Human pancreatic adipocyte spheroids as model to study the role of pancreatic fat in type 2 diabetes

Background and aim: Accumulation of adipocytes in the pancreatic parenchyma is associated with impaired insulin secretion in individuals at high risk for type 2 Diabetes (T2D). However, how the metabolic environment impacts on pancreatic adipocyte biology, and consequently influences islet function, remains largely unexplored. As the in vitro 2D cell culture models fail to recapitulate adipogenesis and function of in situ adipocytes, translational approaches are consequently hampered. To address this limitation, we aim to generate an in vitro 3D model of pancreatic adipocytes (adipocyte spheroids), that mimics the specific features of in situ pancreatic adipocytes.

Methods: Mature adipocytes and stromal vascular fraction (SVF) cells were obtained following collagenase digestion of peripancreatic fat biopsies from non-diabetic patients (n=8). SVF-derived preadipocytes were differentiated in vitro for 19 days using either conventional 2D monolayer or 3D spheroid culture. The expression of adipogenic and inflammatory markers from in vitro differentiated adipocytes and native pancreatic adipocytes was analysed by RT-qPCR. Lipolytic activity, assessed by free fatty acid (FFA) release and western blot of P-PKA/PKA and PHSL/HSL, was analysed in both cultured models. Lipid droplets and infiltration of CD68+ macrophages were visualized by immunofluorescence.

Results: Pancreatic adipocyte spheroids have a higher mRNA level of adipocyte differentiation markers, such as PPARG (12-fold) and ADIPOQ (50-fold), compared to 2D cultured adipocytes. These levels are similar to those detected in mature adipocytes isolated from the pancreatic biopsies. Pancreatic adipocyte spheroids are rich in multilocular lipid droplets and express perilipin1. The differentiation capacity of pancreatic spheroids, indicated by the ADIPOQ mRNA levels, negatively correlates with donor body weight (BMI) (p=0,01).

Stimulation of lipolysis with isoproterenol (beta-adrenergic agonist, 1 µM) and forskolin (adenylate cyclase activator, 5 µM) increased FFA release by 56- and 112-fold respectively in adipocyte spheroids, while in 2D culture, FFA release was increased by 2.7- and 3.8-fold, respectively. The higher lipolytic rates of pancreatic adipocytes spheroids coincides with higher pHSL/HSL protein levels.

Inflammatory markers (IL6 and MCP1) are downregulated during adipocyte differentiation in spheroids, whereas their levels remain constant in adipocyte 2D culture. IL1B mRNA is detected in pancreatic adipocyte spheroids, which confirms the CD68+ cell population observed in spheroids.

Conclusion: Pancreatic adipocyte spheroids exhibit long-term viability and closely resemble the in situ mature pancreatic adipocytes, representing an optimal tool to study the role of pancreatic fat for islet dys-/function and T2D.

 

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Publication History

Article published online:
18 April 2024