How Does Inflammation in the Adipose Tissue Contribute to Diabetes? Kiana Khosravian, Jan Heinrichsdorff

Olefsky Lab University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0378

Introduction: Type 2 Diabetes is a metabolic disorder due to the development of insulin resistance in cells throughout the body. We are studying how inflammation in adipose tissue contributes to insulin resistance and the onset of diabetes. Excess lipids in the cells of adipose tissue (lipid overload) cause the cells to leak the excess fatty acids leading to higher DAG (diacylglycerols) levels, a type of secondary messenger to accumulate in muscle cells. The accumulation of DAG interferes with the insulin receptor signaling and inhibits insulin from regulating glucose levels1. We use conditional mouse models to knock out certain inflammatory signaling molecules in the adipose tissue and investigate how this affects insulin resistance in adipose tissue and other insulin sensitive organs. To study the adipose tissue of the mice we have to collect cell cultures which we turned into histology sections. We looked at histology sections of fat tissue and assessed the number of fat cells and the number of apoptotic cells in wild type mice versus knockout mice to see if there is a difference in the number of cells.

Conditional Mouse Models to Delete a Gene in Adipose Tissue

The Cre-Lox System was used to knock out a certain

gene in the mouse’s genome.

DNA from mouse tails was isolated and used for PCR

(Polymerase Chain Reaction) genotyping.

Gel Electrophoresis was used to separate the DNA and to determines which mice were wildtype and which have the

gene deleted.

Mouse # 767 is Cre+ which means that Cre is expressed and will delete the targeted gene

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Ladder

L 756 763 767 768 770 778 785 798 813 814 815 816 861 862

864 865 866 867 868 869 870 - +

778 785 798 813 814 815 816 861 862 864 784 865 866 867 W+

868 865 870 756 763 767 768 770 W+

Cell Culture of Peritoneal Macrophages

1 •  We injected 3% thioglycolate into the peritoneal cavity of the mice which irritates the

mice cells and attracts macrophages.

2•  Two days later, the macrophages that accumulated because of the injection were

flushed out of the peritoneal cavity.

3 •  The collected cells were then washed and the red blood cells were lysed.

4 •  The cells were counted and plated at a density of 2.5 X 105 cells per well in a 24 well

plate. Medium DEMEM 10% FCS P/S

Histology

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There was not much difference between the cell counts of the wildtype and knockout mice. This means The obese knockout mice have the same amount of cells compared to obese wild type mice.

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We used TUNNEL assay to label the dead, apoptotic cells. There was more apoptosis in the fat tissue of obese knockout mice compared to the obese wild type mice.

References:

1.  Taubes, G (2009) Prosperity’s Plague. SCIENCE 325, 256-260.

2.  http://en.wikipedia.org/wiki/Cre-Lox_recombination

Mice with a band of DNA amplified in our PCR similar to 867 show that their gene has been floxed which means that a particular piece of DNA has been inserted with a section of loxP on either side of it. The larger the DNA the slower it migrates on the gel so this indicated that there is a larger piece of DNA which contains the loxP site.

Here are some pictures of the cells I cultured. Unfortunately after three days the wells without antibiotics were infected with bacteria.

Conclusion: We can conclude that the knockout gene affects cell viability.

Schematic Diagram of Cre-Lox System2.

Cre is a DNA recombinase protein which catalyzes the recombination of DNA between specific target sites. LoxP is a sequence of DNA that flanks two sides of a DNA section which you would recombine with Cre. LoxP are the specific target sites. The Cre is able to only affect the adipose tissue in a mouse because we place it after a certain promoter (AP2) in the DNA that is specific to the cells in adipose tissue.

N=6

N=6

Apoptotic Cell

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