Molecular mechanisms of insulin-induced rat brown adipose tissue structural remodelling

U svetlu sve većeg broja dokaza koji ukazuju na prisustvo metabolički aktivnog mrkog masnog tkiva (BAT – brown adipose tissue, engl.) kod odraslih ljudi, kao i na njegovu potencijalnu ulogu u sprečavanju razvoja gojaznosti, insulinske rezistence i metaboličkog sindroma uopšte, sve je više studija o rasvetljavanju metaboličkog značaja ovog tkiva, kao i njegovoj termogenoj stimulaciji u svrhu terapije navedenih poremećaja. Insulin se, kao važan anabolički hormon, smatra značajnim modulatorom strukturne organizacije i funkcije BAT. Cilj ovog istraživanja bio je rasvetljavanje molekulskih osnova insulinomindukovanog strukturnog remodeliranja BAT putem identifikacije mehanizama koji regulišu insulinom-stimulisanu proliferaciju i diferencijaciju ćelija, kao i identifikacije mehanizama koji učestvuju u oštećenjima ćelija i njihovom umiranju. Pacovi soja Wistar su jednom dnevno tretirani fiziološkom (0.4 IU/kg telesne mase) i suprafiziološkom (4 IU/kg telesne mase) dozom insulina, jedan (akutan tretman) ili tri (hroničan tretman) dana. Kao fiziološka kontrola, dve grupe pacova su u istom trajanju tretirane fiziološkim rastvorom (akutna i hronična kontrola). Tri sata nakon poslednje doze, životinje su žrtvovane, a interskapularni depo BAT je izolovan, izmeren i pripremljen za tehnike svetlosne i elektronske mikroskopije. Analiza tkiva izvršena je korišćenjem metoda histohemijskog i imunohistohemijskog bojenja, metoda konfokalne i transmisione elektronske mikroskopije i elektron-disperzivne analize X-zracima (EDX) hemijskog sastava tkiva kao i metoda stereološke i morfometrijske analize. Rezultati su pokazali postojanje strukturnog i funkcijskog remodeliranja BAT, gde dominiraju procesi hipertrofije i hiperplazije ćelija, praćene povećanjem termogenog kapaciteta mrkih adipocita (raste ekspresija dekuplujućeg proteina 1 (UCP1 – uncoupling protein 1, engl.), udeo mitohondrija, procesi mitohondriogeneze). S druge strane, primetno je da se, naročito pri hroničnim i tretmanima visokom dozom insulina, javljaju citotoksični i inhibitorni efekti na pojedine ćelije ili klastere ćelija: raste učestalost ćelijske smrti, ekstravazacija eritrocita i njihovo uklanjanje od strane mrkih adipocita i makrofaga, (evidentno toksično po adipocite sudeći po rastu: ekspresije enzima antioksidativne odbrane; lipidne peroksidacije i nitrozilacije proteina, učestalosti lipofuscinogeneze). Takođe se pri hronično visokoj hiperinsulinemiji smanjuje ekspresija regulatora termogeneze (PGC-1α - peroxisome proliferator-activated receptor – γ coactivator - 1α, engl.) ukazujući da produženo izlaganje visokim dozama insulina, može da inhibira termogeni odgovor tkiva, o čemu svedoči i povećana učestalost mitohondrijalnih oštećenja. Povećana ekspresija proinflamatornih citokina (TNF-α - tumor-necrosis factor – α, engl., interleukina 6), nukleusnog faktora – kB (NF-kB) i makrofagnog inflamatornog proteina - 3β (MIP-3β) u pojedinačnim zrelim, multilokulusnim adipocitima, preadipocitima i unilokulusnim adipocitima ukazuje na njihovu potencijalnu ulogu u lokalnom smanjenju insulinske senzitivnosti. U vezi sa hiperplazijom ćelija, primećeno je da su adipogeneza i angiogeneza u BAT vremenski i prostorno povezane, tj. da postoji udruženost navedenih procesa, koja ukazuje na potrebu udruženog nastanka adipocita i endotelskih ćelija, u svrhu efikasnog strukturno-funkcijskog remodeliranja tkiva. Sumarno, može se zaključiti da hiperinsulinemija ima značajan efekat na BAT – potvrđeno je anaboličko dejstvo insulina i važnost njegove uloge u stimulaciji BAT, ali je i pokazana toksičnost visoke doze i hroničnog izlaganja na ćelije BAT; što je sve doprinelo rasvetljavanju molekulskih mehanizama u osnovi strukturnog remodeliranja ovog tkiva. Takođe, pokazano je da se visoka plastičnost BAT ogleda u postojanju funkcionalnih klastera ćelija, u kojima se odvijaju svi važni tkivni procesi (adipo/angiogeneza, ćelijska smrt, eritrofagocitoza), što ukazuje na pravilnu uređenost tkivne organizacije i strukturnog remodeliranja, kao preduslova za pravilno funkcionisanje tkiva, kako u fiziološkim, tako i u uslovima izmenjene tkivne homeostaze, što je pokazano na modelu hiperinsulinemije.There is an increasing amount of evidence that indicates presence of brown adipose tissue (BAT) in adult humans, and its potential role in prevention of obesity, insulin resistance and metabolic syndrom, in general. That is why number of studies on elucidation of the metabolic importance of this tissue, along with its thermogenic stimulation in order to treat these disorders, increases. As an important anabolic hormone, insulin is considered as a major modulator of BAT structure and function. The aim of this study was to elucidate the molecular basis of insulin-induced structural remodeling of BAT through the identification of mechanisms that regulate insulin-stimulated proliferation and differentiation and through the identification of mechanisms involved in cell damage and death. Wistar strain rats were treated intraperitoneally with physiological (0.4 IU/kg BW) or supraphysiological (4 IU/kg BW ) dose of insulin for one (acute treatment) or three days (chronic treatment), respectively. Two groups of rats served as physiological controls, and were treated with saline (acute and chronic control). Three hours after the last injection the animals were sacrificed and the interscapular portion of BAT was removed, its weight was measured and it was processed for electron and light microscopic examinations. Methods of histochemical staining, immunohistochemical labeling, confocal and transmission electron microscopy, tissue electron-dispersive X-ray microanalysis (EDX) and stereological and morphometric analyses were performed. The results showed the existence of structural and functional remodeling of BAT, based on adipocyte hypertrophy and hyperplasia which are followed by increasing of thermogenic capacity (expression of uncoupling protein 1 (UCP1), cellular proportion of mitochondria, mitochondrial biogenesis). On the other hand, it is obvious that, especially after chronic and high-dose treatments, insulin cytotoxic and inhibitory effects on individual cells or cell clusters occur: incidence of cell death increases, as well as red blood cell extravasation and their removal by brown adipocytes and macrophages (which is evidently toxic for the adipocyte, since expression of antioxidative defense enzymes, lipid peroxidation, protein nitrosilation and lipofuscin formation were increased). Also, during chronically high hyperinsulinemia, expression of peroxisome proliferator-activated receptor - γ coactivator - 1α (PGC-1α), regulator of thermogenesis decreases, indicating that prolonged exposure to high doses of insulin can inhibit thermogenic response of BAT, as evidenced by the increased frequency of mitochondrial damage in brown adipocytes. Increased expression of proinflammatory cytokines (tumornecrosis factor - α (TNF-α), interleukin 6), nuclear factor – kB (NF-kB) and macrophage inflammatory protein - 3β (MIP-3β) in individual mature, multilocular adipocytes, preadipocytes and unilocular adipocytes, demonstrates their potential role in local decrease of insulin sensitivity. Regarding hyperplasia of cells, it was noticed spaciotemporal association between processes of adipogenesis and angiogenesis, which means that there is a connection between these processes, demonstrating the need for the coupled differentiation of adipocytes and endothelial cells, for the purpose of efficient structural and functional remodeling of the tissue. In summary, it can be concluded that hyperinsulinemia has a significant impact on BAT – we confirmed the anabolic effect of insulin, and the importance of its role in BAT stimulation, but also demonstrated the toxicity of high dose and chronic exposure to insulin on BAT cells, all of which contribute to understanding the molecular mechanisms underlying structural remodeling of the tissue. In addition, it was shown that the high plasticity of BAT is reflected in the existence of functional clusters of cells, which take place in all the important processes in the tissue (adipo/angiogenesis, cell death, erythrophagocytosis), indicating the proper arrangement of tissue organization and structural remodeling as a prerequisite for the proper functioning of the tissue under physiological conditions and during altered tissue homeostasis, as shown on the model of hyperinsulinemia

EPS-SJ Exopolisaccharide Produced by the Strain Lactobacillus paracasei subsp paracasei BGSJ2-8 Is Involved in Adhesion to Epithelial Intestinal Cells and Decrease on E-coil Association to Caco-2 Cells

The aim of this study was to determine the role of an exopolysaccharide produced by natural dairy isolate Lactobacillus paracasel subsp. paracasel BGSJ2-8, in the adhesion to intestinal epithelial cells and a decrease in Escherichia coil's association with Caco-2 cells. Annotation of the BGSJ2-8 genome showed the presence of a gene cluster, epsSJ, which encodes the biosynthesis of the strain-specific exopolysaccharide EPS-SJ, detected as two fractions (P1 and P2) by size exclusion chromatography (SEC) coupled with multi angle laser light scattering (MALLS) detection. SEC MALLS analysis revealed that an EPS-SJ(-) mutant (EPS7, obtained by insertion mutagenesis of the glps_2198 gene encoding primary glycosyltransferase) does not produce the P2 fraction of EPS-SJ. Transmission electron microscopy showed that EPS7 mutant has a thinner cell wall compared to the EPS-SJ(+) strain BGSJ2-83 (a plasmid free derivative of BGSJ2-8). Interestingly, strain BGSJ2-83 showed higher adhesion to Caco-2 epithelial intestinal cell line than the EPS7 mutant. Accordingly, BGSJ2-83 effectively reduced E. coil ATCC25922's association with Caco-2 cells, while EPS7 did not show statistically significant differences. In addition, the effect of EPS-SJ on the proliferation of lymphocytes in gastrointestinal associated lymphoid tissue (GALT) was tested and the results showed that the reduction of GALT lymphocyte proliferation was higher by BGSJ2-83 than by the mutant. To the best of our knowledge this is the first report indicating that the presence of EPS (EPS-SJ) on the surface of lactobacilli can improve communication between bacteria and intestinal epithelium, implying its possible role in gut colonization

Protein expression of ubiquitin in interscapular brown adipose tissue during acclimation of rats to cold: the impact of (NO)-N-center dot

In this study, the effects of l-arginine-nitric-oxide ((NO)-N-a (TM))-producing pathway on protein content of ubiquitin, as an important component of ubiquitin-proteasome system for protein removal, were investigated. We showed that l-arginine markedly decreased ubiquitin protein content in interscapular brown adipose tissue, both in thermogenic inactive (at room temperature) and thermogenic active (on cold) states; while in l-NAME-treated groups this effect was abolished. This result suggests that nitric oxide ((NO)-N-a (TM)), besides well established roles, is involved in this aspect of structure remodeling, as well.Ministry of Education and Science of the Republic of Serbia [173055

Interscapular brown adipose tissue metabolic reprogramming during cold acclimation: Interplay of HIF-1 alpha and AMPK alpha

Background: Brown adipose tissue thermogenic program includes complex molecular and structural changes. However, energetic aspects of this process are poorly depicted. Methods: We investigated time-dependent reprogramming of interscapular brown adipose tissue (IBAT) energy metabolism during cold-acclimation, as well as the effects of nitric oxide ((center dot)NO) on those changes. Rats were exposed to cold (4 +/- 1 degrees C) for periods of 1, 3, 7, 12, 21. and 45 days, and divided into three groups: control, treated with L-arginine, and treated with N(omega)-nitro-L-arginine methyl ester (L-NAME). Results: In the early phase of cold-acclimation (up to 7 days), the protein levels of all metabolic parameters and oxidative phosphorylation components were below the control. However, metabolic parameters and respiratory chain components entered a new homeostatic level in the late phase of cold-acclimation. These changes were accompanied with increased protein levels of phospho-AMP-dependent protein kinase-alpha (phospho-AMPK alpha) on the first day of cold-acclimation, and hypoxia-inducible factor-1 alpha (HIF-1 alpha) throughout early cold-acclimation. L-arginine positively affected protein expression of enzymes involved in glucose metabolism and beta-oxidation of fatty acids in the early phase of cold-acclimation, and oxidative phosphorylation components throughout cold-acclimation. In contrast, L-NAME had the opposite effects. Conclusion: Results suggest that IBAT structural remodeling is followed by energy metabolism reprogramming, which control might be orchestrated by the action of AMPK alpha and HIF-1 alpha. Data also indicated the involvement of L-arginine-(center dot)NO in the regulation of IBAT metabolism. General significance: Results obtained in this study might be of great importance for elucidating regulatory pathways governing energy metabolism in both physiological and pathophysiological states. (C) 2011 Elsevier B.V. All rights reserved.Ministry of Education and Science of the Republic of Serbia [173055

Protein expression of ubiquitin in interscapular brown adipose tissue during acclimation of rats to cold: the impact of (NO)-N-center dot

In this study, the effects of l-arginine-nitric-oxide ((NO)-N-a (TM))-producing pathway on protein content of ubiquitin, as an important component of ubiquitin-proteasome system for protein removal, were investigated. We showed that l-arginine markedly decreased ubiquitin protein content in interscapular brown adipose tissue, both in thermogenic inactive (at room temperature) and thermogenic active (on cold) states; while in l-NAME-treated groups this effect was abolished. This result suggests that nitric oxide ((NO)-N-a (TM)), besides well established roles, is involved in this aspect of structure remodeling, as well.Ministry of Education and Science of the Republic of Serbia [173055

Regulatory role of PGC-1 alpha/PPAR signaling in skeletal muscle metabolic recruitment during cold acclimation

This study examined the molecular basis of energy-related regulatory mechanisms underlying metabolic recruitment of skeletal muscle during cold acclimation and possible involvement of the L-arginine/nitric oxide-producing pathway. Rats exposed to cold (4 +/- 1 degrees C) for periods of 1, 3, 7, 12, 21 and 45 days were divided into three groups: untreated, L-arginine treated and N-omega-nitro-L-arginine methyl ester (L-NAME) treated. Compared with controls (22 +/- 1 degrees C), there was an initial increase in the protein level of 5'-AMP-activated protein kinase alpha (day 1), followed by an increase in peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) and peroxisome proliferator-activated receptors (PPARs): PPAR alpha and PPAR gamma from day 1 and PPAR delta from day 7 of cold acclimation. Activation of the PGC-1 alpha/PPAR transcription program was accompanied by increased protein expression of the key metabolic enzymes in beta-oxidation, the tricarboxylic acid cycle and oxidative phosphorylation, with the exceptions in complex I (no changes) and ATP synthase (decreased at day 1). Cold did not affect hexokinase and GAPDH protein levels, but increased lactate dehydrogenase activity compared with controls (1-45 days). L-arginine sustained, accelerated and/or intensified cold-induced molecular remodeling throughout cold acclimation. L-NAME exerted phase-dependent effects: similar to L-arginine in early cold acclimation and opposite after prolonged cold exposure (from day 21). It seems that upregulation of the PGC-1 alpha/PPAR transcription program early during cold acclimation triggers the molecular recruitment of skeletal muscle underlying the shift to more oxidative metabolism during prolonged cold acclimation. Our results suggest that nitric oxide has a role in maintaining the skeletal muscle oxidative phenotype in late cold acclimation but question its role early in cold acclimation.Ministry of Education, Science and Technological development of the Republic of Serbia [173054, 173055

The impact of cold acclimation and hibernation on antioxidant defenses in the ground squirrel (Spermophilus citellus): An update

Any alteration in oxidative metabolism is coupled with a corresponding response by an antioxidant defense (AD) in appropriate subcellular compartments. Seasonal hibernators pass through circannual metabolic adaptations that allow them to either maintain euthermy (cold acclimation) or enter winter torpor with body temperature falling to low values. The present study aimed to investigate the corresponding pattern of AD enzyme protein expressions associated with these strategies in the main tissues involved in whole animal energy homeostasis: brown and white adipose tissues (BAT and WAT, respectively), liver, and skeletal muscle. European ground squirrels (Spermophilus citellus) were exposed to low temperature (4 +/- 1 C) and then divided into two groups: (1) animals fell into torpor (hibernating group) and (2) animals stayed active and euthermic for 1, 3, 7, 12, or 21 days (cold-exposed group). We examined the effects of cold acclimation and hibernation on the tissue-dependent protein expression of four enzymes which catalyze the two-step detoxification of superoxide to water: superoxide dismutase 1 and 2 (SOD 1 and 2), catalase (CAT), and glutathione peroxidase (GSH-Px). The results showed that hibernation induced an increase of AD enzyme protein expressions in BAT and skeletal muscle. However, AD enzyme contents in liver were largely unaffected during torpor. Under these conditions, different WAT depots responded by elevating the amounts of specific enzymes, as follows: SOD 1 in retroperitoneal WAT, GSH-Px in gonadal WAT, and CAT in subcutaneous WAT. Similar perturbations of AD enzymes contents were seen in all tissues during cold acclimation, often in a time-dependent manner. It can be concluded that BAT and muscle AD capacity undergo the most dramatic changes during both cold acclimation and hibernation, while liver is relatively unaffected by either condition. Additionally, this study provides a basis for further metabolic study that will illuminate the causes of these tissue-specific AD responses, particularly the novel finding of distinct responses by different WAT depots in hibernators. (C) 2013 Elsevier Inc. All rights reserved.Ministry of Education, Science and Technological Development of the Republic of Serbia [173055

Regulatory role of PGC-1 alpha/PPAR signaling in skeletal muscle metabolic recruitment during cold acclimation

This study examined the molecular basis of energy-related regulatory mechanisms underlying metabolic recruitment of skeletal muscle during cold acclimation and possible involvement of the L-arginine/nitric oxide-producing pathway. Rats exposed to cold (4 +/- 1 degrees C) for periods of 1, 3, 7, 12, 21 and 45 days were divided into three groups: untreated, L-arginine treated and N-omega-nitro-L-arginine methyl ester (L-NAME) treated. Compared with controls (22 +/- 1 degrees C), there was an initial increase in the protein level of 5'-AMP-activated protein kinase alpha (day 1), followed by an increase in peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) and peroxisome proliferator-activated receptors (PPARs): PPAR alpha and PPAR gamma from day 1 and PPAR delta from day 7 of cold acclimation. Activation of the PGC-1 alpha/PPAR transcription program was accompanied by increased protein expression of the key metabolic enzymes in beta-oxidation, the tricarboxylic acid cycle and oxidative phosphorylation, with the exceptions in complex I (no changes) and ATP synthase (decreased at day 1). Cold did not affect hexokinase and GAPDH protein levels, but increased lactate dehydrogenase activity compared with controls (1-45 days). L-arginine sustained, accelerated and/or intensified cold-induced molecular remodeling throughout cold acclimation. L-NAME exerted phase-dependent effects: similar to L-arginine in early cold acclimation and opposite after prolonged cold exposure (from day 21). It seems that upregulation of the PGC-1 alpha/PPAR transcription program early during cold acclimation triggers the molecular recruitment of skeletal muscle underlying the shift to more oxidative metabolism during prolonged cold acclimation. Our results suggest that nitric oxide has a role in maintaining the skeletal muscle oxidative phenotype in late cold acclimation but question its role early in cold acclimation.Ministry of Education, Science and Technological development of the Republic of Serbia [173054, 173055

The impact of cold acclimation and hibernation on antioxidant defenses in the ground squirrel (Spermophilus citellus): An update

Any alteration in oxidative metabolism is coupled with a corresponding response by an antioxidant defense (AD) in appropriate subcellular compartments. Seasonal hibernators pass through circannual metabolic adaptations that allow them to either maintain euthermy (cold acclimation) or enter winter torpor with body temperature falling to low values. The present study aimed to investigate the corresponding pattern of AD enzyme protein expressions associated with these strategies in the main tissues involved in whole animal energy homeostasis: brown and white adipose tissues (BAT and WAT, respectively), liver, and skeletal muscle. European ground squirrels (Spermophilus citellus) were exposed to low temperature (4 +/- 1 C) and then divided into two groups: (1) animals fell into torpor (hibernating group) and (2) animals stayed active and euthermic for 1, 3, 7, 12, or 21 days (cold-exposed group). We examined the effects of cold acclimation and hibernation on the tissue-dependent protein expression of four enzymes which catalyze the two-step detoxification of superoxide to water: superoxide dismutase 1 and 2 (SOD 1 and 2), catalase (CAT), and glutathione peroxidase (GSH-Px). The results showed that hibernation induced an increase of AD enzyme protein expressions in BAT and skeletal muscle. However, AD enzyme contents in liver were largely unaffected during torpor. Under these conditions, different WAT depots responded by elevating the amounts of specific enzymes, as follows: SOD 1 in retroperitoneal WAT, GSH-Px in gonadal WAT, and CAT in subcutaneous WAT. Similar perturbations of AD enzymes contents were seen in all tissues during cold acclimation, often in a time-dependent manner. It can be concluded that BAT and muscle AD capacity undergo the most dramatic changes during both cold acclimation and hibernation, while liver is relatively unaffected by either condition. Additionally, this study provides a basis for further metabolic study that will illuminate the causes of these tissue-specific AD responses, particularly the novel finding of distinct responses by different WAT depots in hibernators. (C) 2013 Elsevier Inc. All rights reserved.Ministry of Education, Science and Technological Development of the Republic of Serbia [173055

Lipofuscin accumulation in brown adipocytes of hyperinsulinaemic rats

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