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Relevance to the Human Situation (L-Cell In Vivo)

Despite the rodent origin of GLUTag cells, they are the most extensively used in vitro model for the study of GLP-1 release. They have been demonstrated to respond to the same stimuli that trigger GLP-1 secretion in humans and they have been used to test potential therapeutic agents (i.e. GPR119 agonists). The difficulties in obtaining primary L-cell cultures from humans, as well as the homogeneity of the GLUTag cells (i.e. single-cell clone) as compared to other L-cell models, have made GLUTag cells an invaluable tool for the characterization of the intra-cellular pathways that govern GLP-1 secretion.

General Protocol

Maintenance of the GLUTag Cell Line

GLUTag cells are routinely grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), whereas supplementation with antibiotics, normally penicillin and streptomycin, is optional but not required. Cells must be trypsinized and passaged every 3–4 days when they reach 60–80 % confluence. The presence of floating cells may be indicative of contamination or overgrowth. There is no standard glucose concentration to grow the cells but a final concentration within the range 5–25 mmol/L is commonly used. It has recently been reported that cells grown with 25 mmol/L of glucose show increased reactive oxygen species production, upregulated proglucagon, prohormone convertase 1/3 and glucokinase content, and elevated basal secretion of GLP-1 as compared to cells maintained under 5 mmol/L conditions, suggesting enhanced metabolic activity. However, no difference in cell viability was found between the two conditions, and the cells grown with 25 mmol/L glucose were more resistant to a further metabolic insult (Puddu et al. 2014). Moreover, cells grown at either a low (5 mmol/L) or high (25 mmol/L) concentration of glucose have been demonstrated to respond to stimuli in a similar way, and no differences in GLP-1 secretion or production were found in cells exposed to low or high glucose for 2 h (Reimann and Gribble 2002), which is the standard incubation period to examine the GLUTag cells secretory response to stimuli.

Performance of Secretion and Expression Assays

To study the effects of different treatments on GLP-1 release (secretion assays), cells can be cultured in 6or 24-well plates coated with either poly-D-lysine or Matrigel® to avoid detachment of cells during the washes. A 2-day period between plating and the secretion assay is recommended in order to allow the cells to recover from trypsin and to reach the proper size and confluence. When the cells are too confluent (over 80 %), they tend to grow on top of each other, forming clumps. This should be avoided since it is associated with higher basal levels of secretion (personal observations). During the test period of 2 h, GLUTag cells should be maintained in either FBS-free DMEM or DMEM with a very low concentration of FBS (0.5 %), containing the treatments tested at different doses; buffers such as Krebs– Ringer are also acceptable. To study the effects of treatments on gene expression or protein content, larger surface areas, such as 10 cm culture plates, may be required and longer incubation periods are recommended.

A recent study has shown that GLUTag cells possess an endogenous metabolic clock and that the activity of the cells can be synchronized after overnight incubation with a low concentration of FBS (0.5 %) followed by a 1-h shock with 10 % FBS and 10 μmol/L forskolin (Gil-Lozano et al. 2014). Synchronized GLUTag cells show a rhythm in clock genes expression as well as in their GLP-1 response to secretagogues, with higher responses at 4 h after synchronization and lack of effect at 16 h. This protocol may be adopted to obtain maximum secretory responses to a particular treatment.

 
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