Cheng Mao Lin

Epidermal growth factor activation of intestinal glutamine transport is mediated by mitogen-activated protein kinases

Glutamine is an essential nutrient for gut functions, but the regulation of its uptake by intestinal mucosal cells is poorly understood. Given the pivotal role of epidermal growth factor (EGF) in regulating gut metabolism, growth, and differentiation, this in vitro study was designed to investigate the intracellular signaling pathways involved in the regulation of EGF-mediated intestinal glutamine transport in intestinal epithelia. Continuous incubation with EGF (>30 hours, 100 ng/ml) stimulated glutamine transport activity across intestinal epithelial Caco-2 cell apical membrane. Exposure to EGF for 48 hours resulted in an increase in transport activity (50%) and glutamine transport system B gene ATB0 mRNA levels (ninefold). EGF stimulated glutamine transport activity by increasing the glutamine transporter maximal velocity (Vmax) without altering the transporter apparent affinity (Km). Furthermore, EGF stimulated both intracellular protein kinase C and mitogen-activated protein kinase MEK1/2 activities. The EGF-stimulated glutamine transport activity was attenuated individually by the specific protein kinase C inhibitor chelerythrine chloride and the mitogen-activated protein kinase MEK1 inhibitor PD 98059. These data suggest that EGF activates glutamine transport activity across intestinal epithelial membrane via a signaling mechanism that involves activation of protein kinase C and the mitogen-activated protein kinase MEK1/2 cascade. EGF activates glutamine transport via alterations in transporter mRNA levels and the number of functional copies of transporter units.

Activation of Intestinal Arginine Transport by Protein Kinase C Is Mediated by Mitogen-Activated Protein Kinases

L-Arginine uptake by the small intestine can play a pivotal role in regulating nitric oxide synthesis and immune functions in catabolic states. We previously showed that protein kinase C (PKC) activation stimulates intestinal brush-border membrane arginine transport. However, the signaling pathways implicated in this activation have not been studied. The purpose of this study was to investigate the intracellular signal transduction pathways involved in the protein kinase C stimulation of arginine transport across the apical membrane of intestinal epithelial Caco-2 cells. [3H]-L-arginine transport activity, Northern blot analysis of mRNA levels of the intestinal arginine transporter CAT1,and Western blot analysis of the mitogen-activated protein (MAP) kinases phospho-p44/42 activity and phospho-MEK1/2 were measured in cultured Caco-2 cells treated with phorbol ester (phorbol 12-myristate 13-acetate, TPA; 0 to 0.5_mol/L), and the MEK1 inhibitor PD 98059 (0 to 50_ mol/L). Phorbol ester stimulated intestinal arginine transport activity. Arginine transporter gene CAT1 mRNA, phospho-p44/42, and phospho-MEK1/2 levels were stimulated in phorbol ester-treated cells, compared with the control group. Phorbol ester stimulation of arginine transport activity and transporter CAT1 mRNA levels was blocked by PD 98059. These data suggest that phorbol ester stimulates arginine transport in Caco-2 cells via signaling pathways that lead to increased transcription and/or stabilization of CAT1 mRNA. Protein kinase C and MAP kinases MEK1/2 and p44/42 are key intracellular regulators involved in this signal transduction cascade.

Metabolic acidosis stimulates intestinal glutamine absorption.

Glutamine is an essential nutrient for cell integrity during acidotic states such as shock, but the effect of extracellular pH on intestinal mucosal cell glutamine uptake is poorly understood. The purpose of this in vitro study was to investigate the intracellular signaling pathways involved in controlling intestinal glutamine transport during acidosis. Lowering the pH in the cell culture medium resulted in an increase in glutamine transport activity in a time- and pH-dependent fashion. Chronic acidosis (pH 6.6 for 48 hours) resulted in a twofold increase in glutamine transport activity (1.63 ± 0.25 nmole/mg protein/ minute in acidosis vs. 0.78 ± 0.11 nmole/mg protein/minute in control) and a threefold increase in glutamine transport gene ATB messenger RNA levels. This acidosis-induced increase in glutamine transport activity was due to a stimulation of transporter maximal transport capacity (Vmax 13.6 ± 0.73 nmole/mg protein/minute in acidosis vs. 6.3 ± 0.46 nmole/mg protein/minute in control) rather than a change in transporter affinity (Km = 0.23 ± 0.02 mmol/L glutamine in acidosis vs. 0.19 ± 0.02 mmol/ L glutamine in control). This acidosis-stimulated glutamine transport activity was blocked by actinomycin-D or cycloheximide. Cellular mitogen-activated protein kinase (MAPK) MEK1/2 and p42/44 levels were elevated in acidotic cells, and the acidosis-induced glutamine transport activity was blocked by the MAPK MEK 1 inhibitor PD 98059. Acidosis stimulates glutamine transport in Caco-2 cells via signaling pathways that lead to transcription of the glutamine transporter gene and translation of functional transporters. Mitogen-activated protein kinases are key intracellular regulators involved in this signal transduction cascade. An increased availability of glutamine to cells subjected to redox stress may help in maintaining cellular integrity.

Regulation of amino acid arginine transport by lipopolysaccharide and nitric oxide in intestinal epithelial IEC-6 cells

As a precursor for nitric oxide (NO) synthesis and an immune-enhancing nutrient, amino acid L-arginine plays a critical role in maintaining intestine mucosal integrity and immune functions in sepsis. However, the relationship between intestinal arginine transport and NO synthesis in sepsis remains unclear. In the present study, we investigated the effects of lipopolysaccharide (LPS) and NO on the arginine transport in cultured rat intestinal epithelial IEC-6 cell. Near-confluent IEC-6 cells were incubated with LPS (0-50 μg/ml) in serum-free Dulbecco’s modified Eagles’s medium, in the presence and absence of the NO donor sodium nitroprusside (SNP, 0–500 μmol/L) and the inducible nitric oxide synthase (iNOS) inhibitor N-ω-nitro-L-arginine (NNA, 0–1000 μmol/L) for various periods of time (0-48 hours). Arginine transport activity, arginine transporter CAT1 mRNA and protein levels were measured with transport assay, Northern blot analysis, and Western blot analysis, respectfully. LPS increased arginine transport activity in a time- and dose-dependent fashion. Prolonged incubation of LPS (24 hours, 25 μg/ml) resulted in a 3-fold increase of arginine transport activity (control: 28 ±5; LPS: 92 ±20 pmol/mg/ min, P < 0.05), with the System y+ as the predominant arginine transport system, and a 2-fold increase of System y+CAT1 mRNA and transporter protein levels (P < 0.05). LPS increased the arginine transport System y+ maximal velocity (Vmax, control: 1484 ±180; LPS: 2800 ±230 pmol/mg/min, P<0.05) without affecting the transport affinity (Km, control: 76 ±8; LPS: 84 ±14 μmol/L, p = NS). The LPSinduced arginine transport activity was blocked by sodium nitroprusside (SNP) (control: 25 ±6; LPS: 97 ±26*; SNP: 22 ±0.4+; LPS+SNP: 33 ±10.3+ pmole/mg/min, *P < 0.01 and +p = NS, compared with control). In contrary, the LPS-induced arginine transport activity was further augmented by NNA (control: 18 ±3.2; LPS: 59 ±2.7*; NNA: 26.3 ±5.8; LPS + NNA: 127 ±18+ pmol/mg/min; *P < 0.01 compared with control and +P < 0.01 compared with control or LPS). LPS-stimulates arginine transport activity in IEC-6 cells via a mechanism that involves increase of transport System y+ mRNA levels and transporter protein levels. The LPS-stimulated arginine transport activity is regulated by the availability of nitric oxide.

Epidermal growth factor regulation of system L alanine transport in undifferentiated and differentiated intestinal Caco-2 cells

Epidermal growth factor (EGF) in intestinal lumen regulates many gut epithelial cell functions. Influenced by growth factors at various differentiation stages, enterocytes execute the major task of absorbing nutrient amino acids. The purpose of this study was to investigate the effects of EGF on Na+-independent L-alanine transport in intestinal epithelial cells. Na+-independent [3H]-L-alanine transport was measured in the differentiating Caco-2 cells. In both the undifferentiated and differentiated states, L-alanine uptake occurred via a single saturable Na+-independent system L plus simple passive diffusion. System L activity decreased as the cells progressed from the undifferentiated to the differentiated state. Prolonged incubation with EGF (>30 hours) resulted in a 70% increase in system L activity in both undifferentiated and differentiated cells. EGF stimulated the system L Vmax without affecting Km. System L activity stimulation was inhibited by chelerythrine chloride, cycloheximide, or actinomycin D. These data suggest that intestinal epithelial cell differentiation is associated with a decrease in system L transport capacity. EGF activates system L transport activity through a signaling mechanism involving protein kinase C, independent of cell differentiation state. Both cell differentiation and EGF regulation of system L activity occur via alteration of functional copies of the system L transporter.

Specific Reversible Stimulation of System y+ l-Arginine Transport Activity in Human Intestinal Cells

Abstractl-Arginine, which is intimately involved in cellular immune functions and nitric oxide biology, is transported by intestinal cells largely via transport System y+. The gut epithelium is exposed to various luminal amino acids at any given time, and therefore the purpose of this study was to study the regulation of luminal arginine transport by other amino acids. System y+l-arginine transport activity was measured in Caco-2 monolayers exposed to various amino acids. l-arginine and/or other System y+ substrates specifically upregulated System y+ transport activity twofold after 1 hour, with a response noted as early as 5 minutes. Non-System y+ substrates did not affect l-arginine absorption. Kinetic analysis indicated that l-arginine exposure increased both System y+ Km and Vmax. Neither cycloheximide nor actinomycin affected this stimulation, indicating that the regulation did not involve transcription or translation. The System y+ substrate activation effect was reversible. l-arginine transport activity returned to baseline within 3 hours when cells were reincubated in amino acid-free media. These data indicate that System y+ arginine transport activity is rapidly and reversibly activated by System y+ substrates via a mechanism consistent with transmembrane stimulation. These findings identify a mechanism by which luminal nutrients regulate arginine uptake by the gut.

Insulin-like growth factor-2 activation of intestinal glutamine transport is mediated by mitogen-activated protein kinases

Insulin-like growth factor-2 (IGF-2) plays a pivotal role in regulating intestinal epithelial metabolism, growth, and proliferation, but its regulatory effects on mucosal cell amino acid transport have not been well studied. The purpose of this in vitro study was to investigate the regulatory mechanisms and intracellular signaling pathways involved in the regulation of IGF-2 on glutamine transport in cultured intestinal cells. Continuous incubation with IGF-2 stimulated glutamine transport activity in cultured IEC-6 cells in a dose- and time-dependent fashion. Prolonged incubation (up to 48 hours) resulted in a 50% increase in transport activity (0.81 ± 0.21 nmole/mg protein/min in IGF-2 cells vs. 0.57 ±0.15 nmole/ mg protein/min in control cells) and a threefold increase in glutamine transporter ATB0 mRNA levels. IGF-2 stimulated transport activity by increasing transport maximal capacity (Vmax 4.31 ± 0.36 nmole/ mg protein/min in IGF-2 cells vs. 2.51 ± 0.23 nmole/mg protein/min in control cells) without affecting the transport affinity (Km 0.31 ± 0.03 mmol/L glutamine in IGF-2 cells vs. 0.28 ± 0.03 mmol/L glutamine in control cells). This IGF-2-induced glutamine transport activity was attenuated by actinomycin-D or cycloheximide. The levels of mitogen-activated protein kinases p42/44, MEK1/2, and p38 as well as protein kinase C levels were elevated in IGF-2-treated cells and inhibitors of mitogen-activated protein kinase MEK1 (PD 98059), mitogen-activated protein kinase p38, and protein kinase C (chelerythrine chloride) individually attenuated the IGF-2-induced glutamine transport. These data suggest that IGF-2 stimulates intestinal glutamine uptake in cultured rat intestinal epithelial cells via a mechanism that involves transcription and translation of the transporter. Activation of mitogen-activated protein kinases and protein kinase C cascades are involved in the regulation. This increase in glutamine uptake may occur to support intestinal cell growth and proliferation.