Effects of hyperinsulinemia on lipolytic function of three-dimensional adipocyte/endothelial co-cultures

Abstract

The increased incidence of both type 2 diabetes mellitus and obesity has prompted the need to develop physiologically relevant adipose tissue models for controlled study of both normal and diseased adipose functions. Insulin resistance, characteristic of both type 2 diabetes mellitus and obesity, is often preceded by hyperinsulinemia. We propose here a three-dimensional (3D) co-culture adipose tissue model to study the effects of high insulin exposure, which enabled the study of physiological cell responses to hyperinsulinemic conditions. Two-dimensional adipocyte studies were initially conducted to establish a baseline control in which insulin levels were established. Adipocytes and endothelial cells were subsequently co-cultured on 3D porous silk fibroin scaffolds in normal or high insulin concentrations, and their physiological responses were assessed with respect to lipogenesis and lipolysis. High insulin levels stimulated both an increase in triglyceride accumulation and a decrease in lipolysis levels compared to that of normal insulin conditions. In contrast, adipocyte monocultures did not exhibit any differences between insulin levels. The ability of this 3D system to elicit physiological responses to hyperinsulinemia in co-culture serves as a significant step forward in adipose tissue engineering. The development of physiologically relevant 3D in vitro adipose tissue models presents promise for the study of disease mechanisms as well as in assessing therapeutic treatments.

FIG. 1.

Schematic of adipose tissue outcome measures. Adipogenic differentiation is initiated upon inhibition of both Wnt and Hedgehog (Hh) signaling. PPARγ and C/EBPα are key transcriptional regulators that promote expression of adipogenic genes such as GAPDH, Glut4, FABP4, and ACS. Triglyceride (TG) synthesis subsequently occurs, causing mature adipokines to be secreted (leptin and adiponectin) and extracellular matrix (ECM) production. TG breakdown, or lipolysis, is mediated by 2 key lipases (adipose triglyceride lipase [ATGL] and hormone sensitive lipase [HSL]), producing glycerol and fatty acids.

FIG. 2.

Two-dimensional adipogenic-differentiated hASCs. hASCs were cultured for 12 days in the medium containing 10 μM insulin (A–D) or 1 μM insulin (E–H) (n = 3), and secreted glycerol (mg/mL) was measured at each time point (I). Scale bar = 50 μm. Original magnification, 10 × . Significant difference between insulin levels are labeled as ⁺p < 0.05; significant differences from day 0 levels are labeled as *p < 0.05.

FIG. 3.

DNA content of 3D constructs in the medium containing 10 or 1 μM insulin. (A) Adipocyte/endothelial co-cultures, (B) adipocyte monocultures, and (C) endothelial monocultures were evaluated (n = 3). Significant differences from day 0 levels are labeled as *p < 0.05.

FIG. 4.

Triglyceride accumulation and secreted glycerol measurements of 3D constructs cultured in medium containing 10 μM or 1 μM insulin. (A, D) adipocyte/endothelial co-cultures, (B, E) adipocyte monocultures, and (C, F) endothelial monocultures were evaluated (n = 3). Significant differences from day 0 levels labeled as *p < 0.05.

FIG. 5.

Real time reverse transcription (RT)-PCR measurements for glut4 and CD31 transcript expression of 3D constructs cultured in medium containing 10 μM or 1 μM insulin. (A, D) adipocyte/endothelial co-cultures, (B, E) adipocyte monocultures, and (C, F) endothelial monocultures were evaluated (n = 3). Significant differences from day 0 levels are labeled as *p < 0.05.