Abstract
At present, in western countries, 20% of the population are considered obese with 50% classified as overweight. Obesity can be defined as hyperplasia and hypertrophy of adipose tissue. The extensive health complications coupled with the economic burden associated with obesity has led to increased research into the factors regulating fat development, formation (adipogenesis), maintenance and conversion.Carbonic anhydrase 3 (CA3) is the least understood and investigated of all the Carbonic anhydrase isoforms. It has a number of unique properties that allow it to be distinguished from other family members. This protein has been demonstrated to constitute extremely high levels of the soluble fraction of adipocytes (25%) and has been shown to be down regulated in obese rodent models. It has been implicated as having a role in lipid metabolism and has recently been shown to have novel antioxidant properties.
We re-confirm in this study that Ca3 expression is indeed differentiation dependant via utilisation of an in vitro model of murine adipogenesis, the 3T3-L1 cell line. We demonstrate for the first time that ectopic expression of Ca3 in 3T3-L1 cells results in a completely abolished ability to undergo differentiation evidenced by lack of accumulated lipids, coupled with marked reductions in adipogenic genes; Ppary2, C/ebpa, Fabp4 and Leptin. Next we investigated whether the repression of differentiation was dependant on the antioxidant activity of Ca3. This avenue of investigation revealed the same inability to accumulate lipids as the Ca3 over-expressing cell populations, as well as similar reductions in the adipogenic genes investigated. These results suggest that Ca3 is a negative regulator of adipogenesis and that the repression occurs in an antioxidant independent manner. These data suggests a potential molecular mechanism to prevent the over accumulation of lipids characteristic of obesity. However, abolishing the ability of white adipose tissue to accumulate lipids could result in lipotoxicity and as such this mechanism would have to be utilised in conjunction with either an increase in beige or brown adipose tissue or exercise/diet regime in order to be a viable therapeutic method.
The transcription factor Evi-1 has been recently demonstrated to be required for adipogenesis; it also displays a reciprocal relationship with Ca3 in Rati fibroblasts. Ectopic expression of Evi-1 in 3T3-L1 cells resulted in accelerated differentiation evidenced by enhanced expression of Ppary2, C/ebpa, Fabp4, Leptin and Ca3. In an attempt to elucidate the domain/s of Evi-1 responsible for this enhanced differentiation ectopic expression of Evi1 naturally occurring splice variants (Rp+, A105 and A324) as well as a mutant version of Evi-1 (which had both CtBP binding sites mutated) were examined for their ability to enhance differentiation in 3T3-L1 cells. We demonstrate the A324 had a slightly diminished ability to enhance differentiation in comparison to the Rp-t- and A105 splice variants, suggesting that the zinc fingers truncated in the 1st zinc finger domain in the A324 splice variant are required for the full function of Evi-1 mediated acceleration of differentiation. The CtBP Evi-1 mutant showed no enhanced differentiation and therefore CtBP binding is essential for enhanced adipogenesis. These data suggest that Evi-1 may be involved in the commitment or development of white adipose tissue.
| Date of Award | 2015 |
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| Original language | English |
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| Supervisor | Christopher Bartholomew (Supervisor) & Annette Graham (Supervisor) |