Figure 3. Type 2 diabetes in KKA^y mice suppresses hepatic levels of flotillin-1 in association with hypertriglyceridemia.

Protein and RNA were extracted from liver tissues of 12-week-old phenotypically lean KK mice (controls) and their hyperphagic, centrally obese, T2DM KKA^y littermates. Panel A: Immunoblots in triplicate for hepatic flotillin-1 protein and, as a loading control, GAPDH. Each lane represents a sample from a different animal. Panel B: Hepatic flotillin-1 mRNA levels normalized to β-actin (Actb) mRNA levels, as assessed by quantitative reverse transcription polymerase chain reactions (qRT-PCR). Shown are means ± SEMs, n=6-8 per group, ^**p< 0.01 (two-tailed Student’s t-test). Panels C,D: The same liver samples used in Panels A,B were analyzed for their contents of the syndecan-1 HSPG by immunoblot (Panel C) and the syndecan-1 core protein mRNA by qRT-PCR (Panel D, p﹥0.5 by the two-tailed Student’s t-test). Panel E: Syndecan-1 was immunoprecipitated from mouse liver extracts (IP: SDC1, with nonimmune IgG as the negative control), and the IP pellets were subjected to electrophoretic separation. The upper two immunoblots were performed to detect flotillin-1 (IB: Flotillin-1) in the immunoprecipitate, and then the same membranes were stripped and reprobed to detect syndecan-1 (IB: SDC1). The lower three immunoblots show the contents of syndecan-1, flotillin-1, and GAPDH in liver tissue homogenates from the same experiment, i.e., the starting material for each of the co-immunoprecipitations, arranged in the same order as in the upper immunoblots. Panel F: Plot of plasma triglyceride concentrations versus hepatic Flot1 mRNA levels for all control KK mice and all T2DM KKA^y mice. All data in this figure are representatives of three independent experiments.