Factors that affect the adipocytic differentiation of adipose-derived mesenchymal stem cells

McCaffrey, Caitlin (2022-04)

Thesis (MMed)--Stellenbosch University, 2022.

Thesis

Background – Increased adiposity, particularly the accumulation of visceral adipose tissue is linked to the onset and progression of the metabolic syndrome. Despite the known pathogenicity of excessive visceral fat accrual, a paucity of knowledge on the mechanism behind its expansion exists. We hypothesise that hypertrophy and cellular dysfunction of subcutaneous adipocytes leads to the secretion of endocrine factors which stimulate adipogenesis in visceral adipose-derived mesenchymal stem cells (AD-MSCs) and that this communication might be affected by diet. We also postulate that the endocrine-induced adipogenesis of visceral AD-MSCs is amplified in individuals prescribed the PPARγ agonist, rosiglitazone for treatment of type II diabetes mellitus. Methods – Male Wistar rats at 3-weeks of age were fed either control lab chow, a high-fat diet (HFD), or an elevated sugar diet (ESD) (n = 7/group) ad libitum for 16 weeks and euthanised. The rat weights and endpoint fasting blood glucose were recorded and bilateral inguinal subcutaneous and retroperitoneal visceral adipose tissue depots excised. Histological studies of the tissue allowed adipocyte structure to be observed and measured. AD-MSCs from each depot were also isolated and expanded in vitro. The in vitro morphology of AD-MSCs was characterised, proliferation monitored, and the expression of the MSC markers (CD90+ and CD45-) measured. Adipogenesis was induced in the AD-MSCs, and conditioned media (CM) – containing the secretome of the mature adipocytes – was collected. Naïve AD-MSCs isolated from both adipose depots of age-matched control rats (n = 3) were then treated with the subcutaneous- and visceral conditioned media alone or in combination with either standard growth media (SGM) or adipocyte differentiation media (AM). This was repeated with and without the addition of 1μM rosiglitazone (Rosi). The effects of these treatments were qualitatively assessed by microscopy of oil red O-stained lipids and quantitatively assessed by in-silico micrograph- and spectrophotometric analyses. Results – The findings of this study indicate that feeding male Wistar rats a HFD or an ESD increases the mass of the inguinal and retroperitoneal adipose depots and cause hypertrophy in the constituent adipocytes. The diets also affected the MSC marker expression of the AD-MSCs from both adipose depots, having elevated the expression of CD90 in the HFD AD-MSCs but decreased the CD90 expression in the ESD AD-MSCs. The CM samples collected from the chow-fed control, HFD, and ESD adipocytes stimulated lipid accrual in the visceral AD-MSCs when added alone or in combination with SGM or AM, but the most significant adipogenic effects were induced by the ESD visceral CM in all three formulations. Only the control subcutaneous- and ESD visceral CM, when supplemented with AM, were able to elicit significant lipid accrual in the subcutaneous AD-MSCs. Following Rosi supplementation, the visceral AD-MSCs treated with HFD- and control CM treatments accumulated significant quantities of intracellular lipids. None of the CM+Rosi treatments stimulated significant lipid accrual in the naïve subcutaneous AD-MSCs. The CM and Rosi experiments revealed that subcutaneous AD-MSCs are less susceptible to CM-induced adipogenesis than visceral AD-MSCs. Conclusion – This study provides evidence for a possible endocrine communication between the subcutaneous and visceral adipose depots which appears to be amplified by the chronic consumption of an elevated sugar diet as well as by supplementation with rosiglitazone. These novel findings may contribute towards elucidating the underlying mechanisms behind the pathogenic expansion of visceral adipose tissue.

Agtergrond – ‘n Toename in adipositeit, spesifiek die ophoping van visserale adipose weefsel is verwant aan die intrede en verloop van die metaboliese sindroom. Ten spyte van die bekende patogeniteit betrokke by die akkumulasie van oortollige visserale vet, is daar gebrekkige kennis aangaande die meganisme van uitsetting van hierdie tipe vet. Ons hipotese is dat hipertrofie en sellulêre disfunksie van subkutane adiposiete die afskeiding van endokriene faktore tot gevolg het, wat adipogenese in visserale adipose-afgeleide mesenchimale stamselle (AD-MSCs) stimuleer en dat hierdie kommunikasie deur dieet beïnvloed kan word. Ons postuleer ook dat die endokriene-geïnduseerde adipogenese van visserale AD-MSCs versterk word in individue wat die PPARγ agonis rosiglitasoon, vir die behandeling van tipe II-diabetes mellitus, voorgeskryf is. Metodes - Drie week oud manlike Wistar rotte is óf kontrole laboratorium rotkos, óf 'n hoë-vet dieet (HFD), óf 'n verhoogde suiker dieet (ESD) (n = 7 / groep) ad libitum gevoed vir 16 weke waarna hulle uitgesit is. Die liggaamsgewigte en eindpunt vastende bloedglukose vlakke van die rotte is aangeteken. Bilaterale subkutane inguinale en viscerale retroperitoneale adipose weefsel depots is chirurgies verwyder. Histologiese studies van die weefsel is onderneem om adiposiet-struktuur waar te neem en te meet. AD-MSCs is ook vanuit elke depot geïsoleer en in vitro uitgebrei. Hierdie AD-MSCs is gekarakteriseer in terme van in vitro morfologie, proliferasie en die uitdrukking van MSC merkers (CD90 + en CD45-). Adipogenese is geïnduseer in die AD-MSCs en gekondisioneerde media (KM) – wat die sekretoom van volwasse adiposiete bevat – versamel. Naïewe AD-MSCs wat geïsoleer is vanuit beide adipose depots van ouderdom-ooreenstemmende kontrole rotte (n = 3) is vervolgens behandel met die gekondisioneerde media afkomstig vanaf die subkutane- en visserale selle alleen of in kombinasie met óf standaard groei media (SGM) of adiposiet differensiasie media (AM). Dit is herhaal met en sonder die toevoeging van 1μM rosiglitasoon (Rosi). Die gevolge van hierdie behandelings is kwalitatief geassesseer deur mikroskopie van olie rooi o-gekleurde lipiede en gekwantifiseer met behulp van in-silico mikrograaf- en spektrofotometriese ontledings. Resultate - Die bevindings van hierdie studie dui daarop dat die voeding van manlike Wistar rotte met 'n HFD of 'n ESD die massa van die inguinale en retroperitoneale adipose depots verhoog en ook hipertrofie veroorsaak in die adiposiete waaruit hierdie weefsels saamgestel is. Hierdie diëte het ook die MSC- merkeruitdrukking in die AD-MSCs van beide adipose depots beïnvloed. Die uitdrukking van CD90 is in die HFD AD-MSCs verhoog, terwyl CD90-uitdrukking in die ESD AD-MSCs verminder is. Alle KM monsters wat versamel is vanaf rotkos-gevoerde kontrole, HFD, en ESD adiposiete het lipied akkumulasie in die visserale AD-MSCs gestimuleer, maar die mees uitgesproke adipogeniese effekte is veroorsaak deur die ESD visserale KM. Die CM monsters versamel uit die chow-gevoed beheer, HFD, en ESD adipocytes gestimuleer lipied aanwas in die viscerale AD-MSCs wanneer alleen of in kombinasie met SGM of AM bygevoeg, maar die belangrikste adipogene effekte is veroorsaak deur die ESD viscerale CM in al drie formulerings. Slegs die beheer subkutane- en ESD viscerale CM, wanneer dit met AM aangevul is, kon beduidende lipied-aanwas in die subkutane AD- MSC's ontlok. Na aanleiding van Rosi-aanvulling het die viscerale AD-MSC's wat met HFD- en beheer CM-behandelings behandel is, beduidende hoeveelhede intrasellulêre lipiede opgehoop. Nie een van die CM + Rosi behandelings gestimuleer beduidende lipied aanwas in die naïewe subkutane AD-MSCs. Die CM- en Rosi-eksperimente het aan die lig gebring dat subkutane AD-MSC's minder vatbaar is vir CM-geïnduseerde adipogenese as viscerale AD-MSC's. Gevolgtrekking – Hierdie studie bied bewyse vir moontlike endokriene kommunikasie tussen die subkutane en viscerale adipose depots wat blyk te wees versterk deur die chroniese verbruik van 'n verhoogde suiker dieet sowel as deur aanvulling met rosiglitazone. Hierdie nuwe bevindings kan bydra tot die verduideliking van die onderliggende meganismes agter die patogene uitbreiding van viscerale vetweefsel.

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