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.

SummaryAs 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 N ω -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. Coldinduced enhancement of number of mitochondrial profiles per cell profile was additionally increased in L-argininetreated rats. Ultrastructural examinations of L-argininetreated 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.

SUMMARYEarly 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 Larginine/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 N -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 phasedependent 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.

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