hyperplasia and mitochondrial-remodeling, while L-NAME had an opposite effects. Results show that NO improves thermogenesis-related mitochondriogenesis, angiogenesis and tissue hyperplasia, positively affecting molecular basis of these processes, suggesting that NO is an essential regulator of IBAT thermogenic-program operating, at genes, proteins and tissue structure levels.

l-Arginine supplementation induces glutathione synthesis in interscapular brown adipose tissue through activation of glutamate-cysteine ligase expression: The role of nitric oxide.

We examined whether nitric oxide (NO) in vivo could induce interscapular brown adipose tissue (IBAT) glutathione synthesis. Data show that NO induces in vivo IBAT glutathione synthesis through activation of glutamate-cysteine ligase (GCL) mRNA and protein expression. This NO effect appeared to be mediated by nuclear factor-kappaB (NF-kappaB) activation. We have also observed a complex series of in vivo cellular responses during chronic inhibition of NO synthesis, suggesting that regulatory pathways unrelated to GCL alteration underlie glutathione level increase induced by N(omega)-nitro-l-arginine methyl ester (l-NAME). In general, glutathione synthesis in IBAT seemed to be finely tuned by NO to provide glutathione for either mediating the effects of NO, or for preventing potential nitrosative stress.

Leptin immunoexpression and innervation in rat interscapular brown adipose tissue of cold-acclimated rats: the effects of L-arginine and L-NAME.

The aim of the present study was to explore the effect of nitric oxide on leptin immunoexpression and innervation in interscapular brown adipose tissue (IBAT) of room- and cold- acclimated rats. Animals acclimated both to room-temperature (22 +/- 1 degrees C) and cold (4 +/- 1 degrees C) were treated with L-arginine, a substrate for nitric oxide synthases (NOSs), or N?-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOSs, for 45 days. Leptin expression and localization in brown adipocytes was studied by immunohistochemistry, and innervation stained by the Bodian method. Strong leptin immunopositivity was observed in brown adipocytes cytoplasm of all room-acclimated groups, but nuclear leptin positivity was found only in L-NAME treated rats. In cold-acclimated control and L-NAME treated rats leptin immunopositivity was absent, while L-arginine treatment reversed the cold-induced suppression of leptin expression. Comparing to control, L-arginine, and even more L-NAME, at 22 +/- 1 degrees C induced greater innervation. In conclusion, L-arginine treatment changes leptin expression pattern on cold in rat IBAT.

White adipocytes transdifferentiation into brown adipocytes induced by triiodothyronine

The uncontrolled expansion of white adipose tissue (WAT) seen in obesity predisposes affected individuals to health complications. Another type of fat, brown adipose tissue (BAT) has an opposing physiological function because it allows dissipation instead of storage of energy. Recent studies on the transcriptional control of adipocyte differentiation offer a new perspective on conversion between brown and white adipocytes. The ability of the adipose organ to interconvert its main cytotypes in order to meet changing metabolic needs is highly pertinent to the physiopathology of obesity and related to therapeutic strategies [1]. Thyroid hormones (TH) have an important role in modulation of brown adipose tissue (BAT) thermogenic function. The main objective of this work was to investigate capability of triiodothyronine to induce structural and functional changes of rat white adipocytes in retroperitoneal white adipose tissue (RWAT).