ALA Treatment Research Articles

Treatment of sarcopenia and glucose intolerance through mitochondrial activation by 5-aminolevulinic acid

Recently, sarcopenia has attracted attention as therapeutic target because it constitutes a risk factor for metabolic and cardiovascular diseases. We focused 5-aminolevulinic acid (ALA) which act as electron carriers in the mitochondrial electron transport system. The mice that received ALA for 8 weeks gained muscle strength and endurance, and exhibited increased muscle mass and mitochondrial amount. Administration of ALA to sarcopenia mice aged 100 weeks and chronic kidney disease (CKD) model mice also increased muscle mass and improved physical performance. Metabolome analysis revealed increased branched-chain amino acids (BCAAs) levels in the skeletal muscle of ALA-treated mice. Quantitative PCR analysis revealed decreased expression levels in branched-chain amino acid transaminases (BCATs) that degrade BCAAs and other muscle-degrading factors, and increased levels of mitochondria-activating factors. We also studied in cultured myocytes and obtained compatible results. ALA-treated mice tended to increase body weight, but reduced blood glucose level. These suggested that ALA treatment not only activated muscle mitochondria but also enhanced muscle mass through an increase in BCAAs contents, as to improve muscle strength, endurance and glucose tolerance in mice. In these ways, muscle mitochondrial activation with ALA is suggested to be useful for the treatment of sarcopenia and glucose intolerance.

Chronic treatment of (R)-α-lipoic acid reduces blood glucose and lipid levels in high-fat diet and low-dose streptozotocin-induced metabolic syndrome and type 2 diabetes in Sprague-Dawley rats.

(R)‐ α ‐lipoic acid (ALA), an essential cofactor in mitochondrial respiration and a potential antioxidant, possesses a wide array of metabolic benefits including anti‐obesity, glucose lowering, insulin‐sensitizing, and lipid‐lowering effects. In this study, the curative effects of ALA (100 mg/kg) on a spectrum of conditions related to metabolic syndrome and type 2 diabetes (T2D) were investigated in a high‐fat diet (HFD)‐fed and low‐dose streptozotocin (STZ)‐induced rat model of metabolic syndrome and T2D. The marked rise in the levels of glucose, triglycerides, total‐cholesterol, LDL‐cholesterol, and VLDL‐cholesterol in the blood of HFD‐fed and low‐dose STZ‐injected rats were significantly reduced by ALA treatment. Furthermore, ALA treatment significantly increased the serum HDL‐cholesterol levels and tended to inhibit diabetes‐induced weight reduction. Mathematical computational analysis revealed that ALA also significantly improved insulin sensitivity and reduced the risk of atherosclerotic lesions and coronary atherogenesis. This study provides scientific evidence to substantiate the use of ALA to mitigate the glucose and lipid abnormality in metabolic syndrome and T2D.

Promotive effects of 5-aminolevulinic acid on fruit quality and coloration of Prunus persica (L.) Batsch

5-Aminolevulinic acid (ALA) is an essential precursor of all tetrapyrrole compounds in plants. Given its function as a regulator of plant growth, ALA has been widely applied to crops to enhance production. However, successful application of ALA to improve the fruit quality and coloration of Prunus persica (L.) Batsch has rarely been reported. The objective of this study was to investigate the effects of exogenous application of ALA on fruit quality and anthocyanin accumulation of fruit skin in P. persica. We measured the contents of vitamin C (Vc), soluble sugar, and titratable acid, the activity of antioxidant enzymes, and fruit mass. The content of anthocyanin in peach skin and the relative expression level of structural genes and transcription factor genes involved in anthocyanin synthesis were analyzed. Six-year-old peach trees were sprayed with 0 (control), 200, and 400mg/L ALA. Results showed that treatment with 200 and 400mg/L ALA significantly enhanced the contents of Vc and soluble sugar, the activity of antioxidant enzymes, and the ratio of soluble sugar to titratable acid in fruit flesh. In addition, the content of titratable acid in fruit flesh treated with 200 and 400mg/L ALA were significantly decreased relative to those in the control. However, ALA treatment did not affect fruit mass. The results of the qRT-PCR analysis indicated that the expression levels of six structural genes (CHS, CHI, F3H, DFR, LDOX, and UFGT) and two transcription factors (MYB10 and WD40) involved in anthocyanin biosynthesis were all significantly upregulated by ALA treatment. However, no significant difference was observed in the expression level of bHLH3 between the ALA-treated and control fruit skin. Taken together, our findings indicate that exogenous ALA treatment promotes fruit quality and anthocyanin accumulation in peach skin, and thereby enhances peach coloration by inducing the expression of key genes and transcription factors related to anthocyanin biosynthesis.

Supplementation of linoleic acid (C18:2n-6) or α-linolenic acid (C18:3n-3) changes microbial agonist-induced oxylipid biosynthesis

Oxylipids are derived from polyunsaturated fatty acids (PUFA) in cellular membranes and the relative abundance or balance may contribute to disease pathogenesis. Previous studies documented unique oxylipid profiles from cows with either coliform or Streptococcus uberis mastitis, suggesting that lipid mediator biosynthesis may be dependent on the type of microbial-derived agonist. Changing the fatty acid content of peripheral blood leukocytes also may be critical to the relative expression of oxylipid profiles and the outcome of bacterial infection. No information is available in dairy cows describing how changing cellular PUFA content will modify oxylipids in the context of a microbial agonist challenge. Therefore, the hypothesis for the current study was that PUFA supplementation would change bovine leukocyte fatty acid content and respective oxylipid profiles from ex vivo microbial agonist-challenged leukocytes. Fatty acid content of leukocytes and plasma was quantified in (1) samples from cows not supplemented with PUFA, (2) cows supplemented with linoleic acid (LnA), and (3) cows supplemented with α-linolenic acid (ALA). Plasma oxylipids were assessed after S. uberis or lipopolysaccharide exposure and was compared with unstimulated oxylipid profiles. Fatty acid supplementation with ALA significantly increased ALA content of blood leukocytes and plasma relative to LnA. Fatty acid supplementation affected several S. uberis-induced oxylipids, but only S. uberis-induced 15-oxoETE was greater with ALA supplementation compared with LnA. Notably, only LPS-induced 5,6 LXA4 was altered with fatty acid supplementation, but no significant effect of LnA vs. ALA treatment was identified. Future studies are needed to understand how leukocyte activation and membrane PUFA availability collectively contribute to differential oxylipid profiles.

Nematicidal Effects of 5-Aminolevulinic Acid on Plant-Parasitic Nematodes

Plant-parasitic nematodes are important agricultural pests and often cause serious crop losses. Novel, environmental friendly nematicides are urgently needed because of the harmful effects of some existing nematicides on human health. 5-Aminolevulinic acid (ALA) was reported as a potential biodegradable herbicide, insecticide, or plant-growth promoting agent. Lack of information on ALA against plant-parasitic nematodes prompted this investigation to determine the effects of ALA on Meloidogyne incognita, Heterodera glycines, Pratylenchus coffeae, and Bursaphelenchus xylophilus. A series of in vitro assays and one greenhouse trial were conducted to examine the nematicidal effects of ALA. The results demonstrated that ALA exhibited a strong effect of suppression against the four nematodes tested. ALA also inhibited hatching of M. incognita and H. glycines. Results from the greenhouse experiment indicated that treatment of soil with 6.0 mM ALA significantly reduced the root-gall index (RGI) and egg mass number per root system compared with the uninoculated control (P ≤ 0.05). The metabolism assays indicated that ALA treatment significantly altered the nematode metabolism including the total protein production, malondialdehyde (MDA) content, and oxidase activities. This study suggested that ALA is a promising nematicide against plant-parasitic nematodes.

Ginsenoside Rb2 enhances the anti-inflammatory effect of ω-3 fatty acid in LPS-stimulated RAW264.7 macrophages by upregulating GPR120 expression.

Recent studies confirm that chronic low-grade inflammation is closely associated with metabolic syndromes, and anti-inflammatory therapy is a potential approach for treating cardiovascular diseases and type 2 diabetes. Accumulating evidence suggests that GPR120 activation is a feasible solution to ameliorating chronic inflammation and improving glucose metabolism. In this study we investigated whether ginsenoside Rb2 (Rb2), which exhibited regulatory activities in glucose and lipid metabolism, affected GPR120 expression in lipopolysaccharide (LPS)-activated mouse macrophage RAW264.7 cells, and examined the contribution of GPR120 activation to reducing the LPS-induced inflammatory response. LPS (100 ng/mL) activated the macrophages, resulting in dramatic increases in TNF-α, IL-6, IL-1β and NO production. Treatment with a ω-3 fatty acid α-linolenic acid (ALA, 50 μmol/L) produced moderate reduction in LPS-stimulated inflammatory cytokines and NO production (TNF-α and IL-6 were decreased by 46% and 42%, respectively). Pre-incubation with Rb2 (1 or 10 μmol/L) for 12 h before ALA treatment dramatically amplified the inhibitory effects of ALA (TNF-α and IL-6 were decreased by 74% and 86%, respectively). Compared to the treatment with ALA alone, pre-incubation with Rb2 resulted in a more prominent reduction in LPS-stimulated expression of iNOS and COX-2 and LPS-stimulated IKK/NF-κB phosphorylation and MAPK pathway activation. Rb2 (0.1-100 μmol/L) dose- and time-dependently increased both mRNA and protein expression of GPR120 in RAW264.7 cells, but treatment with Rb2 alone did not exert anti-inflammatory effect in LPS-activated RAW264.7 cells. In RAW264.7 cells transfected with GPR120 shRNA, the ameliorating effects of Rb2 on LPS-induced inflammation were abolished. In conclusion, Rb2 exerts anti-inflammatory effect in LPS-stimulated mouse macrophage RAW264.7 cells in vitro by increasing GPR120 expression and subsequently enhancing ω-3 fatty acid-induced GPR120 activation.

Applications of 5-aminolevulinic acid on the physiological and biochemical characteristics of strawberry fruit during postharvest cold storage

ABSTRACT: The compound 5-aminolevulinic acid (ALA) is a key precursor in the biosynthesis of porphyrins, such as chlorophyll, heme and phytochromobilin, and has multiple physiological effects on plants. Varying concentrations of ALA (50mg L-1, 100mg L-1, and 150mg L-1) and water (control) were applied to white stage 'Sweet Charlie' strawberry fruit. All ALA treatments delayed senescence and improved the qualities of strawberries fruit during storage. Among the treatments, 150mg L-1 ALA was the most effective dosage concentration. Exogenously applied ALA significantly reduced the decay index, respiration rate, O2 - production rate (O2 -), H2O2 and malondialdehyde (MDA) content, increased superoxide dismutase (SOD), ascorbate peroxidase activities (APX), total soluble solids (TSS) content, titratable acidity (TA) and anthocyanin content during the initial stage of storage. These results supported the pre harvest application of ALA as a beneficial strategy for the prevention of postharvest decay of strawberry fruit.

Proteomics and SSH Analyses of ALA-Promoted Fruit Coloration and Evidence for the Involvement of a MADS-Box Gene, MdMADS1.

Skin color is a key quality attribute of fruits and how to improve fruit coloration has long been a major concern. 5-Aminolevulinic acid (ALA), a natural plant growth regulator, can significantly increase anthocyanin accumulation in fruit skin and therefore effectively improve coloration of many fruits, including apple. However, the molecular mechanism how ALA stimulates anthocyanin accumulation in fruit skin remains unknown. Here, we investigated the impact of ALA on apple skin at the protein and mRNA levels. A total of 85 differentially expressed proteins in apple skins between ALA and water treatment (control) were identified by complementary gel-based and gel-free separation techniques. Most of these differentially expressed proteins were up-regulated by ALA. Function analysis suggested that 87.06% of the ALA-responsive proteins were associated with fruit ripening. To further screen ALA-responsive regulators, we constructed a subtracted cDNA library (tester: ALA treatment; driver: control) and obtained 104 differentially expressed unigenes, of which 38 unigenes were indicators for the fruit ripening-related genes. The differentially changed proteins and transcripts did not correspond well at an individual level, but showed similar regulated direction in function at the pathway level. Among the identified fruit ripening-related genes, the expression of MdMADS1, a developmental transcription regulator of fruit ripening, was positively correlated with expression of anthocyanin biosynthetic genes (MdCHS, MdDFR, MdLDOX, and MdUFGT) in apple skin under ALA treatment. Moreover, overexpression of MdMADS1 enhanced anthocyanin content in transformed apple calli, which was further enhanced by ALA. The anthocyanin content in MdMADS1-silenced calli was less than that in the control with ALA treatment, but higher than that without ALA treatment. These results indicated that MdMADS1 is involved in ALA-induced anthocyanin accumulation. In addition, anthocyanin-related verification in apple calli suggested that the regulation of MdMADS1 on anthocyanin biosynthesis was partially independent of fruit ripening process. Taken together, our findings provide insight into the mechanism how ALA regulates anthocyanin accumulation and add new information on transcriptase regulators of fruit coloration.

Mechanism of alpha-lipoic acid in treating TNBS-induced colitis in mice

Objective: To investigate whether α-lipoic acid (ALA) regulates autophagy in the pathogenesis of colitis through the mammalian target of rapamycin (mTOR) signaling pathway. Methods: Balb/c mouse colitis model was induced with 2, 4, 6-trinitrobenzenesulfonic acid (TNBS). Male Balb/c mice (22-25 g) were divided into four groups: control group, ALA group, colitis group, and ALA treatment group, with 10 mice in each group. ALA was administered at a dose of 80 mg/kg once a day for 7 days. The colon weight index, the disease activity index (DAI) and the histologic degeneration score (HDS) of colon tissue in each group were calculated. The autophagy gene Beclin-1 mRNA level was detected by RT-PCR. The levels of mTOR, phosphorylated mTOR (p-mTOR), and Beclin-1 proteins were detected by Western blot. Results: Compared with the control group, there was no statistically significant difference in colon weight index in the ALA group[(9.21±0.57)vs(8.91±0.91)g/kg, P>0.05], but increased in the colitis group [(12.65±1.33)g/kg, P<0.05] and decreased in the ALA treatment group [(10.04±1.02)g/kg, P<0.05]; there were no significant differences in DAI or HDS in the ALA group, but significantly increased in the colitis group, and decreased in the ALA treatment group (all P<0.05). The Beclin-1 mRNA and protein levels showed no significant differences between the control and the ALA groups (1.00±0.12 vs 1.05±0.05, 1.00±0.11 vs 1.00±0.06). However, the expression of Beclin-1 was significantly decreased in the colitis group compared to the control group (0.51±0.07 vs 1.00±0.12, 0.52±0.07 vs 1.00±0.11, both P<0.05), but significantly increased in the ALA treatment group compared to the colitis group (1.75±0.09 vs 0.51±0.07, 1.82±0.14 vs 0.52±0.07, both P<0.05). The mTOR total protein levels were not significantly different among the four groups, but the p-mTOR level was significantly higher in the colitis group than in the control group (3.07±0.20 vs 1.00±0.07), and reduced in the ALA treatment group than in the colitis group (1.49±0.11 vs 3.07±0.20) (all P<0.05). Conclusion: ALA may improve the TNBS-induced colitis in mice by inhibiting the phosphorylation of mTOR to promote autophagy.

Effects of 5-aminolevulinic acid treatment on photosynthesis of strawberry

Effects of root treatment with 5-aminolevulinic acid (ALA) on leaf photosynthesis in strawberry (Fragaria ananassa Duch.) plants were investigated by rapid chlorophyll fluorescence and modulated 820 nm reflection using 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) and methyl viologen (MV). Our results showed that ALA treatments increased the net photosynthetic rate and decreased the intercelluar CO2 concentration in strawberry leaves. Under DCMU treatment, trapping energy for QA reduction per PSII reaction center increased greatly, indicating DCMU inhibited electron transfer from QA −. The maximum photochemical efficiency of PSII (Fv/Fm) decreased under the DCMU treatment, while a higher Fv/Fm remained in the ALA-pretreated plants. Not only the parameters related to a photochemical phase, but also that one related to a heat phase remained lower after the ALA pretreatment, compared to the sole DCMU treatment. The MV treatment decreased PSI photochemical capacity. The results of modulated 820 nm reflection analysis showed that DCMU and MV treatments had low re-reduction of P700 and plastocyanin (PSI). However, the strawberry leaf discs pretreated with ALA exhibited high re-reduction of PSI under DCMU and MV treatments. The results of this study suggest that the improvement of photosynthesis by ALA in strawberry was not only related to PSII, but also to PSI and electron transfer chain.

Comparison of sensory tests and neuronal quantity of peripheral nerves between streptozotocin (STZ)-induced diabetic rats and paclitaxel (PAC)-treated rats

Although diabetic peripheral neuropathy (DPN) and chemotherapy-induced peripheral neuropathy (CIPN) are different disease entities, they share similar neuropathic symptoms that impede quality of life for these patients. Despite having very similar downstream effects, there have been no direct comparisons between DPN and CIPN with respect to symptom severity and therapeutic responses. We compared peripheral nerve damage due to hyperglycemia with that caused by paclitaxel (PAC) treatment as represented by biochemical parameters, diverse sensory tests, and immunohistochemistry of cutaneous and sciatic nerves. The therapeutic effects of alpha-lipoic acid and DA-9801 were also compared in the two models. Animals were divided into seven groups (n = 7–10) as follows: normal, diabetes (DM), DM + alpha-lipoic acid 100 mg/kg (ALA), DM + DA-9801 (100 mg/kg), paclitaxel-treated rat (PAC), PAC + ALA (100 mg/kg), and PAC + DA-9801 (100 mg/kg). The sensory thresholds of animals to mechanical, heat, and pressure stimuli were altered by both hyperglycemia and PAC when compared with controls, and the responses to sensory tests were different between both groups. There were no significant differences in the biochemical markers of blood glutathione between DM and PAC groups (p > .05). Quantitative comparisons of peripheral nerves by intraepidermal nerve fiber density (IENFD) analysis indicated that the DM and PAC groups were similar (6.18 ± 1.03 vs. 5.01 ± 2.57). IENFD was significantly improved after ALA and DA-9801 treatment in diabetic animals (7.6 ± 1.28, 7.7 ± 1.28, respectively, p < .05) but did not reach significance in the PAC-treated groups (6.05 ± 1.76, 5.66 ± 1.26, respectively, p > .05). Sciatic nerves were less damaged in the PAC-treated groups compared with the DM groups with respect to axonal diameter and area (8.60 ± 1.14 μm vs. 6.66 ± 1.07 μm, and 59.04 ± 15.16 μm2 vs. 35.71 ± 11.2 μm2, respectively, p < .05). Based on these results, the neuropathic manifestation and therapeutic responses of DPN may be different from other peripheral neuropathies. Therefore, specific pathogenic consideration according to peripheral neuropathy classification in addition to common treatments needs to be developed for management strategies of peripheral neuropathies.

α-lipoic acid exerts neuroprotective effects on neuronal cells by upregulating the expression of PCNA via the P53 pathway in neurodegenerative conditions.

Oxidative stress appears to be a central event responsible for the degeneration of dopaminergic neurons in Parkinson's disease (PD). 1-methyl-4‑phenyl-1,2,3,6-tetrahydropyridine or its toxic metabolite 1‑methyl‑4‑phenylpyridinium (MPP+) are classical widely‑used pharmacological and toxic agents to model PD; they cause the production of reactive oxygen species by inhibiting mitochondrial complex I, leading to DNA oxidative damage and subsequent neuronal death. Previous findings have suggested that proliferating cell nuclear antigen (PCNA), a critical regulatory protein for DNA repair, is involved in dopaminergic neuron damage in the MPP+‑induced PD model. The naturally occurring dithiol compound, α‑lipoic acid (ALA) has been reported to provide neuroprotection in invitro models of PD. The molecular mechanism by which ALA reduces neuronal death in PD remains to be fully elucidated. The present study aimed to analyze the ability of ALA to protect neuronal PC12 cells from the toxicity induced by MPP+, and the molecular mechanism underlying these actions using MTT and lactate dehydrogenase cytotoxicity assays, Hoechst33258 staining and western blot analysis. The results demonstrated that ALA efficiently increased the production of PCNA in MPP+‑treated PC12 cells. Accordingly, ALA treatment attenuated MPP+‑induced toxicity in the PC12 cells, and reduced cell apoptosis. The increase in the expression levels of PCNA by ALA in the MPP+‑treated PC12 cells appeared to be mediated by repression of the p53 protein, as the expression of p53 was increased by MPP+‑treatment and reduced by ALA. Taken together, these results indicated that ALA protected dopaminergic neurons against MPP+‑induced neurotoxicity through its ability to upregulate the DNA repair protein, PCNA, via the P53 pathway.

Effects of α-Linolenic Acid in Frozen-thawed Boar Spermatozoa

The aim of this study was to evaluate effect of α-linolenic acid (ALA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved in 20% egg yolk freezing extender containing ALA (0, 3, 5, and 10 ng/mL) with 0.05% ethanol. The frozen-boar spermatozoa were thawed at 37.5℃ for 45 sec in water-bath. The spermatozoa samples were evaluated the plasma membrane integrity, acrosome reaction, and mitochondrial integrity using flow cytometry. In results, population of live sperm with intact plasma membrane was significantly higher in control and 3 ng/mL ALA treatment group than ethanol group (p<0.05). In contract, dying sperms were higher in ethanol group than 3 ng/mL ALA treatment (p<0.05). Acrosomal membrane damage in all sperm population was reduced in 3 ng/mL ALA groups compared with ethanol treatment (p<0.05). However, acrosome damage in live sperm population was no significant difference among the all treatment groups. Mitochondrial integrity was not influenced by ALA treatments in both of live and all sperm population. In conclusion, this results show that supplement of ALA during the cryopreservation process could reduce the membrane damages including plasma and acrosomal membrane, whereas ALA did not influence to mitochondria in boar spermatozoa. Therefore, these results suggest that ALA can protect against the membrane damage derived cryo-stress, and cryopreservation efficiency of boar semen would be improved by use of ALA.

Interaction of drought and 5-aminolevulinic acid on growth and drought resistance of Leymus chinensis seedlings

Leymus chinensis is a dominant grass species in the Inner Mongolia steppes owing to its high vegetative productivity, good palatability for cattle, and abundant horizontally creeping rhizomes. Drought is generally regarded as one of the main environmental issues that is becoming a daunting challenge to the growth of plants, and ultimately results in land degradation. Drought stress influences plant growth and development by modulating physiological and biochemical events; however, some growth substances such as 5-aminolevulinic acid (5-ALA) show potential to ameliorate the damaging effects of drought. The objective of this study was to evaluate the response and mechanism of L. chinensis seedlings under drought stress, the effects of 5-ALA application, and the interaction of drought stress and 5-ALA application. Healthy and uniform-size seeds of L. chinensis were collected in a natural community of the Ecological Experimental Station in the Xilingole grassland. A potting experiment was carried out to determine the influence of exogenously applied 5-ALA at various concentrations (10mg/L, 50mg/L, and 100mg/L) under different soil water regimes (50% and 80% soil relative water content) on the morphological and physiological attributes of L. chinensis plants from June to November 2014. The seeds were grown in a biochemical incubator, and then the seedlings were transferred to pots. Water and Hoagland nutrient solution was applied to ensure an adequate nutrient supply at 5-day intervals. When seedlings attained a height of 18–21cm, 5-ALA was applied at different concentrations. Water spray was applied to L. chinensis plants as a control treatment. A second and third spray of 5-ALA was applied at 1-day intervals to exploit the full potential of 5-ALA application. Simultaneously, drought stress treatment was imposed using 50% soil relative water content; 80% soil relative water content was treated as the control. Therefore, there were eight treatments with three replications established in a random complete block design. The sampling for morphological, physiological, and biochemical attributes was conducted after 15days of drought stress, respectively. The results showed that 5-ALA could promote the growth of L. chinensis seedlings, including plant height, leaf area, plant water content, biomass, root activity, and photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoid), under a soil relative water content of 80%. Among all concentrations, 10mg/L 5-ALA proved to have the best effect on growth. Drought stress (50% soil relative water content) hampered plant growth. However, treatments of 5-ALA ameliorated the damaging effect of drought stress on seedlings, and improved the morphological index, biomass, plant water content, root activity, photosynthetic pigments, osmotic adjustments, and antioxidase activities viz. peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and ascorbate (APX), but reduced malondialdehyde (MDA) content and electrical conductivity. Among all concentrations, 50mg/L 5-ALA proved to have the best effect under drought stress. In summary, 5-ALA application improved the performance of L. chinensis by modulating growth and other morphological and physiological traits. However, the effect of 5-ALA on L. chinensis was concentration-dependent. Furthermore, significant interactions between drought stress and 5–ALA treatment (at a given spraying concentration) were observed with respect to leaf area, leaf width, fresh weight, root activity, chlorophyll b, carotenoids, MDA, proline, soluble protein, SOD, APX, and GR.

α-Linolenic acid increases the G0/G1 switch gene 2 mRNA expression in peripheral blood mononuclear cells from obese patients: a pilot study

BackgroundRecent evidence has demonstrated that the G0/G1 switch gene 2 (G0S2) is an important negative regulator of the rate-limiting lipolytic enzyme adipose triglyceride lipase-mediated lipolysis. It has been revealed that α-linolenic acid (ALA), a plant-based essential omega-3 polyunsaturated fatty acids, reduces adipose tissue lipolysis. However, it is not known whether G0S2 is implicated in ALA-induced inhibition of lipolysis. The purpose of this pilot study is to investigate the effect of ALA on G0S2 gene expression in peripheral blood mononuclear cells (PBMC) of obese patients and the potential influence of G0S2 gene expression in ALA-induced inhibition of lipolysis.MethodsA total of 26 obese patients were randomly assigned to be treated with or without ALA treatment (~4.0 g daily) for 12 weeks: the ALA-treated group (n = 14) or the untreated control group (n = 12). Plasma triglyceride (TG), free fatty acids (FFA), glycerol, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), as well as the mRNA expression levels of proliferator-activated receptor gamma (PPAR-γ), G0S2, and G protein-coupled receptor 120 (GPR120) in PBMC were repeatedly examined from fasting obese patients before and after ALA treatment.ResultsALA significantly decreased plasma TG, FFA, glycerol, IL-6, and TNF-α levels and increased the mRNA expression levels of PPAR-γ, G0S2, and GPR120 in PBMC, compared with the untreated control group. In obese patients from the ALA-treated group, decreased plasma FFA (a biomarker for lipolysis) level was significantly correlated with increased PPAR-γ (a functional omega-3 fatty acids receptor) and G0S2 (a direct target gene of PPAR-γ) mRNA expression in PBMC, while decreased plasma FFA level was not correlated with increased GPR120 (another functional omega-3 fatty acids receptor) mRNA expression in PBMC.ConclusionThis study shows that ALA increases G0S2 gene expression in PBMC in parallel with the decrease of plasma FFA level in obese patients. Increased G0S2 gene expression might contribute to the beneficial anti-lipolytic effect of ALA in obese patients.

Prophylactic and abundant intake of α-lipoic acid causes hepatic steatosis and should be reconsidered in usage as an anti-aging drug.

The majority of research has suggested that α-lipoic acid (ALA) is a potential therapeutic agent for chronic diseases associated with oxidative stress, including atherosclerosis, diabetes, and neurodegeneration. Therefore, a nutritional supplementation with ALA is recommended although the effects of a short- and long-term intake of ALA on central organs in healthy individuals are not studied in detail yet. Therefore, liver tissue of 4 and 74 weeks ALA-treated healthy C57BL6/J mice with respect to lipid metabolism was analyzed. In doing so, it was shown that short-term and long-term ALA treatment caused a marked increase of β-oxidation, as indicated by a significant rise of mRNA expression of fgf21, pparα, and its target genes, for example, acox1, cpt1α, and cpt2, as well as of Fgf21 plasma concentration. Glycolytic activity, as assessed by pklr1 mRNA expression and pyruvate kinase activity, was also found increased. In addition, it was shown that both short- and long-term ALA treatment increased cholesterol content, induced systemic triglyceridemia, and enhanced rxrα and lxrα mRNA expression. Despite the fact that short-term ALA intake reduced lipogenesis, as given by significant declines of fas and srebp1c mRNA expression, and that a long-term ALA intake induced a significant rise of these lipogenic genes, both treatment regimen caused fat accumulation. This, however, was more pronounced upon long-term ALA intake, leading to hepatic steatosis and liver injury, as indicated by increased inflammation and disruption of the general hepatic architecture. In summary, the prophylactic and abundant use of ALA under healthy conditions should be considered with caution.

Influence of exogenous 5-aminolevulinic acid on chlorophyll synthesis and related gene expression in oilseed rape de-etiolated cotyledons under water-deficit stress

5-aminolevulinic acid (ALA) is an essential precursor for the biosynthesis of tetrapyrrols such as heme and chlorophyll (Chl). Previous studies have focused mainly on promotive effects of exogenous ALA on plant growth, while regulatory mechanisms affecting Chl biosynthesis have been only partially discussed. In the present study, the ameliorative role of exogenous ALA was investigated on Chl and endogenous ALA biosynthesis in six-day-old etiolated oilseed rape (Brassica napus L.) cotyledons during the de-etiolation stage. We showed that exogenously applied ALA of a low dosage enhanced Chl and ALA accumulation in cotyledons, while 600 µM ALA treatment inhibited the accumulation of Chl and ALA severely. However, the gene expression levels of glutamyl-tRNA reductase (HEMA) and glutamate-1-semialdehyde aminotransferase (GSA) were not affected under either low or high ALA concentrations. Furthermore, water deficit induced by polyethylene glycol 6000 (PEG) suppressed the Chl and ALA accumulation in cotyledons, while the inhibition was partially alleviated in the cotyledons pretreated with ALA. The decrease in Chl biosynthesis induced by PEG stress was assumed to be related to downregulation of HEMA and Mg-chelatase ChlH (ChlH), and upregulation of ferrochelatase (FC) genes. Moreover, exogenously applied ALA did not show any effect on the expression of Chl synthesis-related genes under the PEG treatment. These results showed a difference in suppressing ALA synthesis due to the high concentration of ALA and PEG. Exogenously applied ALA did not affect the expression of HEMA and GSA, thus exogenous ALA regulated Chl synthesis not via the regulation of transcriptional level in ALA biosynthesis. However, the inhibition in Chl and endogenous ALA accumulation by the PEG treatment may be attributed to downregulation of HEMA and ChlH, and upregulation of FC.

Promotive effects of 5-Aminolevulinic acid on growth, photosynthetic gas exchange, chlorophyll, and antioxidative enzymes under salinity stress in Prunnus persica (L.) Batseh Seedling

The physiological characteristics of peach seedlings (Prunnus persica L. Batseh) under salt stress were investigated to ameliorate the effects of 5-aminolevulinic acid (ALA). Physiological indices of salt-stressed peach seedlings treated with exogenous ALA were investigated; these indices included stem diameter, plant height, leaf area, aboveground and underground weights, net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO2 concentration (Ci), stomatal conductance (Gs), root vitality, and membrane permeability. The contents of chlorophyll, malondialdehyde (MDA), soluble sugars, soluble proteins, and proline as well as the activities of antioxidant enzymes were evaluated. Results showed that the physiological characteristics of peach seedlings under NaCl stress (100 mM) were inhibited. Peach seedlings treated with100 mM NaCl and 200mg/l ALA for every germination index recovered. Moreover, peach seedlings treated with 200mg/l ALA exhibited enhanced physiological characteristics. Treatment with exogenous ALA increased stem diameter, plant height, leaf area, aboveground and underground weights, net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO2 concentration (Ci), stomatal conductance (Gs), and root vitality. The treatment also increased the contents of chlorophyll, soluble sugars, soluble proteins, and proline but decreased membrane permeability and MDA content. Furthermore, treatment with ALA significantly enhanced the activities of superoxide dismutase, peroxidase, and catalase. These findings indicate that exogenous ALA treatment could improve the growth and relieve the salt stress injury of peach seedlings by increasing photochemical efficiency, osmotic content, and antioxidant enzyme activity.

δ-Aminolevulinic acid (ALA) 유도체들의 미토콘드리아 탈분극 유도에 의한 인간 섬유아세포의 세포분열 억제

δ-Aminolevulinic acid (ALA) is an endogenous metabolite formed in the mitochondria from succinyl-CoA and glycine, and plays a key role in the living body as an intermediate of the compound in the porphyrin biosynthesis pathway. ALA has been commonly used in photodynamic therapy for several years, because ALA is of interest as a biodegradable mediator, a growth regulator, and an effective agent used in dermatology. Here, we determined which ALA derivatives were the most effective for the inhibition of the cell proliferation and growth of human fibroblast. As a result, we found that the treatment of ALA derivatives including ALA, ALAP (ALA phosphate salt), MAL (Methyl 5-aminolevulinate hydrochloride salt), PBGL (phophobilinogen lactam) and PBGH (phophobilinogen-HCl) could attenuate cell proliferation of human fibroblast cells. Among them, PBGH was the most effective derivative. In addition, PBGH treatment could induce mitochondrial membrane depolarization, leading to cell death of human fibroblast. These results suggest that mitochondrial membrane depolarization induced by ALA and PBGH treatment might be responsible for inhibition of cell proliferation and death. Taken together, our results propose the possibility that PBGH can be used as one of the effective drugs in human skin disease, psoriasis.

Delta-Aminolevulinate Preferentially Induces Gamma-Globin Expression in Erythroid Cells through Activation of NRF2 Stress Response

Fetal hemoglobin (HbF,α2γ2) is an important genetic modifier for β-hemoglobinopathies including sickle cell disease (SCD) and β-Thalassemia which occur as a result of mutations in the adult β-globin gene. δ-aminolevulinate (ALA), the first product synthesized in the mammalian heme biosynthesis pathway, has been reported to induce hemoglobin production. In this study, we determined the effects of ALA on γ- and β-globin transcription and HbF production in an erythroid cell line and primary erythroid progenitors. We demonstrated that ALA increased heme levels by 50% with 48 hour treatment in KU812 erythroleukemia cells. By reverse transcription-real time polymerase chain reaction (RT-qPCR), we detected an induction of γ-globin transcription in a time- and dose-dependent manner with 2.4-fold elevation at the 2mM ALA concentration. Moreover, we showed that HbF protein level increased dramatically by 22.9-fold at 48 hour ALA treatment by western blot analysis with HbF specific antibody. By contrast, ALA treatment did not change β-globin transcription level. Subsequent studies in primary erythroid progenitors derived from CD34+ stem cells, we observed γ-globin transcriptional activation up to 6.5-fold by day 22 in culture with 48hour ALA treatment, whereas β-globin transcription was not significantly changed. Studies to determine molecular mechanisms demonstrated that addition of succinylacetone (SA), a specific inhibitor of heme biosynthesis, blocked the ALA-mediated induction of γ-globin transcription and HbF synthesis in KU812 cells detected by RT-qPCR, western blot and enzyme-linked immunosorbent assay. Using 2'-7'-Dichlorodihydrofluorescein diacetate with flow cytometer detection, we observed a 3-fold increase of reactive oxygen species such as hydrogen peroxide with 24-hour ALA treatment in KU812 cells which was inhibited by the addition of SA. In addition, western blot analysis detected increased nuclear translocation of NRF2, a master transcription factor controlling the cellular antioxidant response. Using chromatin immunoprecipitation, we demonstrated that NRF2-associated enrichment of γ-globin proximal promoter chromatin was enhanced 10-fold in the region of the antioxidant responsive element. Furthermore, in KU812 cells transiently transfected with a wild-type and ARE-mutated γ-globin promoter luciferase reporters followed by ALA treatment, we detected activation of the wild-type γ-globin promoter-driven luciferase reporter. As predicted, deletion of the ARE in the γ-globin promoter not only significantly reduced the basal activity by 10-fold but also abolished the ALA-induced promoter activity. These data support a role of ALA in activating heme production and γ-globin expression through an NRF2 activation mechanism. Our results provide evidence that ALA and the heme biosynthesis pathway are novel targets for therapeutic potential in treating β-hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.