Modeling Adipokine and Insulin-Mediated Crosstalk Between Adipocytes and Beta Cells Using Flow-Enabled Microfluidics

Abstract

Obesity-associated beta cell dysfunction is a crucial factor in the pathogenesis of Type 2 Diabetes (T2D), driven by a dysfunctional crosstalk between adipose tissues and pancreatic beta cells. Traditional culture systems cannot capture this crosstalk in a dynamic and controlled manner. A flow-enabled microfluidic is developed with an embedded micro-Tesla (µTesla) pump to assess the adipocyte-beta cell crosstalk. This recirculating system allows them to study the transport of soluble-factor-based between cultured 3T3 L1 adipocytes and INS1 beta cells. It is found that flow-enabled incubation with elevated glucose and insulin increased the levels of adipocyte-derived secretions of IL-6, TNF-α, and adiponectin in the media. In turn, adipocyte-derived IL-6 enhanced beta-cell insulin secretions in the same media, establishing a feed-forward loop. This mechanism can contribute to the hyperinsulinemia and pro-inflammatory conditions characteristic of obesity-related T2D. The findings highlight the advantages of flow-enabled microfluidics in modeling adipocyte-beta cell crosstalk in obesity, providing novel insights into obesity-associated beta cell dysfunction.

Keywords: 3T3‐L1; INS‐1 beta cells; adipocytes; co‐culture; diabetes; microfluidics.

Figure 1

Fabrication of a Flow‐Enabled Microfluidic Device Utilizing µTesla Pumps A) The device was fabricated using Fused Deposition Modeling (FDM) to print the master template, over which polydimethylsiloxane (PDMS) was poured and cured. Once cured, the PDMS layer was peeled off and bonded to a glass substrate. B) The device design was created using Autodesk Inventor software and manufactured using the FDM method. µTesla pumps were integrated into the device, and the PDMS layer and the pumps were bonded to a glass substrate. C) µTesla pump comprises a rotor and housing of 10 mm size and is printed using a 0.15 mm nozzle following the FDM approach. Two magnets were polarized into the rotor before being assembled into the housing. D) These images illustrate injecting cell culture medium into the embedded µTesla pumps, connected via silicone tubes to the cell chambers, enabling media flow over the cultured cells. Cells were introduced into the chamber through the designated pore (^*) and incubated overnight to facilitate attachment. Subsequently, the µTesla pump was connected to the cell chamber through a secondary pore (^**), establishing a continuous media flow over the cells. The symmetrical design of the device allows us to run two experiments simultaneously with closed‐loop flow, providing a platform to study the bidirectional crosstalk between adipocytes and beta cells.

Figure 2

Timeline for the adipocyte differentiation and experimental setup 3T3‐L1 preadipocytes were differentiated into adipocytes in a microfluidic chamber. On the 6th day of the 3T3‐L1 differentiation, beta INS‐1 cells were plated in another chamber for 3 days until they became confluent. The flow of media was started on the 3rd day of 3T3‐L1 adipocyte differentiation for adipocyte monoculture with flow (MC+F) and on the 9th day for INS‐1 cell MC+F and the co‐culture of 3T3‐L1 adipocytes and INS‐1 beta cell with flow (CC+F). For the monoculture condition (MC), no flow was applied (static condition).

Figure 3

Molecular diffusion modeling in Transwells versus a closed‐loop microfluidic device. In the microfluidic setup, adipocytes are placed in the left chamber and beta cells in the right, with left‐to‐right recirculating flow at 950 µL min⁻¹ shown at 0 s A), 250 s B), and 500 s C). The simulation accounts for the delay time associated with fluid recirculation through the pump system. For comparison, the Transwell configuration with adipocytes in the insert and beta cells at the bottom is shown at corresponding time points: 0 s D), 250 s E), and 500 s F).

Figure 4

Co‐culture with flow improves the metabolic activities of adipocytes and beta cells A) Cellular ATP (red) and ROS (green) productions in adipocytes (top) and INS‐1 beta cells (bottom). Images were taken for 24 h post the 3 days of INS‐1 culture and post‐differentiation (9 days) for adipocytes, and then quantified using image J. B) Quantified ATP production in adipocytes. n = 3 independent experiments. C) Quantified ROS production in adipocytes. n = 3 independent experiments. D) Quantified ATP production in INS‐1 beta cells. n = 3 independent experiments. E) Quantified ROS production in INS‐1 beta cells. n = 3 independent experiments. Two‐way ANOVA with Tukey multiple comparisons test was used for statistical analyses. ^* p< 0.05, ^** p< 0.01, ^*** p< 0.001, ^**** p< 0.0001. n stands for different experiments conducted in separate days.

Figure 5

Adipocyte‐beta cell co‐culture under flow responds to hyperglycemia A) Adipocyte IL‐6 response to glucose incubation in MC+F and CC+F conditions. Adipocytes were incubated with different concentrations of glucose (5.5, 12.5, 25 mm) for 24 h under MC+F and co‐culture with beta cells with flow (CC+F) and IL‐6 levels in the media were measured. B) Adipocyte TNF‐α levels in adipocyte MC+F and adipocyte‐beta cell CC+F in response to increasing concentrations of glucose. C) Adiponectin levels in adipocyte MC+F and adipocyte‐beta cell CC+F in response to increasing glucose concentration. D) Insulin levels in INS‐1 cell MC+F and adipocyte‐beta cell CC+F in response to glucose. Two‐way ANOVA with Tukey multiple comparison tests was used. Data are presented as means ± SEM, n = 3 independent experiments. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001. n stands for different experiments conducted in separate days.

Figure 6

Adipocyte‐beta cell co‐culture with flow maintains a robust response to glucose for up to 3 days A) IL‐6 levels in adipocyte‐beta cell CC+F in response to different glucose concentrations. Measured on days 1, 2, and 3. B) TNF‐α levels in adipocyte‐beta cell CC+F in response to different glucose concentrations. C) Adiponectin levels in adipocyte‐beta cell CC+F in response to different glucose concentrations. D) Insulin levels in adipocyte‐beta cell CC+F cultured in the media with different glucose concentrations for up to day 3. Assays were performed with media collected every day, 24 h after daily medium change. After media collection, samples were frozen at −20 °C until used for ELISA assays. Two‐way ANOVA with Tukey multiple comparisons test was used for statistical analysis. Data is presented as means ± SEM, n = 3 independent experiments. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001, ns: no significance. n stands for different experiments conducted in separate days.