Oral Administration of Nicotinamide Mononucleotide Increases Nicotinamide Adenine Dinucleotide Level in an Animal Brain

As a redox-sensitive coenzyme, nicotinamide adenine dinucleotide (NAD⁺) plays a central role in cellular energy metabolism and homeostasis. Low NAD⁺ levels are linked to multiple disease states, including age-related diseases, such as metabolic and neurodegenerative diseases. Consequently, restoring/increasing NAD⁺ levels in vivo has emerged as an important intervention targeting age-related neurodegenerative diseases. One of the widely studied approaches to increase NAD⁺ levels in vivo is accomplished by using NAD⁺ precursors, such as nicotinamide mononucleotide (NMN). Oral administration of NMN has been shown to successfully increase NAD⁺ levels in a variety of tissues; however, it remains unclear whether NMN can cross the blood–brain barrier to increase brain NAD⁺ levels. This study evaluated the effects of oral NMN administration on NAD⁺ levels in C57/B6J mice brain tissues. Our results demonstrate that oral gavage of 400 mg/kg NMN successfully increases brain NAD⁺ levels in mice after 45 min. These findings provide evidence that NMN may be used as an intervention to increase NAD⁺ levels in the brain.     

Age-Dependent Decline of NAD+—Universal Truth or Confounded Consensus?

Nicotinamide adenine dinucleotide (NAD⁺) is an essential molecule involved in various metabolic reactions, acting as an electron donor in the electron transport chain and as a co-factor for NAD⁺-dependent enzymes. In the early 2000s, reports that NAD⁺ declines with aging introduced the notion that NAD⁺ metabolism is globally and progressively impaired with age. Since then, NAD⁺ became an attractive target for potential pharmacological therapies aiming to increase NAD⁺ levels to promote vitality and protect against age-related diseases. This review summarizes and discusses a collection of studies that report the levels of NAD⁺ with aging in different species (i.e., yeast, C. elegans, rat, mouse, monkey, and human), to determine whether the notion that overall NAD⁺ levels decrease with aging stands true. We find that, despite systematic claims of overall changes in NAD⁺ levels with aging, the evidence to support such claims is very limited and often restricted to a single tissue or cell type. This is particularly true in humans, where the development of NAD⁺ levels during aging is still poorly characterized. There is a need for much larger, preferably longitudinal, studies to assess how NAD⁺ levels develop with aging in various tissues. This will strengthen our conclusions on NAD metabolism during aging and should provide a foundation for better pharmacological targeting of relevant tissues.     

Effect of H<Subscript>2</Subscript>S on the circadian rhythm of mouse hepatocytes

Background  

Dysregulation of circadian rhythms can contribute to diseases of lipid metabolism. NAD-dependent deacetylase sirtuin-1(SIRT1) is an important hub which links lipid metabolism with circadian clock by its deacetylation activity depends on intracellular NAD⁺/NADH content ratio. Hydrogen sulfide (H₂S) is an endogenous reductant which can affect the intracellular redox state. Therefore, we hypothesized that exogenous H₂S can affect the expression of circadian clock genes mediated by sirt1 thereby affecting body's lipid metabolism. And also because the liver is a typical peripheral circadian clock oscillator that is intimately linked to lipid metabolism. Thus the effect of H₂S were observed on 24-hour dynamic expression of 4 central circadian clock genes and sirt1gene in primary cultured hepatocytes.     

Nicotinamide Riboside and Dihydronicotinic Acid Riboside Synergistically Increase Intracellular NAD+ by Generating Dihydronicotinamide Riboside

Through evolution, eukaryote organisms have developed the ability to use different molecules as independent precursors to generate nicotinamide adenine dinucleotide (NAD⁺), an essential molecule for life. However, whether these different precursors act in an additive or complementary manner is not truly well understood. Here, we have evaluated how combinations of different NAD⁺ precursors influence intracellular NAD⁺ levels. We identified dihydronicotinic acid riboside (NARH) as a new NAD⁺ precursor in hepatic cells. Second, we demonstrate how NARH, but not any other NAD⁺ precursor, can act synergistically with nicotinamide riboside (NR) to increase NAD⁺ levels in cultured cells and in mice. Finally, we demonstrate that the large increase in NAD⁺ prompted by the combination of these two precursors is due to their chemical interaction and conversion to dihydronicotinamide riboside (NRH). Altogether, this work demonstrates for the first time that NARH can act as a NAD⁺ precursor in mammalian cells and how different NAD⁺ precursors can interact and influence each other when co-administered.     

Cross-Sectional Associations between Dietary Daily Nicotinamide Intake and Patient-Reported Outcomes in Colorectal Cancer Survivors, 2 to 10 Years Post-Diagnosis

Supplementation with nicotinamide adenine dinucleotide (NAD⁺) precursors including dietary nicotinamide has been found to boost tissue NAD⁺ levels and ameliorate oxidative stress-induced damage that contributes to aging and aging-related diseases. The association between dietary NAD⁺ precursors and patient-reported health-related outcomes in cancer survivors has not been investigated. This study aimed to determine associations of dietary nicotinamide intake with different patient-reported outcomes in colorectal cancer survivors, 2 to 10 years post-diagnosis. A total of 145 eligible participants were recruited into this cross-sectional study. Dietary nicotinamide intake level was calculated based on data from 7-day food diaries. Fatigue was assessed with the Checklist Individual Strength (CIS), which is a subscale of the cancer-specific European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC), and anxiety and depression were assessed with Hospital Anxiety and Depression Scale (HADS). Oxidative stress marker serum protein carbonyl contents and serum NAD⁺ levels were measured. A hierarchical linear regression model with confounder adjustment was performed to analyze the association of nicotinamide intake, serum protein carbonyl contents, and NAD⁺ levels with patient-reported outcomes. The median values of daily nicotinamide intake for male and female participants were 19.1 and 14.4 mg, respectively. Daily dietary nicotinamide intake was associated with a lower level of fatigue (β: −14.85 (−28.14, −1.56)) and a lower level of anxiety and depression (β: −4.69 (−8.55, −0.83)). Subgroup analyses by sex showed that a beneficial association between nicotinamide intake and patient-reported outcomes was mainly found in men. To conclude, our findings suggested that higher dietary NAD⁺ precursor nicotinamide intake was cross-sectionally associated with less patient-reported outcomes in CRC survivors.     

A Combination of Nicotinamide and D-Ribose (RiaGev) Is Safe and Effective to Increase NAD+ Metabolome in Healthy Middle-Aged Adults: A Randomized,Triple-Blind,Placebo-Controlled,Cross-Over Pilot Clinical Trial

Nicotinamide adenine dinucleotide (NAD⁺) is an essential cofactor required for proper functioning of all cells and its decline is correlated with advancing age and disease. This randomized, triple-blind, placebo-controlled, crossover pilot study assessed the efficacy and safety of a combination of nicotinamide with D-ribose (RiaGev) for NAD metabolome enhancement and related benefits in healthy middle-aged adults. Supplementing with 1520 mg RiaGev twice daily for 7 days significantly increased the NAD⁺ metabolome in blood, especially NADP⁺ by 27% compared to the placebo group (p = 0.033) and over the baseline (p = 0.007). Increases in glutathione and high energy phosphates were also observed in the blood. Seven-day supplementation with RiaGev significantly (p = 0.013) reduced overall blood glucose without significant changes in insulin secretion (p = 0.796), suggesting an improved insulin sensitivity and glucose tolerance. The waking salivary cortisol of the subjects steadily and significantly decreased (p = 0.026) in the RiaGev group in contrast to the placebo. Subjects in the RiaGev group showed less fatigue, improved mental concentration and motivation over the baseline (p = 0.015, 0.018, and 0.012, respectively) as observed through the Checklist Individual Strength (CIS) questionnaire. There were no clinically relevant adverse events, or alterations in hematology, electrolytes, liver, and kidney markers pre- and post-supplementation. RiaGev appears to be safe and efficacious in increasing NAD⁺ metabolome in healthy middle-aged adults, as shown by this study.     

Role of CD38 in Adipose Tissue: Tuning Coenzyme Availability?

Nicotinamide adenine dinucleotide (NAD⁺) is a fundamental molecule in the regulation of energy metabolism, representing both a coenzyme and a substrate for different NAD⁺ degrading enzymes. Among these enzymes, CD38 can be seen under two perspectives: as the enzyme synthesizing Ca²⁺-mobilizing second messenger, starting from NAD⁺, and as the major NAD⁺-consumer, to be inhibited to increase NAD⁺ levels. Indeed, the regulation of NAD⁺ availability is a key event during different processes. In this review, we examine the recent studies related to the modulation of CD38 expression and activity, and the consequent changes in NAD(P)(H), in adipose tissue, during inflammation and cold-induced thermogenesis.     

Dietary Cocoa Flavanols Enhance Mitochondrial Function in Skeletal Muscle and Modify Whole-Body Metabolism in Healthy Mice

Mitochondrial dysfunction is widely reported in various diseases and contributes to their pathogenesis. We assessed the effect of cocoa flavanols supplementation on mitochondrial function and whole metabolism, and we explored whether the mitochondrial deacetylase sirtuin-3 (Sirt3) is involved or not. We explored the effects of 15 days of CF supplementation in wild type and Sirt3^-/- mice. Whole-body metabolism was assessed by indirect calorimetry, and an oral glucose tolerance test was performed to assess glucose metabolism. Mitochondrial respiratory function was assessed in permeabilised fibres and the pyridine nucleotides content (NAD⁺ and NADH) were quantified. In the wild type, CF supplementation significantly modified whole-body metabolism by promoting carbohydrate use and improved glucose tolerance. CF supplementation induced a significant increase of mitochondrial mass, while significant qualitative adaptation occurred to maintain H₂O₂ production and cellular oxidative stress. CF supplementation induced a significant increase in NAD⁺ and NADH content. All the effects mentioned above were blunted in Sirt3^-/- mice. Collectively, CF supplementation boosted the NAD metabolism that stimulates sirtuins metabolism and improved mitochondrial function, which likely contributed to the observed whole-body metabolism adaptation, with a greater ability to use carbohydrates, at least partially through Sirt3.     

Colon carcinogenesis and the role of dehydrogenase enzyme activity: Inhibition of tumorigenesis by pyrazole

Dimethylhydrazine and the products of its metabolism, azoxymethane and methylazoxymethanol, are potent inducers of colon tumors in experimental animals. The marked sensitivity of the colon to these substances, as compared with the other segments of intestine, appears to be related to the presence of dehydrogenase enzymes in colonic epithelium able to activate methylazoxymethanol. Results of several studies indicate that the organotropic effects of this carcinogen are related to NAD⁺/NADP⁺-dependent dehydrogenase enzyme activity and that the product of this reaction, the aldehydic form of methylazoxymethanol, is the agent responsible for the observed biologic effects. Rats treated with pyrazole, an inhibitor of NAD⁺-dependent alcohol dehydrogenase, prior to their receiving methylazoxymethanol, are protected against the acute toxicity induced by this agent and do not develop any intestinal tumors. In related studies aimed at determining the mechanism of resistance to colon tumor induction in Lobund Wistar rats, it was found that this phenomenon is not due to differences in metabolic activation of carcinogen.     

The effect of antioxidant supplementation on fatigue during exercise: potential role for NAD+(H)

This study compared serum pyridine levels (NAD⁺ /NADH) in trained (n = 6) and untrained (n = 7) subjects after continuous progressive exercise at 50%, 70% then 95% of physical work capacity until fatigue (TTF) after consuming a placebo or antioxidant (AO) cocktail (Lactaway^©). An increase of 17% in TTF was observed in AO as compared to placebo (p = 0.032). This was accompanied by a significant increase in serum NAD⁺ levels (p = 0.037) in the AO supplemented group post exercise. The increases in NAD⁺ and improved endurance reflect lower oxidative stress-induced suppression of aerobic respiration.     

Nicotinamide Riboside for the Prevention and Treatment of Doxorubicin Cardiomyopathy. Opportunities and Prospects

Despite the progress in the development of new anticancer strategies, cancer is rapidly spreading around the world and remains one of the most common diseases. For more than 40 years, doxorubicin has been widely used in the treatment of solid and hematological tumors. At the same time, the problem of its cardiotoxicity remains unresolved, despite the high efficiency of this drug. Symptomatic therapy is used as a treatment for side-effects of doxorubicin or pathological conditions that have already appeared in their background. To date, there are no treatment methods for doxorubicin cardiomyopathy as such. A drug such as nicotinamide riboside can play an important role in solving this problem. Nicotinamide riboside is a pyridine nucleoside similar to vitamin B3 that acts as a precursor to NAD⁺. There is no published research on nicotinamide riboside effects on cardiomyopathy, despite the abundance of works devoted to the mechanisms of its effects in various pathologies. The review analyzes information about the effects of nicotinamide riboside on various experimental models of pathologies, its role in the synthesis of NAD⁺, and also considers the possibility and prospects of its use for the prevention of doxorubicin cardiomyopathy.     

Design, synthesis and X-ray crystallographic study of NAmPRTase inhibitors as anti-cancer agents

NAmPRTase (PBEF/Visfatin) plays a pivotal role in the salvage pathway of NAD⁺ biosynthesis. NAmPRTase has been an attractive target for anti-cancer agents that induce apoptosis of tumor cells via a declining plasma NAD⁺ level. In this report, a series of structural analogs of FK866 (1), a known NAmPRTase inhibitor, was synthesized and tested for inhibitory activities against the proliferation of cancer cells and human NAmPRTase. Among them, compound 7 showed similar anti-cancer and enzyme inhibitory activities to compound 1. Further investigation of compound 7 with X-ray analysis revealed a co-crystal structure in complex with human NAmPRTase, suggesting that Asp219 in the active site of the enzyme could contribute to an additional interaction with the pyrrole nitrogen of compound 7.     

Synergistic Effect of β-Lapachone and Aminooxyacetic Acid on Central Metabolism in Breast Cancer

The compound β-lapachone, a naturally derived naphthoquinone, has been utilized as a potent medicinal nutrient to improve health. Over the last twelve years, numerous reports have demonstrated distinct associations of β-lapachone and NAD(P)H: quinone oxidoreductase 1 (NQO1) protein in the amelioration of various diseases. Comprehensive research of NQO1 bioactivity has clearly confirmed the tumoricidal effects of β-lapachone action through NAD⁺-keresis, in which severe DNA damage from reactive oxygen species (ROS) production triggers a poly-ADP-ribose polymerase-I (PARP1) hyperactivation cascade, culminating in NAD⁺/ATP depletion. Here, we report a novel combination strategy with aminooxyacetic acid (AOA), an aspartate aminotransferase inhibitor that blocks the malate-aspartate shuttle (MAS) and synergistically enhances the efficacy of β-lapachone metabolic perturbation in NQO1⁺ breast cancer. We evaluated metabolic turnover in MDA-MB-231 NQO1⁺, MDA-MB-231 NQO1⁻, MDA-MB-468, and T47D cancer cells by measuring the isotopic labeling of metabolites from a [U-¹³C]glucose tracer. We show that β-lapachone treatment significantly hampers lactate secretion by ~85% in NQO1⁺ cells. Our data demonstrate that combinatorial treatment decreases citrate, glutamate, and succinate enrichment by ~14%, ~50%, and ~65%, respectively. Differences in citrate, glutamate, and succinate fractional enrichments indicate synergistic effects on central metabolism based on the coefficient of drug interaction. Metabolic modeling suggests that increased glutamine anaplerosis is protective in the case of MAS inhibition.     

The Role of Palmitoleic Acid in Regulating Hepatic Gluconeogenesis through SIRT3 in Obese Mice

Hepatic gluconeogenesis is a crucial process to maintain glucose level during starvation. However, unabated glucose production in diabetic patients is a major contributor to hyperglycemia. Palmitoleic acid is a monounsaturated fatty acid (16:1n7) that is available from dietary sources. Palmitoleic acid exhibits health beneficial effects on diabetes, insulin resistance, inflammation, and metabolic syndrome. However, the mechanism by which palmitoleate reduces blood glucose is still unclear. SIRT3 is a key metabolism-regulating NAD⁺-dependent protein deacetylase. It is known that fasting elevates the expression of SIRT3 in the liver and it regulates many aspects of liver’s response to nutrient deprivation, such as fatty acid oxidation and ketone body formation. However, it is unknown whether SIRT3 also regulates gluconeogenesis. Our study revealed that palmitoleic acid reduced hepatic gluconeogenesis and the expression of SIRT3 under high-fat diet conditions. Overexpression of SIRT3 in the liver and hepatocytes enhanced gluconeogenesis. Further study revealed that SIRT3 played a role in enhancing the activities of gluconeogenic enzymes, such as PEPCK, PC, and MDH2. Therefore, our study indicated that under a high-fat diet, palmitoleic acid decreased gluconeogenesis by reducing enzymatic activities of PEPCK, PC, and MDH2 by down-regulating the expression of SIRT3.     

Nicotinamide Riboside Supplementation to Suckling Male Mice Improves Lipid and Energy Metabolism in Skeletal Muscle and Liver in Adulthood

Nicotinamide riboside, an NAD⁺ precursor, has been attracting a lot of attention in recent years due to its potential benefits against multiple metabolic complications and age-related disorders related to NAD⁺ decline in tissues. The metabolic programming activity of NR supplementation in early-life stages is much less known. Here, we studied the long-term programming effects of mild NR supplementation during the suckling period on lipid and oxidative metabolism in skeletal muscle and liver tissues using an animal model. Suckling male mice received a daily oral dose of NR or vehicle (water) from day 2 to 20 of age, were weaned at day 21 onto a chow diet, and at day 90 were distributed to either a high-fat diet (HFD) or a normal-fat diet for 10 weeks. Compared to controls, NR-treated mice were protected against HFD-induced triacylglycerol accumulation in skeletal muscle and displayed lower triacylglycerol levels and steatosis degree in the liver and distinct capacities for fat oxidation and decreased lipogenesis in both tissues, paralleling signs of enhanced sirtuin 1 and AMP-dependent protein kinase signaling. These pre-clinical findings suggest that mild NR supplementation in early postnatal life beneficially impacts lipid and energy metabolism in skeletal muscle and liver in adulthood, serving as a potential preventive strategy against obesity-related disorders characterized by ectopic lipid accumulation.     

A Theacrine-Based Supplement Increases Cellular NAD+ Levels and Affects Biomarkers Related to Sirtuin Activity in C2C12 Muscle Cells In Vitro

There is evidence in rodents to suggest that theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (termed NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~2 mM theacrine) for 3 and 24 h (n = 6 treatment wells per time point). Relative to control (CTL)-treated cells, NAD3 treatments increased (p < 0.05) Sirt1 mRNA levels at 3 h, as well as global sirtuin activity at 3 and 24 h. Follow-up experiments comparing 24 h NAD3 or CTL treatments indicated that NAD3 increased nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 protein levels (p < 0.05). Cellular nicotinamide adenine dinucleotide (NAD⁺) levels were also elevated nearly two-fold after 24 h of NAD3 versus CTL treatments (p < 0.001). Markers of mitochondrial biogenesis were minimally affected. Although these data are limited to select biomarkers in vitro, these preliminary findings suggest that a theacrine-based supplement can modulate select biomarkers related to NAD⁺ biogenesis and sirtuin activity. However, these changes did not drive increases in mitochondrial biogenesis. While promising, these data are limited to a rodent cell line and human muscle biopsy studies are needed to validate and elucidate the significance of these findings.     

Phenotype of the anti-Rickettsia CD8 T cell response suggests cellular correlates of protection for the assessment of novel antigens

The obligately intracellular bacteria Rickettsia infect endothelial cells and cause systemic febrile diseases that are potentially lethal. No vaccines are currently available and current knowledge of the effective immune response is limited. Natural and experimental rickettsial infections provide strong and cross-protective cellular immunity if the infected individual survives the acute infection. Although resistance to rickettsial infections is attributed to the induction of antigen-specific T cells, particularly CD8⁺ T cells, the identification and validation of correlates of protective cellular immunity against rickettsial infections, an important step toward vaccine validation, remains a gap in this field. Here, we show that after a primary challenge with Rickettsia typhi in the C3H mouse model, the peak of anti-Rickettsia CD8⁺ T cell-mediated responses occurs 7 days post-infection (dpi), which coincides with the beginning of rickettsial clearance. At this time point, both effector-type and memory-type CD8⁺ T cells are present, suggesting that 7 dpi is a valid time point for the assessment of CD8⁺ T cell responses of mice previously immunized with protective antigens. Based on our results, we suggest four correlates of cellular protection for the assessment of protective rickettsial antigens: (1) production of IFN-γ by antigen-experienced CD3⁺CD8⁺CD44^high cells, (2) production of Granzyme B by CD27^lowCD43^low antigen-experienced CD8⁺ T cells, (3) generation of memory-type CD8⁺ T cells [Memory Precursor Effector Cells (MPECs), as well as CD127^highCD43^low, and CD27^highCD43^low CD8⁺ T cells], and (4) generation of effector-like memory CD8⁺ T cells (CD27^lowCD43^low). We propose that these correlates could be useful for the general assessment of the quality of the CD8⁺ T cell immune response induced by novel antigens with potential use in a vaccine against Rickettsia.     

Niacin Deficiency Alters p53 Expression and Impairs Etoposide-Induced Cell Cycle Arrest and Apoptosis in Rat Bone Marrow Cells

One focus of chemoprevention research is the interaction of nutrients with specific molecular targets associated with the maintenance of genomic stability. This study tested the impact of dietary niacin status on bone marrow NAD⁺ and poly(ADP-ribose) (pADPr) levels, p53 expression, and etoposide (ETO)-induced apoptosis and cell cycle arrest. After 3 wk on niacin-deficient (ND), pair-fed niacin-replete (PF), or nicotinic acid–supplemented (4 g/kg diet) (NA) diets, Long-Evans rats were gavaged with ETO (25 mg/kg) or vehicle. ND and NA diets caused a 72% decrease and a 240% increase in bone marrow NAD⁺, respectively. Basal and ETO-induced pADPr levels differed dramatically among ND, PF, and NA diets (undetectable, 42 and 216 fmol/million cells, respectively; basal and undetectable, 119 and 484 fmol/million cells, respectively, following ETO). ND diet alone caused overexpression of two distinct isoforms of p53. Levels of p53 in PF and NA marrow increased in response to ETO treatment, but this did not occur in ND bone marrow. Quantitative polymerase chain reaction of regular and alternative spliced variants of p53 mRNA revealed that niacin deficiency actually decreased both forms of p53 message, implicating protein stability in the accumulation of p53 in ND marrow. ETO-induced apoptosis (TUNEL) was suppressed during niacin deficiency and enhanced by supplementation. G1 arrest was also impaired in ND bone marrow relative to PF and NA. Despite a poor G1 arrest, p21 ^waf1 was overexpressed in the ND bone marrow and dramatically induced following ETO treatment. In conclusion, dietary niacin deficiency causes changes in NAD⁺ and pADPr metabolism, alters p53 expression, and impairs cellular responses to DNA damage.     

Neurotensin- and EGF-Induced Metabolic Activation of Colon Carcinoma Cells Is Diminished by Dietary Flavonoid Cyanidin but Not by Its Glycosides

Dietary polyphenols, including anthocyanidins and their glycosides anthocyanins, are suggested to be involved in the protective effects of fruits and vegetables against cancer. Very few data are available concerning the effects of anthocyanidins/anthocyanins on cellular processes induced by growth factors such as neurotensin and epidermal growth factor (EGF), which are implicated in the pathophysiology of colon cancer. Here, we show that neurotensin and EGF caused an increase in the extracellular acidification rate, which could reflect the activity of cellular metabolism, in the human carcinoma cell line HT29 clone 19A. Neurotensin and EGF also caused a strong rise in the intracellular Ca²⁺concentration, induced phosphorylation of extracellular signal-regulated kinases (ERK1 and ERK2), and stimulated growth of human carcinoma cells. Cyanidin (10 μM), but not its glycosides cyanin and idaein, was able to inhibit the neurotensin- and EGF-induced increased rate of extracellular acidification. In contrast to N-ethyl-N-isopropyl amiloride, an inhibitor of Na⁺/H⁺ exchange, cyanidin did not alter the rate of intracellular pH recovery of cells loaded by NH₃/NH⁴⁺, indicating that cyanidin inhibits cellular metabolism, rather than directly altering Na⁺/H⁺ exchange. Cyanidin, but not cyanin and idaein, was able to inhibit an increase in intracellular Ca²⁺ concentration induced by neurotensin. Neurotensin- and EGF-induced phosphorylation of ERKs was not affected by cyanidin, cyanin, and idaein at ?100 μM. Only cyanidin (100 μM), but not cyanin and idaein, was able to inhibit cellular growth induced by EGF. Thus these findings suggest that a dietary polyphenol cyanidin, but not its glycosides, is a potent inhibitor of mitogen-induced metabolic activity, increase in free intracellular Ca²⁺, and cellular growth of cultured colon carcinoma cells.