Publikationsdatenbank

Influence of magnesium on endocrine regulation and energy metabolism in bovine adipocytes (2022)

Art

Hochschulschrift

Autor

Becker, Sandra Karolina (WE 2)

Quelle

Berlin: Mensch und Buch Verlag, 2022 — VI, 127 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/37268

Kontakt

Institut für Veterinär-Physiologie

Oertzenweg 19 b
14163 Berlin
+49 30 838 62600
physiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

High performance cattle go through a tremendous increase in energy requirements from about 3 weeks before to 3 weeks after calving. Feed intake at the onset of lactation is insufficient, leading to a negative energy balance (NEB) which is compensated for by the depletion of fat reserves in the body. Furthermore, a physiological insulin deficiency or insulin resistance or both occur in tissues such as the liver, muscle, or adipose tissue in order to supply insulinindependent tissues such as the mammary gland with sufficient glucose for lactose production. This state promotes metabolic dysfunctions with diseases like ketosis, milk fever, or inflammatory reactions like mastitis. As the main cells engaged in fat metabolism of cattle, adipocytes not only play an essential role in energy balance, but also have a whole-body influence on the health of cattle. Magnesium as the second most abundant intracellular cation plays a prominent role in the insulin signaling pathway. Magnesium homeostasis is tightly regulated with a reciprocal relationship between insulin and magnesium. The main aim of the present study was to investigate and to understand the interaction between insulin and magnesium on bovine adipocytes, with the hypothesis that magnesium could have positive effects on animal health by improving the insulin sensitivity of adipocytes. To facilitate this research, a cell culture protocol for preadipocyte differentiation into bovine adipocytes was established. Mesenchymal stem cells derived from subcutaneous adipose tissue in the neck region of calves were shown to yield large numbers of preadipocytes when cultured as explants. Cells showed the expression of stem cell markers, namely CD73, CD90, and CD105.in immunohistochemical and qRT-PCR studies. A higher expression of stem cell markers in preadipocytes was dependent on fetal bovine serum (FBS). In contrast, bovine serum lipids (BSL) increased adipocyte differentiation, with downregulation of stem cell markers and upregulation of adipocyte markers, e.g., FABP4. Thus, FBS is essential for the cultivation of mesenchymal stem cells by promoting cell replication and the expression of stem cell markers, whereas BSL counteracts this and promotes differentiation. Furthermore, it could be shown that important media additives for induction, e.g., dexamethasone, insulin, rosiglitazone, IBMX, and biotin, could be reduced to 30% of the original value derived from literature without reducing induction performance. The addition of ascorbic acid as a relevant supplement for collagen synthesis and as an antioxidant showed that, together with BSL, differentiation was most successful under these conditions. Ascorbic acid had outstanding effects on the expression of the adipocyte marker FABP4 and on LPS and the protein expression of FAS. During differentiation adipocytes increasingly formed intracellular fat vacuoles, as evidenced by Nile red staining. Because of lower density of the fat that they contained, loss of cells from the bottom of the cell culture flasks occurred. Coating of culture plates with poly-L-lysine or gelatin caused significantly better adherent cell numbers than flasks with collagen coating or without coating. To investigate the influence and interplay of magnesium and insulin on bovine adipocytes, cells were exposed to various magnesium and insulin concentrations in a two-factorial design. Lipid accumulation increased with increasing insulin concentrations, veryfying that insulin acts as an adipogenic and protective factor for adipocytes. Severe magnesium deficiency (0.1 mM) led to lower lipogenesis of cells, independently of insulin concentration. A similar pattern was seen for glucose uptake where insulin also had a promoting effect, but magnesium starvation at 0.1 mM reduced glucose uptake. Nevertheless, this effect was not as pronounced as in lipid accumulation; hence, we can assume that insulin-independent glucose uptake mechanisms are also present, for example, via the glucose transporter GLUT1, which is also influenced by magnesium. To investigate the interplay of glucose and lipid metabolism further, a glycerol-3-phosphate dehydrogenase (GPDH) assay was performed. Contrary to expectations, the highest activity of GPDH was detectable in cells grown at a low physiological insulin concentration of 25 pM and in those cultured at the highest magnesium concentration of 3 mM. Considering that cattle have low insulin and glucose concentrations in the blood after calving, the present results suggest that a high supply of magnesium might promote an increased incorporation of glycerol into fat cells in vivo, thereby, decreasing the danger of fatty liver syndrome. In the last part of this work, the so-called magnesium-responsive genes were investigated by RT-qPCRs. The transporters SLC41A2, TRPM6, TRPM7, and CNNM2 showed no significant changes in their expression patterns in bovine adipocytes and thus were not affected by the concentrations of insulin or magnesium. While the expression of SLC41A1 was found to be decreased at low magnesium concentrations, presumably in order to keep the intracellular magnesium content constant. Furthermore, an inhibitory effect of insulin was shown. SLC41A3 showed a reduced expression in cells in culture medium with 250 pM insulin, similar to SLC41A1, and in low magnesium concentrations. This reduction in expression might keep mitochondrial magnesium concentration constant at low magnesium availability. The magnesium transporter MAGT1, which transports magnesium into the cell, was expressed to a greater extent at low magnesium concentrations, showing an opposite and traceable expression pattern to SLC41A1. NIPA1 also showed upregulation at low magnesium concentrations, although this effect was neutralized at high insulin concentrations. These results show that magnesium has an essential influence on insulin signalling pathways and thus fat metabolism in bovine adipocytes providing the foundation for future investigations for potential therapeutic approaches to prevent excessive lipomobilization together with possible associated diseases in cattle.