Presentations

Abstract

Introduction:

Approximately 1 in 10 Americans are diabetic, with over 90% of diabetic individuals having type II diabetes (Centers for Disease Control and Prevention 2023). Due to preexisting knowledge on the role of CHIP (carboxyl terminus of Hsc/Hsp70-interacting protein) in multiple metabolic cascades (Zhang et al. 2020), and its proven ability to lower blood glucose levels when overexpressed (Ali et al. 2021), we chose to examine CHIP's involvement in glucose regulation.

Methods:

Mice: Wild-type and CHIP-deficient mice were generated and maintained on a mixed genetic background of C57BL/6 and 129SvEv (C57BL/6 ×129), or by repeatedly backcrossing the 129SvEv mouse strain with 129Sv/C57B6 mice carrying a single mutated CHIP allele. Mice in the present study were 3-3.5 months old.

--Glucose and insulin tolerance tests: n = 15/20 or 5/6 in wild-type/CHIP-/- mice for glucose or insulin tolerance bolus, respectively.

--Gastrocnemius fluorescence microscopy: Fasted mice received an IP injection of 20% D-glucose (2 g glucose/kg body mass). Mice were sacrificed 40 minutes after injection, and muscles were dissected and flash-frozen in liquid nitrogen. Protein concentration was quantified using Protein Assay Dye and normalized to wild-type gastrocnemius values per strain of mice.

-Creating skeletal muscle cell lines for stains: Lentiviral transduction of control or CHIP shRNA in mouse skeletal muscle C2C12 cells (creating shCONT and shCHIP cell lines) to compare cells containing CHIP (shCONT) with CHIP-deficient cells.

-Immunoblots and fluorescence: Cell lysates were collected and subsequently separated into NP-40-soluble and -insoluble fractions by centrifugation. Protein lysates were separated using Bis-Tris 4-12% SDS-PAGE, transferred to polyvinylidene fluoride membranes, and immunoblotted using standard chemiluminescence technique. Primary antibodies included: α-tubulin, β-tubulin, Glut4, CHIP, stathmin, phosphorylated stathmin at serine 16, and myc. All cells and muscle fiber preparations were mounted with glass cover slips using Vectashield mounting media with DAPI. Micrographs were obtained on a Nikon Eclipse E800 upright fluorescent microscope utilizing QCapture software.

Results:

Figure 1. Blood glucose levels in 129SvEv mice measured at the indicated time points after (A) glucose or (B) insulin bolus injection represented by the mean ± SEM.

Figure 2. Representative immunoblot analysis of phosphorylated stathmin at serine 16 (S16), total stathmin, and β-actin.

Figure 3. Fluorescent micrographs of C2C12 control cells (shCONT) or cells with reduced CHIP expression (shCHIP). After serum starvation (0 min) or stimulation (30 min) with complete media plus insulin, cells were fixed and visualized for nuclei (DAPI), phalloidin staining (F-actin), and endogenous Glut4 expression.

Figure 4. Representative fluorescent micrographs of individual gastrocnemius muscle fibers isolated from wild-type and CHIP-deficient fasted mice or fasted mice given a glucose bolus (40 min) were stained for α-tubulin (top). False color overlays of α-tubulin (orange) and the nuclear counterstain DAPI (blue) are provided (bottom).

Figure 5. Role of CHIP in insulin-mediated glucose uptake cascade.

Conclusion:

We found that CHIP -/- mice demonstrate a Type II diabetes-like phenotype, including poor glucose tolerance, decreased sensitivity to insulin, and decreased insulin-stimulated glucose uptake in isolated skeletal muscle, characteristic of insulin resistance. In CHIP-deficient C2C12 cells, glucose/insulin stimulation fails to induce translocation of Glut4 to the plasma membrane. This impairment in Glut4 translocation in CHIP-deficient cells is accompanied by decreased tubulin polymerization associated with decreased phosphorylation of stathmin, a microtubule-associated protein required for polymerization-dependent protein trafficking within the cell.

These data describe a novel role for CHIP in sustaining the glucose/insulin-induced phosphorylation signaling cascade in a manner necessary for signaling to the interior of the cell such that the microtubule polymerization required for glucose transporter translocation occurs, thus promoting whole-body glucose homeostasis and sensitivity to insulin.

This research will hopefully drive future studies to investigate how CHIP's conserved role in glucose signaling presents in humans and the potential for CHIP to serve as a measure of relative risk for type II diabetes in a clinical setting.