Enterocytic differentiation and glucose utilization in the human colon tumor cell line Caco‐2: Modulation by forskolin

Abstract

The human colon cancer line Caco-2 exhibits after confluency a concomitant increase of glycogen accumulation and an enterocytic differentiation. The purpose of this work was to investigate whether forskolin (FK), an activator of adenylate cyclase, would induce a permanent glycogenolysis and, if so, whether it would result in modifications of the differentiation pattern of the cells. FK activates adenylate cyclase in Caco-2 cells with an ED50 of 7 X 10(-6)M. Three different treatment protocols with FK (10(-5)M) were applied: 1) the cells were treated during all the time in culture (20 days); 2) the treatment was started after confluency; 3) the treatment was interrupted after confluency. The presence of FK results in a permanent stimulation of cAMP accumulation (10 to 20 fold the basal values) and in a permanently reduced glycogen content (30 or 50% of the control values). The rates of glucose consumption are increased three and five fold in protocols 1 and 3 respectively. These metabolic changes are associated with morphological changes (tightening of the intercellular spaces and shortening of the brush border microvilli) and with a dual inhibition of the activities of brush border hydrolases: a) an inhibition of the post-confluent increase of activity of sucrase, aminopeptidase N and alkaline phosphatase in the brush border enriched fraction; b) an inhibition of the post-confluent increase of activity of sucrase in the cell homogenate. A comparison of the results obtained in each protocol shows that the morphological modifications and the decrease of the enzyme activities in the brush border fraction are regularly associated with an increased cAMP accumulation, whereas the inhibition of the differentiation of sucrase is a direct consequence of the increase in glucose consumption and decrease in glycogen stores.

Regulation ofα1-proteinase inhibitor release by proinflammatory cytokines in human intestinal epithelial cells

“…Caco-2 cells originate from human colonic adenocarcinoma, and therefore their use in such types of study may raise queries about their suitability to resemble normal enterocytes. However, Caco-2 cells are the only cell line described to date able to differentiate spontaneously into an enterocyte-like phenotype in culture [23]. Such differentiated Caco-2 cells express many characteristics of normal enterocytes, such as polarization of the cell layer with the formation of domes and the presence of an apical brush border membrane enriched in hydrolases typical for normal enterocytes, such as sucrase-isomaltase, lactase, amino-peptidase N and dipeptidylpeptidase IV [24].…”

Transcytosis of Gastrointestinal Epithelial Cells by Escherichia coli K1

“…Both gentamicin protection assays and transcytosis assays using Caco-2 cells were performed at different times (4, 7, 11, 14, and 17 d) to assay the effects of polarization and differentiation upon invasion. Brush border enzymes were harvested from Caco-2 cells at each of these times according to the method of Rousset et al (11), with the modification that cells were disrupted by passage through a French pressure cell rather than by tissue grinding and sonication. Briefly, the cells from two 25-cm 2 flasks were harvested with trypsin/EDTA and washed three times in PBS at 4°C.…”

“…Most important in evaluating invasion of Caco-2 cells appears to be the length of time they are grown in culture, which regulates their degree of differentiation and polarization (11,18). Proliferation usually does not begin until after a lag period of 48 h. Caco-2 cells typically require 5 to 6 d in culture to become confluent.…”

Activation of Transepithelial lon Transport by Secretin in Human Intestinal Caco-2 Cells.

“…In spite of the origin of Caco-2 cells, they differentiate after confluence under standard culture conditions into a polarized monolayer with the properties of small intestinal epithelial cells [15,16]. Namely, confluent Caco-2 cells have domes and tight junctions, and possess brush-border microvilli in the apical membranes [16,17]. The activities of several kinds of transporters, such as the sodium-dependent phosphate transporter [18], the sodium-dependent glucose transporter [19], the proton-dependent dipeptide transporter [20], and the proton-dependent amino acid transporter [21], all of which are present in the apical membranes in the small intestine, have been found in confluent Caco-2 cells.…”