COBRE Pilot Project for Brian Finlin: Center for Research in Obesity and Cardiovascular Disease

During the development of obesity, adipose dysfunction occurs and contributes to insulin resistance. The development of adipose dysfunction with obesity is complex, and recent studies suggest that increased TGF-â signaling could play an important role in this process. Thrombospondin-1 (TSP-1) is a TGF-â activating protein, and we have found that its expression correlates with body mass index and inversely correlates with insulin sensitivity in the subcutaneous adipose of humans (13). Furthermore, TSP-1 expression is inhibited by treatment with the insulin sensitizer pioglitazone (13). Others have found that TSP-1 is up regulated in mouse white adipose tissue (WAT) by obesity (14) and that TGF-â1 is increased in WAT by obesity (16). These results suggest that TGF-â signaling should be increased in WAT with obesity and recent studies document increased SMAD phosphorylation in the WAT of obese mice (16). Furthermore, inhibition of TGF-â signaling by the 1D11 neutralizing antibody or by SMAD3 knockout protected mice from diet induced obesity by “browning” the white adipose tissue of the mice. The mechanism is thought to involve increasing the expression of the transcriptional coactivator PGC-1á since SMAD3 was shown to be a negative regulator of PGC-1á expression (16). Interestingly, our preliminary data confirms that inhibiting TGF-â signaling in differentiated human adipocytes by the pharmacological inhibitor SB505124 induces PGC-1á mRNA expression. We hypothesize that increased TGF-â signaling in white adipose with obesity causes increased fibrosis, reduced PGC-1á and reduced UCP-1 expression hence less “browning”, and impaired WAT function. The SMAD3 knockout mice and TGF-â neutralization are whole body approaches to inhibiting TGF-â that would affect other organ systems. Therefore, to gain understanding into the specific function of TGF-â in adipose, we intend to study adipose-specific TGF-â1 knockout mice. The purpose of this COBRE pilot grant is to develop the mouse model that will be studied as part (Aim 4) of the Phase II COBRE project (PI: Cassis, Lisa A./project 5: Finlin, Brian S.). This aim will complement our studies of the TGF-â activating protein TSP-1 in the first three aims of the project. We intend to develop this mouse model by crossing floxed TGF-â1 mice (1) and adiponectin Cre mice (4), which are both available from the Jackson Labs. These mice will be crossed with each other until male TGF-â1fl/fl adipo Cre1/0 mice can be bred with femalefl/fl mice. The offspring of these mice will be control TGF- â1fl/fl mice and TGF-â1fl/fl adipo Cre1/0 mice in which TGF-â1 will be specifically reduced in adipocytes. Ultimately, we will analyze these mice and the adipo-Cre mice, which will be analyzed to control for any background phenotypes. The goal of this pilot project is to generate the TGF-â1fl/fl mice and TGF-â1fl/fl adipo Cre1/0 mice and then to verify the adipocyte-specific knockdown of TGF-â1. We will study these mice as a part of the following specific aim for the Phase II COBRE project: Specific Aim 4. Determine whether adipose knockout of TGF-â1 increases adipose tissue browning and/or reduces fibrosis in response to high fat feeding. We will specifically look at the effects of TGF-â signaling on adipose without the complications of whole-body knockout of TSP-1 (Aim 1) or SMAD3 (16). We hypothesize that adipose specific TGF-â1 knockout mice will have improved metabolic parameters in response to high fat feeding. We will measure glucose and insulin tolerance in TGF-â knockout mice and littermate controls that have been challenged with a high fat diet. If the mice display improved metabolic function, they will be further characterized by hyperinsulinemiceuglycemic clamp. We will extensively characterize the white adipose ECM and capillary density of these mice and will determine whether brown fat gene expression (e.g. PRDM-16 and PGC-1á) increases in WAT.