Defining Nkx6.1 binding partners in young and aged pancreatic islet
Sean Kang firstname.lastname@example.org
Dr. Jeffery Tessem Nutrition, Dietetics, and Food Science
To determine binding partners of the transcription factor Nkx6.1 in the β-cell, and define the
differences in their expression and binding in adolescent and aged rat islets.
Diabetes is an increasing epidemic that deserves more attention. Those affected by diabetes
are restricted to either strict diets or remedial insulin supplementation. Both Type 1 and Type 2
diabetes are directly related to a decrease in functional β-cell mass. Exciting research has revealed
Nkx6.1 to be an influential transcription factor which induces β-cell proliferation, protects against
apoptosis and enhances insulin secretion when overexpressed 1 . Our recent findings indicate
significant β-cell proliferation is limited to β-cells from adolescent rats, with a severe decrease in
proliferation as the animal ages. 2 Nkx6.1 expression is present in every stage of development, but its
downstream functions are impaired in aged animals. Our preliminary data demonstrates that while
Nkx6.1 can be overexpressed in aged β-cells, it fails to induce expression of critical downstream
genes. This crucial difference leads us to hypothesize that there are binding partners of Nkx6.1 whose
expression in the β-cell are altered as animal ages. Our goal is to identify and quantify Nkx6.1binding
partners that are present in the 832/13 β-cell cell line. We will then validate their expression in
young and aged islets. These findings could potentially lead to therapeutic procedures to induce
proliferative, enhance insulin secretion, and increase survival of β-cells.
Increased functional β-cell mass would alleviate many diabetic symptoms. Preliminary
research had confirmed Nkx6.1 as a β-cell transcription factor that can increase proliferation, cell
survival and insulin secretion in adolescent β-cells 1 . While the effects of Nkx6.1 overexpression in
adolescent β-cells are clear, Nkx6.1 fails to induce the same effects in β-cells from adult animals (Fig 1A). When overexpressing Nkx6.1 in adolescent or adult islets, the level of Nkx6.1 is comparable, however
induction of proliferation does not occur. This data suggests that there are critical differences between adolescent and adult β-cells that affect the ability of Nkx6.1 to enhance functional β-cell mass. We hypothesized that Nkx6.1 may have binding partners present in adolescent β-cells that are absent in adult β-cells, whose absence impedes the function of Nkx6.1 to enhance functional β-cell mass.
We sought to identify cooperative binding partners that are necessary for Nkx6.1 to bind to its activation site and induce transcription of critical genes. We conducted several different co-immunoprecipitation procedures. Agarose beads equipped with antibody to immunoprecipitate Nkx6.1 and any associated binding partners. Several repetitions showed promise (Fig 2). The bands and accompanying schmears indicates that Nkx6.1 and its accompanying proteins were successfully precipitated out of the mixture of cell particles. This procedure, however, yielded very small quantities of precipitant. After reexamination of the procedure we decided to implement A/G magnetic beads as our main
coimmunoprecipitation procedure. Magnetic beads are useful as a sustainable source for multiple trials. With the amount of protein needed and the small amounts obtained from each trial, this change in procedure was a logical move.
Multiple trials were conducted with the new magnetic
beads yielding minimal amounts of precipitant. This may
have occurred through a variety of reasons. The beads may
not have been made correctly. The solvents used may not
have been made correctly. Contamination in the lab may
have affected initial protein concentration.
Despite the difficulties, this project had shown some
promise and should be revisited.
1. Tessem JS, Schisler JC, Fueger PT, Babu DA, Hohmeier HE, Lu D, Becker TC, Naziruddin B, Levy M,
Mirmira RG, et al. Stimulation of human and rat islet beta-cell proliferation with retention of function by the homeodomain transcription factor Nkx6.1. Molecular and Cellular Biology. 2008 Mar;28
(10):3465-76. < website >
2. Hobson A, Draney C, Stratford A, Becker TC, Lu D, Arlotto M, Tessem JS. Aurora Kinase A is critical for the Nkx6.1 mediated β-cell proliferation pathway. 2015 Jan; 7(1):e1027854.