AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing's syndrome.

Chronic exposure to glucocorticoid hormones, resulting from either drug treatment or Cushing's syndrome, results in insulin resistance, central obesity, and symptoms similar to the metabolic syndrome. We hypothesized that the major metabolic effects of corticosteroids are mediated by changes in the key metabolic enzyme adenosine monophosphate-activated protein kinase (AMPK) activity. Activation of AMPK is known to stimulate appetite in the hypothalamus and stimulate catabolic processes in the periphery. We assessed AMPK activity and the expression of several metabolic enzymes in the hypothalamus, liver, adipose tissue, and heart of a rat glucocorticoid-excess model as well as in in vitro studies using primary human adipose and primary rat hypothalamic cell cultures, and a human hepatoma cell line treated with dexamethasone and metformin. Glucocorticoid treatment inhibited AMPK activity in rat adipose tissue and heart, while stimulating it in the liver and hypothalamus. Similar data were observed in vitro in the primary adipose and hypothalamic cells and in the liver cell line. Metformin, a known AMPK regulator, prevented the corticosteroid-induced effects on AMPK in human adipocytes and rat hypothalamic neurons. Our data suggest that glucocorticoid-induced changes in AMPK constitute a novel mechanism that could explain the increase in appetite, the deposition of lipids in visceral adipose and hepatic tissue, as well as the cardiac changes that are all characteristic of glucocorticoid excess. Our data suggest that metformin treatment could be effective in preventing the metabolic complications of chronic glucocorticoid excess.

LPS induces interleukin-6 and interleukin-8 but not tumor necrosis factor-alpha in human adipocytes.

Adipose tissue-derived cytokines are presumably involved in obesity-associated pathologies including type 2 diabetes and atherosclerosis. Here we studied the lipopolysaccharide (LPS)-induced expression dynamics of tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), IL-8 and IL-10 in human adipose tissue biopsies, in preadipocyte-derived adipocytes, and in mesenchymal stem cell (MSC)-derived adipocytes. TNFalpha, IL-6, IL-8 and IL-10 secretions by adipose tissue explants were increased 5.5-, 19.5-, 3.5- and 12.5-fold, respectively, by LPS (1 microg/mL) administration. Concordantly, IL-6 and IL-8 release was dose-dependently induced in MSC-derived adipocytes by LPS (>10 pg/mL). In contrast, TNFalpha and IL-10 remained undetectable even at the highest LPS dose (1 microg/mL) after 24h. In MSC- and preadipocyte-derived adipocytes, respectively, exposure to LPS evoked a weak and transient induction of TNFalpha mRNA whereas induction of IL-6 and IL-8 mRNA were pronounced and sustained for at least 24h. Basal glucose uptake, lipolysis and IL-6 mRNA were induced by exogenous TNFalpha (10 ng/mL) but not by IL-6 (10 ng/mL), IL-8 (100 ng/mL) and IL-10 (20 ng/mL). In this adipocyte model TNFalpha induces well known metabolic effects, but together with previous reports these data suggest that inflammation-induced TNFalpha may derive from non-adipocyte sources in adipose tissue, likely to be macrophages.

Extraction of high-integrity RNA suitable for microarray gene expression analysis from long-term stored human thyroid tissues.

INTRODUCTION: Isolation of high-quality RNA from fresh-frozen thyroid tissues stored for more than a decade would open novel options for gene expression profiling. Herein, we describe successful extraction of high-integrity RNA from human thyroid tissues that were stored for more than a decade. METHODS: Seventy-nine samples (15 goitres, 20 follicular adenomas, 30 papillary carcinomas, 14 follicular carcinomas) that were shock-frozen in isopentane and stored for a median of 11 years (range 1-16 years) were processed using standard precipitation and column filtration techniques. RNA integrity was assessed by electrophoresis using the RNA integrity number (RIN) algorithm and by gene expression profiling determining the 3'/5' ratio of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and the percentage of transcripts detected on the Affymetrix U133 2.0 human genome GeneChip. RESULTS: The median RNA yield was 1.9 microg/mg tissue (papillary carcinoma 2.1 microg/mg, range 0.2-7.2 microg/mg; follicular carcinoma 2.4 microg/mg, range 0.2-3.2 microg/mg; goitre 1.4 microg/mg, range 0.1-5.4 microg/mg; follicular adenoma 1.6 microg/mg, range 0.1-6.2 microg/mg; p = 0.46) with an 8.6 (7.3-9.8) median RIN. The median GAPDH gene 3'/5' ratio was 1.43 (1.34-1.52) and the median percentage of present calls was 48.1% (42.7-52.0%). CONCLUSIONS: Age and entity independent RNA suitable for expression profiling can be extracted from long-term stored fresh-frozen human thyroid tissues.

Multipotential nestin and Isl-1 positive mesenchymal stem cells isolated from human pancreatic islets.

Mesenchymal cells in the developing pancreas express the neural stem cell marker nestin and the transcription factor islet-1 (Isl-1). Using defined culture conditions we isolated on a single cell basis nestin producing cells from human pancreatic islets. These cells were immortalized with lentiviral vectors coding for telomerase and mBmi. They are positive for Isl-1 and nestin and have the potential to adopt a pancreatic endocrine phenotype with expression of critical transcription factors including Ipf-1, Isl-1, Ngn-3, Pax4, Pax6, Nkx2.2, and Nkx6.1 as well as the islet hormones insulin, glucagon, and somatostatin. In addition, they can be differentiated into human albumin producing cells in vivo when grafted into a SCID mouse liver. In accordance with a mesenchymal phenotype, the cells were also able to adopt adipocytic or osteocytic phenotypes in vitro. In conclusion, cultured pancreatic islets contain nestin and Isl-1 positive mesenchymal stem cells with multipotential developmental capacity.

Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells.

Mesenchymal stem cells (MSC) from mouse bone marrow were shown to adopt a pancreatic endocrine phenotype in vitro and to reverse diabetes in an animal model. MSC from human bone marrow and adipose tissue represent very similar cell populations with comparable phenotypes. Adipose tissue is abundant and easily accessible and could thus also harbor cells with the potential to differentiate in insulin producing cells. We isolated human adipose tissue-derived MSC from four healthy donors. During the proliferation period, the cells expressed the stem cell markers nestin, ABCG2, SCF, Thy-1 as well as the pancreatic endocrine transcription factor Isl-1. The cells were induced to differentiate into a pancreatic endocrine phenotype by defined culture conditions within 3 days. Using quantitative PCR a down-regulation of ABCG2 and up-regulation of pancreatic developmental transcription factors Isl-1, Ipf-1, and Ngn3 were observed together with induction of the islet hormones insulin, glucagon, and somatostatin.

Cytokine-induced metabolic effects in human adipocytes are independent of endogenous nitric oxide.

Nitric oxide (NO) has been recognized as a potential mediator of inflammation-induced metabolic alterations, including insulin resistance. However, expression mechanisms and potential roles of endothelial and inducible NO synthases (eNOS and iNOS, respectively) in human adipocytes are poorly understood. In the present study, we aimed to analyze several aspects of NO-related gene expression and metabolite synthesis in basal and inflammation-activated human adipocyte models. eNOS mRNA was highly expressed in omental and to a lesser extent in human subcutaneous adipose tissue biopsies, but not in purified adipocytes, in mesenchymal stem cell (MSC)- and in preadipocyte-derived adipocytes, respectively. Trace amounts of iNOS mRNA were detected in adipose tissue samples of donors with abdominal infection, as opposed to noninfected subjects. Interferon-gamma, in combination with interleukin-1beta or lipopolysaccharide, evoked a transient (4 h < time < 24 h) iNOS mRNA expression in human MSC and preadipocyte-derived adipocytes, respectively. This induction was preceded by cytokine-specific mRNAs. In addition, it was accompanied by an activation of the tetrahydrobiopterin synthesis pathway and by inhibition of peroxisome proliferator-activated receptor-gamma2. In contrast to murine 3T3-L1-derived adipocytes, iNOS protein and NO oxidation products remained undetectable in iNOS mRNA-positive human adipocytes. Accordingly, coadministration of NOS inhibitors (i.e., Nomega-nitro-L-arginine methyl ester, Nomega-monomethyl-L-arginine, and 1400W) had no effects on insulin-mediated glucose uptake and lipolysis. We conclude that, in human adipocytes, endogenous NO is not involved in metabolic regulation during either basal or cytokine-activated conditions.

Autocrine/paracrine role of inflammation-mediated calcitonin gene-related peptide and adrenomedullin expression in human adipose tissue.

Human adipose tissue is a contributor to inflammation- and sepsis-induced elevation of serum procalcitonin (ProCT). Several calcitonin (CT) peptides, including ProCT, CT gene-related peptide (CGRP), and adrenomedullin (ADM) are suspected mediators in human inflammatory diseases. Therefore, we aimed to explore the expression, interactions, and potential roles of adipocyte-derived CT peptide production. Expression of CT peptide-specific transcripts was analyzed by RT-PCR and quantitative real-time PCR in human adipose tissue biopsies and three different inflammation-challenged human adipocyte models. ProCT, CGRP, and ADM secretions were assessed by immunological methods. Adipocyte transcriptional activity, glycerol release, and insulin-mediated glucose transport were studied after exogenous CGRP and ADM exposure. With the exception of amylin, CT peptides were expressed in adipose tissue biopsies from septic patients, inflammation-activated mature explanted adipocytes, and macrophage-activated preadipocyte-derived adipocytes. ProCT and CGRP productions were significantly augmented in IL-1beta and lipopolysaccharide-challenged mesenchymal stem cell-derived adipocytes but not in undifferentiated mesenchymal stem cells. In contrast, ADM expression occurred before and after adipogenic differentiation. Interferon-gamma coadministration inhibited IL-1beta-mediated ProCT and CGRP secretion by 78 and 34%, respectively but augmented IL-1beta-mediated ADM secretion by 50%. Exogenous CGRP and ADM administration induced CT, CGRP I, and CGRP II mRNAs and dose-dependently (10(-10) and 10(-6) m) enhanced glycerol release. In contrast, no CGRP- and ADM-mediated effects were noted on ADM, TNFalpha, and IL-1beta mRNA abundances. In summary, CGRP and ADM are two differentially regulated novel adipose tissue secretion factors exerting autocrine/paracrine roles. Their lipolytic effect (glycerol release) suggests a metabolic role in adipocytes during inflammation.

Somatostatin is expressed and secreted by human adipose tissue upon infection and inflammation.

Somatostatin (SRIF) is a well-known neuroendocrine secretion product. SRIF expression and secretion are induced after inflammation in murine macrophages and in endotoxin-injected sheep and pigs. Because adipocytes have been demonstrated to produce numerous cytokines and peptide hormones, we investigated the expression of SRIF and its receptors (SSTR1-5) in human adipose tissue after inflammatory stimulation in vitro and in tissues from patients with septic disease.Preadipocyte-derived adipocytes, mesenchymal stem cell-derived adipocytes, and mature explanted adipocytes expressed SRIF-mRNA after endotoxin [lipopolysaccharide (LPS)] or IL-1beta treatments. LPS- and IL-1beta-mediated SRIF-mRNA induction was blocked by pretreatment with dexamethasone. Using cocultures and quantitative real-time PCR, we demonstrate adipocyte SRIF induction by secretion factors from activated peripheral blood mononuclear cell-derived macrophages. In contrast to basal adipocytes, SRIF protein was detected in culture supernatants of LPS-treated and of combined TNFalpha/IL-1beta/LPS-treated adipocytes. SRIF protein was visualized by immunohistochemistry in explanted minced adipose tissue after overnight incubation in culture medium supplemented with combined IL-1beta and LPS. In septic patients, expression of SRIF-mRNA and SRIF protein was found in visceral, but not in sc, adipose tissue. Adipocyte mRNA abundance of SSTR 1-5 was differentially regulated by inflammatory treatments.Thus, human visceral adipose tissue secretes SRIF during inflammation and sepsis and expresses several SSTRs. It is tempting to speculate that visceral adipose tissue-derived SRIF plays a modulatory role in the immunological and metabolic response to inflammation.

Expression and secretion of procalcitonin and calcitonin gene-related peptide by adherent monocytes and by macrophage-activated adipocytes.

OBJECTIVE: To explore the roles of peripheral blood mononuclear cells (PBMCs) and PBMC-derived macrophages in sepsis-related increased procalcitonin and calcitonin gene-related peptide (CGRP) I production. DESIGN: Prospective, in vitro primary human cell culture study and human tissue samples gene expression analysis. SETTING: University hospital research laboratories. PATIENTS: Cells from healthy donors and septic patients. INTERVENTIONS: PBMCs were obtained from healthy donors. Isolation of pure monocyte cultures was performed by magnetic depletion of nonmonocyte cells from PBMCs. Adipose tissue biopsies and circulating leukocytes were collected from septic patients. Expressions of calcitonin messenger RNA and CGRP I messenger RNA were analyzed using reverse transcriptase-polymerase chain reaction and quantitative real-time polymerase chain reaction. Supernatant procalcitonin and CGRP protein content were determined by ultrasensitive chemiluminometric and radioimmunoassays, respectively. MEASUREMENTS AND MAIN RESULTS: PBMCs expressed and secreted procalcitonin and CGRP within 3-5 hrs after adherence to endothelial cells or plastic surfaces. This induction was transient, as it was not detectable after 18 hrs. No calcitonin or CGRP I messenger RNA was observed in leukocytes obtained from septic patients with markedly increased serum procalcitonin concentrations. Stimulation with cytokines, endotoxin, or Escherichia coli did not induce expression of calcitonin and CGRP I messenger RNA in PBMC-derived macrophages. However, inflammatory factors released from activated macrophages induced a marked expression of procalcitonin and CGRP in co-cultured human adipocytes. CONCLUSIONS: The adhesion-induced, transient expression and secretion of procalcitonin and CGRP in vitro may play an important role during monocyte adhesion and migration in vivo. PBMC-derived macrophages may contribute to the marked increase in circulating procalcitonin by recruiting parenchymal cells within the infected tissue, as exemplified with adipocytes.

In vitro and in vivo calcitonin I gene expression in parenchymal cells: a novel product of human adipose tissue.

Circulating levels of calcitonin precursors (CTpr), including procalcitonin (ProCT), increase up to several thousand-fold in human sepsis, and immunoneutralization improves survival in two animal models of this disease. Herein, we analyzed inflammation-mediated calcitonin I gene (CALC I) expression in human adipocyte primary cultures and in adipose tissue samples from infected and noninfected patients with different levels of serum ProCT. In ex vivo differentiated adipocytes, the expression of CT mRNA increased 24-fold (P < 0.05) after the administration of Escherichia coli endotoxin (lipopolysaccharide) and 37-fold (P < 0.05) after IL-1beta administration by 6 h. ProCT protein secretion into culture supernatant increased 13.5-fold (P < 0.01) with lipopolysaccharide treatment and 15.2-fold (P < 0.01) with IL-1beta after 48 h. In coculture experiments, adipocyte CT mRNA expression was evoked by E. coli-activated macrophages in which CT mRNA was undetectable. The marked IL-1beta-mediated ProCT release was inhibited by 89% during coadministration with interferon-gamma (IFNgamma). In patients with infection and markedly increased serum ProCT, CT mRNA was detected in adipose tissue biopsies. Hence, we demonstrate that ProCT, which is suspected to mediate deleterious effects in sepsis and inflammation, is a novel product of adipose tissue secretion. The inhibiting effect of IFNgamma on IL-1beta-induced CT mRNA expression and on ProCT secretion might explain previous observations that serum ProCT concentrations increase less in systemic viral compared with bacterial infections.