Investigation of the role of L-type Ca2+ channels in the mobilisation of lipophilic compounds from white adipocytes: a possible aetiology of metabolic disorders
Publication Date
July 18, 2023
Creator
Abstract
Voltage gated calcium channels (CaVs) are transmembrane proteins which allow the entry of calcium ions into cells in response to membrane depolarisation. They are readily expressed in excitable cells where their molecular and functional expression are well characterised, however they are less characterised in unexcitable cells such as white fat adipocytes (WFAs). The physiological role of WFAs has been recognised to transcend its central role as the main energy storage of the body. The need for intracellular calcium in physiological processes mediated by the adipose tissue has been recognised. Furthermore, the aetiology of metabolic disorders such as type 2 diabetes and obesity involves derangements in physiological processes of WFAs such as lipolysis, as well as exposure to highly lipophilic obesogenic and diabetogenic compounds, which can be released into the general circulation during lipolysis. This study thus aimed to characterise CaV channels in WFAs and determine the contribution of intracellular calcium in adipocyte lipolysis and release of fat stored compounds.
Molecular expression and proteomic analysis were respectively determined by western blotting and tandem mass spectrometry. Epifluorescent microscopy using the calcium specific dye, Fluo 4 for intracellular calcium imaging was used to study functional expression. Fluorescent measurement of nile red release and free fatty acid analysis were employed to monitor lipophilic compound release from the fat cell as well as lipolysis under calcitropic conditions.
A CaV1.2 α1 subunit of ~290 kDa, a splice isoform of the canonical CaV1.2 α1 subunit, was differentially expressed in the plasma membrane of rat visceral and sub-cutaneous WFAs and 3T3-L1 adipocytes. This large protein was absent in undifferentiated 3T3-L1 cells, instead a protein of ~210 kDa, similar to that found in the control rat brain tissue, was detected. Also, the Cav3.1 α1 subunit of ~265 kDa was detected only in undifferentiated 3T3-L1 adipocytes. Proteomic analysis also revealed the presence of α2/δ1 subunits of CaV1.2 in the primary adipocytes. Calcium imaging confirmed that the detected proteins were indeed functional and were of the L-type, based on the pharmacology of their response to L-type antagonists. Intracellular calcium influx in CD1 male mice visceral WFAs was insensitive to regulation by conditions previously shown to modulate the adipocyte membrane potential. However, hormonal stimulation by recombinant human growth hormone and the full-length parathyroid hormone (1-84) led to increases in calcium influx in these cells. Nile red fluorescent measurements and free fatty acid analysis showed that conditions previously shown to unequivocally increase intracellular calcium had no effect on release of fat stored lipophiles and on lipolysis. This study has confirmed the molecular identity of WFA CaV channels as CaV1.2 and increased our understanding of how these channels are modulated in the fat cell. Knowledge of the role of these channels in the release of fat stored lipophiles is however still lacking.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
72247
UoN Repository URI
Except where otherwise noted, this item's license is described as
File(s)![Thumbnail Image]()
Name
PhD Thesis_final ver2. Nneoma Akaniro-Ejim.pdf
Type
Full-text
Description
Examined
Size
4.44 MB
Format
Adobe PDF
Checksum (MD5)
6a36a024ef120e0db0a7c0038f79c934