Gene Expression Profiling in the Type 1 Diabetes Rat Diaphragm

Background

Respiratory muscle contractile performance is impaired by diabetes, mechanisms of which included altered carbohydrate and lipid metabolism, oxidative stress and changes in membrane electrophysiology. The present study examined to what extent these cellular perturbations involve changes in gene expression.

Methodology/Principal Findings

Diaphragm muscle from streptozotocin-diabetic rats was analyzed with Affymetrix gene expression arrays. Diaphragm from diabetic rats had 105 genes with at least ±2-fold significantly changed expression (55 increased, 50 decreased), and these were assigned to gene ontology groups based on over-representation analysis using DAVID software. There was increased expression of genes involved in palmitoyl-CoA hydrolase activity (a component of lipid metabolism) (P = 0.037, n = 2 genes, fold change 4.2 to 27.5) and reduced expression of genes related to carbohydrate metabolism (P = 0.000061, n = 8 genes, fold change −2.0 to −8.5). Other gene ontology groups among upregulated genes were protein ubiquitination (P = 0.0053, n = 4, fold change 2.2 to 3.4), oxidoreductase activity (P = 0.024, n = 8, fold change 2.1 to 6.0), and morphogenesis (P = 0.012, n = 10, fold change 2.1 to 4.3). Other downregulated gene groups were extracellular region (including extracellular matrix and collagen) (P = 0.00032, n = 13, fold change −2.2 to −3.7) and organogenesis (P = 0.032, n = 7, fold change −2.1 to −3.7). Real-time PCR confirmed the directionality of changes in gene expression for 30 of 31 genes tested.

Conclusions/Significance

These data indicate that in diaphragm muscle type 1 diabetes increases expression of genes involved in lipid energetics, oxidative stress and protein ubiquitination, decreases expression of genes involved in carbohydrate metabolism, and has little effect on expression of ion channel genes. Reciprocal changes in expression of genes involved in carbohydrate and lipid metabolism may change the availability of energetic substrates and thereby directly modulate fatigue resistance, an important issue for a muscle like the diaphragm which needs to contract without rest for the entire lifetime of the organism.     

Molecular basis of dysfunctional Kv channels in small coronary artery smooth muscle cells of streptozotocin-induced diabetic rats

We have previously shown that diabetes impaired cAMP-mediated endothelium independent vasodilation of rat small coronary arteries. Inhibition of Kv channel activity plays an important role in the decrease of cAMP mediated vasodilation. The present study investigated the effect of streptozotocin (STZ)-induced diabetes on mRNA and protein expressions of Kv1.2 and Kv1.5 channels in vascular smooth muscle cells of rat small coronary artery using RT-PCR, Western blot and immunohistochemistry methods. STZ-induced diabetes obviously impaired mRNA expression of Kv1.2 and Kv1.5 channel. The mRNA levels of Kv1.2 channel were 0.65 +/- 0.08 and 1.02 +/- 0.17 in STZ rats and control rats, respectively (n = 7, P < 0.05). Whereas the levels of Kv1.5 channel were 0.58 +/- 0.05 and 0.94 +/- 0.13 in STZ rats and control rats, respectively (n = 7, P < 0.05). Western blotting analysis showed that protein expression of Kv1.2 channel was decreased significantly but not Kv1.5 channel. Protein expressions of Kv1.2 channel were 0.49 +/- 0.04 and 0.70 +/- 0.06 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 0.61 +/- 0.12 and 0.59 +/- 0.14 in STZ rats and control rats, respectively (n = 5, P > 0.05). Immunohistochemistry identification indicated that immunological reaction of Kv1.2 channel protein was attenuated, but Kv1.5 channel protein was not altered. Positive staining intensity normalized by gray values of Kv1.2 channel were 173 +/- 13 and 131 +/- 11 in STZ rats and control rats, respectively (n = 5, P < 0.05), but those of Kv1.5 channel were 139 +/- 16 and 141 +/- 12 in STZ rats and control rats, respectively (n = 5, P > 0.05). These results suggested that impairment of cAMP-mediated endothelium independent vasodilation of rat small coronary artery by STZ-induced diabetes was resulted from decrease of mRNA and protein expressions of Kv channels, and which eventually leads to a reduced current from Kv channels.     

Identification of a molecular signature in human type 1 diabetes mellitus using serum and functional genomics

Understanding active proinflammatory mechanisms at and before type 1 diabetes mellitus (T1DM) onset is hindered in humans, given that the relevant tissues are inaccessible and pancreatic immune responses are difficult to measure in the periphery by traditional approaches. Therefore, we investigated the use of a sensitive and comprehensive genomics strategy to investigate the presence of proinflammatory factors in serum. The sera of recent onset diabetes patients (n = 15, 12 possessing and 3 lacking islet cell autoantibodies), long-standing diabetes patients (n = 12), "at risk" siblings of diabetes patients (n = 9), and healthy controls (n = 12) were used to induce gene expression in unrelated, healthy PBMC. After culture, gene expression was measured with microarrays and normalized expression data were subjected to hierarchical clustering and multidimensional scaling. All recent onset sera induced an expression signature (192 UniGenes; fold change: >1.5, p < 0.01; false discovery rate: <0.01) that included IL-1 cytokine family members and chemokines involved in monocyte/macrophage and neutrophil chemotaxis, as well as numerous receptors and signaling molecules. This molecular signature was not induced with the sera of healthy controls or long standing diabetes patients, where longitudinal analysis of "at risk" siblings (n = 3) before and after onset support the hypothesis that the signature emerges years before onset. This study supports prior investigations of serum that reflect disease processes associated with progression to T1DM. Identification of unique inflammatory mediators may improve disease prediction beyond current islet autoantibodies. Furthermore, proinflammatory serum markers may be used as inclusion criteria or endpoint measures in clinical trials aimed at preventing T1DM.     

Differential effects of saturated and unsaturated fatty acid diets on cardiomyocyte apoptosis, adipose distribution, and serum leptin

Fatty acids are the primary fuel for the heart and are ligands for peroxisome proliferator-activated receptors (PPARs), which regulate the expression of genes encoding proteins involved in fatty acid metabolism. Saturated fatty acids, particularly palmitate, can be converted to the proapoptotic lipid intermediate ceramide. This study assessed cardiac function, expression of PPAR-regulated genes, and cardiomyocyte apoptosis in rats after 8 wk on either a low-fat diet [normal chow control (NC); 10% fat calories] or high-fat diets composed mainly of either saturated (Sat) or unsaturated fatty acids (Unsat) (60% fat calories) (n = 10/group). The Sat group had lower plasma insulin and leptin concentrations compared with the NC or Unsat groups. Cardiac function and mass and body mass were not different. Cardiac triglyceride content was increased in the Sat and Unsat groups compared with NC (P < 0.05); however, ceramide content was higher in the Sat group compared with the Unsat group (2.9 +/- 0.2 vs. 1.4 +/- 0.2 nmol/g; P < 0.05), whereas the NC group was intermediate (2.3 +/- 0.3 nmol/g). The number of apoptotic myocytes, assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining, was higher in the Sat group compared with the Unsat group (0.28 +/- 0.05 vs. 0.17 +/- 0.04 apoptotic cells/1,000 nuclei; P < 0.04) and was positively correlated to ceramide content (P < 0.02). Both high-fat diets increased the myocardial mRNA expression of the PPAR-regulated genes encoding uncoupling protein-3 and pyruvate dehydrogenase kinase-4, but only the Sat diet upregulated medium-chain acyl-CoA dehydrogenase. In conclusion, dietary fatty acid composition affects cardiac ceramide accumulation, cardiomyocyte apoptosis, and expression of PPAR-regulated genes independent of cardiac mass or function.     

Reduced neuronal nitric oxide synthase expression contributes to cardiac oxidative stress and nitroso-redox imbalance in ob/ob mice.

Disruption of leptin signaling in the heart may contribute to obesity-related cardiac disease, as leptin deficient (oblob) mice display cardiac hypertrophy, increased cardiac apoptosis and reduced survival. Since leptin maintains a tonic level of neuronal nitric oxide synthase (NOS1) expression in the brain, we hypothesized that leptin deficiency would decrease NOS1 cardiac expression, in turn activating xanthine oxidoreductase (XOR) and creating nitroso-redox imbalance. We studied 2- to 6-month-old oblob (n=26) and C57Bl/6 controls (n=27). Cardiac NOS1 protein abundance (P<0.01) and mRNA expression (P=0.03) were reduced in oblob (n=10 and 6, respectively), while NOS3 protein abundance and mRNA expression were unaltered. Importantly, cardiac NOS1 protein abundance was restored towards normal in oblob mice after leptin treatment (n=3; P<0.05 vs leptin untreated oblob mice). NO metabolite (nitrite and nitrate) production within the myocardium was also reduced in oblob mice (n=5; P=0.02). Furthermore, oxidative stress was increased in oblob mice as GSH/GSSG ratio was decreased (n=4; P=0.02). Whereas XOR activity measured by Amplex Red fluorescence was increased (n=8; P=0.04), XOR and NADPH oxidase subunits protein abundance were not changed in oblob mice (n=6). Leptin deficiency did not disrupt NOS1 subcellular localization, as NOS1 co-localized with ryanodine receptor but not with caveolin-3. In conclusion, leptin deficiency is linked to decreased cardiac expression of NOS1 and NO production, with a concomitant increase in XOR activity and oxidative stress, resulting in nitroso-redox imbalance. These data offer novel insights into potential mechanisms of myocardial dysfunction in obesity.     

CYP2R1-, CYP27B1- and CYP24-mRNA expression in German type 1 diabetes patients

1,25(OH)(2)D(3) and 25(OH)D(3) have been associated with type 1 diabetes. Diverse enzymes are involved in the synthesis of these metabolites: the 25-Vitamin-D-hydroxylase (CYP2R1), the 25-hydroxyvitamin-D(3)-1-alpha-hydroxylase (CYP27B1) and the 25(OH)D(3)-24-hydroxylase (CYP24) among others. Serum levels of 25(OH)D(3) and 1,25(OH)(2)D(3) were investigated in type 1 diabetes patients (n=173) and the mRNA expression of the CYP2R1, CYP27B1 and CYP24 genes in type 1 diabetes patients (n=33) and healthy controls (n=23). These parameters were correlated with the -1260 (C/A) polymorphism in the CYP27B1 gene. Lower expression of CYP27B1 mRNA in comparison with healthy controls (1.7165 versus 1.7815, P=0.0268) was found. Additionally, patients carrying the genotype CC possessed a reduced amount of CYP27B1 mRNA compared to healthy controls (1.6855 versus 1.8107, respectively, P=0.0220). The heterozygosity rate of the -1260 C/A polymorphism was more frequent in patients with normal levels of 1,25(OH)(2)D(3) (> or =19.9 pmol/ml) than in whose with a level of less than 19.9 pmol/ml (46.7% versus 22.2%, P=0.0134). No correlation with serum levels of 25(OH)D(3) was found. Thus, CYP27B1 gene could play a functional role in the pathogenesis of type 1 diabetes through modulation of its mRNA expression and influence serum levels of 1,25(OH)(2)D(3) via the -1260 C/A polymorphism.     

Effects of pre-, peri-, and postmyocardial infarction treatment with losartan in rats: effect of dose on survival, ventricular arrhythmias, function, and remodeling

Angiotensin receptor blockers (ARBs) reduce adverse left ventricular (LV) remodeling and improve LV function and survival when started postmyocardial infarction (MI). ARBs also reduce ventricular arrhythmias during ischemia-reperfusion injury when started pre-MI. No information exists regarding their efficacy and safety when started pre-MI and continued peri- and post-MI. We evaluated whether the ARB losartan improves the outcome when started pre-MI and continued peri- and post-MI. Male Wistar rats (n = 502) were treated for 7 days pre-MI with losartan at a high dose (30 mg.kg(-1).day(-1)), progressively increasing dose (3 mg.kg(-1).day(-1) increased to 10 mg.kg(-1).day(-1) 10 days and 30 mg.kg(-1).day(-1) 20 days post-MI), or no treatment. Ambulatory systolic blood pressure and Holter monitoring were performed for 24 h post-MI. Echocardiography was done 30 days post-MI, and LV remodeling, cardiac hemodynamics, and fetal gene expression were assessed 38 days post-MI. High-dose losartan reduced 24-h post-MI survival compared with the progressive dose and control (21.9% vs. 36.6% and 38.1%, P = 0.033 and P = 0.009, respectively). This was associated with greater hypotension in the high dose and no change in ventricular arrhythmias in all groups. In 24-h post-MI survivors, the progressive dose group had reduced mortality from 24 h to 38 days (8.5% vs. 28.6% for control vs. 38.9% for high dose, P = 0.032 and P = 0.01, respectively). Survivors of both losartan groups demonstrated improved LV remodeling, cardiac hemodynamics, preserved GLUT-4, and reduced cardiac fetal gene expression. Pretreatment with ARBs does not reduce 24-h post-MI ventricular arrhythmias or survival, and high doses increase mortality by causing excessive hypotension. In 24-h post-MI survivors, progressively increasing doses of losartan have multiple beneficial effects, including improved survival.     

Transcriptomic responses of Synechocystis sp. PCC 6803 encapsulated in silica gel

Global gene expression of Synechocystis sp. PCC 6803 encapsulated in silica gel was examined by microarray analysis. Cultures were encapsulated in gels derived from aqueous precursors or from alkoxide precursors and incubated under constant light for 24?h prior to RNA extraction. Cultures suspended in liquid media were exposed to 500?mM salt stress and incubated under identical conditions for comparison purposes. The expression of 414 genes was significantly altered by encapsulation in aqueous-derived gels (fold change ≥1.5 and P value?相似文献   

Testosterone and interleukin-1β increase cardiac remodeling during coxsackievirus B3 myocarditis via serpin A 3n

Myocarditis and dilated cardiomyopathy (DCM) are often caused by viral infections and occur more frequently in men than in women, but the reasons for the sex difference remain unclear. The aim of this study was to assess whether gene changes in the heart during coxsackievirus B3 (CVB3) myocarditis in male and female BALB/c mice predicted worse DCM in males. Although myocarditis (P = 4.2 × 10(-5)) and cardiac dilation (P = 0.008) were worse in males, there was no difference in viral replication in the heart. Fibrotic remodeling genes, such as tissue inhibitor of metalloproteinase (TIMP)-1 and serpin A 3n, were upregulated in males during myocarditis rather than during DCM. Using gonadectomy and testosterone replacement, we showed that testosterone increased cardiac TIMP-1 (P = 0.04), serpin A 3n (P = 0.007), and matrix metalloproteinase (MMP)-8 (P = 0.04) during myocarditis. Testosterone increased IL-1β levels in the heart (P = 0.02), a cytokine known to regulate cardiovascular remodeling, and IL-1β in turn increased cardiac serpin A 3n mRNA (P = 0.005). We found that 39 of 118 (33%) genes identified in acute DCM patients were significantly altered in the heart during CVB3 myocarditis in mice, including serpin A 3n (3.3-fold change, P = 0.0001). Recombinant serpin A 3n treatment induced cardiac fibrosis during CVB3 myocarditis (P = 0.0008) while decreasing MMP-3 (P = 0.04) and MMP-9 (P = 0.03) levels in the heart. Thus, serpin A 3n was identified as a gene associated with fibrotic cardiac remodeling and progression to DCM in male myocarditis patients and mice.     

Na+-Glucose Cotransporter SGLT1 Protein in Salivary Glands: Potential Involvement in the Diabetes-Induced Decrease in Salivary Flow

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na⁺-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.     

Cardiac function and metabolism in Type 2 diabetic mice after treatment with BM 17.0744, a novel PPAR-alpha activator

Hearts from diabetic db/db mice, a model of Type 2 diabetes, exhibit left ventricular failure and altered metabolism of exogenous substrates. Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) ligands reduce plasma lipid and glucose concentrations and improve insulin sensitivity in db/db mice. Consequently, the effect of 4- to 5-wk treatment of db/db mice with a novel PPAR-alpha ligand (BM 17.0744; 25-38 mg x kg(-1) x day(-1)), commencing at 8 wk of age, on ex vivo cardiac function and metabolism was determined. Elevated plasma concentrations of glucose, fatty acids, and triacylglycerol (34.0 +/- 3.6, 2.0 +/- 0.4, and 0.9 +/- 0.1 mM, respectively) were reduced to normal after treatment with BM 17.0744 (10.8 +/- 0.6, 1.1 +/- 0.1, and 0.6 +/- 0.1 mM). Plasma insulin was also reduced significantly in treated compared with untreated db/db mice. Chronic treatment of db/db mice with the PPAR-alpha agonist resulted in a 50% reduction in rates of fatty acid oxidation, with a concomitant increase in glycolysis (1.7-fold) and glucose oxidation (2.3- fold). Correction of the diabetes-induced abnormalities in systemic and cardiac metabolism after BM 17.0744 treatment did not, however, improve left ventricular contractile function.     

Effects of streptozotocin-induced diabetes and physical training on gene expression of titin-based stretch-sensing complexes in mouse striated muscle

In striated muscle, a sarcomeric noncontractile protein, titin, is proposed to form the backbone of the stress- and strain-sensing structures. We investigated the effects of diabetes, physical training, and their combination on the gene expression of proteins of putative titin stretch-sensing complexes in skeletal and cardiac muscle. Mice were divided into control (C), training (T), streptozotocin-induced diabetic (D), and diabetic training (DT) groups. Training groups performed for 1, 3, or 5 wk of endurance training on a motor-driven treadmill. Muscle samples from T and DT groups together with respective controls were collected 24 h after the last training session. Gene expression of calf muscles (soleus, gastrocnemius, and plantaris) and cardiac muscle were analyzed using microarray and quantitative PCR. Diabetes induced changes in mRNA expression of the proteins of titin stretch-sensing complexes in Z-disc (MLP, myostatin), I-band (CARP, Ankrd2), and M-line (titin kinase signaling). Training alleviated diabetes-induced changes in most affected mRNA levels in skeletal muscle but only one change in cardiac muscle. In conclusion, we showed diabetes-induced changes in mRNA levels of several fiber-type-biased proteins (MLP, myostatin, Ankrd2) in skeletal muscle. These results are consistent with previous observations of diabetes-induced atrophy leading to slower fiber type composition. The ability of exercise to alleviate diabetes-induced changes may indicate slower transition of fiber type.