Regular ArticleRetardation of the Senescence of Cultured Human Diploid Fibroblasts by Carnosine
Abstract
We have examined the effects of the naturally occurring dipeptide carnosine (β-alanyl-L-histidine) on the growth, morphology, and lifespan of cultured human diploid fibroblasts. With human foreskin cells, HFF-1, and fetal lung cells, MRC-5, we have shown that carnosine at high concentrations (20-50 mM) in standard medium retards senescence and rejuvenates senescent cultures. These late-passage cultures preserve a nonsenescent morphology in the presence of carnosine, in comparison to the senescent morphology first described by Hayflick and Moorhead. Transfer of these late-passage cells in medium containing carnosine to unsupplemented normal medium results in the appearance of the senescent phenotype. The serial subculture of cells in the presence of carnosine does not prevent the Hayflick limit to growth, although the lifespan in population doublings as well as chronological age is often increased. This effect is obscured by the normal variability of human fibroblast lifespans, which we have confirmed. Transfer of cells approaching senescence in normal medium to medium supplemented with carnosine rejuvenates the cells but the extension in lifespan is variable. Neither D -carnosine, (β-alanyl-D-histidine), homocarnosine, anserine, nor β-alanine had the same effects as carnosine on human fibroblasts. Carnosine is an antioxidant, but it is more likely that it preserves cellular integrity by its effects on protein metabolism.
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Whole-cell biotransformation for large scale production of carcinine in Escherichia coli
2022, Journal of BiotechnologyCarcinine is a natural imidazole-containing peptide derivative. It is widely used in the cosmetics industry as anti-aging supplement with antioxidant, anti-glycation and glycation reversal functions, and it also has a notable pharmacological effect as anti-tumor drug and in protection against retinopathy. However, a technological method for synthesis and production of carcinine has not been established. In this study, a whole-cell transformation system converting β-alanine and histamine to carcinine by the enzymes Ebony and phosphopantetheine transferase (Sfp) has been developed. The results revealed that the catalytic efficiency of the strain containing the fusion protein of Ebony and Sfp (Sfp-glycine-serine-glycine-Ebony, SGE) in Escherichia coli W3110 (WSGE strain) is significantly higher (7.45 mM) than the combinatorial strain of pET28a-ebony and pACYCDuet-sfp in E. coli BL21(DE3) (BSE strain) (2.17 mM). Under the optimal reaction conditions (25 ℃, pH 7.0, 12.5 g/L wet cells, 20 mM β-alanine and 40 mM histamine), the carcinine can be quickly synthesized within 24 h up to a concentration of 22.63 mM. To achieve a continuous and efficient conversion of the precursors, a batch-feeding catalysis was designed. With this system, β-alanine (40 mM) and histamine (40 mM) could be completely transformed to carcinine (40.34 mM) in 36 h with a productivity of 0.204 g/L h reaching a titer of 7.34 g/L. Hence, the batch-feeding whole-cell biocatalysis is a promising technology for the high yield production of carcinine which can promote the industrial production of carcinine.
Histidine dipeptides are key regulators of excitation-contraction coupling in cardiac muscle: Evidence from a novel CARNS1 knockout rat model
2021, Redox BiologyHistidine-containing dipeptides (HCDs) are abundantly expressed in striated muscles. Although important properties have been ascribed to HCDs, including H+ buffering, regulation of Ca2+ transients and protection against oxidative stress, it remains unknown whether they play relevant functions in vivo. To investigate the in vivo roles of HCDs, we developed the first carnosine synthase knockout (CARNS1−/−) rat strain to investigate the impact of an absence of HCDs on skeletal and cardiac muscle function. Male wild-type (WT) and knockout rats (4 months-old) were used. Skeletal muscle function was assessed by an exercise tolerance test, contractile function in situ and muscle buffering capacity in vitro. Cardiac function was assessed in vivo by echocardiography and cardiac electrical activity by electrocardiography. Cardiomyocyte contractile function was assessed in isolated cardiomyocytes by measuring sarcomere contractility, along with the determination of Ca2+ transient. Markers of oxidative stress, mitochondrial function and expression of proteins were also evaluated in cardiac muscle. Animals were supplemented with carnosine (1.8% in drinking water for 12 weeks) in an attempt to rescue tissue HCDs levels and function. CARNS1−/− resulted in the complete absence of carnosine and anserine, but it did not affect exercise capacity, skeletal muscle force production, fatigability or buffering capacity in vitro, indicating that these are not essential for pH regulation and function in skeletal muscle. In cardiac muscle, however, CARNS1−/− resulted in a significant impairment of contractile function, which was confirmed both in vivo and ex vivo in isolated sarcomeres. Impaired systolic and diastolic dysfunction were accompanied by reduced intracellular Ca2+ peaks and slowed Ca2+ removal, but not by increased markers of oxidative stress or impaired mitochondrial respiration. No relevant increases in muscle carnosine content were observed after carnosine supplementation. Results show that a primary function of HCDs in cardiac muscle is the regulation of Ca2+ handling and excitation-contraction coupling.
L-histidine and L-carnosine accelerate wound healing via regulation of corticosterone and PI3K/Akt phosphorylation in D-galactose-induced aging models in vitro and in vivo
2019, Journal of Functional FoodsImpaired skin wound healing in the elderly can lead to medical issues and increased mortality. Although l-histidine and l-carnosine are potent anti-aging amino acids, the wound healing effects of these amino acids in aging remain to be elucidated. Here, we investigated the regenerative potential of l-histidine and l-carnosine in in vitro and in vivo aging models. l-histidine (1 mM), l-carnosine (10 mM), or a combination improved proliferation, migration, senescence, and epithelial-mesenchymal transition (EMT) in d-galactose-induced aged keratinocytes. An in vivo mouse aging model was established with injection of d-galactose (s.c. 500 mg/kg) daily for eight weeks. Supplementation with l-histidine (2 g/L), l-carnosine (2 g/L), or a combination improved collagen and wound healing with EMT markers, E-cadherin, N-cadherin, and MMP-2. These effects were concomitant with reduced circulating levels of corticosterone and increased PI3K/Akt phosphorylation. These results suggest that l-histidine and l-carnosine have the potential to facilitate wound healing in aging skin.
Pivotal role of carnosine in the modulation of brain cells activity: Multimodal mechanism of action and therapeutic potential in neurodegenerative disorders
2019, Progress in NeurobiologyCarnosine (β-alanyl-l-histidine), a dipeptide, is an endogenous antioxidant widely distributed in excitable tissues like muscles and the brain. Although discovered more than a hundred years ago and having been extensively studied in the periphery, the role of carnosine in the brain remains mysterious. Carnosinemia, a rare metabolic disorder with increased levels of carnosine in urine and low levels or absence of carnosinase in the blood, is associated with severe neurological symptoms in humans. This review deals with the role of carnosine in the brain in both physiological and pathological conditions, with a focus on preclinical evidence suggesting a high therapeutic potential of carnosine in neurodegenerative disorders. We review carnosine and carnosinemia’s discoveries and the extensive research on the role and benefits of carnosine in the periphery. We then turn to carnosine’s biochemistry and distribution in the brain. Using an array of recent observations as a foundation, we draw a parallel with the role of carnosine in muscles and speculate on the role of carnosine in promoting the metabolic support of neurons by glial cells. Finally, carnosine has been shown to exert a multimodal activity including inhibition of protein cross-linking and aggregation of amyloid-β and related proteins, free radical generation, nitric oxide detoxification, and an anti-inflammatory activity. It could thus play an important role in the prevention and treatment of neurodegenerative diseases such as Alzheimer’s disease. We discuss the potential of carnosine in this context and speculate on new preclinical research directions.
β-Alanine supplementation reduces anxiety and increases neurotrophin expression in both young and older rats
2019, Nutrition ResearchThe effect of 30 days of β-alanine supplementation (100 mg/kg) on behavioral response and expression of brain-derived neurotrophic factor (BDNF), neuropeptide Y (NPY), and markers of inflammation was examined in both young (4 months) and older (14 months) rats. We hypothesized that animals fed β-alanine would experience reduced inflammation and an enhanced neurotrophin and behavioral response. Animals were assigned to either a control group, in which young or older rats were fed regular chow and water, or a β-alanine group, in which rats were fed regular chow and provided β-alanine in their water. Behavior measures were conducted following the 30-day supplementation period, which included spatial learning, memory, and an anxiety index. Hippocampal expressions of BDNF, NPY, glial fibrillary acidic protein, nuclear factor–κB p50 and p65 subunits, tumor necrosis factor–α, and cyclooxygenase-2 were also analyzed. Learning ability was reduced (P = .001) and anxiety index was higher (P = .001) in older compared to young rats. Similarly, BDNF and NPY expressions were reduced and all inflammatory markers were elevated (P < .05) in the older animals. β-Alanine increased BDNF expressions in the cornu ammonis area 1 (P = .003) and 3 (P < .001) subregions of the hippocampus. BDNF expression for younger rats in the β-alanine group was also significantly greater than younger rats in the control group in cornu ammonis area 3. Learning for young animals fed β-alanine was significantly better than all other groups. Significant reductions in anxiety were noted in both older and younger rats fed β-alanine compared to age-matched controls. Results indicated that β-alanine ingestion in both young and older rats was effective in attenuating anxiety and augmenting BDNF expression in the hippocampus.
Carnosine research in relation to aging brain and neurodegeneration: A blessing for geriatrics and their neuronal disorders
2020, Archives of Gerontology and GeriatricsCarnosine, an endogenous dipeptide (β-Ala-l-His), is enriched in prefrontal cortex and olfactory bulb of the brain, blood and also in muscle. It has mainly antioxidant and antiglycating properties which makes this molecule unique. Its content reduces during aging and aging-induced neurodegenerative diseases. Aging is a progressive biological process that leads to develop the risk factors of diseases and death. During aging the morphological, biochemical, cellular and molecular changes occur in brain and blood including other tissues. The objective of this review is to combine the updated information from the existing literature about the aging-induced neurodegeneration and carnosine research to meet the lacuna of mechanism of carnosine. The grey matter and white matter loses its normal ratio in aging, and hence the brain volume and weight. Different aging related neurodegenerative disorders arise due to loss of neurons, and synapses as a result of proteinopathies in some cases. Carnosine, being an endogenous biomolecule and having antioxidant, antiglycating properties has shown its potency to counteract erroneous protein biosynthesis, stress, activated microglial and astrocyte activity, and different neurodegenerative disorders. It (carnosine) can also inhibit the metal ion-induced degeneration by acting as a metal chelator. In this review the trends in carnosine research in relation to aging brain and neurodegeneration have been discussed with a view to its (carnosine) eligibility (including its mechanism of action) to be used as a promising neurotherapeutic for the betterment of elderly populations of our society at the national and international levels in near future.