Dissertation, Funktionelle Charakterisierung des Jmjd1c Gens in der Adipogenese

Mesenchymale Stammzellen können sich durch den Einfluss verschiedener Transkriptionsfaktor zu Osteoblasten, Adipozyten, Chondrozyten oder Myoblasten differenzieren. Während sie sich unter Runx2-Einfluss entlang der osteoblastären Linie differenzieren, entwickeln sie sich bei vorliegendem PPARγ entlang des adipogenen Differenzierungswegs. Das Gleichgewicht zwischen beiden Faktoren und ihr Zusammenspiel stellen einen wichtigen Bereich in der Osteoporoseforschung dar. In dieser Dissertation wurde durch Runx2-Suppression bzw. Runx2-Überexpression die Rolle dieses Faktors in pHOB und SCP1-Zellen erfasst und die Interaktion zwischen Runx2 und PPARγ untersucht. Der Runx2-Knockdown’ erfolgte mittels RNA-Interferenz, die Runx2-Überexpression durch ein Runx2 exprimierendes Plasmid. In RT-PCRs wurden mRNA-Messungen durchgeführt. Die Proteinbestimmung erfolgte im ‚Westernblot’. Der funktionelle Einfluss der Runx2-Überexpression auf die PPARγ-Transkription wurde durch Kotransfektion des an Luziferase gekoppelten PPARg-Promotorgens erfasst. Die funktionelle Aktivität des PPARg-Proteins wurde durch die Transfektion des an Luziferase gekoppelten PPRE-Gens gemessen. Promotoraktivität und Funktionalität der Proteine wurden in Luziferase-Reportergenassays erfasst. Unter basalen Kulturbedingungen differenzierten sich pHOB osteogen. Durch zweimalige siRunx2-Transfektion gelang auf mRNA-Ebene eine suffiziente Runx2-Suppression über 29 Tage auf durchschnittlich 10,1%. Neben einer Steigerung der PPARγ-mRNA nach sieben Tagen konnte darunter auch eine Suppression der osteogenen Differenzierungsmarker OC und AP beobachtet werden. Ein ‚Rescue’ der supprimierten Runx2-Genexpression konnte durch osteogene Stimulation nicht erreicht werden. In den Runx2-/PPARγ-Interaktionsversuchen wurden SCP1-Zellen adipogen stimuliert, um die PPARγ2-mRNA und PPARγ-Promotoraktivität zu erhöhen. Darunter konnte ebenfalls eine gesteigerte Funktionalität des PPARγ-Proteins beobachtet werden. Durch Runx2-Überexpression wurde in SCP1-Zellen die PPARγ-Promotoraktivität und somit der Beginn der mRNA-Synthese gehemmt. Die PPARγ2-mRNA hingegen blieb unbeeinflusst. Die zentrale Rolle des Runx2 in der osteogenen Differenzierung scheint durch den Einfluss auf die osteogenen Marker OC und AP in pHOB bestätigt zu werden. Auch der Einfluss auf die adipogene Differenzierung erfolgt über Runx2. Im Rahmen dieser Dissertation konnte erstmalig die Hemmung des PPARγ-Promotors durch Runx2 beschrieben werden. Hierdurch werden die PPARγ-Transkription und somit voraussichtlich die Interaktion zwischen Adipogenese und Osteogenese beeinflusst.

Todas as adaptações adquiridas através do treinamento físico são reversíveis durante a inatividade. Reduções significativas no consumo máximo de oxigênio (VO2max) são observadas dentro de duas a quatro semanas de destreinamento. Por outro lado, as consequências do destreinamento sobre o tecido adiposo são pouco estudadas. O objetivo foi investigar os efeitos de destreinamento físico sobre o metabolismo e celularidade do tecido adiposo periepididimal. Métodos e Resultados: Ratos Wistar machos, com idade de 6 semanas, foram divididos em 3 grupos: treinado (T) durante 12 semanas; destreinados (D), (treinados por 8 semanas e destreinados por 4 semanas), e sedentário (S) pareados por idade. O treinamento consistiu em sessões de esteira rolante (1h/dia, 5d/semk, 50 60% da capacidade máxima). A análise morfométrica do tecido PE revelou diferenças significativas entre os grupos. A área seccional dos adipócitos do grupo D foi significativamente maior que a T e S (3474 µm2 ± 68,8 µm2 vs 1945,7 µm2 ± 45,6 µm2 vs 2492,4 µm2 ± 49,08 µm2, respectivamente). Em comparação com as células dos animais do grupo T as células do D apresentaram 48% de aumento na capacidade de realizar a lipogênese, espontaneamente ou insulina estimulada. Já lipólise basal não se alterou. A redução de 15% na apoptose foi observada nos grupos T e D em relação ao S. Algumas expressões de genes foram alterados em D vs S: a adiponectina aumentou 3 vezes e PPAR-gama aumentou 2 vezes. O gene do Pref-1 foi 3 vezes maior em T vs S. Estes resultados sugerem fortemente que a adipogênese foi estimulada neste grupo. Conclusões: o destreinamento causa aumento significativo no tamanho dos adipócitos e na capacidade lipogênica. Como a apoptose celular na gordura PE foi reduzida em D e T, estes resultados sugerem que as alterações do tecido adiposo após destreinamento podem ser potencialmente obesogenicas. / All adaptations acquired through physical training are reversible during inactivity. Significant reductions in maximal oxygen uptake (VO2Max) are observed within two-four weeks of detraining. Conversely, the consequences of detraining on adipose tissue are poorly known. The objective was to investigate the physical detraining effects on metabolism and cellularity of rat periepididymal adipose tissue. Methods and Results: Male Wistar rats, ageing 6 weeks, were divided in 3 groups: trained (T) for 12 weeks; detrained (D), (trained for 8 weeks and detrained for 4 weeks), and age-matched sedentary (S). Training consisted in treadmill running sessions (1h/day, 5d/week, 50 60% of the maximal capacity). The morphometric analysis of PE tissue disclosed significant differences between the groups. The adipocyte sectional area of group D was significantly bigger than T and S (3474 µm2 ± 68,8 µm2 vs 1945,7 µm2 ± 45,6 µm2 vs 2492,4 µm2 ± 49,08 µm2, respectively). Compared to T the cells of D animals showed 48% increased ability to perform: lipogenesis, either spontaneously or insulin stimulated and isoproterenol-stimulated lipolysis. Basal lipolysis did not change. A 15% reduction in apoptosis was observed in groups T and D in relation to S. Some gene expressions were changed in D vs S: adiponectin (3-fold up) and PPAR-gamma (2-fold up). PREF-1 gene was 3-fold higher in T vs S. These results strongly suggest that adipogenesis was stimulated in this group. Conclusions: Detraining causes significant increase in adipocyte size and lipogenic capacity. As PE fat cell apoptosis was reduced in D and T, these results suggest that adipose tissue changes following detraining can potentially be obesogenic.

Todas as adaptações adquiridas através do treinamento físico são reversíveis durante a inatividade. Reduções significativas no consumo máximo de oxigênio (VO2max) são observadas dentro de duas a quatro semanas de destreinamento. Por outro lado, as consequências do destreinamento sobre o tecido adiposo são pouco estudadas. O objetivo foi investigar os efeitos de destreinamento físico sobre o metabolismo e celularidade do tecido adiposo periepididimal. Métodos e Resultados: Ratos Wistar machos, com idade de 6 semanas, foram divididos em 3 grupos: treinado (T) durante 12 semanas; destreinados (D), (treinados por 8 semanas e destreinados por 4 semanas), e sedentário (S) pareados por idade. O treinamento consistiu em sessões de esteira rolante (1h/dia, 5d/semk, 50 60% da capacidade máxima). A análise morfométrica do tecido PE revelou diferenças significativas entre os grupos. A área seccional dos adipócitos do grupo D foi significativamente maior que a T e S (3474 µm2 ± 68,8 µm2 vs 1945,7 µm2 ± 45,6 µm2 vs 2492,4 µm2 ± 49,08 µm2, respectivamente). Em comparação com as células dos animais do grupo T as células do D apresentaram 48% de aumento na capacidade de realizar a lipogênese, espontaneamente ou insulina estimulada. Já lipólise basal não se alterou. A redução de 15% na apoptose foi observada nos grupos T e D em relação ao S. Algumas expressões de genes foram alterados em D vs S: a adiponectina aumentou 3 vezes e PPAR-gama aumentou 2 vezes. O gene do Pref-1 foi 3 vezes maior em T vs S. Estes resultados sugerem fortemente que a adipogênese foi estimulada neste grupo. Conclusões: o destreinamento causa aumento significativo no tamanho dos adipócitos e na capacidade lipogênica. Como a apoptose celular na gordura PE foi reduzida em D e T, estes resultados sugerem que as alterações do tecido adiposo após destreinamento podem ser potencialmente obesogenicas. / All adaptations acquired through physical training are reversible during inactivity. Significant reductions in maximal oxygen uptake (VO2Max) are observed within two-four weeks of detraining. Conversely, the consequences of detraining on adipose tissue are poorly known. The objective was to investigate the physical detraining effects on metabolism and cellularity of rat periepididymal adipose tissue. Methods and Results: Male Wistar rats, ageing 6 weeks, were divided in 3 groups: trained (T) for 12 weeks; detrained (D), (trained for 8 weeks and detrained for 4 weeks), and age-matched sedentary (S). Training consisted in treadmill running sessions (1h/day, 5d/week, 50 60% of the maximal capacity). The morphometric analysis of PE tissue disclosed significant differences between the groups. The adipocyte sectional area of group D was significantly bigger than T and S (3474 µm2 ± 68,8 µm2 vs 1945,7 µm2 ± 45,6 µm2 vs 2492,4 µm2 ± 49,08 µm2, respectively). Compared to T the cells of D animals showed 48% increased ability to perform: lipogenesis, either spontaneously or insulin stimulated and isoproterenol-stimulated lipolysis. Basal lipolysis did not change. A 15% reduction in apoptosis was observed in groups T and D in relation to S. Some gene expressions were changed in D vs S: adiponectin (3-fold up) and PPAR-gamma (2-fold up). PREF-1 gene was 3-fold higher in T vs S. These results strongly suggest that adipogenesis was stimulated in this group. Conclusions: Detraining causes significant increase in adipocyte size and lipogenic capacity. As PE fat cell apoptosis was reduced in D and T, these results suggest that adipose tissue changes following detraining can potentially be obesogenic.

Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, we set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. We quantified by optical diffraction tomography that differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, we dissect relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. Our work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders.

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