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  • 1.
    de Jong, Jasper M. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Cannon, Barbara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Nedergaard, Jan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Promotion of lipid storage rather than of thermogenic competence by fetal versus newborn calf serum in primary cultures of brown adipocytes2018Inngår i: Adipocyte, ISSN 2162-3945, E-ISSN 2162-397X, Vol. 7, nr 3, s. 166-179Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Much current understanding of brown adipocyte development comes from in-vitro cell models. Serum type may affect the behavior of cultured cells and thus conclusions drawn. Here, we investigate effects of serum type (fetal bovine versus newborn calf) on responses to differentiation inducers (the PPAR agonist rosiglitazone or the neurotransmitter norepinephrine) in cultured primary brown adipocytes. Lipid storage was enhanced by fetal versus newborn serum. However, molecular adipose conversion (Pparg2 and Fabp4 expression) was not affected by serum type. Rosiglitazone-induced (7-days) expression of thermogenic genes (i.e. Ucp1, Pgc1a, Dio2 and Elovl3) was not systematically affected by serum type. However, importantly, acute (2h) norepinephrine-induced thermogenic gene expression was overall markedly higher (and adipose genes somewhat lower) in cells cultured in newborn serum. Thus, newborn serum promotes thermogenic competence, and the use of fetal serum in brown adipocyte cultures (as is often routine) counteracts adequate differentiation. Agents that counteract this inhibition may therefore confoundingly be ascribed genuine thermogenic competence-inducing properties.

  • 2. Isidor, Marie S.
    et al.
    Winther, Sally
    Basse, Astrid L.
    Petersen, M. Christine H.
    Cannon, Barbara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Nedergaard, Jan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Hansen, Jacob B.
    An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes2016Inngår i: Adipocyte, ISSN 2162-3945, E-ISSN 2162-397X, Vol. 5, nr 2, s. 175-185Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high-and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to beta-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo browning. In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.

  • 3.
    Kamble, Prasad G.
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk diabetologi och metabolism.
    Pereira, Maria J
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk diabetologi och metabolism.
    Gustafsson, Stefan
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Molekylär epidemiologi.
    Lundkvist, Per
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk diabetologi och metabolism.
    Castillejo-Lopez, Casimiro
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Molekylär epidemiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Fall, Tove
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Molekylär epidemiologi.
    Ingelsson, Erik
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Molekylär epidemiologi.
    Eriksson, Jan W.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk diabetologi och metabolism.
    Role of peroxisome proliferator-activated receptor gamma Pro12Ala polymorphism in human adipose tissue: assessment of adipogenesis and adipocyte glucose and lipid turnover.2018Inngår i: Adipocyte, ISSN 2162-3945, E-ISSN 2162-397X, Vol. 7, nr 4, s. 285-296Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Protective mechanisms of peroxisome proliferator-activated receptor gamma (PPARγ) Pro12Ala polymorphism in type 2 diabetes (T2D) are unclear. We obtained adipose tissue (AT) before and 3 h after oral glucose (OGTT) in carriers and non-carriers of the Ala allele (12 Pro/Pro, 15 Pro/Ala, and 13 Ala/Ala). Adipogenesis, adipocyte glucose uptake and lipolysis as well as PPARγ target genes expression were investigated and compared between the genotype groups. On fasting and post-OGTT, neither basal nor insulin-stimulated adipocyte glucose uptake differed between genotypes. Compared to fasting, a decreased hormone-sensitive lipase gene expression in Pro/Pro (p<0.05) also accompanied with a higher antilipolytic effect of insulin post-OGTT (p<0.01). The adipocyte size was similar across groups. Preadipocyte differentiation rates between Pro/Pro and Ala/Ala were unchanged. In conclusion, no major differences in AT differentiation, glucose uptake, lipolysis or expression of PPARγ target genes were observed between different PPARγ Pro12Ala genotypes. Albeit small, our study may suggest that other pathways in AT or effects exerted in other tissues might contribute to the Pro12Ala-mediated protection against T2D.

  • 4. Taube, Magdalena
    et al.
    Andersson-Assarsson, Johanna C
    Lindberg, Kristin
    Pereira, Maria J
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk diabetologi och metabolism.
    Gäbel, Markus
    Svensson, Maria K
    Eriksson, Jan W
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk diabetologi och metabolism.
    Svensson, Per-Arne
    Evaluation of reference genes for gene expression studies in human brown adipose tissue2015Inngår i: Adipocyte, ISSN 2162-3945, E-ISSN 2162-397X, Vol. 4, nr 4, s. 280-285Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Human brown adipose tissue (BAT) has during the last 5 year been subjected to an increasing research interest, due to its putative function as a target for future obesity treatments. The most commonly used method for molecular studies of human BAT is the quantitative polymerase chain reaction (qPCR). This method requires normalization to a reference gene (genes with uniform expression under different experimental conditions, e.g. similar expression levels between human BAT and WAT), but so far no evaluation of reference genes for human BAT has been performed. Two different microarray datasets with samples containing human BAT were used to search for genes with low variability in expression levels. Seven genes (FAM96B, GNB1, GNB2, HUWE1, PSMB2, RING1 and TPT1) identified by microarray analysis, and 8 commonly used reference genes (18S, B2M, GAPDH, LRP10, PPIA, RPLP0, UBC, and YWHAZ) were selected and further analyzed by quantitative PCR in both BAT containing perirenal adipose tissue and subcutaneous adipose tissue. Results were analyzed using 2 different algorithms (Normfinder and geNorm). Most of the commonly used reference genes displayed acceptably low variability (geNorm M-values <0.5) in the samples analyzed, but the novel reference genes identified by microarray displayed an even lower variability (M-values <0.25). Our data suggests that PSMB2, GNB2 and GNB1 are suitable novel reference genes for qPCR analysis of human BAT and we recommend that they are included in future gene expression studies of human BAT.

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