ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs

Key to realizing the diagnostic and therapeutic potential of human brown/brite adipocytes is the identification of a renewable, easily accessible and safe tissue source of progenitor cells, and an efficacious in vitro differentiation protocol. We show that macromolecular crowding (MMC) facilitates brown adipocyte differentiation in adult human bone marrow mesenchymal stem cells (bmMSCs), as evidenced by substantially upregulating uncoupling protein 1 (UCP1) and uncoupled respiration. Moreover, MMC also induced ‘browning’ in bmMSC-derived white adipocytes. Mechanistically, MMC creates a 3D extracellular matrix architecture enshrouding maturing adipocytes in a collagen IV cocoon that is engaged by paxillin-positive focal adhesions also at the apical side of cells, without contact to the stiff support structure. This leads to an enhanced matrix-cell signaling, reflected by increased phosphorylation of ATF2, a key transcription factor in UCP1 regulation. Thus, tuning the dimensionality of the microenvironment in vitro can unlock a strong brown potential dormant in bone marrow

Ca2+-associated triphasic pH changes in mitochondria during brown adipocyte activation

Objective: Brown adipocytes (BAs) are endowed with a high metabolic capacity for energy expenditure due to their high mitochondria content. While mitochondrial pH is dynamically regulated in response to stimulation and, in return, affects various metabolic processes, how mitochondrial pH is regulated during adrenergic stimulation-induced thermogenesis is unknown. We aimed to reveal the spatial and temporal dynamics of mitochondrial pH in stimulated BAs and the mechanisms behind the dynamic pH changes. Methods: A mitochondrial targeted pH-sensitive protein, mito-pHluorin, was constructed and transfected to BAs. Transfected BAs were stimulated by an adrenergic agonist, isoproterenol. The pH changes in mitochondria were characterized by dual-color imaging with indicators that monitor mitochondrial membrane potential and heat production. The mechanisms of pH changes were studied by examining the involvement of electron transport chain (ETC) activity and Ca2+ profiles in mitochondria and the intracellular Ca2+ store, the endoplasmic reticulum (ER). Results: A triphasic mitochondrial pH change in BAs upon adrenergic stimulation was revealed. In comparison to a thermosensitive dye, we reveal that phases 1 and 2 of the pH increase precede thermogenesis, while phase 3, characterized by a pH decrease, occurs during thermogenesis. The mechanism of pH increase is partially related to ETC. In addition, the pH increase occurs concurrently with an increase in mitochondrial Ca2+. This Ca2+ increase is contributed to by an influx from the ER, and it is further involved in mitochondrial pH regulation. Conclusions: We demonstrate that an increase in mitochondrial pH is implicated as an early event in adrenergically stimulated BAs. We further suggest that this pH increase may play a role in the potentiation of thermogenesis

A new zearalenone biodegradation strategy using non-pathogenic Rhodococcus pyridinivorans K408 strain.

Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17β-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17β-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to the feeding did not display any estrogenic effect neither on uterine weight nor on the expression of estrogen-regulated genes. Consequently, the identification of Rhodococcus pyridinivorans K408 strain in ZEA biodegradation proved to be a very efficient biological tool that is able to eliminate the complete estrogenic effects of ZEA. It is also remarkable that this biotransformation pathway of ZEA did not result in any residual estrogenic effects

Blood plasma ZEA concentrations in <i>Experiment 1</i>.

<p>Blood plasma ZEA concentrations in female immature rats after 3-day exposure to different dosage of ZEA (0.1–10 mg/kg bw) was measured by competitive fluorescent-microsphere based immunoassay (CFIA). The results show that there was a dose dependent increase in plasma ZEA concentration following oral administration. Blood plasma levels showed a strong positive correlation (R  = 0.919186) with uterus weight in a dose dependent manner. (**) Significantly different from control (p<0.001) (n = 6–7). (***) Significantly different from control (p<0.001) (n = 6–7).</p

Remained estrogen effect in the zearalenone metabolism of <i>Rhodococcus pyridinivorans</i> analysed by the bioluminescens yeast reporter system (BLYES).

<p>The results show that the incubation with <i>Rhodococcus pyridinivorans</i> K408 decreased the estrogenic activity of zearalenone. After 5 days, remained estrogen effect was 81.75% compared to control. The results of the BLYES based estrogenic biotest showed good correlation with ZEA concentration measurement via HPLC-FLD.</p

Gene expression of apelin, aquaporin 5, complement component 2 and calbindin 3 in <i>Experiment 1</i>.

<p>Gene expression of apelin (APLN) and aquaporin 5 (AQP5) decreased and complement component 2 (C2) and calbindin 3 (CALB3) increased significantly both by E2 0.04 mg/kg bw and ZEA 10 mg/kg bw 3-day-long treatment. (*) Significantly different from control (p<0.05). (**) Significantly different from control (p<0.01) (n = 6–7). (***) Significantly different from control (p<0.001) (n = 6–7).</p

Gene expression of apelin, aquaporin 5, complement component 2, and calbindin 3 in <i>Experiment 2</i>.

<p>Gene expression of apelin (APLN) and aquaporin 5 (AQP5) decreased and complement component 2 (C2) and calbindin 3 (CALB3) increased significantly after 3-day ZEA treatment. There is no significant difference between control and ZEA + <i>R. pyridinivorans</i> K408 treatment in folding changes. (*) Significantly different from control (p<0.05). (**) Significantly different from control (p<0.01). (#) Significantly different from ZEA (p<005). (###) Significantly different from ZEA (p<0,001) (n = 6–7).</p