Corticosteroid-binding-globulin (CBG)-deficient mice show high pY216-GSK3ß and phosphorylated-Tau levels in the hippocampus.

Corticosteroid-binding globulin (CBG) is the specific carrier of circulating glucocorticoids, but evidence suggests that it also plays an active role in modulating tissue glucocorticoid activity. CBG polymorphisms affecting its expression or affinity for glucocorticoids are associated with chronic pain, chronic fatigue, headaches, depression, hypotension, and obesity with an altered hypothalamic pituitary adrenal axis. CBG has been localized in hippocampus of humans and rodents, a brain area where glucocorticoids have an important regulatory role. However, the specific CBG function in the hippocampus is yet to be established. The aim of this study was to investigate the effect of the absence of CBG on hippocampal glucocorticoid levels and determine whether pathways regulated by glucocorticoids would be altered. We used cbg-/- mice, which display low total-corticosterone and high free-corticosterone blood levels at the nadir of corticosterone secretion (morning) and at rest to evaluate the hippocampus for total- and free-corticosterone levels; 11ß-hydroxysteroid dehydrogenase expression and activity; the expression of key proteins involved in glucocorticoid activity and insulin signaling; microtubule-associated protein tau phosphorylation, and neuronal and synaptic function markers. Our results revealed that at the nadir of corticosterone secretion in the resting state the cbg-/- mouse hippocampus exhibited slightly elevated levels of free-corticosterone, diminished FK506 binding protein 5 expression, increased corticosterone downstream effectors and altered MAPK and PI3K pathway with increased pY216-GSK3ß and phosphorylated tau. Taken together, these results indicate that CBG deficiency triggers metabolic imbalance which could lead to damage and long-term neurological pathologies.

FGF15 improves outcomes after brain dead donor liver transplantation with steatotic and non-steatotic grafts in rats.

BACKGROUND & AIMS: Donation after brain death (DBD) grafts are associated with reduced graft quality and function post liver transplantation (LT). We aimed to elucidate i) the impact of FGF15 levels on DBD grafts; ii) whether this impact resulted from altered intestinal FXR-FGF15; iii) whether administration of FGF15 to donors after brain death could confer a benefit on graft function post LT; and iv) whether FGF15 affects bile acid (BA) accumulation. METHODS: Steatotic and non-steatotic grafts from DBD donors and donors without brain death were transplanted in rats. FGF15 was administered alone or combined with either a BA (cholic acid) or a YAP inhibitor. RESULTS: Brain death induced intestinal damage and downregulation of FXR. The resulting reduced intestinal FGF15 was associated with low hepatic FGF15 levels, liver damage and regenerative failure. Hepatic FGFR4-Klb - the receptor for FGF15 - was downregulated whereas CYP7A1 was overexpressed, resulting in BA accumulation. FGF15 administration to DBD donors increased hepatic FGFR4-Klb, reduced CYP7A1 and normalized BA levels. The benefit of FGF15 on liver damage was reversed by cholic acid, whereas its positive effect on regeneration was maintained. YAP signaling in DBD donors was activated after FGF15 treatment. When a YAP inhibitor was administered, the benefits of FGF15 on regeneration were abolished, whereas its positive effect on hepatic damage remained. Neither the Hippo-YAP-BA nor the BA-IQGAP1-YAP axis was involved in the benefits of FGF15. CONCLUSION: Alterations in the gut-liver axis contribute to the reduced quality of DBD grafts and the associated pathophysiology of LT. FGF15 pre-treatment in DBD donors protected against damage and promoted cell proliferation. LAY SUMMARY: After brain death, potential liver donors have reduced intestinal FXR, which is associated with reduced intestinal, circulatory and hepatic levels of FGF15. A similar reduction in the cell-surface receptor complex Fgfr4/Klb is observed, whereas CYP7A1 is overexpressed; together, these molecular events result in the dangerous accumulation of bile acids, leading to damage and regenerative failure in brain dead donor grafts. Herein, we demonstrate that when such donors receive appropriate doses of FGF15, CYP7A1 levels and hepatic bile acid toxicity are reduced, and liver regeneration is promoted.

The Effect of Fibroblast Growth Factor 15 Signaling in Non-Steatotic and Steatotic Liver Transplantation from Cardiocirculatory Death.

We elucidate the relevance of fibroblast growth factor 15 (FGF15) in liver transplantation (LT) using rats with both steatotic and non-steatotic organs from donors after cardiocirculatory death (DCD). Compared to LT from non-DCDs, the induction of cardiocirculatory death (CD) increases hepatic damage, proliferation, and intestinal and circulatory FGF15. This is associated with high levels of FGF15, bilirubin and bile acids (BAs), and overexpression of the enzyme involved in the alternative BA synthesis pathway, CYP27A1, in non-steatotic livers. Furthermore, CD activates the proliferative pathway, Hippo/YAP, in these types of liver. Blocking FGF15 action in LT from DCDs does not affect CYP27A1 but causes an overexpression of CYP7A, an enzyme from the classic BA synthesis pathway, and this is related to further accumulation of BAs and exacerbated damage. FGF15 inhibition also impairs proliferation without changing Hippo/YAP. In spite of worse damage, steatosis prevents a proliferative response in livers from DCDs. In steatotic grafts, CD does not modify CYP7A1, CYP27A1, BA, or the Hippo/YAP pathway, and FGF15 is not involved in damage or proliferation. Thus, endogenous FGF15 protects against BA accumulation and damage and promotes regeneration independently of the Hippo/YAP pathway, in non-steatotic LT from DCDs. Herein we show a minor role of FGF15 in steatotic LT from DCDs.

Corticosteroid-Binding Globulin is expressed in the adrenal gland and its absence impairs corticosterone synthesis and secretion in a sex-dependent manner.

Corticosteroid-binding globulin (CBG) is synthesized by the liver and secreted into the bloodstream where binds to glucocorticoids. Thus CBG has the role of glucocorticoid transport and free hormone control. In addition, CBG has been detected in some extrahepatic tissues without a known role. CBG-deficient mice show decreased total corticosterone levels with missing of classical sexual dimorphism, increased free corticosterone, higher adrenal gland size and altered HPA axis response to stress. Our aim was to ascertain whether CBG deficiency could affect the endocrine synthetic activity of adrenal gland and if the adrenal gland produces CBG. We determined the expression in adrenal gland of proteins involved in the cholesterol uptake and its transport to mitochondria and the main enzymes involved in the corticosterone, aldosterone and catecholamine synthesis. The results showed that CBG is synthesized in the adrenal gland. CBG-deficiency reduced the expression of ACTH receptor, SRB1 and the main genes involved in the adrenal hormones synthesis, stronger in females resulting in the loss of sexual dimorphism in corticosteroid adrenal synthesis, despite corticosterone content in adrenal glands from CBG-deficient females was similar to wildtype ones. In conclusion, these results point to an unexplored and relevant role of CBG in the adrenal gland functionality related to corticosterone production and release.

EGF-GH Axis in Rat Steatotic and Non-steatotic Liver Transplantation From Brain-dead Donors.

BACKGROUND: We evaluated the potential dysfunction caused by changes in growth hormone (GH) levels after brain death (BD), and the effects of modulating GH through exogenous epidermal growth factor (EGF) in steatotic and nonsteatotic grafts. METHODS: Steatotic and nonsteatotic grafts from non-BD and BD rat donors were cold stored for 6 hours and transplanted to live rats. Administration of GH and EGF and their underlying mechanisms were characterized in recipients of steatotic and nonsteatotic grafts from BD donors maintained normotensive during the 6 hours before donation. Circulating and hepatic GH and EGF levels, hepatic damage, and regeneration parameters were evaluated. Recipient survival was monitored for 14 days. Somatostatin, ghrelin, and GH-releasing hormones that regulate GH secretion from the anterior pituitary were determined. The survival signaling pathway phosphoinositide-3-kinase/protein kinase B that regulates inflammation (suppressors of cytokine signaling, high-mobility group protein B1, oxidative stress, and neutrophil accumulation) was evaluated. RESULTS: BD reduced circulating GH and increased GH levels only in steatotic livers. GH administration exacerbated adverse BD-associated effects in both types of graft. Exogenous EGF reduced GH in steatotic livers, thus activating cell proliferation and survival signaling pathways, ultimately reducing injury and inflammation. However, EGF increased GH in nonsteatotic grafts, which exacerbated damage. The benefits of EGF for steatotic grafts were associated with increased levels of somatostatin, a GH inhibitor, whereas the deleterious effect on nonsteatotic grafts was exerted through increased amounts of ghrelin, a GH stimulator. CONCLUSIONS: GH treatment is not appropriate in rat liver transplant from BD donors, whereas EGF (throughout GH inhibition) protects only in steatotic grafts.

Relevance of VEGFA in rat livers subjected to partial hepatectomy under ischemia-reperfusion.

We examined the effects of VEGFA on damage and regeneration in steatotic and non-steatotic livers of rats submitted to PH under I/R, and characterized the underlying mechanisms involved. Our results indicated that VEGFA levels were decreased in both steatotic and non-steatotic livers after surgery. The administration of VEGFA increased VEGFA levels in non-steatotic livers, reducing the incidence of post-operative complications following surgery through the VEGFR2-Wnt2 pathway, independently of Id1. Unexpectedly, administration of VEGFA notably reduced VEGFA levels in steatotic livers, exacerbating damage and regenerative failure. After exogenous administration of VEGFA in steatotic animals, circulating VEGFA is sequestered by the high circulating levels of sFlt1 released from adipose tissue. Under such conditions, VEGFA cannot reach the steatotic liver to exert its effects. Consequently, the concomitant administration of VEGFA and an antibody against sFlt1 was required to avoid binding of sFlt1 to VEGFA. This was associated with high VEGFA levels in steatotic livers and protection against damage and regenerative failure, plus improvement in the survival rate via up-regulation of PI3K/Akt independently of the Id1-Wnt2 pathway. The current study highlights the different effects and signaling pathways of VEGFA in liver surgery requiring PH and I/R based in the presence of steatosis. KEY MESSAGES: VEGFA administration improves PH+I/R injury only in non-steatotic livers of Ln animals. VEGFA benefits are exerted through the VEGFR2-Wnt2 pathway in non-steatotic livers. In Ob rats, exogenous VEGFA is sequestered by circulating sFlt1, exacerbating liver damage. Therapeutic combination of VEGFA and anti-sFlt1 is required to protect steatotic livers. VEGFA+anti-sFlt1 treatment protects steatotic livers through a VEGFR2-PI3K/Akt pathway.

Use of Steatotic Grafts in Liver Transplantation: Current Status.

In the field of liver transplantation, the demand for adequate allografts greatly exceeds the supply. Therefore, expanding the donor pool to match the growing demand is mandatory. The present review summarizes current knowledge of the pathophysiology of ischemia/reperfusion injury in steatotic grafts, together with recent pharmacological approaches aimed at maximizing the utilization of these livers for transplantation. We also describe the preclinical models currently available to understand the molecular mechanisms controlling graft viability in this specific type of donor, critically discussing the heterogeneity in animal models, surgical methodology, and therapeutic interventions. This lack of common approaches and interventions makes it difficult to establish the pathways involved and the relevance of isolated discoveries, as well as their transferability to clinical practice. Finally, we discuss how new therapeutic strategies developed from experimental studies are promising but that further studies are warranted to translate them to the bedside.

The effect of cortisol in rat steatotic and non-steatotic liver transplantation from brain-dead donors.

In the present study, we examined the effects of cortisol on steatotic and non-steatotic liver grafts from brain-dead donors and characterized the underlying mechanisms involved. Non-steatotic liver grafts showed reduced cortisol and increased cortisone levels in association with up-regulation of enzymes that inactivate cortisol. Conversely, steatotic liver grafts exhibited increased cortisol and reduced cortisone levels. The enzymes involved in cortisol generation were overexpressed, and those involved in cortisol inactivation or clearance were down-regulated in steatotic liver grafts. Exogenous administration of cortisol negatively affected hepatic damage and survival rate in non-steatotic liver transplantation (LT); however, cortisol treatment up-regulated the phosphoinositide 3-kinase (PI3K)-protein kinase C (PKC) pathway, resulting in protection against the deleterious effects of brain-dead donors on damage and inflammatory response in steatotic LT as well as in increased survival of recipients. The present study highlights the differences in the role of cortisol and hepatic mechanisms that regulate cortisol levels based on the type of liver. Our findings suggest that cortisol treatment is a feasible and highly protective strategy to reduce the adverse effects of brain-dead donor livers in order to ultimately improve liver graft quality in the presence of steatosis, whereas cortisol treatment would not be recommended for non-steatotic liver grafts.

Modulation of SHBG binding to testosterone and estradiol by sex and morbid obesity.

OBJECTIVE: Sex hormone-binding globulin (SHBG) binds and transports testosterone and estradiol in plasma. The possibility that SHBG is a mixture of transporting proteins has been postulated. We analyzed in parallel the effects of obesity status on the levels and binding capacity of circulating SHBG and their relationship with testosterone and estradiol. DESIGN: Anthropometric measures and plasma were obtained from apparently healthy young (i.e. 35 ± 7 years) premenopausal women (n = 32) and men (n = 30), with normal weight and obesity (BMI >30 kg/m2). METHODS: SHBG protein (Western blot), as well as the plasma levels of testosterone, estradiol, cortisol and insulin (ELISA) were measured. Specific binding of estradiol and testosterone to plasma SHBG was analyzed using tritium-labeled hormones. RESULTS: Significant differences in SHBG were observed within the obesity status and gender, with discordant patterns of change in testosterone and estradiol. In men, testosterone occupied most of the binding sites. Estrogen binding was much lower in all subjects. Lower SHBG of morbidly obese (BMI >40 kg/m2) subjects affected testosterone but not estradiol. The ratio of binding sites to SHBG protein levels was constant for testosterone, but not for estradiol. The influence of gender was maximal in morbid obesity, with men showing the highest binding/SHBG ratios. CONCLUSIONS: The results reported here are compatible with SHBG being a mixture of at least two functionally different hormone-binding globulins, being affected by obesity and gender and showing different structure, affinities for testosterone and estradiol and also different immunoreactivity.

Altered lipid partitioning and glucocorticoid availability in CBG-deficient male mice with diet-induced obesity.

OBJECTIVE: To evaluate how deficiency in corticosteroid-binding globulin (CBG), the specific carrier of glucocorticoids, affects glucocorticoid availability and adipose tissue in obesity. METHODS: C57BL/6 (WT) and CBG-deficient (KO) male mice were fed during 12 weeks with standard or hyperlipidic diet (HL). Glucocorticoid availability and metabolic parameters were assessed. RESULTS: Body weight and food intake were increased in KO compared with WT mice fed a standard diet and were similar when fed a HL diet. Expression of CBG was found in white adipose tissue by immunochemistry, real-time PCR, and Western blot. In obesity, the subcutaneous depot developed less in KO mice compared with WT, which was associated with a minor adipocyte area and peroxisome proliferator-activated receptor-γ expression. Conversely, the epididymal depot displayed higher weight and adipocyte area in KO than in WT mice. CBG deficiency caused a fall of hepatic 11ß-hydroxysteroid dehydrogenase type 2 expression and an increase in epidymal adipose tissue, particularly in HL mice. CONCLUSIONS: Deficiency in CBG drives lipid partitioning from subcutaneous to visceral adipose depot under a context of lipid excess and differentially modulates 11ß-hydroxysteroid dehydrogenase type 2 expression.

Data related to inflammation and cholesterol deposition triggered by macrophages exposition to modified LDL.

This article supports experimental evidence on the time-dependent effect on gene expression related to inflammation and cholesterol deposition in lipid-loaded cells. The cells employed were human monocytes THP1 line transformed into macrophages by treatment with phorbol esters. Macrophages were treated at different times with oxidized low density lipoprotein (Ox-LDL) and then gene expression was measured. We also include data about the different types of oxidized lipoprotein obtained (low, media or high oxidation) for differential exposure with Cu ions. These data include characterization to lipid and protein peroxidative damage and also quantification of cell viability by exposure to native and modified LDL. The present article complements data published in "Decreased OxLDL uptake and cholesterol efflux in THP1 cells elicited by cortisol and by cortisone through 11ß-hydroxysteroid dehydrogenase type 1" Ledda et al. (in press) [1].

Decreased OxLDL uptake and cholesterol efflux in THP1 cells elicited by cortisol and by cortisone through 11ß-hydroxysteroid dehydrogenase type 1.

BACKGROUND AND AIMS: Data about glucocorticoids role in the development of atherosclerosis are controversial showing different effects in human than in experimental animal models. Atherosclerosis is the result of a chronic inflammatory response to an injured endothelium where an uncontrolled uptake of OxLDL by macrophages triggers the development of foam cells, the main component of fatty streaks in atherosclerotic plaque. There are few data about the direct effect of glucocorticoids in macrophages of atherosclerotic plaque. The aim of the study was to elucidate the role of glucocorticoids in the development of foam cells in atherosclerosis initiation. METHODS: For this purpose we used THP1 cells differentiated to macrophages with phorbol esters and incubated with OxLDL alone or with cortisol or cortisone. THP1 cells were also incubated with cortisone plus an inhibitor of 11ß-hydroxysteroid dehydrogenase 1 (11ßHSD1) activity to determine the role of this enzyme on glucocorticoid action in this process. RESULTS: Ours results showed that cortisol and cortisone decreased significantly the inflammation promoted by OxLDL, and also diminished the expression of genes involved in influx and efflux of cholesterol resulting in a reduced lipid accumulation. Likewise cortisol and cortisone decreased 11ßHSD1 expression in THP1 cells. The presence of the inhibitor of 11ßHSD1 abolished all the effects elicited by cortisone. CONCLUSION: Our results indicate a direct effect of glucocorticoids on macrophages braking atherosclerosis initiation, reducing pro-inflammatory markers and OxLDL uptake and cholesterol re-esterification, but also inhibiting cholesterol output. These effects appear to be mediated, at least in part, by 11ßHSD1 activity.

New Roles for Corticosteroid Binding Globulin and Opposite Expression Profiles in Lung and Liver.

Corticosteroid-binding globulin (CBG) is the specific plasma transport glycoprotein for glucocorticoids. Circulating CBG is mainly synthesized in liver but, its synthesis has been located also in other organs as placenta, kidney and adipose tissue with unknown role. Using an experimental model of acute pancreatitis in cbg-/- mice we investigated whether changes in CBG affect the progression of the disease as well as the metabolism of glucocorticoids in the lung. Lack of CBG does not modify the progression of inflammation associated to pancreatitis but resulted in the loss of gender differences in corticosterone serum levels. In the lung, CBG expression and protein level were detected, and it is noteworthy that these showed a sexual dimorphism opposite to the liver, i.e. with higher levels in males. Reduced expression of 11ß-HSD2, the enzyme involved in the deactivation of corticosterone, was also observed. Our results indicate that, in addition to glucocorticoids transporter, CBG is involved in the gender differences observed in corticosteroids circulating levels and plays a role in the local regulation of corticosteroids availability in organs like lung.