Interplay of Glucagon-Like Peptide-1 and Transforming Growth Factor-β Signaling in Insulin-Positive Differentiation of AR42J Cells

Abstract

The differentiation of pancreatic exocrine AR42J cells into insulin-expressing endocrine cells has served as an important model for both endogenous in vivo ␤-cell differentiation as well as potential application to ␤-cell engineering of progenitor cells. Exogenous activin, possibly working through intracellular smad 2 and/or smad 3, as well as exogenous exendin-4 (a long-acting glucagon-like peptide-1 agonist) have both been shown to induce insulin-positive/endocrine differentiation in AR42J cells. In this study, we present evidence of significant interplay and interdependence of these two pathways as well as potential synergy between the pathways. In particular, insulin-positive differentiation seems to entail an exendin-4 -induced drop in smad 2 and elevation in smad 3 in RNA levels. The latter appears to be dependent on endogenous transforming growth factor (TGF)-␤ isoform release by the AR42J cells and may serve as a mechanism to promote ␤-cell maturation. The drop in smad 2 may mediate early endocrine commitment. The coapplication of exogenous exendin-4 and, specifically, low-dose exogenous TGF-␤1 led to a dramatic 20-fold increase in insulin mRNA levels, supporting a novel synergistic and codependent relationship between exendin-4 signaling and TGF-␤ isoform signaling. Diabetes 53: 2824 -2835, 2004 M echanisms underlying the differentiation of pancreatic precursor cells into insulin-positive cells are of paramount importance toward our goal of engineering cells to become glucose-responsive ␤-cells for the curative therapy of diabetes. Over the last several years, a well-validated model of such differentiation has been developed using rat AR42J pancreatic epithelial cells (1-3). Early reports used either activin, or activin in combination with hepatocyte growth factor or ␤-cellulin, to generate either endocrine (activin alone) or insulin-positive (activin in combination) cell lines. Activins are members of the transforming growth factor (TGF)-␤ superfamily that form hetero-or homodimers and bind specific type I and type II TGF-␤ superfamily receptors (4). In most cases, activin ligand signaling induces phosphorylation and activation of smad 2 and/or smad 3 transcription factors to initiate downstream signaling (5). Based on the above initial observations of the effects of activin, subsequent AR42J studies showed a potential role for intracellular smad signaling in mediating these activin-induced pathways. Early findings were that smad 2 was necessary for insulin-positive differentiation, but forced overexpression of smad 2 in AR42J cells was not sufficient to induce insulin-positive differentiation (6). More recently, given the incretin (insulin release) and insulinotrophic (insulin cell growth) effects of glucagon-like peptide-1 (GLP-1) (7-15), either GLP-1 protein or a long-acting form of GLP-1 (exendin-4) was used to induce an endocrine phenotype in AR42J cells. Here, multiple islet cell types were seen, with expression of cell-specific markers and the acquisition of an endocrine morphology in culture...

Mechanisms of action of glucagon-like peptide 1 in the pancreas

“…All of the NFAT sites studied in the rat I insulin promoter are responsive to the combination of glucose and GLP-1, although NFAT2 was relatively insensitive to GLP-1 alone (Lawrence et al, 2002) Schema outlining the signaling mechanisms reported to be involved in GLP-1R-induced differentiation/neogenesis of pancreatic precursor cells, proliferation and in the prevention of apoptosis. Dashed lines indicate mechanisms that are either not fully delineated or in the case of the cAMP activation of the MEK pathway are complex and are shown completely in Fig 2. The mechanism shown for involvement of BMP and TGFβ signaling pathways in differentiation is after Gittes and co-workers Yew et al, 2004). PKB is shown as inhibiting FoxO1 (by phosphorylation).…”

“…The Gittes laboratory (Yew et al, 2004) have expanded on these observations and performed a dose-response curve for Ex-4 (1 pM -100 nM) conversion by quantifying insulin II, pdx-1, and IAPP mRNA levels (Yew and colleagues supplemented their media with 20% FBS). They found that 5 pM of Ex-4 was the most effective dose over a 3 day treatment period.…”

Activation of GLP-1 and gastrin signalling induces in vivo reprogramming of pancreatic exocrine cells into beta cells in mice

“…1a, b), newly generated beta cells of Insulin-Timer embryos [24] and in acinar cells of the human adult pancreas [23], we hypothesised that GLP-1 may function in non-beta cells, including acinar and duct cells, and we activated the GLP-1 signalling pathway by the administration of exendin-4, a long-acting GLP1R agonist, and by overexpressing GLP1R in exocrine cells. Whereas in vitro studies have shown that GLP-1 and exendin-4 induce beta cell differentiation in the pancreatic tumour cell line AR42J [7,9], our present study revealed that exendin-4 itself fails to initiate beta cell neogenesis in SOX9-lineage exocrine cells in vivo, even after the overexpression of GLP1R (Fig. 3f-i).…”

“…Among the many humoral factors that regulate pancreas development, glucagon-like peptide-1 (GLP-1) and its analogue, exendin-4, have been reported to affect beta cell differentiation under in vitro and ex vivo conditions [7][8][9]. In addition, previous in vivo studies showed that the co-activation of GLP-1 and gastrin signalling increased the number of beta cells in mouse models of diabetes [10][11][12].…”

Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination

“…Although our results indicate that bon and snail can be regulated by Smad3, our data do not rule out co-regulation by Smad2, and regulation of these genes most likely occurs via cooperation between these two Smads. Nevertheless, there are structural/functional differences between both Smads, several of which appear to distinguish their actions in vitro (Yew et al, 2004;Uemura et al, 2005;Ju et al, 2006) and in vivo (Dunn et al, 2005;Wang et al, 2006). In addition, the ratio of Smad2 versus Smad3 influences their respective roles as effectors (Dunn et al, 2004;Kim et al, 2005).…”