Abstract Aims/hypothesis Pathological cardiac hypertrophy is an early phenotype in both types 1 and 2 diabetes. The primary stimulus for hypertrophic growth in diabetes is yet unknown and may involve ...neurohumoral stimulation of Gq-coupled receptors as well as direct glucose-dependent mechanisms. To discriminate between these hypertrophic stimuli we analyzed hypertrophic signalling pathways in wildtype and Gα11 -knockout mice. Methods Experimental diabetes was induced in wildtype and knockout mice by intraperitoneal injection of streptozotocin. 8 weeks after induction of diabetes myocardial function and structure was assessed by echocardiography before sacrifice. To identify prohypertrophic signalling pathways expression and translocation of protein kinase C isoforms α, βII , δ, ε and ζ were analyzed by immunohistochemical staining and immunoblot analysis after tissue fractionation. Changes in calcineurin signalling were identified by immunoblot analysis and functional assays. Expression levels of transcription factors GATA4 and NF-κB were quantified by real-time RT-PCR. Results Diabetic wildtype mice developed myocardial hypertrophy with preserved cardiac function. Calcineurin signalling was not different between the two groups. However, diabetic wildtype mice showed increased protein levels of PKC-α and PKC-ζ, translocation of PKC-α, -δ and -ε to cellular membranes and higher levels of NF-κB expression. In contrast, diabetic Gα11 -knockout mice showed no altered phenotype and no changes in NF-κB or PKC expression, although translocation of PKC-ε occurred as in wildtypes. Conclusions Gα11 is essential for the development of cardiac hypertrophy in type 1-diabetes. Stimulation of hypertrophic signalling through PKC-α, PKC-δ, PKC-ζ, and NF-κB appears to be receptor-dependent, whereas PKC-ε is activated by hyperglycemia, independent of Gα11.
Neurohumoral stimulation of Gq-coupled receptors has been proposed as a central mechanism in the pathogenesis of diabetic heart disease. The resulting contractile dysfunction is closely related to ...abnormal intracellular Ca(2+) handling with functional defects of the sarcoplasmic reticulum (SR). The present study was therefore designed to determine the role of G(q)-protein signaling via G(alpha)(11) and G(alpha)(q) in diabetes for the induction of functional and structural changes in the Ca(2+) release complex of the SR. An experimental type 1-diabetes was induced in wild type, G(alpha)(11) knockout, and G(alpha)(11/q)-knockout mice by injection of streptozotocin. Cardiac morphology and function was assessed in vivo by echocardiography. SR Ca(2+) leak was tested in vitro based on a (45)Ca(2+) assay and protein densities as well as gene expression of ryanodine receptor (RyR2), FKBP12.6, sorcin, and annexin A7 were analyzed by immunoblot and RT-PCR. In wild type animals 8 weeks of diabetes resulted in cardiac hypertrophy and SR Ca(2+) leak was increased. In addition, diabetic wild type animals showed reduced protein levels of FKBP12.6 and annexin A7. In G(alpha)(11)- and G(alpha)(11/q)-knockout animals, however, SR Ca(2+) release and cardiac phenotype remained unchanged upon induction of diabetes. Densities of the proteins that we presently analyzed were also unaltered in G(alpha)(11)-knockout mice. G(alpha)(11/q)-knockout animals even showed increased expression of sorcin and annexin A7. Thus, based on the present study we suggest a signaling pathway via the G(q)-proteins, G(alpha)(11) and G(alpha)(q), that could link increased neurohumoral stimulation in diabetes with defective RyR2 channel function by regulating protein expression of FKBP12.6, annexin A7, and sorcin.
Neurohumoral stimulation of Gq-coupled receptors has been proposed as a central mechanism in the pathogenesis of diabetic heart disease. The resulting contractile dysfunction is closely related to ...abnormal intracellular Ca.sup.2+ handling with functional defects of the sarcoplasmic reticulum (SR). The present study was therefore designed to determine the role of G.sub.q-protein signaling via Galpha.sub.11 and Gaq in diabetes for the induction of functional and structural changes in the Ca.sup.2+ release complex of the SR. An experimental type 1-diabetes was induced in wild type, Galpha.sub.11 knockout, and Galpha.sub.11/q- knockout mice by injection of streptozotocin. Cardiac morphology and function was assessed in vivo by echocardiography. SR Ca.sup.2+ leak was tested in vitro based on a sup.45Ca.sup.2+ assay and protein densities as well as gene expression of ryanodine receptor (RyR2), FKBP12.6, sorcin, and annexin A7 were analyzed by immunoblot and RT-PCR. In wild type animals 8 weeks of diabetes resulted in cardiac hypertrophy and SR Ca.sup.2+ leak was increased. In addition, diabetic wild type animals showed reduced protein levels of FKBP12.6 and annexin A7. In Galpha.sub.11- and Galpha.sub.11/q-knockout animals, however, SR Ca.sup.2+ release and cardiac phenotype remained unchanged upon induction of diabetes. Densities of the proteins that we presently analyzed were also unaltered in Galpha.sub.11-knockout mice. Galpha.sub.11/q-knockout animals even showed increased expression of sorcin and annexin A7. Thus, based on the present study we suggest a signaling pathway via the G.sub.q-proteins, Galpha.sub.11 and Galpha.sub.q, that could link increased neurohumoral stimulation in diabetes with defective RyR2 channel function by regulating protein expression of FKBP12.6, annexin A7, and sorcin. Keywords Ryanodine receptor * Type 1-diabetes * Knockout mice * Sorcin * Annexin A7
Neurohumoral stimulation of Gq-coupled receptors has been proposed as a central mechanism in the pathogenesis of diabetic heart disease. The resulting contractile dysfunction is closely related to ...abnormal intracellular Ca²⁺ handling with functional defects of the sarcoplasmic reticulum (SR). The present study was therefore designed to determine the role of Gq-protein signaling via Gα₁₁ and Gαq in diabetes for the induction of functional and structural changes in the Ca²⁺ release complex of the SR. An experimental type 1-diabetes was induced in wild type, Gα₁₁ knockout, and Gα₁₁/q-knockout mice by injection of streptozotocin. Cardiac morphology and function was assessed in vivo by echocardiography. SR Ca²⁺ leak was tested in vitro based on a ⁴⁵Ca²⁺ assay and protein densities as well as gene expression of ryanodine receptor (RyR2), FKBP12.6, sorcin, and annexin A7 were analyzed by immunoblot and RT-PCR. In wild type animals 8 weeks of diabetes resulted in cardiac hypertrophy and SR Ca²⁺ leak was increased. In addition, diabetic wild type animals showed reduced protein levels of FKBP12.6 and annexin A7. In Gα₁₁- and Gα₁₁/q-knockout animals, however, SR Ca²⁺ release and cardiac phenotype remained unchanged upon induction of diabetes. Densities of the proteins that we presently analyzed were also unaltered in Gα₁₁-knockout mice. Gα₁₁/q-knockout animals even showed increased expression of sorcin and annexin A7. Thus, based on the present study we suggest a signaling pathway via the Gq-proteins, Gα₁₁ and Gαq, that could link increased neurohumoral stimulation in diabetes with defective RyR2 channel function by regulating protein expression of FKBP12.6, annexin A7, and sorcin.
Cytochrome P45011B1 (11 beta-hydroxylase) was detected in the human adrenal cortex and in human adenomas by in situ hybridization methods. Specific riboprobes were generated by in vitro transcription ...of 11 beta-hydroxylase--specific synthetic oligonucleotides with attached T7 and SP6 polymerase promotors. 35S- and digoxigenin-labeled riboprobes were hybridized to sections of an aldosterone-producing adenoma (APA), the non-tumour portion of the corresponding adrenal gland, and two adenomas not related to hyperaldosteronism using standard protocols and varying washing conditions. After exposure of the radiolabeled sections to X-ray film, the signals were quantified and compared by statistical tests. Following autoradiography or immunohistochemical detection of the digoxigenin cytochrome P45011B1 mRNA was clearly localized in the zona fasciculata/reticularis of non-tumour portion of an human adrenal with an APA. Zona glomerulosa, medulla and connective tissue were free of label. As revealed by the semi-quantitative analysis, 11 beta-hydroxylase mRNA signals in the APA were significantly lower than those in the attached non-tumour portion and the other two adenomas. The results confirm known observations on the occurrence of cytochrome P45011B1 in the adrenal cortex of other species, but show, contrary to several immunohistochemical studies, that the enzyme is obviously not expressed in the zona glomerulosa.
Cytochrome P45011BI (11(3-hydroxylase) was detected in the brain of male rats by in situ hybridization methods. Normal Sprague-Dawley rats were compared to the transgenic strain TGR(mRen2)27, ...characterized by the expression of the murine
Ren-2
d
renin gene and the development of severe hypertension. Specific riboprobes were generated by in vitro transcription of a 152 base-pair long cDNA template.
35S-labeled riboprobes were hybridized to cryostat sections from adrenal glands and from two different levels of the brain using standard protocols and varying washing conditions. After exposure of the radiolabeled sections to X-ray film, the signals were quantified and compared. Following autoradiography and counterstaining, cytochrome P45011B1 mRNA was clearly localized in the zona fasciculata/reticularis of the adrenal cortex and in distinct layers of the cerebral cortex. High signal densities were obtained in the layers 11-1V of the neocortex and in the layer 11 of the piriform cortex, although the concentrations of cytochrome P45011B1 mRNA were remarkably lower in the central nervous system as compared to adrenal glands. As revealed by the semi-quantitative analysis, there was a slight increase in adrenal 11β-hydroxylase mRNA in the transgenic rats, whereas the brain seems to express nearly the same amount of this enzyme in both strains. The cytochrome P4501IB1 mRNA expression in distinct cells, probably nerve cells, and especially in regions with high densities of glucocorticoid receptors points to a possible function of brain derived corticosterone in receptor activation.
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