By: Rhiannon Bates
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Editor's Note: Today we have an article from a special guest talking about her research. If you'd like to write for my blog, contact me. Or just make a git pull request.
Hello everyone! I'm Rhiannon, and I will be taking over Jake's blog for today. I'd like to introduce myself first and give you a glimpse into the work I do. I'm currently a PhD student studying Biomedical Sciences with a research focus in neuroregulation of adipose tissue and adeno-associated (AAV) viral design. I also have an MPH in veterinary public health.
My current research project began when Lei Cao (my PI) told me that environmentally enriched mice, mice that are able to socialize and exercise in a large space, are leaner and have remodeled adipose tissue. This is in comparison to mice in standard housing with access to exercise (wheels, etc). Enriched environments have numerous beneficial effects, including improved metabolism and healthy aging. My lab focuses on the metabolic, immune, and hormonal effects via the HSA (hypothalamic sympathetic adrenal) axis. This knowledge is important in understanding mechanisms behind physiologic changes that dramatically improve metabolism and longevity and resistance to pathologies, including cancer. Those of you who know Jake well will know his love for fat. Adipose tissue is innervated and has many roles in metabolic function, communication to the brain, and peripheral tissues. Plus it tastes good.
BDNF, brain derived neurotropic factor, is upregulated in environmentally enriched mice and has been shown to be the molecule behind many of these physiological changes in enriched mice. Mice are used extensively in biomedical research due to their physiological similarity to humans and ease of experimentation. My lab has studied this extensively in the past. qPCR (quantitative PCR) results show that a small molecule, adipsin (otherwise known as complement factor D) is also upregulated in enriched mice, but not mice solely receiving exercise. Adipsin upregulated expression begins early on (around 6 days in mice) and is sustained for months. It is found in adipose tissue regardless of sex, age, and strain of mice. Through my own work the last month or so I have found that adipsin is highly concentrated in the serum of enriched animals, but less concentrated in BTBR mice, which is a model for autism. This may have something to do with the finding that BTBR mice have reduced BDNF signaling. I am interested in understanding the role of BDNF on adipsin expression.
Not much is known about the role adipsin plays when secreted from adipose tissue, but it is a crucial component of the complement system (involved in immune defense against bacterial infections). Recent studies have shown adipsin modulates beta-cell function, which are the cells that produce insulin, indicating it has a role in metabolism. Adipsin stimulates glucose transport for triglyceride accumulation in fat and inhibits lipolysis. In diabetic or obese mice, there is a low level of adipsin expression. Patients with type 2 diabetes with beta-cell failure have low adipsin expression. It is thought that adipsin sustains insulin production and secretion in diabetes because in diabetic mice with injected adipsin, they have lowered fasting glucose levels, increased insulin, and increased glucose clearance. Before you run out and purchase adipsin for increasing your metabolic strength, let me do my job to understand the role it plays within the entire body. For safety, you know.
In obese humans, adipsin is highly expressed at first due to the increased fat mass, then declines with adipose dysfunction. I would like to know how the timing of adipsin expression occurs and the mechanism of its regulation. Another interesting finding from the literature is that in humans, adipsin levels decline during pregnancy, but the mechanism behind this is unclear.
To begin my experiments into adipsin's role, I've been working on designing a virus to deliver adipsin cDNA specifically to adipose tissue. This involves mutating capsid proteins for entering a host cell, and using bacteria to amplify my plasmid DNA. This involves a lot more work than what I just described, most of which is pipetting tiny amounts (microliters) of clear liquid into tiny amounts of another clear liquid. Somehow after pipetting things, results occur and I can verify that the viral construct contains my DNA. My DNA of adipsin was designed by introducing start and stop codons and sequences that enzymes will recognize to cut and insert into a vector plasmid for transfer. I also used some of my lab's money to sequence it. Hopefully I can pay it back to them when I'm rich and famous.
Plasmids are used in gene therapy to carry promoters, antibiotic resistance elements, and other useful sequences to isolate bacteria colonies that contain the plasmid. These plasmids are extracted and packaged into AAV, which can then be transfected into HEK193 cells. By doing so, I can make sure these viruses are able to get inside cells and insert their DNA. GFP, green fluorescent protein, is also contained in the plasmid and used to visualize if the cells took up the plasmid. AAV vectors transiently express the DNA, but lentiviruses can permanently express whatever gene you want to have expressed in a cell. However, using those viruses has random integration into the genome, and could lead to cellular transformation with unintended consequences. Gene therapy for humans relies on AAV mostly, as this is the safest method so far.
Currently I'm working really hard at molecular biology before setting up my in vivo experiments, which I'll be designing a micro-RNA targeting adipsin for destruction in order to knock it out of mice. This past weekend was my birthday and this coming weekend I'm on vacation, though... But in the next several months I will have more information about adipsin. Stay tuned!