Increased SGK1 activity potentiates mineralocorticoid/NaCl-induced kidney injury.

Serum and glucocorticoid-regulated kinase 1 (SGK1) stimulates aldosterone-dependent renal Na+ reabsorption and modulates blood pressure. In addition, genetic ablation or pharmacological inhibition of SGK1 limits the development of kidney inflammation and fibrosis in response to excess mineralocorticoid signaling. In this work, we tested the hypothesis that a systemic increase in SGK1 activity would potentiate mineralocorticoid/salt-induced hypertension and kidney injury. To that end, we used a transgenic mouse model with increased SGK1 activity. Mineralocorticoid/salt-induced hypertension and kidney damage was induced by unilateral nephrectomy and treatment with deoxycorticosterone acetate and NaCl in the drinking water for 6 wk. Our results show that although SGK1 activation did not induce significantly higher blood pressure, it produced a mild increase in glomerular filtration rate, increased albuminuria, and exacerbated glomerular hypertrophy and fibrosis. Transcriptomic analysis showed that extracellular matrix- and immune response-related terms were enriched in the downregulated and upregulated genes, respectively, in transgenic mice. In conclusion, we propose that systemically increased SGK1 activity is a risk factor for the development of mineralocorticoid-dependent kidney injury in the context of low renal mass and independently of blood pressure.NEW & NOTEWORTHY Increased activity of the protein kinase serum and glucocorticoid-regulated kinase 1 may be a risk factor for accelerated renal damage. Serum and glucocorticoid-regulated kinase 1 expression could be a marker for the rapid progression toward chronic kidney disease and a potential therapeutic target to slow down the process.

SGK1 activation exacerbates diet-induced obesity, metabolic syndrome and hypertension.

The serum- and glucocorticoid-induced kinase 1 (SGK1) is a transcriptional target of steroid hormones including glucocorticoids or aldosterone in addition to other stimuli such as glucose. SGK1 is activated via phosphoinositide 3-kinase, placing it downstream of insulin signaling. SGK1 participates in the upregulation of kidney Na+ reabsorption by aldosterone and has been linked to obesity-related hypertension in humans. We hypothesized that a systemic increase in SGK1 activity may trigger a multiplicity of mechanisms leading to simultaneous development of the main conditions that characterize the metabolic syndrome (MetS), including hypertension. We used a transgenic mouse model made with a bacterial artificial chromosome containing the whole mouse Sgk1 gene modified to introduce an activating point mutation. Wild type or transgenic 14-week-old male mice were fed with standard chow diet or high-fat diet for up to 18 weeks. Development of the main features of MetS and hepatic steatosis were monitored, and in vitro adipocyte differentiation was studied. Our results show that transgenic animals under high-fat diet rapidly and markedly develop MetS characterized by obesity, glucose intolerance, insulin resistance, dyslipidemia and hypertension. In addition, SGK1 gain-of-function accelerates the development of hepatic steatosis. Our study suggests that inappropriate SGK1 activity represents a risk factor in developing MetS with hypertension and related end-organ damage. Our data support SGK1 as a possible therapeutic target in MetS and related complications and provides a useful gain-of-function model for pre-clinical drug testing.

Synthetic biology: insights into biological computation.

Organisms have evolved a broad array of complex signaling mechanisms that allow them to survive in a wide range of environmental conditions. They are able to sense external inputs and produce an output response by computing the information. Synthetic biology attempts to rationally engineer biological systems in order to perform desired functions. Our increasing understanding of biological systems guides this rational design, while the huge background in electronics for building circuits defines the methodology. In this context, biocomputation is the branch of synthetic biology aimed at implementing artificial computational devices using engineered biological motifs as building blocks. Biocomputational devices are defined as biological systems that are able to integrate inputs and return outputs following pre-determined rules. Over the last decade the number of available synthetic engineered devices has increased exponentially; simple and complex circuits have been built in bacteria, yeast and mammalian cells. These devices can manage and store information, take decisions based on past and present inputs, and even convert a transient signal into a sustained response. The field is experiencing a fast growth and every day it is easier to implement more complex biological functions. This is mainly due to advances in in vitro DNA synthesis, new genome editing tools, novel molecular cloning techniques, continuously growing part libraries as well as other technological advances. This allows that digital computation can now be engineered and implemented in biological systems. Simple logic gates can be implemented and connected to perform novel desired functions or to better understand and redesign biological processes. Synthetic biological digital circuits could lead to new therapeutic approaches, as well as new and efficient ways to produce complex molecules such as antibiotics, bioplastics or biofuels. Biological computation not only provides possible biomedical and biotechnological applications, but also affords a greater understanding of biological systems.

Glucose homeostasis changes and pancreatic ß-cell proliferation after switching to cyclosporin in tacrolimus-induced diabetes mellitus.

BACKGROUND: Switching to cyclosporine A may result in a reversion of tacrolimus-induced diabetes mellitus. However, mechanisms underlying such a reversion are still unknown. METHODS: Obese Zucker rats were used as a model for tacrolimus-induced diabetes mellitus. A cohort of 44 obese Zucker rats received tacrolimus for 11 days (0.3mg/kg/day) until diabetes development; then: (a)22 rats were euthanized at day 12 and were used as a reference group (tacrolimus-day 12), and (b)22 rats on tacrolimus were shifted to cyclosporin (2.5mg/kg/day) for 5 days (tacrolimus-cyclosporin). An additional cohort of 22 obese Zucker rats received the vehicle for 17 days and were used as a control group. All animals underwent an intraperitoneal glucose tolerance test at the end of the study. RESULTS: ß-cell proliferation, apoptosis and Ins2 gene expression were evaluated. Compared to rats in tacrolimus-day 12 group, those in tacrolimus-cyclosporin group showed a significant improvement in blood glucose levels in all assessment points in intraperitoneal glucose tolerance test. Diabetes decreased from 100% in tacrolimus-day 12 group to 50% in tacrolimus-cyclosporin group. Compared to tacrolimus-day 12 group, rats in tacrolimus-cyclosporin group showed an increased ß-cell proliferation, but such an increase was lower than in rats receiving the vehicle. Ins2 gene expressions in rats receiving tacrolimus-cyclosporin and rats receiving the vehicle were comparable. CONCLUSION: An early switch from tacrolimus to cyclosporin in tacrolimus-induced diabetes mellitus resulted in an increased ß-cell proliferation and reversion of diabetes in 50% of cases.

Cambios en la homeostasis de la glucosa y la proliferación de la célula beta pancreática tras el cambio a ciclosporina en la diabetes inducida por tacrolimus / Glucose homeostasis changes and pancreatic beta-cell proliferation after switching to cyclosporin in tacrolimus-induced diabetes mellitus

Antecedentes: El cambio a ciclosporinaA podría revertir la diabetes inducida por tacrolimus. Sin embargo, los mecanismos de esta reversibilidad se desconocen. Métodos: Usamos como modelo de diabetes inducida por tacrolimus las ratas Zucker obesas. Un grupo de 44 ratas Zucker obesas fue tratado con tacrolimus durante 11 días (0,3mg/kg/día) hasta que desarrollaron diabetes; posteriormente, a)22 fueron sacrificadas a día 12 como grupo referencia (tacrolimus-d12), y b)en otras 22 el tacrolimus fue reemplazado por ciclosporina (2,5mg/kg/día) durante 5 días (tacrolimus-ciclosporina). Veintidós ratas Zucker obesas recibieron vehículo durante 17 días (grupo control). A todos los animales se les realizó una sobrecarga intraperitoneal de glucosa al final del experimento. Resultados: Se analizó la proliferación de la célulaβ, la apoptosis y la expresión del gen Ins2. En el grupo tacrolimus-ciclosporina, los niveles de glucemia mejoraron significativamente en cada punto del test intraperitoneal de glucosa comparados con el grupo tacrolimus-d12. La diabetes se redujo del 100% en los tacrolimus-d12 hasta el 50% en tacrolimus-ciclosporina. La proliferación de las células β en tacrolimus-ciclosporina se incrementó en comparación con tacrolimus-d12, pero fue menor que en los tratados con vehículo. La expresión génica de Ins2en tacrolimus-ciclosporina fue comparable a los tratados con el vehículo. Conclusión: El cambio temprano de tacrolimus por ciclosporina en la diabetes inducida por tacrolimus incrementa la proliferación de la célulaβ y revierte la diabetes en un 50% de los casos (AU)

Background: Switching to cyclosporinA may result in a reversion of tacrolimus-induced diabetes mellitus. However, mechanisms underlying such a reversion are still unknown. Methods: Obese Zucker rats were used as a model for tacrolimus-induced diabetes mellitus. A cohort of 44 obese Zucker rats received tacrolimus for 11 days (0.3mg/kg/day) until diabetes development; then: (a)22 rats were euthanized at day 12 and were used as a reference group (tacrolimus-day 12), and (b)22 rats on tacrolimus were shifted to cyclosporin (2.5mg/kg/day) for 5 days (tacrolimus-cyclosporin). An additional cohort of 22 obese Zucker rats received the vehicle for 17 days and were used as a control group. All animals underwent an intraperitoneal glucose tolerance test at the end of the study. Results: β-cell proliferation, apoptosis and Ins2 gene expression were evaluated. Compared to rats in tacrolimus-day 12 group, those in tacrolimus-cyclosporin group showed a significant improvement in blood glucose levels in all assessment points in intraperitoneal glucose tolerance test. Diabetes decreased from 100% in tacrolimus-day 12 group to 50% in tacrolimus-cyclosporin group. Compared to tacrolimus-day 12 group, rats in tacrolimus-cyclosporin group showed an increased β-cell proliferation, but such an increase was lower than in rats receiving the vehicle. Ins2 gene expressions in rats receiving tacrolimus-cyclosporin and rats receiving the vehicle were comparable. Conclusion: An early switch from tacrolimus to cyclosporin in tacrolimus-induced diabetes mellitus resulted in an increased β-cell proliferation and reversion of diabetes in 50% of cases (AU)

Connexin-dependent signaling in neuro-hormonal systems.

The advent of multicellular organisms was accompanied by the development of short- and long-range chemical signalling systems, including those provided by the nervous and endocrine systems. In turn, the cells of these two systems have developed mechanisms for interacting with both adjacent and distant cells. With evolution, such mechanisms have diversified to become integrated in a complex regulatory network, whereby individual endocrine and neuro-endocrine cells sense the state of activity of their neighbors and, accordingly, regulate their own level of functioning. A consistent feature of this network is the expression of connexin-made channels between the (neuro)hormone-producing cells of all endocrine glands and secretory regions of the central nervous system so far investigated in vertebrates. This review summarizes the distribution of connexins in the mammalian (neuro)endocrine systems, and what we know about the participation of these proteins on hormone secretion, the life of the producing cells, and the action of (neuro)hormones on specific targets. The data gathered since the last reviews on the topic are summarized, with particular emphasis on the roles of Cx36 in the function of the insulin-producing beta cells of the endocrine pancreas, and of Cx40 in that of the renin-producing juxta-glomerular epithelioid cells of the kidney cortex. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Activation of protein kinase C-ζ in pancreatic ß-cells in vivo improves glucose tolerance and induces ß-cell expansion via mTOR activation.

OBJECTIVE: PKC-ζ activation is a key signaling event for growth factor-induced ß-cell replication in vitro. However, the effect of direct PKC-ζ activation in the ß-cell in vivo is unknown. In this study, we examined the effects of PKC-ζ activation in ß-cell expansion and function in vivo in mice and the mechanisms associated with these effects. RESEARCH DESIGN AND METHODS: We characterized glucose homeostasis and ß-cell phenotype of transgenic (TG) mice with constitutive activation of PKC-ζ in the ß-cell. We also analyzed the expression and regulation of signaling pathways, G1/S cell cycle molecules, and ß-cell functional markers in TG and wild-type mouse islets. RESULTS: TG mice displayed increased plasma insulin, improved glucose tolerance, and enhanced insulin secretion with concomitant upregulation of islet insulin and glucokinase expression. In addition, TG mice displayed increased ß-cell proliferation, size, and mass compared with wild-type littermates. The increase in ß-cell proliferation was associated with upregulation of cyclins D1, D2, D3, and A and downregulation of p21. Phosphorylation of D-cyclins, known to initiate their rapid degradation, was reduced in TG mouse islets. Phosphorylation/inactivation of GSK-3ß and phosphorylation/activation of mTOR, critical regulators of D-cyclin expression and ß-cell proliferation, were enhanced in TG mouse islets, without changes in Akt phosphorylation status. Rapamycin treatment in vivo eliminated the increases in ß-cell proliferation, size, and mass; the upregulation of cyclins Ds and A in TG mice; and the improvement in glucose tolerance-identifying mTOR as a novel downstream mediator of PKC-ζ-induced ß-cell replication and expansion in vivo. CONCLUSIONS PKC:-ζ, through mTOR activation, modifies the expression pattern of ß-cell cycle molecules leading to increased ß-cell replication and mass with a concomitant enhancement in ß-cell function. Approaches to enhance PKC-ζ activity may be of value as a therapeutic strategy for the treatment of diabetes.

Mechanisms in the adaptation of maternal ß-cells during pregnancy.

Pancreatic ß-cell mass adapts to changing insulin demands in the body. One of the most amazing reversible ß-cell adaptations occurs during pregnancy and postpartum conditions. During pregnancy, the increase in maternal insulin resistance is compensated by maternal ß-cell hyperplasia and hyperfunctionality to maintain normal blood glucose. Although the cellular mechanisms involved in maternal ß-cell expansion have been studied in detail in rodents, human studies are very sparse. A summary of these studies in rodents and humans is described below. Since ß-cell mass expands during pregnancy, unraveling the endocrine/paracrine/autocrine molecular mechanisms responsible for these effects can be of great importance for predicting and treating gestational diabetes and for finding new cues that induce ß-cell regeneration in diabetes. In addition to the well known implication of lactogens during maternal ß-cell expansion, additional participants are being discovered such as serotonin and HGF. Transcription factors, such as hepatocyte nuclear factor-4α and the forkhead box protein-M1, and cell cycle regulators, such as menin, p27 and p18, are important intracellular signals responsible for these effects. In this article, we summarize and discuss novel studies uncovering molecular mechanisms involved in the maternal ß-cell adaptive expansion during pregnancy.

Disruption of hepatocyte growth factor/c-Met signaling enhances pancreatic beta-cell death and accelerates the onset of diabetes.

OBJECTIVE: To determine the role of hepatocyte growth factor (HGF)/c-Met on ß-cell survival in diabetogenic conditions in vivo and in response to cytokines in vitro. RESEARCH DESIGN AND METHODS: We generated pancreas-specific c-Met-null (PancMet KO) mice and characterized their response to diabetes induced by multiple low-dose streptozotocin (MLDS) administration. We also analyzed the effect of HGF/c-Met signaling in vitro on cytokine-induced ß-cell death in mouse and human islets, specifically examining the role of nuclear factor (NF)-κB. RESULTS: Islets exposed in vitro to cytokines or from MLDS-treated mice displayed significantly increased HGF and c-Met levels, suggesting a potential role for HGF/c-Met in ß-cell survival against diabetogenic agents. Adult PancMet KO mice displayed normal glucose and ß-cell homeostasis, indicating that pancreatic c-Met loss is not detrimental for ß-cell growth and function under basal conditions. However, PancMet KO mice were more susceptible to MLDS-induced diabetes. They displayed higher blood glucose levels, marked hypoinsulinemia, and reduced ß-cell mass compared with wild-type littermates. PancMet KO mice showed enhanced intraislet infiltration, islet nitric oxide (NO) and chemokine production, and ß-cell apoptosis. c-Met-null ß-cells were more sensitive to cytokine-induced cell death in vitro, an effect mediated by NF-κB activation and NO production. Conversely, HGF treatment decreased p65/NF-κB activation and fully protected mouse and, more important, human ß-cells against cytokines. CONCLUSIONS: These results show that HGF/c-Met is critical for ß-cell survival by attenuating NF-κB signaling and suggest that activation of the HGF/c-Met signaling pathway represents a novel strategy for enhancing ß-cell protection.

Survey of the human pancreatic beta-cell G1/S proteome reveals a potential therapeutic role for cdk-6 and cyclin D1 in enhancing human beta-cell replication and function in vivo.

OBJECTIVES: To comprehensively inventory the proteins that control the G1/S cell cycle checkpoint in the human islet and compare them with those in the murine islet, to determine whether these might therapeutically enhance human beta-cell replication, to determine whether human beta-cell replication can be demonstrated in an in vivo model, and to enhance human beta-cell function in vivo. RESEARCH DESIGN AND METHODS: Thirty-four G1/S regulatory proteins were examined in human islets. Effects of adenoviruses expressing cdk-6, cdk-4, and cyclin D1 on proliferation in human beta-cells were studied in both in vitro and in vivo models. RESULTS: Multiple differences between murine and human islets occur, most strikingly the presence of cdk-6 in human beta-cells versus its low abundance in the murine islet. Cdk-6 and cyclin D1 in vitro led to marked activation of retinoblastoma protein phosphorylation and cell cycle progression with no induction of cell death. Human islets transduced with cdk-6 and cyclin D1 were transplanted into diabetic NOD-SCID mice and markedly outperformed native human islets in vivo, maintaining glucose control for the entire 6 weeks of the study. CONCLUSIONS: The human G1/S proteome is described for the first time. Human islets are unlike their rodent counterparts in that they contain easily measurable cdk-6. Cdk-6 overexpression, alone or in combination with cyclin D1, strikingly stimulates human beta-cell replication, both in vitro as well as in vivo, without inducing cell death or loss of function. Using this model, human beta-cell replication can be induced and studied in vivo.

Evaluation of beta-cell replication in mice transgenic for hepatocyte growth factor and placental lactogen: comprehensive characterization of the G1/S regulatory proteins reveals unique involvement of p21cip.

We hypothesized that combined transgenic overexpression of hepatocyte growth factor (HGF) and placental lactogen in islets would lead to even greater increases in beta-cell mass and replication than either growth factor alone. This did not occur, suggesting that beta-cell replication is saturable or subject to molecular restraint. We therefore performed the first comprehensive G(1)/S cell cycle survey in islets, cataloguing the broad range of kinases, cyclins, and kinase inhibitors that control the G(1)/S transition in islets from normal, HGF, placental lactogen, and doubly transgenic mice. Many of the G(1)/S checkpoint regulators (E2Fs; pRb; p107; p130; cyclins D(1),(2),(3), A, and E; cdk-2; cdk-4; p15; p16; p18; p19; p21; p27; MDM2; p53; c-Myc; and Egr-1) are present in the murine islet. Most of these proteins were unaltered by overexpression of HGF or placental lactogen, either alone or in combination. In contrast, p21(cip) was uniquely, dramatically, and reproducibly upregulated in placental lactogen and HGF islets. p21(cip) was also present in, and upregulated in, proliferating human islets, localizing specifically in beta-cells and translocating to the nucleus on mitogenic stimulation. Homozygous p21(cip) loss releases islets from growth inhibition, markedly enhancing proliferation in response to HGF and placental lactogen.