Page 47 - Petelin, Ana. 2021. Ed. Zdravje starostnikov / Health of the Elderly. Proceedings. Koper: University of Primorska Press.
P. 47
ic metabolic functions and is therefore an excellent model for studying the the effect of non-nutritive sweeteners on lipid metabolism in liver cells 45
central role of the liver in lipid metabolism (Javitt, 1990). The study began with
the cultivation of adherent HepG2 liver cells and exposing them to the four dif-
ferent sweeteners to determine cytotoxic concentrations. In the following ex-
periments, the cells were exposed to the highest non-cytotoxic concentration
of each tested compound for 24 hours. We examined the effect of sweeteners
on the expression of five genes related to lipid metabolism using quantitative
polymerase chain reaction (RT-PCR). The genes were: acetyl-CoA carboxylase
alpha (ACACA), carnitine palmitoyltransferase 1A (CPT1A), carnitine palmi-
toyltransferase 2 (CPT2), diacylglycerol O-acyltransferase 2 (DGAT2) and per-
lipin 2 (PLIN2). We also measured the accumulation of OilRed O stained lipid
droplets in the HepG2 liver cell line. All experiments were performed both in
the presence and absence of sodium palmitate.
Results
Gene expression analysis
The cytotoxic effect of selected sweeteners at different concentrations on HepG2
cells after 24 hours of exposure was first studied. All concentrations of eryth-
ritol sweetener solution from 0.02 to 2 mg/ml were found to have a slight pro-
liferative effect on cells. Similar to erythritol, three concentrations of Natreen
Classic sweetener solution, 2 mg/ml, 1 mg/ml and 0.2 mg/ml, had a prolifera-
tive effect on cells. On the other hand, rebaudioside A (active compound in Ste-
via Sugarel) at concentrations of 2 mg/ml and 1 mg/ml had a cytotoxic effect on
cells, while none of the tested concentrations of Huxol Original had either pos-
itive or negative effect on cell viability.
Based on the obtained data, further analyses were performed with the
highest non-cytotoxic concentration. The following concentrations were used:
2 mg/mL for Erythritol, Huxol Original and Natreen Classic and 0.2 mg/ml for
Stevia Sugarel. Analysis of fat accumulation-related gene expression was per-
formed for five genes: ACACA, DGAT2, PLIN2, CPT1A and CPT2. Gene ex-
pression was tested in cells grown in control medium (source of glucose) and
medium with added palmitate (source of glucose and fatty acid). Analysis was
performed by RT-PCR, checking the relative expression for each of the selected
genes relative to the 18S rRNA gene. The results showed increased expression of
four of the five genes. ACACA gene expression was below the detection limit in
most of the analysed samples.
The results of the CPT1A expression analysis showed that sweeteners,
with the exception of one sample (stevia), increased its expression, as shown
graphically in Figure 1. The following changes in gene expression occurred
between cells in medium with sweeteners and cells in control medium with-
out additives: sweetener Natreen Classic 2.2±0.7-fold up-regulation, sweetener
Erythritol 3.0±0.8-fold up-regulation, sweetener Huxol Original 4.8±0.7-fold
up-regulation, sweetener Stevia Sugarel 0.7±0.2-fold down-regulation. Palmi-
central role of the liver in lipid metabolism (Javitt, 1990). The study began with
the cultivation of adherent HepG2 liver cells and exposing them to the four dif-
ferent sweeteners to determine cytotoxic concentrations. In the following ex-
periments, the cells were exposed to the highest non-cytotoxic concentration
of each tested compound for 24 hours. We examined the effect of sweeteners
on the expression of five genes related to lipid metabolism using quantitative
polymerase chain reaction (RT-PCR). The genes were: acetyl-CoA carboxylase
alpha (ACACA), carnitine palmitoyltransferase 1A (CPT1A), carnitine palmi-
toyltransferase 2 (CPT2), diacylglycerol O-acyltransferase 2 (DGAT2) and per-
lipin 2 (PLIN2). We also measured the accumulation of OilRed O stained lipid
droplets in the HepG2 liver cell line. All experiments were performed both in
the presence and absence of sodium palmitate.
Results
Gene expression analysis
The cytotoxic effect of selected sweeteners at different concentrations on HepG2
cells after 24 hours of exposure was first studied. All concentrations of eryth-
ritol sweetener solution from 0.02 to 2 mg/ml were found to have a slight pro-
liferative effect on cells. Similar to erythritol, three concentrations of Natreen
Classic sweetener solution, 2 mg/ml, 1 mg/ml and 0.2 mg/ml, had a prolifera-
tive effect on cells. On the other hand, rebaudioside A (active compound in Ste-
via Sugarel) at concentrations of 2 mg/ml and 1 mg/ml had a cytotoxic effect on
cells, while none of the tested concentrations of Huxol Original had either pos-
itive or negative effect on cell viability.
Based on the obtained data, further analyses were performed with the
highest non-cytotoxic concentration. The following concentrations were used:
2 mg/mL for Erythritol, Huxol Original and Natreen Classic and 0.2 mg/ml for
Stevia Sugarel. Analysis of fat accumulation-related gene expression was per-
formed for five genes: ACACA, DGAT2, PLIN2, CPT1A and CPT2. Gene ex-
pression was tested in cells grown in control medium (source of glucose) and
medium with added palmitate (source of glucose and fatty acid). Analysis was
performed by RT-PCR, checking the relative expression for each of the selected
genes relative to the 18S rRNA gene. The results showed increased expression of
four of the five genes. ACACA gene expression was below the detection limit in
most of the analysed samples.
The results of the CPT1A expression analysis showed that sweeteners,
with the exception of one sample (stevia), increased its expression, as shown
graphically in Figure 1. The following changes in gene expression occurred
between cells in medium with sweeteners and cells in control medium with-
out additives: sweetener Natreen Classic 2.2±0.7-fold up-regulation, sweetener
Erythritol 3.0±0.8-fold up-regulation, sweetener Huxol Original 4.8±0.7-fold
up-regulation, sweetener Stevia Sugarel 0.7±0.2-fold down-regulation. Palmi-
