Tài liệu Báo cáo khoa học: Differentiation stage-dependent preferred uptake of basolateral

Differentiation stage-dependent preferred uptake of

basolateral (systemic) glutamine into Caco-2 cells results

in its accumulation in proteins with a role in cell–cell

interaction

Kaatje Lenaerts, Edwin Mariman, Freek Bouwman and Johan Renes

Maastricht Proteomics Center, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Human Biology,

Maastricht University, the Netherlands

Glutamine has an important function in the small

intestine with respect to maintaining the gut epithelial

barrier in critically ill patients [1,2]. Several studies

performed in different experimental settings reveal

that it serves as an important metabolic fuel for

enterocytes [3], and as a precursor for nucleotides,

amino sugars, proteins and several other molecules

such as glutathione [4,5]. In vitro cell culture studies

demonstrate that glutamine specifically protects intes￾tinal epithelial cells against apoptosis [6,7], has

trophic effects on the intestinal mucosa [8] and pre￾vents tumour necrosis factor (TNF)-alpha induced

bacterial translocation [9]. In experimental models

of critical illness, glutamine was able to attenuate

Keywords

apical and basolateral; barrier function;

clinical nutrition; intestinal cells; protein

turnover

Correspondence

K. Lenaerts, Maastricht Proteomics Center,

Nutrition and Toxicology Research Institute

Maastricht (NUTRIM), Department of

Human Biology, Maastricht University,

PO Box 616, 6200MD, Maastricht,

the Netherlands

Fax: +31 43 3670976

Tel: +31 43 3881509

E-mail: [email protected]

(Received 4 February 2005, revised 22 April

2005, accepted 3 May 2005)

doi:10.1111/j.1742-4658.2005.04750.x

Glutamine is an essential amino acid for enterocytes, especially in states of

critical illness and injury. In several studies it has been speculated that the

beneficial effects of glutamine are dependent on the route of supply (lumi￾nal or systemic). The aim of this study was to investigate the relevance of

both routes of glutamine delivery to in vitro intestinal cells and to explore

the molecular basis for proposed beneficial glutamine effects: (a) by deter￾mining the relative uptake of radiolabelled glutamine in Caco-2 cells;

(b) by assessing the effect of glutamine on the proteome of Caco-2 cells

using a 2D gel electrophoresis approach; and (c) by examining glutamine

incorporation into cellular proteins using a new mass spectrometry-based

method with stable isotope labelled glutamine. Results of this study show

that exogenous glutamine is taken up by Caco-2 cells from both the apical

and the basolateral side. Basolateral uptake consistently exceeds apical

uptake and this phenomenon is more pronounced in 5-day-differentiated

cells than in 15-day-differentiated cells. No effect of exogenous glutamine

supply on the proteome was detected. However, we demonstrated that exo￾genous glutamine is incorporated into newly synthesized proteins and this

occurred at a faster rate from basolateral glutamine, which is in line with

the uptake rates. Interestingly, a large number of rapidly labelled proteins

is involved in establishing cell–cell interactions. In this respect, our data

may point to a molecular basis for observed beneficial effects of glutamine

on intestinal cells and support results from studies with critically ill patients

where parenteral glutamine supplementation is preferred over luminal sup￾plementation.

Abbreviations

CBB, Coomassie brilliant blue; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; IPG, immobilized pH gradient;

LI-cadherin, liver-intestine cadherin; PTFE, polytetrafluoroethylene.

3350 FEBS Journal 272 (2005) 3350–3364 ª 2005 FEBS