Alkaline phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1

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Abstract

Objective

As alkaline phosphatase may play a role in cell differentiation, our aim was to study the possible role of this enzyme in the differentiation of preadipocytes (3T3-L1 cells) into adipocytes.

Research Methods and Procedures

3T3-L1 cells were grown in medium containing insulin, dexamethasone and IBMX to induce adipogenesis. Adipogenesis was measured using the triglyceride-specific dye, oil red O at 0, 3, 7 and 11 days after initiation of adipogenesis in the presence or absence of the alkaline phosphatase inhibitors, levamisole, histidine and Phe-Gly-Gly. Intracellular localisation of the enzyme was detected using ELF-phosphatase, a fluorescent substrate and alkaline phosphatase gene expression was assessed using RT-PCR.

Results

Alkaline phosphatase activity was detected in untransformed cells (1.91±0.62 mU/mg protein) and activity increased 11.5±1.4-fold after 11 days treatment with transformation medium and 5.3±0.3-fold in transformation medium containing levamisole (p<0.05). Triglyceride content of cells increased 3.1±0.2-fold after 11 days treatment with transformation medium and 2.1±0.3-fold in the presence of levamisole (p<0.005). Histidine inhibited adipogenesis and alkaline phosphatase to a greater extent than did levamisole, but Phe-Gly-Gly had no effect on these variables. Alkaline phosphatase was localised around the lipid droplets of the cells. Gene expression of alkaline phosphatase increased during adipogenesis.

Discussion

This study demonstrates that tissue-nonspecific alkaline phosphatase is present in 3T3-L1 cells and that it may play a role in the control of adipogenesis.

Introduction

The differentiation of preadipocytes into mature, triglyceride containing adipocytes, a process termed adipogenesis, has been extensively studied using the murine fibroblast-like cell line, 3T3-L1 [5]. The control of adipogenesis is complex, involving the interaction of a number of intracellular signalling pathways and the activation of numerous transcription factors [5], [6], particularly peroxisome proliferator-activated receptor γ (PPARγ) [7].

Alkaline phosphatase is expressed across a large number of different species and within mammals is present in a wide variety of tissues [1]. There are four human alkaline phosphatase isozymes, three of which, termed intestinal, germ cell and placental, are expressed in a tissue-specific manner. The fourth isozyme is expressed in a number of tissues and is termed tissue-nonspecific alkaline phosphatase [2]. Each of these four isozymes is encoded by a separate gene. The four enzymes can be differentiated from each other not only by their gene sequences but also by their biochemical response to specific inhibitors. Thus, the tissue-nonspecific isozyme is inhibited by levamisole but not by L-phenylalanyl-glycyl-glycine (Phe-Gly-Gly) whilst the reverse is true for the tissue-specific forms [3], [4].

The endogenous substrates of alkaline phosphatase and its exact function are unknown [1], [2]. However, there is some experimental evidence that the enzyme is involved in tissue development [8], [9] and cellular differentiation [10], [11], although its precise role in these cellular events is not known. Given the proposed role of alkaline phosphatase in cellular differentiation [10], [11] and its reported presence in rat adipose tissue [12], adipocyte precursor cells in human bone marrow [13] and rabbit preadipocytes [14] the aim of this study was to investigate whether alkaline phosphatase was expressed in the 3T3-L1 cell line and if so what role it may play in adipogenesis.

Section snippets

Cell Culture

The 3T3-L1 cells were grown at 37 °C in a humidified atmosphere of 5% CO2 in air in 25-cm2 tissue culture flasks containing Dulbecco's Modified Eagle's Medium (Invitrogen Corporation, Paisley, Scotland) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen) and 2 mmol/l glutamine (Invitrogen) (maintenance medium). When the cells reached confluence, 1.70 μmol/l insulin (Novo-Nordisk, Copenhagen, Denmark), 0.50 mmol/l IBMX

Alkaline phosphatase activity is present in 3T3-L1 cells and increases during the differentiation process

Alkaline phosphatase activity was detectable in non-differentiated 3T3-L1 cells at a level of 1.91±0.62 mU/mg protein (mean±S.E.M. of four experiments). After 3, 7, 11 and 17 days treatment with differentiation medium the levels of alkaline phosphatase activity were 10.94±2.89 (n=3), 17.16±6.03 (n=4), 22.97±9.19 (n=4) and 31.2±0.73 mU/mg protein (n=2), respectively, and the level of alkaline phosphatase activity was significantly different from the level in non-differentiated cells from day 7

Discussion

The present study has shown that an alkaline phosphatase isozyme that is biochemically inhibited by levamisole and histidine but not by Phe-Gly-Gly is expressed in the 3T3-L1 cell line. This is characteristic of the murine tissue-nonspecific isozyme of alkaline phosphatase [20], [21], which shares 90% amino acid-sequence homology with the human isozyme [22]. Confirmation of the identity of the isozyme was obtained from RT-PCR analysis of differentiated 3T3-L1 cells which demonstrated the

Acknowledgements

The authors thank Dr. Eugene Swanepoel and members of the routine laboratory service of the Department of Chemical Pathology, National Health Laboratory Service (NHLS), Johannesburg General Hospital for performing the alkaline phosphatase activity assays. This study was supported by financial assistance from the NHLS and the South African Medical Research Council. Mr. Aus Tariq Ali received postgraduate bursaries from the NHLS and the University of the Witwatersrand.

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