Heme Biosynthesis is Crucial for Cell Survival and Mitochondrial OXPHOS after X Irradiation.

Heme is an essential component of the hemoproteins involved in the mitochondrial electron transport chain (ETC). Cancer cells have been reported to display high heme levels and increased activity of heme-containing proteins. Consistently, inhibition of heme biosynthesis by the ALAD inhibitor succinylacetone (SA) has been shown to reduce tumor cell survival. These observations indicate that heme biosynthesis is essential for cancer cell proliferation. X irradiation has been shown to increase mitochondrial mass, membrane potential, oxygen consumption, reactive oxygen species (ROS) production, and ATP synthesis. This finding suggests that radiation activates mitochondrial oxidative phosphorylation (OXPHOS). However, although heme is an essential component of the mitochondrial ETC, whether radiation influences heme biosynthesis remains unclear. In this study, we evaluated heme biosynthesis activity after X irradiation and examined the effects of heme biosynthesis inhibition by SA on cellular radiosensitivity and mitochondrial OXPHOS function. We demonstrated that X irradiation significantly increased ALAS1 mRNA levels and cellular heme content. Inhibition of heme biosynthesis by SA significantly decreased cellular heme content and sensitized cancer cells to radiation. We also showed that SA reduced cellular ATP levels, mitochondrial membrane potential, and mitochondrial ROS production, suggesting mitochondrial OXPHOS dysfunction. SA decreased the expression of mitochondrial heme-related proteins COX2 and cytochrome c but did not influence COX1 and VDAC expression. These results indicate that inhibition of heme biosynthesis decreased mitochondrial ETC protein expression and OXPHOS activity, which triggered cellular ATP depletion and radiosensitization after X irradiation. In summary, heme biosynthesis is upregulated by X irradiation and is essential for mitochondrial OXPHOS and cell survival.

Impact of a SLC24A5 variant on the retinal pigment epithelium of a Japanese patient with oculocutaneous albinism type 6.

Oculocutaneous albinism (OCA) 6 is a non-syndromic type of OCA that has distinct ocular symptoms and variable cutaneous hypopigmentation. The causative gene of OCA6 is SLC24A5, which encodes NCKX5, a K+ -dependent Na+ /Ca2+ exchanger 5. NCKX5 is involved in the maturation of melanosomes, but its function is still unclear. In this study, we characterized a Japanese patient with OCA6. Genetic analysis revealed compound heterozygous variants in SLC24A5, c.590 + 1dupG, and c.598G>A (p.G200R). To clarify the functional significance of the missense variant, we generated a knock-in (KI) mouse model carrying the mouse homolog of the G200R variant using the CRISPR/Cas9 system. Chemical analysis showed decreased amounts of eumelanin in the hair and skin of KI mice, while levels of benzothiazine units in pheomelanin were significantly increased in their hair. Retinal pigment was also decreased in KI mice. Notably, a histopathologic study revealed a significant pigment loss in the retinal pigment epithelium (RPE) but not in the choroid. Immunohistochemically, the expression of NCKX5 in the RPE was decreased but was maintained in the choroid of KI mice. These findings could explain the difference in phenotypic severity between eye symptoms and hypopigmentation in the skin/hair.

Prevalence and Risk Factors of Silent Cerebral Microbleeds in Patients with Coronary Artery Disease.

OBJECTIVES: Cerebral microbleeds (CMBs), which can be detected by gradient-echo T2*-weighted magnetic resonance imaging (MRI), represent small chronic brain hemorrhages caused by structural abnormalities in cerebral small vessels. CMBs are known to be a potential predictor of future stroke, and are associated with age, various cardiovascular risk factors, cognitive impairment, and the use of antithrombotic drugs. Patients with coronary artery disease (CAD) are at potentially high risk of CMBs due to the presence of coexistent conditions. However, little is known about CMBs in patients with CAD. We aimed to identify the factors associated with the presence of CMBs among patients with CAD. METHODS: We evaluated 356 consecutive patients [mean age, 72 ± 10 years; men = 276 (78%)] with angiographically proven CAD who underwent T2*-weighted brain MRI. The brain MRI was assessed by researchers blinded to the patients' clinical details. RESULTS: CMBs were found in 128 (36%) patients. Among 356 patients, 119 (33%) had previously undergone percutaneous coronary intervention (PCI), and 26 (7%) coronary artery bypass grafting (CABG). There was no significant relationship between CMBs and sex, hypertension, dyslipidemia, diabetes mellitus, anticoagulation therapy, antiplatelet therapy, or prior PCI. CMBs were significantly associated with advanced age, previous CABG, eGFR, non-HDL cholesterol, carotid artery disease, long-term antiplatelet therapy, and long-term dual antiplatelet therapy (DAPT) using univariate logistic regression analysis. The multivariate logistic regression analysis showed that long-term antiplatelet therapy (odds ratio, 1.73; 95% CI, 1.06 - 2.84; P = 0.03) or long-term DAPT (odds ratio, 2.92; 95% CI, 1.39 - 6.17; P = 0.004) was significantly associated with CMBs after adjustment for confounding variables. CONCLUSIONS: CMBs were frequently observed in patients with CAD and were significantly associated with long-term antiplatelet therapy, especially long-term DAPT.

Crosstalk between Metabolic Disorders and Immune Cells.

Metabolic syndrome results from multiple risk factors that arise from insulin resistance induced by abnormal fat deposition. Chronic inflammation owing to obesity primarily results from the recruitment of pro-inflammatory M1 macrophages into the adipose tissue stroma, as the adipocytes within become hypertrophied. During obesity-induced inflammation in adipose tissue, pro-inflammatory cytokines are produced by macrophages and recruit further pro-inflammatory immune cells into the adipose tissue to boost the immune response. Here, we provide an overview of the biology of macrophages in adipose tissue and the relationship between other immune cells, such as CD4+ T cells, natural killer cells, and innate lymphoid cells, and obesity and type 2 diabetes. Finally, we discuss the link between the human pathology and immune response and metabolism and further highlight potential therapeutic targets for the treatment of metabolic disorders.

LAT1 inhibitor JPH203 sensitizes cancer cells to radiation by enhancing radiation-induced cellular senescence.

L-type amino acid transporter 1 (LAT1) is important for transporting neutral amino acids into cells. LAT1 expression is correlated with cancer malignancy, suggesting that LAT1 is a promising target for cancer therapy. JPH203, a potential novel drug targeting LAT1, has been shown to suppress tumor growth in various cancer cell lines. However, a combination study of JPH203 and radiation therapy has not been reported. Here, we examined the effects of JPH203 on radiosensitivity after irradiation in A549 and MIA Paca-2 cells. We showed that X-irradiation increased cellular neutral amino acid uptake via LAT1 in both cell lines. JPH203 inhibited the radiation-induced increase in neutral amino acid uptake. We demonstrated that JPH203, at minimally toxic concentrations, significantly sensitized cancer cells to radiation. JPH203 significantly downregulated mTOR activity and enhanced cellular senescence post-irradiation without reducing ATP and GSH levels. These results indicate that LAT1 inhibition by JPH203 sensitizes cancer cells to radiation by enhancing cellular senescence via mTOR downregulation. Thus, JPH203 may be a potent anti-cancer drug in combination with radiation therapy.

Mode of death in elderly and super-elderly patients with acute heart failure: Insights from Japanese heart failure registry.

BACKGROUND: In Japan, both the prevalence of the elderly and super-elderly and those of acute heart failure (AHF) have been increasing rapidly. METHODS: This registry was a prospective multicenter cohort, which enrolled a total of 1253 patients with AHF. In this study, 1117 patients' follow-up data were available and were categorized into three groups according to age: <75 years old (nonelderly), 75-84 years old (elderly), and ≥ 85 years old (super-elderly). The endpoint was defined as all-cause death and each mode of death after discharge during the 3-years follow-up period. RESULTS: Based on the Kaplan-Meier analysis, a gradually increased risk of all-cause death according to age was found. Among the three groups, the proportion of HF death was of similar trend; however, the proportion of infection death was higher in elderly and super-elderly patients. After adjusting for potentially confounding effects using the Cox and Fine-Gray model, the hazard ratio (HR) of all-cause death increased significantly in elderly and super-elderly patients (HR, 2.60; 95% confidence interval [CI], 1.93-3.54 and HR, 5.04; 95% CI, 3.72-6.92, respectively), when compared with nonelderly patients. The highest sub-distribution HR in detailed mode of death was infection death in elderly and super-elderly patients (HR, 4.25; 95% CI, 1.75-10.33 and HR, 10.10; 95% CI, 3.78-27.03, respectively). CONCLUSIONS: In this population, the risk of all-cause death was found to increase in elderly and super-elderly. Elderly patients and especially super-elderly patients with AHF were at a higher risk for noncardiovascular death, especially infection death.

Association Between Physical Status and the Effects of Combination Therapy With Renin-Angiotensin System Inhibitors and ß-Blockers in Patients With Acute Heart Failure.

Background: This study investigated whether combination therapy (CT) with renin-angiotensin system inhibitors and ß-blockers improved endpoints in acute heart failure (AHF). Methods and Results: AHF patients were recruited to this prospective multicenter cohort study between April 2015 and August 2017. Patients were divided into 3 categories based on ejection fraction (EF), namely heart failure (HF) with reduced EF (HFrEF), HF with midrange EF (HFmrEF), and HF with preserved EF (HFpEF), and a further into 2 groups according to physical status (those who could walk independently outdoors and those who could not). The composite endpoint included all-cause mortality and hospitalization for HF. Data at the 1-year follow-up were available for 1,018 patients. The incidence of the composite endpoint was significantly lower in the CT than non-CT group for HFrEF patients, but not among HFmrEF and HFpEF patients. For patients who could walk independently outdoors, a significantly lower rate of the composite endpoint was recorded only in the HFrEF group. The differences were maintained even after adjustment for comorbidities and prescriptions, with hazard ratios (95% confidence intervals) of 0.39 (0.20-0.76) and 0.48 (0.22-0.99), respectively. Conclusions: In this study, CT was associated with the prevention of adverse outcomes in patients with HFrEF. Moreover, CT prevented adverse events only among patients without a physical disorder, not among those with a physical disorder.

Myeloproliferative neoplasm-driving Calr frameshift promotes the development of pulmonary hypertension in mice.

Frameshifts in the Calreticulin (CALR) exon 9 provide a recurrent driver mutation of essential thrombocythemia (ET) and primary myelofibrosis among myeloproliferative neoplasms (MPNs). Here, we generated knock-in mice with murine Calr exon 9 mimicking the human CALR mutations, using the CRISPR-Cas9 method. Knock-in mice with del10 [Calrdel10/WT (wild-type) mice] exhibited an ET phenotype with increases of peripheral blood (PB) platelets and leukocytes, and accumulation of megakaryocytes in bone marrow (BM), while those with ins2 (Calrins2/WT mice) showed a slight splenic enlargement. Phosphorylated STAT3 (pSTAT3) was upregulated in BM cells of both knock-in mice. In BM transplantation (BMT) recipients from Calrdel10/WT mice, although PB cell counts were not different from those in BMT recipients from CalrWT/WT mice, Calrdel10/WT BM-derived macrophages exhibited elevations of pSTAT3 and Endothelin-1 levels. Strikingly, BMT recipients from Calrdel10/WT mice developed more severe pulmonary hypertension (PH)-which often arises as a comorbidity in patients with MPNs-than BMT recipients from CalrWT/WT mice, with pulmonary arterial remodeling accompanied by an accumulation of donor-derived macrophages in response to chronic hypoxia. In conclusion, our murine model with the frameshifted murine Calr presented an ET phenotype analogous to human MPNs in molecular mechanisms and cardiovascular complications such as PH.

Heterozygous disruption of ALAS1 in mice causes an accelerated age-dependent reduction in free heme, but not total heme, in skeletal muscle and liver.

5-Aminolevulinic acid (ALA) is the rate-limiting intermediate in heme biosynthesis in vertebrate species; a reaction catalyzed by the mitochondrial ALA synthase 1 (ALAS1) enzyme. Previously we reported that knockdown of the ubiquitously expressed ALAS1 gene in mice disrupts normal glucose metabolism, attenuates mitochondrial function and results in a prediabetic like phenotype when animals pass 20-weeks of age (Saitoh et al., 2018). Contrary to our expectations, the cytosolic and mitochondrial heme content of ALAS1 heterozygous (A1+/-) mice were similar to WT animals. Therefore, we speculated that regulatory "free heme" may be reduced in an age dependent manner in A1+/- mice, but not total heme. Here, we examine free and total heme from the skeletal muscle and liver of WT and A1+/- mice using a modified acetone extraction method and examine the effects of aging on free heme by comparing the amounts at 8-12 weeks and 30-36 weeks of age, in addition to the mRNA abundance of ALAS1. We found an age-dependent reduction in free heme in the skeletal muscle and liver of A1+/- mice, while WT mice showed only a slight decrease in the liver. Total heme levels showed no significant difference between young and aged WT and A1+/- mice. ALAS1 mRNA levels showed an age-dependent reduction similar to that of free heme levels, indicating that ALAS1 mRNA expression levels are a major determinant for free heme levels. The free heme pools in skeletal muscle tissue were almost 2-fold larger than that of liver tissue, suggesting that the heme pool varies across different tissue types. The expression of heme oxygenase 1 (HO-1) mRNA, which is expressed proportionally to the amount of free heme, were similar to those of free heme levels. Taken together, this study demonstrates that the free heme pool differs across tissues, and that an age-dependent reduction in free heme levels is accelerated in mice heterozygous for ALAS1, which could account for the prediabetic phenotype and mitochondrial abnormality observed in these animals.

The clinical characteristics and outcomes of heart failure patient with chronic obstructive pulmonary disease from the Japanese community-based registry.

Both heart failure (HF) and chronic obstructive pulmonary disease (COPD) are common diseases, but few studies have assessed the relationship between COPD and outcomes in patients with acute HF, especially in relation to age or ejection fraction (EF). The Kitakawachi Clinical Background and Outcome of Heart Failure Registry was a prospective, multicenter, community-based cohort and enrolled a total of 1,102 patients with acute HF between 2015 and 2017 in this study. The primary endpoint was defined as a composite endpoint that included all-cause mortality and hospitalization for HF. We stratified patients into two groups: those aged ≥ 80 years (elderly) and < 80 years (nonelderly). HF with preserved EF (HFpEF) was defined as EF ≥ 50%, whereas HF with reduced ejection fraction (HFrEF) was defined as EF < 50%. A total of 159 patients (14.4%) with COPD and 943 patients (83.6%) without COPD were included. COPD was found to be independently associated with a higher risk of the composite endpoint (adjusted hazard ratio: 1.42, 95% confidence interval: 1.14-1.77; p = 0.003). During a subgroup analysis, COPD was exposed as an independent risk factor of the composite endpoint in nonelderly patients; however, there was not such a finding observed among elderly patients. Separately, there was a significant association with COPD and the composite endpoint in patients with HFpEF. COPD showed a significantly higher risk of the composite endpoint after discharge in acute HF. However, this heightened risk was observable only in the subgroup of nonelderly patients and those of HFpEF.

Living Alone and Gender Differences in Rehospitalization for Heart Failure After Discharge Among Acute Heart Failure Patients.

Home treatment for heart failure (HF) is one of the most important problems in patients after discharge as a secondary preventive measure for rehospitalization for HF. However, there are no detailed studies on gender differences in sociopsychological factors such as living alone for HF rehospitalization among patients with acute HF (AHF).This prospective multicenter cohort study enrolled patients with AHF between April 2015 and August 2017. Patients of each gender with first AHF were divided into those living alone and those not living alone. The primary endpoint was defined as rehospitalization for HF after discharge. Cox proportional hazard analysis was performed to determine the association between living alone and the endpoint.Overall, 581 patients were included in this study during the 3-year follow-up. The proportion of rehospitalization for HF was significantly higher in patients living alone than in those not living alone among male patients. However, female patients showed no difference in endpoints between the two groups. The difference was independently maintained even after adjusting for differences in social backgrounds in male patients (adjusted hazard ratio (HR) 2.02; 95% confidence interval (CI), 1.07-3.70). In female patients, the HR for rehospitalization for HF showed no difference between the two groups (adjusted HR, 0.99; 95% CI, 0.56-1.69).In this study population, male patients living alone after first AHF discharge had a higher risk of rehospitalization for HF than those not living alone, but these differences were not observed in female patients.

Impact of home- and community-based services in the long-term care insurance system on outcomes of patients with acute heart failure: Insights from the Kitakawachi Clinical Background and Outcome of Heart Failure Registry.

AIM: In Japan, the long-term care insurance (LTCI) system is important for elderly people living at home; however, no clinical studies have revealed a relationship between home- or community-based services and outcomes in patients with acute heart failure (AHF). METHODS: This was a prospective multicenter cohort study of patients with AHF enrolled between April 2015 and August 2017. Patients aged ≥65 years with LTCI were divided into those receiving home- and community-based services (service users) and without home and community-based services (service non-users). The endpoint was defined as a composite endpoint, which included all-cause mortality and hospitalization for heart failure after discharge. Subgroup analyses were performed for elderly patients (<85 years) or super-elderly patients (≥85 years). RESULTS: The study participants were eligible for LTCI two times more than community-dwelling people were. At the 1-year follow-up period, the rate of the composite endpoint showed no significant difference between service users and service non-users among all patients or super-elderly patients. However, in elderly patients, the rate of the composite endpoint was significantly lower among service users than service non-users. The difference was independently maintained even after adjustments for differences in comorbidities or in social backgrounds (adjusted hazard ratio 0.62; 95% confidence interval 0.38-0.99, and adjusted hazard ratio 0.57; 95% confidence interval 0.35-0.90, respectively). CONCLUSIONS: In this study, adverse events following discharge of patients with AHF who used home- and community-based services were prevented only in elderly patients, not in super-elderly patients. Geriatr Gerontol Int 2020; 20: 967-973.

Clinical Characteristics and Outcomes of Heart Failure Patients With Long-Term Care Insurance　- Insights From the Kitakawachi Clinical Background and Outcome of Heart Failure Registry.

BACKGROUND: In Japan, the long-term care insurance (LTCI) system has an important role in helping elderly people, but there have been no clinical studies that have examined the relationship between the LTCI and prognosis for patients with acute heart failure (HF).Methods and Results:This registry was a prospective multicenter cohort, 1,253 patients were enrolled and 965 patients with acute HF aged ≥65 years were comprised the study group. The composite endpoint included all-cause death and hospitalization for HF after discharge. We divided the patients into 4 groups: (i) patients without LTCI, (ii) patients requiring support level 1 or 2, (iii) patients with care level 1 or 2, and (iv) patients with care levels 3-5. The Kaplan-Meier analysis identified a lower rate of the composite endpoint in group (i) than in the other groups. After adjusting for potentially confounding effects using a Cox proportional regression model, the hazard ratio (HR) of the composite endpoint increased significantly in groups (iii) and (iv) (adjusted HR, 1.62; 95% confidence interval [CI], 1.22-1.98 and adjusted HR, 1.62; 95% CI, 1.23-2.14, respectively) when compared with group (i). However, there was no significant difference between groups (i) and (ii). CONCLUSIONS: The level of LTCI was associated with a higher risk of the composite endpoint after discharge in acute HF patients.

Genetic ablation of aldehyde reductase (Akr1a) augments exercise endurance in mice via activation of the PGC-1α-involved pathway.

AIMS: Aldehyde reductase (AKR1A) is involved in the synthesis of ascorbic acid (AsA) as well as the detoxification of aldehydes. AKR1A-/- (KO) mice produce about 10% of the normal amounts of AsA compared to AKR1A+/+ (WT) mice. We investigated physiologic roles of AKR1A in running using the KO mice. MAIN METHODS: The KO mice were subjected to a treadmill test under either restricted AsA production or a sufficiency by supplementation and compared the results with those of WT mice. Contents of glucose, aspartate aminotransferase, AsA and free fatty acids in blood were measured. Glycogen contents were measured in the liver and skeletal muscle, and hepatic proteins were examined by immunoblot analyses. KEY FINDINGS: Running performance was higher in the KO mice than the WT mice irrespective of the AsA status. After the exercise period, blood glucose levels were decreased in the WT mice but were preserved in the KO mice. Liver glycogen levels were also consistently preserved in the KO mice after exercise. Free fatty acid levels tended to be originally high in blood plasma compared to those of the WT mice and were increased to similar extent in them. A key regulator of energy metabolism, PGC-1α, and the products of downstream target genes that encode for glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphatase, were constitutively at high levels in the KO mice. SIGNIFICANCE: The genetic ablation of AKR1A activates the PGC-1α pathway and spare glucose, which would consequently confer exercise endurance.

Karyopherin Alpha 2-Expressing Pancreatic Duct Glands and Intra-Islet Ducts in Aged Diabetic C414A-Mutant-CRY1 Transgenic Mice.

Our earlier studies demonstrated that cysteine414- (zinc-binding site of mCRY1-) alanine mutant mCRY1 transgenic mice (Tg mice) exhibit diabetes characterized by the reduction of ß-cell proliferation and by ß-cell dysfunction, presumably caused by senescence-associated secretory phenotype- (SASP-) like characters of islets. Earlier studies also showed that atypical duct-like structures in the pancreas developed age-dependently in Tg mice. Numerous reports have described that karyopherin alpha 2 (KPNA2) is highly expressed in cancers of different kinds. However, details of the expression of KPNA2 in pancreatic ductal atypia and in normal pancreatic tissues remain unclear. To assess the feature of the expression of KPNA2 in the development of the ductal atypia and islet architectures, we scrutinized the pancreas of Tg mice histopathologically. Results showed that considerable expression of KPNA2 was observed in pancreatic ß-cells, suggesting its importance in maintaining the functions of ß-cells. In mature stages, the level of KPNA2 expression was lower in islets of Tg mice than in wild-type controls. At 4 weeks, the expression levels of KPNA2 in islets of Tg mice were the same as those in wild-type controls. These results suggest that the reduction of KPNA2 might contribute to ß-cell dysfunction in mature Tg mice. Additionally, the formation of mucin-producing intra-islet ducts, islet fibrosis, and massive T cell recruitment to the islet occurred in aged Tg mice. In exocrine areas, primary pancreatic intraepithelial neoplasias (PanINs) with mucinous pancreatic duct glands (PDGs) emerged in aged Tg mice. High expression of KPNA2 was observed in the ductal atypia. By contrast, KPNA2 expression in normal ducts was quite low. Thus, upregulation of KPNA2 seemed to be correlated with progression of the degree of atypia in pancreatic ductal cells. The SASP-like microenvironment inside islets might play stimulatory roles in the formation of ductal metaplasia inside islets and in islet fibrosis in Tg mice.

Interleukin-12p40 variant form reduces Interleukin-12p80 secretion.

IL-12 is a key cytokine for the promotion of CD4+ T cells differentiation to type 1 helper T cells. IL-12 is a heterodimer (IL-12p70) consisting of p40 and p35 subunits, and is mainly secreted from activated antigen-presenting cells, such as macrophages and dendritic cells (DCs). In this study, we found that activated mouse bone marrow-derived DCs (BMDCs) produced a p40 splice variant form mRNA in addition to the conventional p40 mRNA. This p40 variant mRNA was produced by alternative splicing in exon 5, and possessed a premature stop codon. As a result, the p40 variant protein contained 157 amino acids of the N-terminal part of p40 and an additional 10 novel amino acids. When the p40 variant was expressed in HEK-293T cells, it was not secreted from the cells. To investigate the function of the p40 variant, it was co-expressed with p40 and/or p35. The p40 variant did not affect the secretion of IL-12p40 or IL-12p70, or the function of the secreted p70. In contrast, the secretion of IL-12p80, a homodimeric IL-12 with two p40 subunits, was significantly decreased when the p40 variant was expressed. This new splicing variant p40 may act to fine-tune the function of IL-12p80.

A decline in activities of daily living due to acute heart failure is an independent risk factor of hospitalization for heart failure and mortality.

BACKGROUND: Although activities of daily living (ADL) are recognized as being pertinent in averting relevant readmission of heart failure (HF) and mortality, little research has been conducted to assess a correlation between a decline in ADL and outcomes in HF patients. METHODS: The Kitakawachi Clinical Background and Outcome of Heart Failure Registry is a prospective, multicenter, community-based cohort of HF patients. We categorized the patients into four types of ADL: independent outdoor walking, independent indoor walking, indoor walking with assistance, and abasia. We defined a decline in ADL (decline ADL) as downgrade of ADL and others (non-decline ADL) as preservation of ADL before discharge compared with admission. RESULTS: Among 1253 registered patients, 923 were eligible, comprising 98 (10.6%) with decline ADL and 825 (89.4%) with non-decline ADL. Decline ADL exhibited a higher risk of hospitalization for HF and mortality compared with non-decline ADL. A multivariate analysis revealed that decline ADL emerged as an independent risk factor of hospitalization for HF [hazard ratio (HR), 1.42; 95% confidence interval (CI): 1.01-1.96; p=0.046] and mortality (HR, 1.95; 95% CI: 1.23-2.99; p<0.01). Although 66.3% of patients with decline ADL were registered for long-term care insurance, few received daycare services (32.7%) or home-visit medical services (8.2%). CONCLUSIONS: Decline in ADL is a predictor of hospitalization for HF and mortality in HF patients.

A 4-bp deletion promoter variant (rs984225803) is associated with mild OCA4 among Japanese patients.

Oculocutaneous albinism (OCA) type 4 is one of the most common types of albinism among Japanese population. In some patients who were clinically diagnosed with OCA, we have found a heterozygous pathological mutation in the coding region of SLC45A2, the gene responsible for OCA4, not leading to a DNA-based diagnosis. In this study, we evaluated pathological variants in the promoter region of SLC45A2 in these patients. The results indicated that the majority of the patients had a 4-bp deletion in the said region (c.-492_489delAATG; GenBank accession number: NM_016180; rs984225803) in the contralateral allele. These patients displayed a mild phenotype, especially regarding eye manifestations. The results of the luciferase reporter assay and electrophoretic mobility shift assay supported the pathological role of the variant. In addition, four of 220 alleles in Japanese normal control subjects also showed the deletion variant, indicating that this variant could possibly be a skin color-associated variant.

5-aminolevulinic acid (ALA) deficiency causes impaired glucose tolerance and insulin resistance coincident with an attenuation of mitochondrial function in aged mice.

In vertebrates, the initial step in heme biosynthesis is the production of 5-aminolevulinic acid (ALA) by ALA synthase (ALAS). ALA formation is believed to be the rate-limiting step for cellular heme production. Recently, several cohort studies have demonstrated the potential of ALA as a treatment for individuals with prediabetes and type-2 diabetes mellitus. These studies imply that a mechanism exists by which ALA or heme can control glucose metabolism. The ALAS1 gene encodes a ubiquitously expressed isozyme. Mice heterozygous null for ALAS1 (A1+/-s) experience impaired glucose tolerance (IGT) and insulin resistance (IR) beyond 20-weeks of age (aged A1+/-s). IGT and IR were remedied in aged A1+/-s by the oral administration of ALA for 1 week. However, the positive effect of ALA proved to be reversible and was lost upon termination of ALA administration. In the skeletal muscle of aged A1+/-s an attenuation of mitochondrial function is observed, coinciding with IGT and IR. Oral administration of ALA for 1-week brought about only a partial improvement in mitochondrial activity however, a 6-week period of ALA treatment was sufficient to remedy mitochondrial function. Studies on differentiated C2C12 myocytes indicate that the impairment of glucose metabolism is a cell autonomous effect and that ALA deficiency ultimately leads to heme depletion. This sequela is evidenced by a reduction of glucose uptake in C2C12 cells following the knockdown of ALAS1 or the inhibition of heme biosynthesis by succinylacetone. Our data provide in vivo proof that ALA deficiency attenuates mitochondrial function, and causes IGT and IR in an age-dependent manner. The data reveals an unexpected metabolic link between heme and glucose that is relevant to the pathogenesis of IGT/IR.

Over expression of 5-aminolevulinic acid synthase 2 increased protoporphyrin IX in nonerythroid cells.

Protoporphyrin IX has been used as an efficient sensitizer in photodynamic diagnosis, photodynamic therapy, and sonodynamic therapy. The level of protoporphyrin IX is very important for diagnostic or therapy effects. 5-aminolevulinic acid synthase 2 (ALAS2) is the key enzyme upstream of protoporphyrin IX synthesis. To increase protoporphyrin IX accumulation, ALAS2 over-expression transgenic mice were generated. Plasmid containing alas2 gene was transfected in colonic carcinoma cell lines. Both in tissues of transgenic mice and in colonic carcinoma cells, the amount of protoporphyrin IX accumulation did increase. In addition, the level of heme, which is down stream of protoporphyrin IX did not change. Overexpression ALAS2 in nonerythriod cells may become a novel approach to cause protoporphyrin IX accumulation.