Cell lines were procured and grown under the recommended conditions, including 37 °C and 5% CO2. The human intestinal L-cell line NCI-H716 (CCL-251, ATCC, Manassas, VA, USA) was originally isolated from the ascites fluid of a patient with colorectal adenocarcinoma [2]. LS 174 T cells (CL-188, ATCC) are colonic epithelial cells isolated from a patient with colorectal cancer. STC-1 cells (CRL-3254, ATCC) are an intestinal neuroendocrine cell line isolated from a mouse intestinal tumor [16].

The human and mouse L-cells were incubated overnight in low glucose medium without serum, then exposed to. high glucose (25 mM) DMEM with 10% FBS with or without λ-carrageenan (1 μg/ml; Sigma Aldrich, St. Louis MO, USA) for 10 minutes, 1 h, or 24 h. Spent media and cell homogenates were collected. GLP-1 protein levels in the spent media were measured by ELISA (Immuno-Biological Laboratories IBL-America, Minneapolis, MN, USA; #27784). mRNA levels of proglucagon, the precursor of GLP-1, were determined by QRT-PCR, using established procedures with the following primers:

proglucagon (human): left: 5′-CGTTCCCTTCAAGACACAGAGG-3′;

right: 5′-ACGCCTGGAGTCCAGATACTTG -3′;

proglucagon (mouse): left: 5′-CCTTCAAGACACAGAGGAGAACC-3′; and

right: 5′-CTGTAGTCGCTGGTGAATGTGC-3′.

The effect of carrageenan exposure on GLUT2 expression by the human intestinal epithelial cells was tested in an in vitro co-culture model. NCI-H716 human intestinal L-cells were grown on transwell inserts on top of a monolayer of human intestinal epithelial cells (IEC) (LS174T). The L-cells were treated with λ-carrageenan (1 μg/ml) for 10 min, 1 h, and 24 h, and the impact on mRNA expression of GLUT2 by the IEC was measured using the primers:

GLUT2 (human; NM_000340): left-5′-ATGTCAGTGGGACTTGTGCTGC-3′ and

right - 5′-AACTCAGCCACCATGAACCAGG-3′.

Statistics were performed with Microsoft Excel and PRIZM 9.5 software (GraphPad, Boston, MA, USA) using unpaired t-tests, two-tailed with unequal variance for all of the comparisons. Mean values and standard deviations of at least triplicate experiments are presented in the figures. P ≤ 0.05 is considered statistically significant.