Recent Advances of LncRNA H19 in Diabetes LncRNA H19 in Diabetes.

Diabetes mellitus (DM) causes damage to major organs, including the heart, liver, brain, kidneys, eyes, and blood vessels, threatening the health of the individuals. Emerging evidence has demonstrated that lncRNAs has important functions in the pathogenesis of human diseases, such as cancers, neurodegenerative diseases, cardiac fibroblast phenotypes, hypertension, heart failure, atherosclerosis and diabetes. Recently, H19, a lncRNA, has been reported to shown to participate in the regulatory process of muscle differentiation, glucose metabolism, and tumor metastasis, as well as endometrial development. However, the roles of H19 in DM were still not completely understood. This review was conducted to summarize the functions of H19 in diabetes and discuss the challenges and possible strategies of H19 in DM.

Correlation of HMGB1, PON-1, MCP-1, and Periodontal P. gingivalis with Amniotic Fluid Fecal Dye.

BACKGROUND: This paper aims to investigate the correlation between high mobility group protein-1 (HMG-b1), antioxidant enzyme-1 (paraoxon-1, PON-1), monocyte chemoattractant protein-1 (monocyte chemoattractant protein-1, MCP-1), P. gingivalis, and MSAF. MATERIALS AND METHODS: The total sample size comprised of 73 cases in both groups. These patients were further subdivided into 2 groups: the MSAF group and the control group. 38 women were in the MSAF group and 35 women with term amniotic fluid serum were in the control group. The MSAF group was selected as a full-term singleton amniotic fluid fecal infection group. Clinical data were collected, and specimens were collected. Fecal staining of amniotic fluid and full-term amniotic fluid removes the placenta and umbilical cord blood. The expression of HMGB1 in the placenta was observed by immune-histochemical staining of MSAF and control groups. The content of PON-1 in cord blood was determined by ELISA. RESULTS: Correlation between maternal and neonatal clinical data and MSAF was done; MSAF group mean gestational age was 41.38 ± 1.40 weeks; control group mean gestational age was 39.20 ± 1.24 weeks. This study found no correlation between the birth weight, maternal age, sex, first/transmaternal, hyperthyroidism, hypothyroidism, and anemia between the MSAF and control group with nonsignificant P value (P > 0.05). However, the fatal age, gestational diabetes, gestational hypertension, umbilical cord abnormalities, placental abnormalities, and neonatal asphyxia factors were statistically different with a significant P value of <0.05 between both groups. HMGB1 and Periodontal P. gingivalis are mostly expressed in placental trophoblast, vascular endothelial cells, and amniotic epithelial and interstitial cells. After HE staining of 72 placentas by HE in MSAF and control, 6 had acute chorioamnionitis (5.1 control), 32 had chronic (23.9), 35 had abnormal placentas, and three in MSAF had chorionic columnar metaplasia. In immune-histochemistry experiments, the HMGB1 expression intensity of placental tissue was higher in the MSAF group (P < 0.05); however, the level of PON-1 was lower in the MSAF group as compared to the controls (P < 0.05). CONCLUSIONS: Gestational age and placental abnormalities are clinical high-risk factors for MSAF. HMGB1, PON-1, MCP-1, and Periodontal P. gingivalis may be involved in the development of MSAF, suggesting an oxidative/antioxidant imbalance with inflammation, and may be one of the mechanisms for MSAF development.

The Association Between Pancreatic Steatosis and Metabolic Syndrome: A 5-Year Follow-up Study Among a General Chinese Population.

OBJECTIVES: To date, the complete natural history of pancreatic steatosis is unknown. This study aimed to investigate the association of fatty pancreas (FP) in the incidence of metabolic syndrome and its components among Chinese patients with a 5-year follow-up. METHODS: Three independent cross-sectional surveys were carried out in 2013, 2015, and 2018. Fatty pancreas was diagnosed via transabdominal sonography. Logistic regression analysis was used to estimate the correlation between FP and metabolic syndrome. New cases of metabolic syndrome and its components were estimated by Cox proportional hazards models. RESULTS: At baseline, 12,551 individuals classified into FP (n = 1010) and non-FP (n = 11,541) groups were finally enrolled. In cross-sectional analyses, odds ratio of FP was 2.378 (95% confidence interval [CI], 2.085-2.713; P < 0.001). In longitudinal analyses, FP was associated with the occurrence of metabolic syndrome (hazard ratio [HR], 3.179; 95% CI, 2.197-4.6; P < 0.001), type 2 diabetes mellitus (HR, 13.99; 95% CI, 7.865-24.883; P < 0.001), nonalcoholic fatty liver disease (HR, 31.843; 95% CI, 7.73-131.171; P < 0.001), and hypertension (HR, 12.801; 95% CI, 7.323-22.38; P < 0.001). CONCLUSIONS: Pancreatic steatosis is strongly associated with the occurrence of metabolic syndrome and its components such as hypertension and diabetes.

The Functions of Long Non-Coding RNA (lncRNA) H19 in the Heart.

Cardiovascular diseases (CVDs) are major causes of morbidity and mortality worldwide. Great effort has been put into exploring early diagnostic biomarkers and innovative therapeutic strategies for preventing CVD progression over the last two decades. Long non-coding RNAs (lncRNAs) have been identified as novel regulators in cardiac development and cardiac pathogenesis. For example, lncRNA H19 (H19), also known as a fetal gene abundant in adult heart and skeletal muscles and evolutionarily conserved in humans and mice, has a regulatory role in aortic aneurysm, myocardial hypertrophy, extracellular matrix reconstitution, and coronary artery diseases. Yet, the exact function of H19 in the heart remains unknown. This review summarises the functions of H19 in the heart and discusses the challenges and possible strategies of H19 research for cardiovascular disease.

The functions of LncRNA in the heart.

Cardiovascular disease is a major cause of death and disability worldwide. Recently, increasing evidence has demonstrated that various lncRNAs play critical roles in the pathogenesis of cardiovascular diseases, including myocardial ischemia and reperfusion (I/R) injury. LncRNAs are transcripts longer than 200 nucleotides. They are considered a class of dynamic noncoding RNAs known to be involved in physiological and pathological conditions with regulatory and structural roles in numerous biological processes, including genomic imprinting, epigenetic regulation, cell proliferation, development, aging and apoptosis. They are emerging as potential key regulators of a variety of cardiovascular diseases. However, the roles of lncRNAs in the heart function remain largely unknown. The purpose of this review was to summarize the functions of lncRNAs in the heart and discuss the challenges and possible strategies of lncRNA research for cardiovascular disease.

The functions of microRNA-208 in the heart.

Cardiovascular disease is a major chronic complication of obesity and diabetes. Due to several patients with obesity and diabetes, it is necessary to urgently explore early diagnostic biomarkers and innovative therapeutic strategies to prevent the progression of cardiovascular diseases. Recently, microRNAs (also known as miRNAs) have emerged as important players in heart disease and energy regulation. MiRNAs are a group of small, highly conserved non-coding RNA molecules that regulate gene expression by suppressing the translation of messenger RNA of target genes or by promoting mRNA degradation. These act as a class of potential biomarkers and may provide key information in diagnosing common diseases such as tumors, tissue damage, and autoimmune diseases. Among all the known miRNAs, microRNA-208 (miR-208) is specifically expressed in myocardial cells and showed close association with the development of cardiac diseases, such as myocardial hypertrophy, cardiac fibrosis, myocardial infarction, arrhythmia, and heart failure. However, the functions and underlying mechanisms of miR-208 in heart are still unclear. In this review, we highlighted the novel insights of miR-208 functions and associated mechanisms in the regulation of cardiac diseases.

The association between pancreas steatosis and metabolic syndrome: A systematic review and meta-analysis.

OBJECTIVES: Pancreas steatosis is the description of fat accumulation in the pancreatic gland. The prevalence and development mechanisms of pancreatic steatosis in patients with metabolic disorders still remain unclear. The aim of this study is to systematically review the association between pancreatic steatosis and metabolic co-morbidities. METHODS: We performed a systematic search strategy using three electronic databases (MEDLINE, Scopus, and Embase) for relevant studies concerning the associations of pancreatic steatosis with metabolic syndrome (MetS) and its clinical relevance from inception until 30 September 2018. RESULTS: One thousand three hundred fifty one references were identified in the initial search, and a total of 13 studies involving 49 329 subjects were included. This analyses elucidated the presence of non-alcoholic fatty pancreas disease (NAFPD) and was associated with a significant increased risk of metabolic syndrome (RR = 2.25; 95% CI, 2.00-2.53; P < 0.0001; I2  = 42.8%; eight studies included), hypertension (RR = 1.43; 95% CI, 1.08-1.90; P = 0.013; I2  = 94.7%; nine studies included), non-alcoholic fatty liver disease (NAFLD) (RR = 2.49; 95% CI, 2.06-3.02; P < 0.0001; I2  = 96.9%; nine studies included), diabetes mellitus (RR = 1.99; 95% CI, 1.18-3.35; P = 0.01; I2  = 97.6%; 10 studies included), and central obesity (RR = 1.91; 95% CI, 1.67-2.19; P < 0.0001; I2  = 95.9%; six studies included). The association between NAFPD and hyperlipidaemia was not statistically significant (RR = 1.33; 95% CI, 0.82-2.17; P = 0.249; I2  = 97%; five studies included). CONCLUSIONS: The existing evidence indicates that NAFPD is significantly associated with an increased risk of metabolic syndrome and its components. Well-designed prospective cohort studies between pancreatic steatosis and MetS are needed to elaborate the causality in the future.

Sucrose nonfermenting AMPK-related kinase (SNARK) regulates exercise-stimulated and ischemia-stimulated glucose transport in the heart.

The signaling mechanisms mediating myocardial glucose transport are not fully understood. Sucrose nonfermenting AMP-activated protein kinase (AMPK)-related kinase (SNARK) is an AMPK-related protein kinase that is expressed in the heart and has been implicated in contraction-stimulated glucose transport in mouse skeletal muscle. We first determined if SNARK is phosphorylated on Thr208 , a site critical for SNARK activity. Mice were treated with exercise, ischemia, submaximal insulin, or maximal insulin. Treadmill exercise slightly, but significantly increased SNARK Thr208 phosphorylation. Ischemia also increased SNARK Thr208 phosphorylation, but there was no effect of submaximal or maximal insulin. HL1 cardiomyocytes were used to overexpress wild-type (WT) SNARK and to knockdown endogenous SNARK. Overexpression of WT SNARK had no effect on ischemia-stimulated glucose transport; however, SNARK knockdown significantly decreased ischemia-stimulated glucose transport. SNARK overexpression or knockdown did not alter insulin-stimulated glucose transport or glycogen concentrations. To study SNARK function in vivo, SNARK heterozygous knockout mice (SNARK+/- ) and WT littermates performed treadmill exercise. Exercise-stimulated glucose transport was decreased by ~50% in hearts from SNARK+/- mice. In summary, exercise and ischemia increase SNARK Thr208 phosphorylation in the heart and SNARK regulates exercise-stimulated and ischemia-stimulated glucose transport. SNARK is a novel mediator of insulin-independent glucose transport in the heart.

Two novel heterozygous mutations in the CYP17A1 gene in a Chinese patient with 17α-hydroxylase 17,20-lyase deficiency.

Congenital adrenal hyperplasia (CAH) resulting from 17α-hydroxylase/17,20-lyase deficiency (17-OHD) is an uncommon autosomal recessive disease. 17-OHD is caused by mutations in the cytochrome P450c17 gene (CYP17A1), and characterized by hypertension, hypokalemia, female sexual infantilism, or male pseudohermaphroditism. Here we report a case of a 19-year-old Chinese girl (46,XX) with 17-OHD, and analyze her clinical and molecular genetic characteristics. The patient had high blood pressure (147/102 mmHg), irregular menstruation, hypomenorrhea, and normal serum potassium. Physical examination showed that she had normal breasts, armpit hair, pubic hair, and a normal external genital formation. Laboratory investigations revealed low levels of serum cortisol, estradiol, and adrenal androgen, and high levels of adrenocorticotropic hormone (ACTH), progesterone, prolactin (PRL), and renin. However, the levels of blood luteinizing hormone (LH), follicular stimulating hormone (FSH), thyroid stimulating hormone (TSH), aldosterone, and aldosterone/rennin (ARR) were normal. Genomic DNA from the patient and her parents was amplified and sequenced. DNA sequence analysis revealed two novel heterozygous mutations (Ile381Thr/Ser168del) in CYP17A1. The novel heterozygous Ile381Thr mutation (c.1142T>C) was detected in the patient's mother, whereas another novel heterozygous Ser168del mutation (c.503_505delCCT) was detected in her father. In conclusion, we discovered two novel heterozygous CYP17A1 mutations Ile381Thr (c.1142T>C) and Ser168del (c.503_505delCCT) in a Chinese patient with 17-OHD.

The correlation between pancreatic steatosis and metabolic syndrome in a Chinese population.

BACKGROUND: Type 2 diabetes mellitus, obesity and hepatic steatosis showed a strong correlation with metabolic syndrome. However, data on the influence of pancreatic steatosis on metabolic syndrome are lacking. OBJECTIVE: Our aim is to perform the prevalence of pancreatic steatosis in adults and its association with metabolic syndrome in a Chinese population. METHODS: This was a cross-sectional study, randomly selected. A total of 1190 health examination subjects were recruited. Pancreatic steatosis or hepatic steatosis was diagnosed via trans-abdominal sonography. The clinical and metabolic parameters were compared between the two groups, and their associations with pancreatic steatosis were examined. RESULTS: The prevalence of pancreatic steatosis was 30.7%. The presence of pancreatic steatosis was significantly increased by age, gender, central obesity, hepatic steatosis, hypertriglyceridemia and hyperglycemia. In the logistic regression analysis, age (P < 0.05), central obesity (P < 0.01), diabetes (P < 0.05), hypertriglyceridemia (P < 0.05) and hepatic steatosis (P < 0.01) were independently associated with pancreatic steatosis. The number of the parameters of the metabolic syndrome in pancreatic steatosis group was more than that in non-pancreatic steatosis group [(2.5 ± 1.1) vs (1.4 ± 1.2)] (P < 0.01). CONCLUSION: The pancreatic steatosis is strongly associated with the parameters of metabolic syndrome, such as central obesity, diabetes, and hepatic steatosis.

Association of thyroid-stimulating hormone and cardiovascular risk factors.

Thyroid hormone plays an important role in regulating the lipid and glucose metabolism. Previously, much attention has been drawn to define the pathophysiological relationship between thyroid dysfunction and the incidence of cardiovascular diseases (CVDs). While the conditions of overt hypothyroidism and subclinical hypothyroidism were both emphasized, the association between CVD risks and the deregulated circulating thyroid-stimulating hormone (TSH) level remains to be elucidated. Nevertheless, multiple TSH-mediated physiological adaptations, including alteration of the serum lipids, body mass index, blood pressure and insulin sensitivity, have led to the difficulty of clearly examining the association between the TSH level and CVD prevalence. The current review aims to 1) summarize the evidence for the role of thyroid dysfunction and TSH abnormality in CVD pathogenesis and 2) explore the possible underlying molecular mechanisms of TSH-mediated cardiovascular pathology in hopes of providing better therapeutic strategies for the patients with deregulated TSH.

A fibrin matrix promotes the differentiation of EMSCs isolated from nasal respiratory mucosa to myelinating phenotypical Schwann-like cells.

Because Schwann cells perform the triple tasks of myelination, axon guidance and neurotrophin synthesis, they are candidates for cell transplantation that might cure some types of nervous-system degenerative diseases or injuries. However, Schwann cells are difficult to obtain. As another option, ectomesenchymal stem cells (EMSCs) can be easily harvested from the nasal respiratory mucosa. Whether fibrin, an important transplantation vehicle, can improve the differentiation of EMSCs into Schwann-like cells (SLCs) deserves further research. EMSCs were isolated from rat nasal respiratory mucosa and were purified using anti-CD133 magnetic cell sorting. The purified cells strongly expressed HNK-1, nestin, p75(NTR), S-100, and vimentin. Using nuclear staining, the MTT assay and Western blotting analysis of the expression of cell-cycle markers, the proliferation rate of EMSCs on a fibrin matrix was found to be significantly higher than that of cells grown on a plastic surface but insignificantly lower than that of cells grown on fibronectin. Additionally, the EMSCs grown on the fibrin matrix expressed myelination-related molecules, including myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and galactocerebrosides (GalCer), more strongly than did those grown on fibronectin or a plastic surface. Furthermore, the EMSCs grown on the fibrin matrix synthesized more neurotrophins compared with those grown on fibronectin or a plastic surface. The expression level of integrin in EMSCs grown on fibrin was similar to that of cells grown on fibronectin but was higher than that of cells grown on a plastic surface. These results demonstrated that fibrin not only promoted EMSC proliferation but also the differentiation of EMSCs into the SLCs. Our findings suggested that fibrin has great promise as a cell transplantation vehicle for the treatment of some types of nervous system diseases or injuries.

[Expressions and implications of Th1/Th2 cytokines in injured rat brain and spinal cord].

OBJECTIVE: To detect the expression levels of Th1/Th2 cytokines in the acute injury of brain and spinal cord in rats, and explore their possible roles in sustained secondary injury. METHODS: The acute brain and spinal cord injury models in SD rats were established and randomly divided into injury group and lipopolysaccharide (LPS) treatment group, and each group was experimentally intervened in the brain and spinal cord, respectively. The mRNA of Th1/Th2 cytokines were determined by quantitative real-time PCR (qRT-PCR) and analyzed in correlation. RESULTS: Compared with the control group, in injury and LPS treatment groups, the level of IFN-γ (as a Th1 associated cytokine) in brain tissue significantly increased (P<0.05), while the expression of T-bet did not significantly change; in spinal cord, the levels of IFN-γ and transcription factors T-bet, HLX rose significantly (P<0.05). The cytokine IL-4 and transcription factor GATA3 in both injury and LPS treatment groups were as low as those in the control group. CONCLUSION: The expressions of Th1 associated cytokines, especially soluble cytokine IFN-γ, increased in brain and spinal cord injury. The up-regulated T-bet and HLX expressions were only observed in spinal injury.

The regulation and function of the NUAK family.

AMP-activated protein kinase (AMPK) is a critical regulator of cellular and whole-body energy homeostasis. Twelve AMPK-related kinases (ARKs; BRSK1, BRSK2, NUAK1, NUAK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4, and MELK) have been identified recently. These kinases show a similar structural organization, including an N-terminal catalytic domain, followed by a ubiquitin-associated domain and a C-terminal spacer sequence, which in some cases also contains a kinase-associated domain 1. Eleven of the ARKs are phosphorylated and activated by the master upstream kinase liver kinase B1. However, most of these ARKs are largely unknown, and the NUAK family seems to have different regulations and functions. This review contains a brief discussion of the NUAK family including the specific characteristics of NUAK1 and NUAK2.

Fibrin scaffolds containing ectomesenchymal stem cells enhance behavioral and histological improvement in a rat model of spinal cord injury.

Fibrin has been widely used in wound healing. However, its benefit for spinal cord injury (SCI) is limited. In this study, we investigated the impact of fibrin scaffolds containing ectomesenchymal stem cells (EMSCs) on histological and behavioral recovery after SCI and compared it with fibrin alone. To achieve this, EMSCs derived from adult rat nasal respiratory mucosa were cultured, characterized and transfected with green fluorescent protein adenovirus before transplantation. Then, Sprague-Dawley host rats were randomly assigned into four groups: the control group (laminectomy); the SCI group (laminectomy and transection of spinal cords); the fibrin group (fibrin was transplanted immediately after SCI), and the fibrin cell (FC) group (fibrin scaffolds containing EMSCs were transplanted after SCI). Three days after the operation, a TUNEL assay indicated less apoptotic cells in the FC group than in the fibrin group. Two weeks after SCI, fluorescence staining demonstrated not only the survival and migration of EMSCs into the lesion sites, but also a higher number of nerve fibers in the FC group than in the fibrin group. Histological examination including immunohistochemistry and transmission electron microscopy 12 weeks after the operation showed more nerve fibers and a thicker myelin sheath in the FC group compared to the fibrin group. Western blotting confirmed these morphological results. Consistent with the histological results, Basso, Beattie and Bresnahan locomotor scores of the FC group were higher than those of the fibrin group. These results suggest that fibrin scaffolds containing EMSCs can improve the behavioral and histological recovery after SCI better than fibrin alone.

[Culture and induced multilineage differentiation of mesenchymal stem cells derived from human nasal mucosa].

OBJECTIVE: To establish an in vitro method to culture mesenchymal stem cells(MSCs) derived from human nasal mucosa, and explore their stemness and differentiation potential. METHOD: Based on the observation of distribution of MSCs in human nasal mucosa, we cultured and proliferated MSCs in vitro and identified the expression of stem cell markers on them including Nestin, CD133, Vimentin and Sa114 with immunofluorescence. The MSCs were induced to differentiate to osteoblasts with medium containing dexamethasone, ascorbic acid and beta sodium glycerophosphate, and to neurons with Neurobasal medium containing B27, ATRA and TSA. Histochemistry and immunofluorescence were applied to evaluate the differentiation. RESULT: The nestin and vimentin immunofluorescence-positive MSCs existed extensively in human nasal mucosa. While the MSCs were cultured in the osteogenic-inducing medium, activities of alkaline phosphatase were increased significantly, and bone nodules were found on the surface of the osteoblasts by alizarin red staining. After the induction by neural-inducing medium, the MSCs adopted neuron like appearance with many slim protrusions interconnected as a network. The induced cells expressed neural markers NF-200 and BM88 strongly. CONCLUSION: The MSCs derived from human nasal mucosa are multipotent stem cells and can be utilized as seed cells to repair bone or neural injury.

Neuronal progenitor cells seeded in fibrin gel differentiate into ChAT-positive neuron.

Previous studies indicate that neural progenitor cells seeded in fibrin can differentiate into the glia cells and neurons. However, whether fibrin gel can induce differentiation of neural progenitor cells of spinal cord into motoneurons remains to be elucidated. In this study, we prepared a fibrin-based hybrid gel incorporated with laminin and fibronectin using rat fresh plasma as a crosslinking agent. The physical properties of this hybrid gel were examined with electron microscope, as well as its degradation and vascularization after subcutaneous transplantation with histological methods. Immunofluorescence stainings were used to study the proliferation and differentiation of the neural progenitor cells seeded in this 3D hybrid gel. The results indicate that such a hybrid gel possesses the unique physical characteristics, and the ability to promote both the proliferation and differentiation of the neural progenitor cells into neuron, including ChAT-positive motoneuron. Thus, it is suggested that this hybrid gel might be suitable for a mimetic tissue transplant for central nerve system injury, such as injured spinal cord repair.

A novel role for thyroid-stimulating hormone: up-regulation of hepatic 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase expression through the cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate-responsive element binding protein pathway.

Elevated thyroid-stimulating hormone (TSH) and hypercholesterolemia commonly coexist, as typically seen in hypothyroidism, but there is no known mechanism directly linking the two. Here, we demonstrated that in liver cells, TSH promoted the expression of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), a rate-limiting enzyme in cholesterol synthesis, by acting on the TSH receptor in hepatocyte membranes and stimulating the cyclic adenosine monophosphate / protein kinase A / cyclic adenosine monophosphate-responsive element binding protein (cAMP/PKA/CREB) signaling system. In thyroidectomized rats, the production of endogenous thyroid hormone was eliminated and endogenous TSH was suppressed through pituitary suppression with constant administration of exogenous thyroid hormone, and hepatic HMGCR expression was increased by administration of exogenous TSH. These results suggested that TSH could up-regulate hepatic HMGCR expression, which indicated a potential mechanism for hypercholesterolemia involving direct action of TSH on the liver.

Chronic ethanol feeding impairs AMPK and MEF2 expression and is associated with GLUT4 decrease in rat myocardium.

Chronic and heavy alcohol consumption is one of the causes of heart diseases. However, the effects of ethanol on insulin sensitivity in myocardium has been unclear. To investigate the effects of ethanol on the expression of AMP-activated protein kinase (AMPK), myocyte enhancer factor 2 (MEF2) and glucose transporter 4 (GLUT4), all of which are involved in the regulation of insulin sensitivity, in the myocardium, we performed three parts of experiments in vivo and in vitro. I: Rats were injected with 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR, 0.8 mg.kg(-l)) for 2 h. II: Rats received different dose (0.5, 2.5 or 5 g.kg(-l).d(-l)) of ethanol for 22-week. III: Primary neonatal rat cardiomyocytes were isolated and treated with or without 100 mM ethanol or 1 mM AICAR for 4 h. The cardiac protein and mRNA expression of AMPKalpha subunits, MEF2 and GLUT4 were observed by western-blotting and RT-PCR, respectively. Serum TNFalpha levels were assessed by ELISA. The results showed chronic ethanol exposure induced insulin resistance. Ethanol decreased the mRNA levels of AMPKalpha1 and alpha2, the protein levels of total- and phospho-AMPKalpha in cardiomyocytes. Similarly, ethanol showed inhibitory effects on both the mRNA and protein levels of MEF2A and 2D, and GLUT4 in a dose-response-like fashion. Correlation analysis implied an association between phospho-AMPKalpha and MEF2A or MEF2D, and between the levels of MEF2 protein and GLUT4 transcription. In addition, ethanol elevated serum TNFalpha level. Taken together, chronic ethanol exposure decreases the expression of AMPKalpha and MEF2, and is associated with GLUT4 decline in rat myocardium.