Molecular Mechanisms of the Melatonin Receptor Pathway Linking Circadian Rhythm to Type 2 Diabetes Mellitus.

Night-shift work and sleep disorders are associated with type 2 diabetes (T2DM), and circadian rhythm disruption is intrinsically involved. Studies have identified several signaling pathways that separately link two melatonin receptors (MT1 and MT2) to insulin secretion and T2DM occurrence, but a comprehensive explanation of the molecular mechanism to elucidate the association between these receptors to T2DM, reasonably and precisely, has been lacking. This review thoroughly explicates the signaling system, which consists of four important pathways, linking melatonin receptors MT1 or MT2 to insulin secretion. Then, the association of the circadian rhythm with MTNR1B transcription is extensively expounded. Finally, a concrete molecular and evolutionary mechanism underlying the macroscopic association between the circadian rhythm and T2DM is established. This review provides new insights into the pathology, treatment, and prevention of T2DM.

The melatonin receptor 1B gene links circadian rhythms and type 2 diabetes mellitus: an evolutionary story.

Disturbed circadian rhythms have been a risk factor for type 2 diabetes mellitus (T2DM). Melatonin is the major chronobiotic hormone regulating both circadian rhythm and glucose homeostasis. The rs10830963 (G allele) of the melatonin receptor 1B (MTNR1B) gene has the strongest genetic associations with T2DM according to several genome-wide association studies. The MTNR1B rs10830963 G allele is also associated with disturbed circadian phenotypes and altered melatonin secretion, both factors that can elevate the risk of diabetes. Furthermore, evolutionary studies implied the presence of selection pressure and ethnic diversity in MTNR1B, which was consistent with the "thrifty gene" hypothesis in T2DM. The rs10830963 G risk allele is associated with delayed melatonin secretion onset in dim-light and prolonged duration of peak melatonin. This delayed melatonin secretion may help human ancestors adapt to famine or food shortages during long nights and early mornings and avoid nocturnal hypoglycemia but confers susceptibility to T2DM due to adequate energy intake in modern society. We provide new insight into the role of MTNR1B variants in T2DM via disturbed circadian rhythms from the perspective of the "thrifty gene" hypothesis; these data indicate a novel target for the prevention and treatment of susceptible populations with the thrifty genotype.

Shared and specific biological signalling pathways for diabetic retinopathy, peripheral neuropathy and nephropathy by high-throughput sequencing analysis.

OBJECTIVES: We aimed to explore the shared and specific signalling pathways involved in diabetic retinopathy (DR), diabetic peripheral neuropathy (DPN) and diabetic nephropathy (DN). METHODS: Differentially expressed mRNAs and lncRNAs were identified by high-throughput sequencing. Subsequently, functional enrichment analysis, protein-protein interaction (PPI) analysis and lncRNAs-mRNAs networks were conducted to determine the pathogenic mechanisms underlying DR, DPN and DN. RESULTS: Twenty-six biological pathways were shared among DR, DPN and DN groups compared to the type 2 diabetes mellitus (T2DM) group without complications, and most of the shared pathways and core proteins were involved in immune and inflammatory responses of microvascular damage. Cytokine‒cytokine receptor interactions and chemokine signalling pathway were the most significant and specific pathways for DR, and the lncRNA‒mRNA regulatory networks affected DR by targeting these pathways. Sphingolipid metabolism and neuroactive ligand-receptor pathways were found to be specific for the pathogenesis of DPN. Moreover, multiple amino acid metabolic pathways were involved in the occurrence and progression of DN. CONCLUSIONS: Diabetic retinopathy, DPN and DN exhibited commonality and heterogeneity simultaneously. The shared pathologic mechanisms underlying these diabetic complications are involved in diabetic microvascular damage via immune and inflammatory pathways. Our findings predict several biomarkers and therapeutic targets for these diabetic complications.

Upregulation of T Cell Receptor Signaling Pathway Components in Gestational Diabetes Mellitus Patients: Joint Analysis of mRNA and circRNA Expression Profiles.

Objective: Gestational diabetes mellitus (GDM) is one of the most common complications of pregnancy, and its pathogenesis is still unclear. Studies have shown that circular RNAs (circRNAs) can regulate blood glucose levels by targeting mRNAs, but the role of circRNAs in GDM is still unknown. Therefore, a joint microarray analysis of circRNAs and their target mRNAs in GDM patients and healthy pregnant women was carried out. Methods: In this study, microarray analyses of mRNA and circRNA in 6 GDM patients and 6 healthy controls were conducted to identify the differentially expressed mRNA and circRNA in GDM patients, and some of the discovered mRNAs and circRNAs were further validated in additional 56 samples by quantitative realtime PCR (qRT-PCR) and droplet digital PCR (ddPCR). Results: Gene ontology and pathway analyses showed that the differentially expressed genes were significantly enriched in T cell immune-related pathways. Cross matching of the differentially expressed mRNAs and circRNAs in the top 10 KEGG pathways identified 4 genes (CBLB, ITPR3, NFKBIA, and ICAM1) and 4 corresponding circRNAs (circ-CBLB, circ-ITPR3, circ-NFKBIA, and circ-ICAM1), and these candidates were subsequently verified in larger samples. These differentially expressed circRNAs and their linear transcript mRNAs were all related to the T cell receptor signaling pathway, and PCR results confirmed the initial microarray results. Moreover, circRNA/miRNA/mRNA interactions and circRNA-binding proteins were predicted, and circ-CBLB, circ-ITPR3, and circ-ICAM1 may serve as GDM-related miRNA sponges and regulate the expression of CBLB, ITPR3, NFKBIA, and ICAM1 in cellular immune pathways. Conclusion: Upregulation of T cell receptor signaling pathway components may represent the major pathological mechanism underlying GDM, thus providing a potential approach for the prevention and treatment of GDM.

Okadaic acid (OA): Toxicity, detection and detoxification.

Okadaic acid (OA), a potent polyether marine toxin, accumulates in the digestive glands of marine mollusks and therefore can severely threaten the health of humans after ingestion of contaminated shellfish. In vivo and in vitro studies have revealed that exposure of various cells, including human embryonic amniotic cells, hepatocytes, neuroblastoma cells, to OA induces morphological and functional modifications as well as the death of cells. As the number of reports on OA poisoning has increased, this toxin has gradually attracted the public's attention, and researchers are trying to study it. This review summarizes the current literature on the toxicity effects of OA, in addition to its detection and detoxification.

Cdc45/Mcm2-7/GINS complex down-regulation mediates S phase arrest in okadaic acid-induced cell damage.

Okadaic acid (OA) is one of the most common and widespread marine toxins and causes acute gastrointestinal symptoms known as diarrheic shellfish poisoning (DSP) in humans. Although OA is not classified as a typical neurotoxin, an increasing number of studies have reported its neurotoxic effects. However, most of the available studies have focused on OA-induced inhibition of serine/threonine protein phosphatases, while the molecular mechanism of OA-induced neurotoxicity remains largely unclear. To better understand the potentially toxicological profile of OA, cell cycle arrest, DNA damage and alterations in gene expression in the human neuroblastoma cell line SHSY5Y upon OA exposure were determined using flow cytometry, comet assay, and transcriptome microarray. The results showed that OA could induce cell cycle arrest at S phase and might be involved in significant DNA strand breaks. Gene expression profiling indicated that the differentially expressed genes after OA exposure were significantly enriched in the "DNA replication" and "cell cycle" pathways. Real-time PCR result had further validated that down-regulation of the Cdc45/Mcm2-7/GINS complex might be the major factor regulating those alterations. These findings provide new insight into the molecular mechanisms of OA-induced neurotoxicity, and the current data may also provide a basis for future studies.

Microarray Analysis of Long Noncoding RNAs in Female Diabetic Peripheral Neuropathy Patients.

BACKGROUND/AIMS: Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus (DM). Because of its controversial pathogenesis, DPN is still not diagnosed or managed properly in most patients. METHODS: In this study, human lncRNA microarrays were used to identify the differentially expressed lncRNAs in DM and DPN patients, and some of the discovered lncRNAs were further validated in additional 78 samples by quantitative realtime PCR (qRT-PCR). RESULTS: The microarray analysis identified 446 and 1327 differentially expressed lncRNAs in DM and DPN, respectively. The KEGG pathway analysis further revealed that the differentially expressed lncRNA-coexpressed mRNAs between DPN and DM groups were significantly enriched in the MAPK signaling pathway. The lncRNA/mRNA coexpression network indicated that BDNF and TRAF2 correlated with 6 lncRNAs. The qRT-PCR confirmed the initial microarray results. CONCLUSION: These findings demonstrated that the interplay between lncRNAs and mRNA may be involved in the pathogenesis of DPN, especially the neurotrophin-MAPK signaling pathway, thus providing relevant information for future studies.

Gene Expression Profiling Identifies Downregulation of the Neurotrophin-MAPK Signaling Pathway in Female Diabetic Peripheral Neuropathy Patients.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus (DM). It is not diagnosed or managed properly in the majority of patients because its pathogenesis remains controversial. In this study, human whole genome microarrays identified 2898 and 4493 differentially expressed genes (DEGs) in DM and DPN patients, respectively. A further KEGG pathway analysis indicated that DPN and DM share four pathways, including apoptosis, B cell receptor signaling pathway, endocytosis, and Toll-like receptor signaling pathway. The DEGs identified through comparison of DPN and DM were significantly enriched in MAPK signaling pathway, NOD-like receptor signaling pathway, and neurotrophin signaling pathway, while the "neurotrophin-MAPK signaling pathway" was notably downregulated. Seven DEGs from the neurotrophin-MAPK signaling pathway were validated in additional 78 samples, and the results confirmed the initial microarray findings. These findings demonstrated that downregulation of the neurotrophin-MAPK signaling pathway may be the major mechanism of DPN pathogenesis, thus providing a potential approach for DPN treatment.

Toxicogenomic analysis identifies the apoptotic pathway as the main cause of hepatotoxicity induced by tributyltin.

Tributyltin (TBT) is one of the most widely used organotin biocides, which has severe endocrine-disrupting effects on marine species and mammals. Given that TBT accumulates at higher levels in the liver than in any other organ, and it acts mainly as a hepatotoxic agent, it is important to clearly delineate the hepatotoxicity of TBT. However, most of the available studies on TBT have focused on observations at the cellular level, while studies at the level of genes and proteins are limited; therefore, the molecular mechanisms of TBT-induced hepatotoxicity remains largely unclear. In the present study, we applied a toxicogenomic approach to investigate the effects of TBT on gene expression in the human normal liver cell line HL7702. Gene expression profiling identified the apoptotic pathway as the major cause of hepatotoxicity induced by TBT. Flow cytometry assays confirmed that medium- and high-dose TBT treatments significantly increased the number of apoptotic cells, and more cells underwent late apoptosis in the high-dose TBT group. The genes encoding heat shock proteins (HSPs), kinases and tumor necrosis factor receptors mediated TBT-induced apoptosis. These findings revealed novel molecular mechanisms of TBT-induced hepatotoxicity, and the current microarray data may also provide clues for future studies.

Gender-Specific Association of ATP2B1 Variants with Susceptibility to Essential Hypertension in the Han Chinese Population.

Previous genome-wide association studies (GWASs) found that several ATP2B1 variants are associated with essential hypertension (EHT). But the "genome-wide significant" ATP2B1 SNPs (rs2681472, rs2681492, rs17249754, and rs1105378) are in strong linkage disequilibrium (LD) and are located in the same LD block in Chinese populations. We asked whether there are other SNPs within the ATP2B1 gene associated with susceptibility to EHT in the Han Chinese population. Therefore, we performed a case-control study to investigate the association of seven tagSNPs within the ATP2B1 gene and EHT in the Han Chinese population, and we then analyzed the interaction among different SNPs and nongenetic risk factors for EHT. A total of 902 essential hypertensive cases and 902 normotensive controls were involved in the study. All 7 tagSNPs within the ATP2B1 gene were retrieved from HapMap, and genotyping was performed using the Tm-shift genotyping method. Chi-squared test, logistic regression, and propensity score analysis showed that rs17249754 was associated with EHT, particularly in females. The MDR analysis demonstrated that the interaction of rs2070759, rs17249754, TC, TG, and BMI increased the susceptibility to hypertension. Crossover analysis and stratified analysis indicated that BMI has a major effect on the development of hypertension, while ATP2B1 variants have a minor effect.

Tributyltin induces disruption of microfilament in HL7702 cells via MAPK-mediated hyperphosphorylation of VASP.

Tributyltin (TBT) has been widely used for various industrial purposes, and it has toxic effects on multiple organs and tissues. Previous studies have found that TBT could induce cytoskeletal disruption, especially of the actin filaments. However, the underlying mechanisms remain unclear. The aim of the present study was to determine whether TBT could induce microfilament disruption using HL7702 cells and then to assess for the total levels of various microfilament-associated proteins; finally, the involvement of the MAPK pathway was investigated. The results showed that after TBT treatment, F-actin began to depolymerize and lost its characteristic filamentous structure. The protein levels of Ezrin and Cofilin remained unchanged, the actin-related protein (ARP) 2/3 levels decreased slightly, and the vasodilator-stimulated phosphoprotein (VASP) decreased dramatically. However, the phosphorylation levels of VASP increased 2.5-fold, and the ratio of phosphorylated-VASP/unphosphorylated-VASP increased 31-fold. The mitogen-activated protein kinases (MAPKs) ERK and JNK were discovered to be activated. Inhibition of ERK and JNK not only largely diminished the TBT-induced hyperphosphorylation of VASP but also recovered the cellular morphology and rescued the cells from death. In summary, this study demonstrates that TBT-induced disruption of actin filaments is caused by the hyperphosphorylation of VASP through MAPK pathways. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1530-1538, 2016.

Polymorphisms in the CISH gene are associated with susceptibility to tuberculosis in the Chinese Han population.

A recent multi-center case-control study identified several single nucleotide polymorphisms (SNPs) within the cytokine-inducible SRC homology 2 domain (CISH) gene that are associated with susceptibility to tuberculosis (TB) in both African and Asian populations. To acquire a more robust and well-powered estimate of the putative influence of these SNPs on TB susceptibility, we conducted a well-designed case-control study in the Chinese Han population. We genotyped 3 previously identified SNPs within CISH in 600 patients with pulmonary TB and 618 healthy controls, and we calculated the pooled P-values and ORs of several studies that have also been conducted in the Chinese populations. The results of the case-control study showed that the C allele of rs2239751 and the T allele of rs414171 are associated with TB susceptibility, and this association exists only in women and young adults. The pooled analysis indicated that both SNPs are significantly associated with TB in the global populations and Chinese populations. The current study confirms that variants of CISH are associated with susceptibility to TB, suggesting that negative regulators of cytokine signaling may have a role in immunity against TB infection. We hypothesize that CISH and estrogen may interact in the cytokine-dependent regulation of the immune system.

[Advances in genome-wide association study of tuberculosis].

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is one of the oldest and most influential diseases in the history due to its devastating effect on health and high mortality rate worldwide. Tuberculosis causes more human deaths than any other single infectious disease and the incidence of the tuberculosis is increasing dramatically in recent years. Genome-wide association study (GWAS) has been used to delineate the genetic basis of tuberculosis, and several susceptibility genes and loci were found, which provids important clues to the early intervention and treatment of tuberculosis. However, due to difference in the population structure and host-pathogen interactions, GWAS on tuberculosis faces great challenges. In this review, we introduced the achievements of GWAS on tuberculosis, and illustrated challenges and strategies in the future study.

Association between polymorphisms of alpha-adducin gene and essential hypertension in Chinese population.

The association between polymorphisms of α-adducin (ADD1) gene and essential hypertension is still unclear. Thus, we carried out a case-control study and an interaction analysis to test whether ADD1 is a common candidate gene for hypertension in the Chinese population. Blood samples and information including body mass index (BMI), smoking habit, and alcohol abuse were collected. Meanwhile, total cholesterol, high density lipoprotein, triglyceride were measured by automatic biochemistry analyzer. All 6 tag single nucleotide polymorphisms (tagSNPs) within ADD1 gene were genotyped by SNPstream genotyping system. Multifactor dimensionality reduction (MDR) was used to identify the interactions among the SNPs and the non-genetic factors. Results showed that plasma triglyceride, total cholesterol, and BMI were significantly higher in the hypertensive group than in the control group. Result from genotyping indicated that rs4963 was significantly associated with essential hypertension. After stratification by gender, rs4963 was associated with essential hypertension only in males. MDR analysis indicated that interaction among BMI, rs4963, and rs16843452 were involved in susceptibility of hypertension. The present study indicated that rs4963 within ADD1 gene was associated with essential hypertension in Chinese population, which might be related to altered exonic splicing and disrupted gene regulation.

Lack of association between polymorphisms from genome-wide association studies and tuberculosis in the Chinese population.

Genome-wide association studies (GWAS) have identified rs4331426 and rs2057178 as being associated with tuberculosis (TB) in African populations. Both are common single nucleotide polymorphisms (SNPs) in Africans, but they are much rarer in Eurasian populations. In order to corroborate these results, we carried out a case-control study in the Chinese population; these 2 SNPs were genotyped in 600 pulmonary TB patients and 618 healthy controls. The results showed that neither of the SNPs was associated with TB, even after stratification by gender, age, and smear status. Considering the limitation of poor coverage of variations in commercial available genotyping platforms in African populations, further GWAS should be conducted in other populations such as Indian and Chinese. Moreover, future genetic studies on host susceptibility to TB need to take into account all the variables, including host, environment, pathogen, and interactions.

[Application of Tm-shift genotyping method in genetic studies].

Melting Temperature shift (Tm-shift) is a new genotyping method. With two GC-rich tails of unequal length combined to 5'-terminal of allele-specific primers, genotypes can be determined by the distinct Tms of the PCR products with inspection of a melting curve on the real-time PCR machine. In this study, 29 SNPs were genotyped with 2 048 samples by using Tm-shift genotyping method, and the results were assessed by success rate, consistent rate, and accuracy. The results indicated that among 29 SNPs, 27 SNPs could be genotyped by Tm-shift. In other words, the success rate was 93.1%. The accuracy confirmed by direct sequencing was 100%. The consistency was 100% with 3 control samples, and 97% from a replication study in 100 samples. Thus, Tm-shift is a genotyping method with advantages including low cost, high accuracy, stability, reliability, flexible throughput, and easy operation, which can be applied to genetic studies widely.

Genetic adaptation of the hypoxia-inducible factor pathway to oxygen pressure among eurasian human populations.

Research into the mechanisms of human adaptation to the hypoxic environment of high altitude is of great interest to the fields of human physiology and clinical medicine. Recently, the gene EGLN1, from the hypoxia-inducible factor (HIF) pathway, was identified as being involved in the hypoxic adaptation of highland Andeans and Tibetans. Both highland Andeans and Tibetans have adapted to an extremely hypoxic habitat and less attention has been paid to populations living in normoxic conditions at sea level and mild-hypoxic environments of moderate altitude, thus, whether a common adaptive mechanism exists in response to quantitative variations of environmental oxygen pressure over a wide range of residing altitudes is unknown. Here, we first performed a genome-wide association study of 35 populations from the Human Genome Diversity-CEPH Panel who dwell at sea level to moderate altitude in Eurasia (N = 691; 0-2,500 m) to identify the genetic adaptation profile of normoxic and mild-hypoxic inhabitants. In addition, we systematically compared the results from the present study to six previously published genome-wide scans of highland Andeans and Tibetans to identify shared adaptive signals in response to quantitative variations of oxygen pressure. For normoxic and mild-hypoxic populations, the strongest adaptive signal came from the mu opioid receptor-encoding gene (OPRM1, 2.54 × 10(-9)), which has been implicated in the stimulation of respiration, while in the systematic survey the EGLN1-DISC1 locus was identified in all studies. A replication study performed with highland Tibetans (N = 733) and sea level Han Chinese (N = 748) confirmed the association between altitude and SNP allele frequencies in OPRM1 (in Tibetans only, P < 0.01) and in EGLN1-DISC1 (in Tibetans and Han Chinese, P < 0.01). Taken together, identification of the OPRM1 gene suggests that cardiopulmonary adaptation mechanisms are important and should be a focus in future studies of hypoxia adaptation. Furthermore, the identification of the EGLN1 gene from the HIF pathway suggests a common adaptive mechanism for Eurasian human populations residing at different altitudes with different oxygen pressures.