doctoral dissertation
Nejc Umek (Author), Erika Cvetko (Mentor), Danijel Petrovič (Thesis defence commission member), Tomaž Marš (Thesis defence commission member), Barbara Radochová (Thesis defence commission member), Ida Eržen (Co-mentor)

Abstract

Uvod: Presnovne motnje, povezane z debelostjo, so eden glavnih vzrokov obolevnosti in umrljivosti v razvitem svetu. Strukturne in funkcionalne spremembe v skeletnih mišicah in kapilarni mreži so pomembno povezane z mehanizmi zmanjšane občutljivosti na inzulin zaradi debelosti. V poznih fazah debelosti je številska gostota počasnih, oksidativnih, na inzulin občutljivih mišičnih vlaken manjša zaradi pretvorbe v hitra, na inzulin manj občutljiva vlakna. Tudi kopičenje znotrajceličnih lipidnih kapljic v mišičnih vlaknih lahko prispeva k zmanjšanju odzivnosti na inzulin, ni pa še znano, kako sta kopičenje znotrajceličnih lipidnih kapljic in pretvorba med tipi mišičnih vlaken povezana. Pri napredovani debelosti z zmanjšano občutljivostjo na inzulin se gostota kapilarne mreže v skeletnih mišicah zmanjša, zmanjšata pa se tudi bazalni in z inzulinom spodbujen krvni pretok. V zgodnji fazi debelosti z zmanjšano občutljivostjo na inzulin so ugotovili povečano funkcionalno odzivnost žilja in kapilarne mreže na inzulin. Ali je takšen funkcionalni odziv žilja povezan tudi z morfološkimi spremembami v kapilarni mreži in ali pride do sprememb v številski gostoti različnih tipov mišičnih vlaken tudi v zgodnjih fazah debelosti, še ni pojasnjeno. Metode: Osemnajst 54 tednov starih miši (linije C57BL/6JOlaHsd) smo razdelili v dve poskusni skupini po devet miši: na debele miši, hranjene s krmo z višjo vsebnostjo maščob, in na vitke miši, hranjene s standardno krmo. Odzivnost na inzulin smo ocenili z oralnim tolerančnim testom za glukozo in meritvami koncentracije krvne glukoze na tešče. Značilnosti kapilarne mreže smo določali s 3D-analizo 100 µm debelih prečnih rezin mišice gluteus maximus. Z imunofluorescenčnimi tehnikami smo označili endotelij kapilar in bazalno membrano kapilar in mišičnih vlaken. Debele prečne rezine smo zajeli s konfokalnim mikroskopom in analizirali s pomočjo programske opreme Ellipse. Izražanje posameznih izooblik težkih verig miozina v zaporednih 10 µm debelih rezinah mišic gluteus maximus, gastrocnemius in plantaris smo ugotavljali s protitelesi proti izooblikam težkih verig miozina tipa 1, 2a, 2x/d in 2b. Primarna protitelesa smo prikazali s posredno imunoperoksidazno metodo. Vsebnost znotrajceličnih lipidov smo določali na 10 µm debelih rezinah mišic gastrocnemius, plantaris in soleus, obarvanih z barvilom Sudan Black B. Indeks vsebnosti lipidov smo izračunali kot stokrat pomnoženo razmerje med površino preseka mišičnega vlakna, ki so ga prekrivale lipidne kapljice, in površino celotnega preseka mišičnega vlakna. S primerjavo premerov mišičnih vlaken mišice gluteus maximus v 10 µm debelih nativnih tkivnih rezinah in v 100 µm debelih fiksiranih in imunofluorescenčno označenih tkivnih rezinah smo ugotavljali, ali se v debelih rezinah mišična vlakna deformirajo tudi v prečni ravnini. Rezultati: V primerjavi z vitkimi mišmi, hranjenimi s standardno krmo, so imele miši, hranjene s krmo z visoko vsebnostjo maščob, znatno večjo telesno maso (p = 0,0001) in bazalno glikemijo ter znižano toleranco za glukozo (p < 0,05). Gostota kapilarne mreže mišice gluteus maximus je bila pri debelih miših večja kot pri vitkih miših. Okoli majhnih mišičnih vlaken (< 40 µm) smo ugotovili značilno večjo dolžino kapilar na dolžino mišičnih vlaken in dolžino kapilar na površino mišičnih vlaken (p < 0,05), medtem ko se okoli velikih mišičnih vlaken gostota kapilarne mreže ni značilno spremenila. Vijugavost, anizotropija in premer mišičnih vlaken se med poskusnima skupinama niso značilno razlikovali. Pri počasni mišici soleus in hitri mišici gluteus maximus, ki ne nosita telesne teže, smo pri debelih miših ugotovili manjšo številsko gostoto počasnih tipov mišičnih vlaken kot pri vitkih miših (p < 0,05). Pri hitri mišici gastrocnemius in srednje hitri mišici plantaris, ki ne nosita telesne teže, v izražanju težkih verig miozina med poskusnima skupinama nismo ugotovili značilnih razlik. Pri debelih miših sta bila v primerjavi z vitkimi mišmi v hitrih in srednje hitrih mišicah gastrocnemius in plantaris premer mišičnih vlaken in vsebnost znotrajceličnih lipidov znatno večja v mišičnih vlaknih tipa 2a in 2x/d (p < 0,05). Ugotovili smo, da se vzorci debelih prečnih rezin skeletnih mišic med pripravo za konfokalno mikroskopijo v prečni ravnini raztegnejo (p < 0,001), v aksialni smeri pa skrčijo (p < 0,001). Pokazali smo pozitivno korelacijo med velikostjo raztezka v prečni ravnini in obsegom krčenja v aksialni smeri (r = 0,493, p < 0,01), pri čemer je bilo krčenje v aksialni smeri bolj izrazito pri prečno kot pri poševno odrezanih tkivnih rezinah. Zaključki: Pri debelih miših z zmanjšano občutljivostjo na inzulin se je kapilarna mreža okoli majhnih, bolj oksidativnih tipov mišičnih vlaken povečala, kar bi lahko bil zgodnji kompenzacijski mehanizem proti zmanjšanju odzivnosti na inzulin. Številska gostota hitrih tipov mišičnih vlaken se je povečala tako v počasnih kot tudi v hitrih mišicah, ki ne nosijo telesne teže. Nasprotno pa pri hitrih mišicah, ki nosijo telesno težo, nismo opazili značilnih sprememb v številski gostoti različnih tipov mišičnih vlaken. Iz tega sklepamo, da lahko povečana obremenitev mišic zaradi povečane telesne mase preprečuje nastanek sprememb v izražanju težkih verig miozina. Pri debelih miših je bilo kopičenje znotrajceličnih lipidov različno v različnih mišicah in v različnih tipih mišičnih vlaken; večje kopičenje smo opazili tako v hitrih kot počasnih mišicah, in sicer največje v mišičnih vlaknih tipa 1, 2a in 2x/d. Debele tkivne rezine se raztezajo v prečni ravnini in skrčijo v aksialni smeri, pri čemer je velikost raztezka v prečni ravnini delno odvisna od velikosti skrčenja v aksialni smeri. Deformacijo vzorca v aksialni smeri sicer lahko popravimo s kalibracijo, da pa bi dosegli, da bi bila tudi deformacija v prečni ravnini čim manjša, moramo optimizirati histološke protokole in tako maksimalno zmanjšati krčenje v aksialni ravnini.

Keywords

debelost;zmanjšana občutljivost na inzulin;skeletna mišica;kapilarizacija;tip mišičnih vlaken;kopičenje znotrajceličnih lipidov;

Data

Language: English
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL MF - Faculty of Medicine
Publisher: [N. Umek]
UDC: 577.124:616.379+616.74(043.3)
COBISS: 83025155 Link will open in a new window
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Other data

Secondary language: Slovenian
Secondary title: Vpliv motene presnove glukoze na kapilarno mrežo in na izražanje težkih verig miozina v skeletnih mišicah miši
Secondary abstract: Background: Obesity-related metabolic disorders are among the leading causes of morbidity and mortality in the developed world. Structural and functional changes in skeletal muscles and the microvasculature are critically involved in the mediation of obesity-related insulin resistance. A shift from expression of slow to fast type myosin heavy chain isoforms in slow twitch muscles has been shown to contribute to reduced insulin sensitivity in obesity, while in fast-twitch weight-bearing muscles no fibre type shifting was observed. Furthermore, in obesity, accumulation of lipid droplets in skeletal muscle fibres can also contribute to insulin resistance. However, it is not yet clear how intramyocellular lipid accumulation and fibre type changes are associated. Moreover, in advanced obesity with insulin resistance, reduced capillary network density in skeletal muscles and impaired capillary recruitment has been demonstrated. On the other hand, in the early stage of obesity with insulin resistance, an increased functional vascular response to insulin has been described. However, it remains unclear whether such functional vascular response is associated with morphological alterations in the capillary network, or if the switch of fibre types toward fast type isoforms also occurs in fast-twitch, non-weight-bearing muscles in the early stages of obesity with insulin resistance. Methods: The study was carried out using eighteen 54-week-old C57BL/6JOlaHsd mice, divided into two study groups of nine mice each: the high fat diet-induced obese and the standard diet-treated lean groups. Insulin resistance status was assessed by the oral glucose tolerance test and fasting glucose measurements. We determined the capillary network characteristics using 3D analysis of 100 µm thick transverse sections of gluteus maximus muscle, and employed immunofluorescent techniques to mark the capillary endothelium and the basement membrane of capillaries and muscle fibres, which were then captured with a confocal microscope and analysed with the Ellipse software. We used the indirect immunohistochemical method to determine the myosin heavy chain isoform expression in muscle fibres. Antibodies against myosin heavy chain isoforms type 1, 2a, 2x/d and 2b were used to determine the expression of individual myosin heavy chain isoforms in successive 10 µm thick sections of gluteus maximus, gastrocnemius, plantaris and soleus muscles. We performed analysis of intramyocellular lipid content using 10 µm thick sections of gastrocnemius, plantaris and soleus muscles stained with Sudan Black B, and calculated the lipid content index as 100 times the ratio of the cross-sectional area of muscle fibre occupied by lipid droplets to the cross-sectional area of muscle fibre. The deformations of skeletal muscle thick tissue sections in horizontal plane were determined by comparison of gluteus maximus muscle fibre diameters in 10 µm thick native tissue sections to 100 µm thick fixed and immunofluorescently labelled tissue sections, respectively. Results: Compared to the standard diet-treated lean mice, mice fed on a high fat diet had a significantly increased body mass (p = 0.0001) and basal glycaemia, and decreased glucose tolerance (p < 0.05). The obese mice also showed denser capillary network of the gluteus maximus muscle compared to lean mice. Compared to the lean mice, capillary length per muscle fibre length, and capillary length per muscle fibre surface area were significantly larger around small muscle fibres (< 40 µm) (p < 0.05) in the obese mice, while there were no significant differences around large fibres in both groups. Other capillary characterization indices such as tortuosity, anisotropy and fibre diameter did not significantly differ between the study groups. In the slow-twitch soleus and fast-twitch non-weight-bearing gluteus maximus muscles, we noted a shift towards fast type myosin heavy chain isoform expression in the obese mice (p < 0.05), while in the weight-bearing fast-twitch gastrocnemius and intermediate plantaris muscles there were no significant differences in myosin heavy chain expression between the two study groups. Moreover, in obese mice muscle fibre size and intramyocellular lipid content were significantly increased in type 2a and 2x/d muscle fibres (p<0.05), with greater prominence in the fast- and intermediate-twitch than slow-twitch skeletal muscles. During the preparation of thick transverse sections of skeletal muscle for confocal microscopy, we demonstrated significant dilation of the sections in horizontal direction (p < 0.001) and shrinkage in the axial direction (p < 0.001). In addition, we noted a positive correlation between the magnitude of horizontal dilation and the magnitude of shrinkage in the axial direction (r = 0.493, p < 0.01), the latter being more pronounced in transversely than obliquely cut tissue sections. Conclusions: We found a selective increase in capillarisation around small muscle fibres of the more oxidative fibre types in our obese insulin-resistant mice, which could be an early compensatory mechanism ameliorating obesity-related insulin resistance. Our findings further suggest that in obesity with insulin resistance, both slow- and fast-twitch muscles exhibit the tendency for a shift toward fast type myosin heavy chain isoforms, and that increased weight bearing may condition the resistance of fast-twitch muscles to fibre type shifting. Our results also reaffirm that in obesity, intramyocellular lipid accumulation is specific for both skeletal muscle and fibre types, with greater prominence in fast-twitch muscles and muscle fibre types 1, 2a and 2x/d. Finally, our study also provide evidence on horizontal dilation and axial shrinkage of thick transverse sections of skeletal muscle. The magnitude of the former was partially dependent on the latter, suggesting that even though axial shrinkage can be corrected by calibration, histological protocols should be optimised to minimize the axial collapse that could cause horizontal dilation.
Secondary keywords: endokrinologija;patofiziologija;Glucose metabolism disorders;Muscle fibres, skeletal;Insuline resistance;Muscle, skeletal;Anatomy and histology;Capillaries;Obesity;Myosin heavy chains;Intracellular signaling peptides and proteins;Laboratory tecniques and procedures;Animal experimentation;Mice;Motnje metabolizma glukoze;Skeletna mišična vlakna;Odpornost proti inzulinu;Skeletna mišica;Anatomija in histologija;Kapilare;Debelost;Težke verige miozina;Znotrajcelični signalni peptidi in porteini;Laboratorijske tehnike in poskusi;Poskusi na živalih;Miši;Presnovne bolezni;Inzulin;Miozin;Mišične celice;Univerzitetna in visokošolska dela;
Type (COBISS): Dissertation
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Medicinska fak.
Pages: XIV f., 76 f. + [4] f. pril.
ID: 13781568