The production and accumulation of advanced glycation end-products (AGEs) are hypothesized to have a causal role in the development of the complications associated with aging and lifestyle-related diseases, such as diabetes, atherosclerosis and hyperlipidemia. Therefore, it is important to reduce the production and accumulation of AGEs. In the present study, the ability of sodium 4-phenylbutyrate (PBA) on inhibition of glycation was assessed.
Glycation is a non-enzymatic chemical reaction that occurs between a ketone or aldehyde group of fructose or glucose and an amino acid residue or the hydroxy-group of a protein or lipid, and is often referred to as the Maillard reaction. Protein glycation occurs through a complex series of very slow reactions in the body, including the formation of the stable Amadori-lysine products (Schiff bases). These give rise to advanced glycation end-products (AGEs) (
It is hypothesized that the production and accumulation of AGEs have causal roles in the development of the complications associated with aging and lifestyle-related diseases, such as diabetes, atherosclerosis and hyperlipidemia (
In the present study, sodium 4-phenylbutyrate (PBA) was assessed as a potential candidate for use as an anti-glycation agent. PBA is an aromatic fatty acid that acts as a histone deacetylase inhibitor, ammonia scavenger and chemical chaperone (
It was previously reported that PBA may be effective for the treatment of neurodegenerative diseases, including Parkinson's disease, and it can suppress the onset of dextran sulfate sodium-induced colitis (
There are no reports assessing the anti-glycation effects of existing drugs, to the best of our knowledge. Therefore, the aim of the present study was to determine whether PBA inhibited the glycation of proteins
The glycation of albumin was measured
The glycation of collagen was measured using a Collagen Glycation assay kit: Glyceraldehyde (cat. no. AK71; Cosmo Bio., Co., Ltd.), according to the manufacturer's protocol. Briefly, the neutralized collagen solution was cooled and 50 µl was carefully added to each well of a 96-well plate, while maintaining the temperature at <10˚C. Next, the plate was incubated overnight at 37˚C in a humidified atmosphere. Then, PBA, aminoguanidine in DPBS and DPBS alone (as the negative control) were sterilized by filtering using 0.22-µm filters, and 40 µl of each solution was added to the collagen gel. Finally, 10 µl 500 mM glyceraldehyde was added to each well and the contents of the wells were mixed using a plate mixer (Iwaki; AGC Techno Glass Co., Ltd.). After incubation for 24 h at 37˚C in a humidified atmosphere, the concentrations of AGEs was assessed by measuring fluorescence intensity (excitation wavelength, 370 nm; emission wavelength, 440 nm) using a microplate reader. The experiment was repeated three times in duplicate (n=6).
For the
Statistical analysis was performed using GraphPad Prism version 6 (GraphPad Software, Inc.). Data are presented as the mean ± standard error of mean. Data were compared using a one or two-way ANOVA followed by a post-hoc Dunnett's test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.
When the fluorescence intensity of the control samples was defined as 100%, the fluorescence intensities measured when treated with 0.156, 0.625, 2.5, 10 and 40 mM PBA were 92.6, 91.1, 86.6, 73.4 and 57.9%, respectively. The fluorescence intensities measured when treated with 0.156, 0.625, 2.5, 10 and 40 mM aminoguanidine, a known anti-glycation agent, were 85.9, 64.5, 46.0, 18.7 and 2.2%, respectively (
When the fluorescence intensity of the control samples was defined as 100%, the fluorescence intensities when treated with 0.156, 0.625, 2.5, 10 and 40 mM PBA were 88.0, 92.4, 91.2, 79.2 and 63.1%, respectively. The fluorescence intensities associated with aminoguanidines were 74.5, 71.5, 54.9, 21.7 and 3.9%, respectively (
The effect of oral administration of PBA on KK mice was monitored for 8 weeks. In the PBA-treated group, the development of glycosuria was delayed, and the weight gained as well as HbA1c levels were lower when compared with the control group. No glycosuric PBA-treated mice were identified after 1 week, whereas 2 mice in the control group were glycosuric after the same time period. At the end of the experiment, 2 glycosuric PBA-treated mice were identified, whereas all the mice in the control group were glycosuric (
In the present study, the effects of PBA on protein glycation were assessed. The effects of PBA on non-enzymatic glycation
Having established the effects of PBA on protein glycation
In conclusion, PBA may limit the aging process and delay the development of lifestyle-related and other chronic diseases, such as diabetes, atherosclerosis, hyperlipidemia, cardiovascular diseases, cerebrovascular disorders, chronic renal failure and neurodegenerative diseases, which are characterized by the glycation of proteins. Reducing the prevalence of lifestyle-related diseases, which are increasing annually worldwide, may substantially reduce the economic burden on healthcare systems. Although it is necessary to elucidate the mechanism by which PBA reduces glycation in more detail, the method of administration and the side-effects of PBA are well established, as it is a currently used therapeutic. Therefore, administering PBA clinically for alleviating aging and lifestyle related disorders may be an additional use in the relatively near future.
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No funding was received.
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
KO and MN conceived the study and drafted the manuscript. KO acquired the data. KO and MN analyzed the data and revised the manuscript. Both authors read and approved the final manuscript.
The animal protocol was approved by the Experimental Laboratory Animal Committee of Fukuoka University (approval no. 1909069).
Not applicable.
The authors declare that they have no competing interests.
Inhibitory effect of PBA on albumin glycation. The vertical axis shows the fluorescence intensity. The number above each bar is the percentage reduction vs. Control. Data are presented as the mean ± standard error of the mean, and were analyzed using a one-way ANOVA, followed by Dunnett's post-hoc test. n=6. *P<0.01 vs. Control. Control, Dulbecco's PBS only; PBA, sodium 4-phenylbutyrate.
Inhibitory effect of PBA on collagen glycation. The vertical axis shows the fluorescence intensity. The number above each bar is the percentage reduction vs. Control. Data are presented as the mean ± standard error of the mean, and were analyzed using a one-way ANOVA, followed by Dunnett's post-hoc test. n=6. *P<0.01 vs. Control. Control, Dulbecco's PBS only; PBA, sodium 4-phenylbutyrate.
Effect of PBA on the body mass of KK mice. The vertical axis shows the body mass of each group and the horizontal axis shows the number of weeks of the study elapsed. The number of mice which were glycosuria positive at each time point is shown underneath the graph. Data are presented as the mean ± standard error of the mean, and were analyzed using a two-way ANOVA. n=5. *P<0.01 vs. Control. Control, H2O; PBA, sodium 4-phenylbutyrate; po, per oral.
Inhibitory effect of PBA on the increase in blood HbA1c in KK mice. The vertical axis shows the blood HbA1c level as a percentage for each group and the horizontal axis shows the number of weeks of the study elapsed. Data are presented as the mean ± standard error of the mean, and were analyzed using a two-way ANOVA. n=5. *P<0.01 vs. Control. Control, H2O; PBA, sodium 4-phenylbutyrate; po, per oral.
Effect of PBA on the production of AGEs. PBA, sodium 4-phenylbutyrate; AGE, advanced glycation end-product.