Although transplantation of c-kit+ cardiac stem cells (CSCs) has been shown to alleviate left ventricular (LV) dysfunction induced by myocardial infarction (MI), the number of exogenous CSCs ...remaining in the recipient heart following transplantation and their mechanism of action remain unclear. We have previously developed a highly sensitive and accurate method to quantify the absolute number of male murine CSCs in female recipient organs after transplantation. In the present study, we used this method to monitor the number of donor CSCs in the recipient heart after intracoronary infusion. Female mice underwent a 60-min coronary occlusion followed by reperfusion; 2 days later, 100,000 c-kit+/lin- syngeneic male mouse CSCs were infused intracoronarily. Only 12.7% of the male CSCs present in the heart immediately (5 min) after infusion were still present in the heart at 24 h, and their number declined rapidly thereafter. By 35 days after infusion, only ∼ 1,000 male CSCs were found in the heart. Significant numbers of male CSCs were found in the lungs and kidneys, but only in the first 24 h. The number of CSCs in the lungs increased between 5 min and 24 h after infusion, indicating recirculation of CSCs initially retained in other organs. Despite the low retention and rapid disappearance of CSCs from the recipient heart, intracoronary delivery of CSCs significantly improved LV function at 35 days (Millar catheter). These results suggest that direct differentiation of CSCs alone cannot account for the beneficial effects of CSCs on LV function; therefore, paracrine effects must be the major mechanism. The demonstration that functional improvement is dissociated from survival of transplanted cells has major implications for our understanding of cell therapy. In addition, this new quantitative method of stem cell measurement will be useful in testing approaches of enhancing CSC engraftment and survival after transplantation.
A novel haplotype in N -acetyltransferase 2 ( NAT2 ) composed of seven non-coding variants (rs1495741, rs4921913, rs4921914, rs4921915, rs146812806, rs35246381, and rs35570672) has been linked to ...dyslipidemia by multiple, independent genome-wide association studies. The haplotype is located approximately 14 kb downstream of NAT2-coding region (ch8:18,272,377-18,272,881; GRCh38/hg38) and represents a non-coding, intergenic haplotype. Interestingly, the same dyslipidemia NAT2 haplotype is also linked to urinary bladder cancer risk. Dyslipidemia risk alleles are associated with rapid acetylator phenotype, whereas bladder cancer risk alleles are associated with slow acetylator, suggesting that the level of systemic NAT2 activity modifies the risk of these pathologies. We speculate that rs1495741 (and its associated haplotype) belongs to a distal regulatory element of human NAT2 gene (e.g., enhancer or silencer), and the genetic variation at the newly discovered haplotype results in a differential level of NAT2 gene expression. Understanding how this NAT2 haplotype contributes to not only urinary bladder cancer but also to dyslipidemia will ultimately help devise strategies to identify and protect susceptible individuals.
RATIONALE:The effects of c-kit cardiac progenitor cells (CPCs, and adult cell therapy in general) on left ventricular (LV) function have been regarded as modest or inconsistent.
OBJECTIVE:To ...determine whether 3 CPC infusions have greater efficacy than 1 infusion.
METHODS AND RESULTS:Rats with a 30-day-old myocardial infarction received 1 or 3 CPC infusions into the LV cavity, 35 days apart. Compared with vehicle-treated rats, the single-dose group exhibited improved LV function after the first infusion (consisting of CPCs) but not after the second and third (vehicle). In contrast, in the multiple-dose group, regional and global LV function improved by a similar degree after each CPC infusion, resulting in greater cumulative effects. For example, the total increase in LV ejection fraction was approximately triple in the multiple-dose group versus the single-dose group (P<0.01). The multiple-dose group also exhibited more viable tissue and less scar, less collagen in the risk and noninfarcted regions, and greater myocyte density in the risk region.
CONCLUSIONS:This is the first demonstration that repeated CPC administrations are markedly more effective than a single administration. The concept that the full effects of CPCs require repeated doses has significant implications for both preclinical and clinical studies; it suggests that the benefits of cell therapy may be underestimated or even overlooked if they are measured after a single dose, and that repeated administrations are necessary to evaluate the effectiveness of a cell product properly. In addition, we describe a new method that enables studies of repeated cell administrations in rodents.
Heterocyclic amines (HCAs) are mutagenic compounds found in cooked meat. Recent epidemiological studies reported significant associations between dietary HCA exposure and insulin resistance and type ...II diabetes, and we recently reported that HCAs induce insulin resistance and glucose production in human hepatocytes. It is well known that HCAs require hepatic bioactivation by cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase 2 (NAT2). NAT2 expresses a well-defined genetic polymorphism in humans that, depending on the combination of NAT2 alleles, correlates to rapid, intermediate, or slow acetylator phenotype that exhibits differential metabolism of aromatic amines and HCAs. No previous studies have examined the role of NAT2 genetic polymorphism in the context of HCA-mediated induction of glucose production. In the present study, we assessed the effect of three HCAs commonly found in cooked meat (2-amino-3,4-dimethylimidazo4,5-fquinoline MeIQ, 2-amino-3,8-dimethylimidazo4,5-fquinoxaline MeIQx, and 2-amino-1-methyl-6-phenylimidazo4,5-bpyridine PhIP) on glucose production in cryopreserved human hepatocytes with slow, intermediate, or rapid NAT2 acetylator phenotype. HCA treatment did not affect glucose production in slow NAT2 acetylator hepatocytes, while a slight increase in glucose production was observed in intermediate NAT2 acetylators treated with MeIQ or MeIQx. However, significant increases in glucose production were observed in rapid NAT2 acetylators following each HCA. The current findings suggest that individuals who are rapid NAT2 acetylators may be at a greater risk of developing hyperglycemia and insulin resistance following dietary exposure to HCAs.
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•HCAs promote insulin resistance and glucose production in human hepatocytes.•HCA-induced glucose production required N-acetyltransferase 2 (NAT2) metabolism.•Rapid NAT2 acetylators may be more susceptible to the metabolic effect of HCAs.
Arylamine N-acetyltransferase 2 (NAT2) is a phase II metabolic enzyme, best known for metabolism of aromatic amines and hydrazines. Genetic variants occurring in the NAT2 coding region have been ...well-defined and are known to affect the enzyme activity or protein stability. Individuals can be categorized into rapid, intermediate, and slow acetylator phenotypes that significantly alter their ability to metabolize arylamines, including drugs (e.g., isoniazid) and carcinogens (e.g., 4-aminobiphenyl). However, functional studies on non-coding or intergenic variants of
are lacking. Multiple, independent genome wide association studies (GWAS) have reported that non-coding or intergenic variants of
are associated with elevated plasma lipid and cholesterol levels, as well as cardiometabolic disorders, suggesting a novel cellular role of NAT2 in lipid and cholesterol homeostasis. The current review highlights and summarizes GWAS reports that are relevant to this association. We also present a new finding that seven, non-coding, intergenic
variants (i.e., rs4921913, rs4921914, rs4921915, rs146812806, rs35246381, rs35570672, and rs1495741), which have been associated with plasma lipid and cholesterol levels, are in linkage disequilibrium with one another, and thus form a novel haplotype. The dyslipidemia risk alleles of non-coding
variants are associated with rapid NAT2 acetylator phenotype, suggesting that differential systemic NAT2 activity might be a risk factor for developing dyslipidemia. The current review also discusses the findings of recent reports that are supportive of the role of NAT2 in lipid or cholesterol synthesis and transport. In summary, we review data suggesting that human
is a novel genetic factor that influences plasma lipid and cholesterol levels and alters the risk of cardiometabolic disorders. The proposed novel role of NAT2 merits further investigations.
Heterocyclic amines (HCAs) are well-known for their mutagenic properties. One of the major routes of human exposure is through consumption of cooked meat, as certain cooking methods favor formation ...of HCAs. Recent epidemiological studies reported significant associations between dietary HCA exposure and insulin resistance and type II diabetes. However, no previous studies have examined if HCAs, independent of meat consumption, contributes to pathogenesis of insulin resistance or metabolic disease. In the present study, we have assessed the effect of three HCAs commonly found in cooked meat (2-amino-3,4,8-trimethylimidazo4,5-fquinoxaline MeIQ, 2-amino-3,8-dimethylimidazo4,5-fquinoxaline MeIQx, and 2-amino-1-methyl-6-phenylimidazo4,5-bpyridine PhIP) on insulin signaling and glucose production. HepG2 or cryopreserved human hepatocytes were treated with 0–50 μM of MeIQ, MeIQx, or PhIP for 3 days. Treatment of HepG2 cells and hepatocytes with MeIQ and MeIQx resulted in a significant reduction in insulin-induced AKT phosphorylation, suggesting that HCA exposure decreases hepatic insulin signaling. HCA treatment also led to significant increases in expression of gluconeogenic genes,
G6PC
and
PCK1
, in both HepG2 and cryopreserved human hepatocytes. Additionally, the level of phosphorylated FOXO1, a transcriptional regulator of gluconeogenesis, was significantly reduced by HCA treatment in hepatocytes. Importantly, HCA treatment of human hepatocytes led to increases in extracellular glucose level in the presence of gluconeogenic substrates, suggesting that HCAs induce hepatic glucose production. The current findings suggest that HCAs induce insulin resistance and promote hepatic glucose production in human hepatocytes. This implicates that exposure to HCAs may lead to the development of type II diabetes or metabolic syndrome.
Although transplantation of c-kit+ cardiac stem cells (CSCs) alleviates post-myocardial infarction left ventricular dysfunction, there are no reliable methods that enable measurement of the absolute ...number of CSCs that persist in the recipient heart. To overcome this limitation, we developed a highly sensitive and accurate method to quantify the absolute number of murine CSCs after transplantation. This method has two unique features: (1) real-time PCR-based detection of a novel male-specific, multiple-copy gene,
Rbmy
, which significantly increases the sensitivity of detection of male donor cells in a female recipient, and (2) an internal standard, which permits quantification of the absolute number of CSCs as well as the total number of cells in the recipient organ. Female C57BL/6 mice underwent coronary occlusion and reperfusion; 2 days later, 10
5
male mouse CSCs were injected intramyocardially. Tissues were analyzed by real-time PCR at serial time points. In the risk region, >75 % of CSCs present at 5 min were lost in the ensuing 24 h; only 7.6 ± 2.1 % of the CSCs present at 5 min could still be found at 7 days after transplantation and only 2.8 ± 0.5 % (i.e., 1,224 ± 230 cells/heart) at 35 days. Thus, even after direct intramyocardial injection, the total number of CSCs that remain in the murine heart is minimal (at 24 h, ~10 % of the cells injected; at 35 days, ~1 %). This new quantitative method of stem cell detection, which enables measurement of absolute cell number, should be useful to optimize cell-based therapies, not only for CSCs but also for other stem cells and other organs.
We have recently demonstrated that repeated administrations of c-kit
POS
cardiac progenitor cells (CPCs) have cumulative beneficial effects in rats with old myocardial infarction (MI), resulting in ...markedly greater improvement in left ventricular (LV) function compared with a single administration. To determine whether this paradigm applies to other species and cell types, mice with a 3-week-old MI received one or three doses of cardiac mesenchymal cells (CMCs), a novel cell type that we have recently described. CMCs or vehicle were infused percutaneously into the LV cavity, 14 days apart. Compared with vehicle-treated mice, the single-dose group exhibited improved LV ejection fraction (EF) after the 1st infusion (consisting of CMCs) but not after the 2nd and 3rd (vehicle). In contrast, in the multiple-dose group, LV EF improved after each CMC infusion, so that at the end of the study, LV EF averaged 35.5 ± 0.7%
vs.
32.7 ± 0.6% in the single-dose group (
P
< 0.05). The multiple-dose group also exhibited less collagen in the non-infarcted region
vs.
the single-dose group. Engraftment and differentiation of CMCs were negligible in both groups, indicating paracrine effects. These results demonstrate that, in mice with ischemic cardiomyopathy, the beneficial effects of three doses of CMCs are significantly greater than those of one dose, supporting the concept that multiple treatments are necessary to properly evaluate the full therapeutic potential of cell therapy. Thus, the repeated-treatment paradigm is not limited to c-kit
POS
CPCs or to rats, but applies to other cell types and species. The generalizability of this concept dramatically augments its significance.
Autologous transplantation of cardiac progenitor cells (CPCs) alleviates myocardial dysfunction in the damaged heart; however, the mechanisms that contribute to their reparative qualities remain ...poorly understood. In this study, we examined CPC metabolism to elucidate the metabolic pathways that regulate their proliferative capacity. In complete growth medium, undifferentiated CPCs isolated from adult mouse heart proliferated rapidly (Td = 13.8 hours). CPCs expressed the Glut1 transporter and their glycolytic rate was increased by high extracellular glucose (Glc) concentration, in the absence of insulin. Although high Glc concentrations did not stimulate proliferation, glutamine (Gln) increased CPC doubling time and promoted survival under conditions of oxidative stress. In comparison with Glc, pyruvate (Pyr) or BSA‐palmitate, Gln, when provided as the sole metabolic substrate, increased ATP‐linked and uncoupled respiration. Although fatty acids were not used as respiratory substrates when present as a sole carbon source, Gln‐induced respiration was doubled in the presence of BSA‐palmitate, suggesting that Gln stimulates fatty acid oxidation. Additionally, Gln promoted rapid phosphorylation of the mTORC1 substrate, p70S6k, as well as retinoblastoma protein, followed by induction of cyclin D1 and cdk4. Inhibition of either mTORC1 or glutaminolysis was sufficient to diminish CPC proliferation, and provision of cell permeable α‐ketoglutarate in the absence of Gln increased both respiration and cell proliferation, indicating a key role of Gln anaplerosis in cell growth. These findings suggest that Gln, by enhancing mitochondrial function and stimulating mTORC1, increases CPC proliferation, and that interventions to increase Gln uptake or oxidation may improve CPC therapy. Stem Cells 2015;33:2613—2627
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