Specific knock‐down of tissue non‐specific alkaline phosphatase mRNA levels inhibits intracellular lipid accumulation in 3T3‐L1 and HepG2 cells

Abstract

The use of non-specific inhibitors of tissue non-specific alkaline phosphatase (TNSALP) in pre-adipocytes blocks intracellular lipid accumulation. TNSALP is also expressed in hepatocytes, which are known to accumulate lipid in a similar manner to pre-adipocytes. The purpose of this study was to use specific silencing of TNSALP mRNA, using short interfering (si) RNA, to investigate the role of TNSALP in intracellular lipid accumulation in 3T3-L1 and HepG2 cells. Cellular activity of TNSALP was measured using an automated colorimetric assay, and intracellular lipid accumulation was determined using the lipid-specific dye, Oil Red O. Cells were transfected with siRNA directed against TNSALP mRNA, and expression of the TNSALP gene was determined at selected time points postinduction of lipid droplet formation. Expression of the TNSALP gene was inhibited by a maximum of 88 ± 1.9% (P < 0.005 vs. control) 11 days after initiation of lipid droplet formation in the 3T3-L1 cells and 80 ± 8.9% (P < 0.05 vs. control) after 4 days in the HepG2 cells. This led to significant inhibition of both TNSALP activity and intracellular lipid accumulation in both cell lines. These data demonstrates that TNSALP plays an important role in the control of lipid droplet formation in both pre-adipocyte and hepatocyte cell lines.

TNAP: A New Multitask Enzyme in Energy Metabolism

“…On the other hand, Esteve et al, reported that TNAP was particularly expressed in a subpopulation of beige adipocyte progenitors, where its chemical or molecular inhibition reduced the levels of markers associated with adipocyte differentiation (PPARγ2, ChREBP, UCP1, LPL), and decreased TG accumulation [ 95 ]. Similar findings were obtained in adipocyte cultures, with TNAP expression increasing with the differentiation of 3T3-L1 or primary adipocytes, and its inhibition with levamisole decreasing TG accumulation [ 73 , 96 , 97 ]. The substrates and mechanisms through which TNAP may impact adipogenesis are poorly known.…”

“…Like in the intestinal lumen, another possible TNAP substrate in the liver is CD36. In HepG2 hepatocytes cultured in presence of high glucose or FA levels, TNAP expression increased in parallel with that of CD36 [ 71 , 72 ], and TNAP inhibition with levamisole or siRNA reduced triglyceride (TG) accumulation [ 71 , 73 ]. That TNAP controls CD36 activity in hepatocytes however remains to be demonstrated.…”

2-Benzylidenebenzofuran-3(2H)-ones as a new class of alkaline phosphatase inhibitors: synthesis, SAR analysis, enzyme inhibitory kinetics and computational studies

“…General procedures for the synthesis of chalcones (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) and aurones (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) In step 1, a mixture of substituted 2 0 -hydroxyacetophenone (1.0 mmol) and aqueous sodium hydroxide solution (30%, 5.0 mL) was dissolved in distilled methanol (20.0 mL) and stirred for 30 min at room temperature followed by the dropwise addition of substituted aryl aldehyde (1.0 mmol). Subsequently, the reaction mixture was stirred at the same temperature for 3-4 h. The progress of the formation of 2 0 -hydroxychalcone was examined by TLC using cyclohexane : ethyl acetate (3 : 1) as the mobile phase.…”

“…HCl (10%) and poured into the cold water. The separated solid was ltered, washed with H 2 O and the dried product was recrystallized from EtOH to obtain the puried compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). In step 2, Hg(OAc) 2 (1.0 mmol) was added to a solution of 2 0 -hydroxychalcone (1.0 mmol) in pyridine (10 mL) at room temperature and the mixture was reuxed for 2 h. Upon completion of the reaction, as indicated by TLC, the reaction mixture was le to cool, and then poured onto ice-cold water (30 mL) and acidied with dil.…”

“…[1][2][3][4][5][6][7][8][9] They are responsible for the effective removal of phosphate groups from alkaloids, nucleotides and proteins in alkaline medium. 10 APs are dimeric non-specic enzymes with regard to the substrate, and thus hydrolyze various substrates. APs exhibit hydrolytic activity, phosphotransferase activity, and pyrophosphatase activity.…”