Pro-inflammatory chemokines CCL2, chemerin, IP-10 and RANTES in human serum during an oral lipid tolerance test.

BACKGROUND: There is a strong coincidence of obesity and a chronic state of modest inflammation. Secretion of pro-inflammatory cytokines from adipocytes and immune cells represents a key mechanism in this process and is affected by fatty acids. MATERIAL AND METHODS: A study cohort of 100 overnight fasted healthy volunteers underwent an oral lipid tolerance test (OLTT) by ingestion of 160ml of a protein- and sugar-free lipid emulsion of defined composition. Venal blood was drawn at 0h (fasting) and at 2, 4, and 6h after lipid ingestion. Subjects were characterized by anthropometric and standard laboratory parameters. Serum concentrations of CCL2, IP-10, chemerin, and RANTES were measured by enzyme-linked immunosorbent assay (ELISA). Murine 3T3-L1 adipocytes were stimulated with free fatty acids (FA) and with sex steroids and concentrations of CCL2 and chemerin in cell culture supernatants were measured by ELISA. RESULTS: A significant reduction of circulating CCL2, IP-10, and chemerin concentrations was observed as a consequence of triglyceride ingestion whereas RANTES levels were increased. CCL2 serum concentrations were positively correlated with resistin and visfatin levels and with LDL/HDL ratio and negatively with adiponectin. There were significant differences in chemerin and RANTES serum concentrations in female and male subjects. CCL2 secretion from 3T3-L1 adipocytes was inhibited by treatment with linoleic (LA) and oleic acid (OA) whereas chemerin secretion was induced. Chemerin release from 3T3-L1 adipocytes was inhibited by testosterone. CONCLUSIONS: Oral lipid loading is linked to reduced circulating pro-inflammatory chemokines CCL2, IP-10, and chemerin and to increased RANTES levels, suggesting that dietary lipids affect immune function.

Mitotane Inhibits Sterol-O-Acyl Transferase 1 Triggering Lipid-Mediated Endoplasmic Reticulum Stress and Apoptosis in Adrenocortical Carcinoma Cells.

Adrenocortical carcinoma (ACC) is a rare malignancy that harbors a dismal prognosis in advanced stages. Mitotane is approved as an orphan drug for treatment of ACC and counteracts tumor growth and steroid hormone production. Despite serious adverse effects, mitotane has been clinically used for decades. Elucidation of its unknown molecular mechanism of action seems essential to develop better ACC therapies. Here, we set out to identify the molecular target of mitotane and altered downstream mechanisms by combining expression genomics and mass spectrometry technology in the NCI-H295 ACC model cell line. Pathway analyses of expression genomics data demonstrated activation of endoplasmic reticulum (ER) stress and profound alteration of lipid-related genes caused by mitotane treatment. ER stress marker CHOP was strongly induced and the two upstream ER stress signalling events XBP1-mRNA splicing and eukaryotic initiation factor 2 A (eIF2α) phosphorylation were activated by mitotane in NCI-H295 cells but to a much lesser extent in four nonsteroidogenic cell lines. Lipid mass spectrometry revealed mitotane-induced increase of free cholesterol, oxysterols, and fatty acids specifically in NCI-H295 cells as cause of ER stress. We demonstrate that mitotane is an inhibitor of sterol-O-acyl-transferase 1 (SOAT1) leading to accumulation of these toxic lipids. In ACC tissue samples we show variable SOAT1 expression correlating with the response to mitotane treatment. In conclusion, mitotane confers adrenal-specific cytotoxicity and down-regulates steroidogenesis by inhibition of SOAT1 leading to lipid-induced ER stress. Targeting of cancer-specific lipid metabolism opens new avenues for treatment of ACC and potentially other types of cancer.

Suspected metastatic adrenocortical carcinoma revealing as pulmonary Kaposi sarcoma in adrenal Cushing's syndrome.

BACKGROUND: Kaposi sarcoma (KS) is a malignant disease most commonly diagnosed in the setting of a human immunodeficiency virus (HIV) infection and in patients receiving immunosuppressive treatment. Pulmonary KS has never been reported in association with endogenous Cushing's syndrome (CS). CASE PRESENTATION: A 60-year-old woman presented with symptoms and signs of CS. Adrenal CS was confirmed by standard biochemical evaluation. Imaging revealed a right adrenal lesion (diameter 3.5 cm) and multiple pulmonary nodules, suggesting a cortisol-secreting adrenal carcinoma with pulmonary metastases. The patient underwent right adrenalectomy with a pathohistological diagnosis of an adrenal adenoma. Subsequent thoracoscopic wedge resection of one lung lesion revealed pulmonary KS with positive immunostaining for human herpes virus 8 (HHV-8). HIV-serology was negative. Hydrocortisone replacement was initiated for secondary adrenal insufficiency after surgery. Post-operative follow up imaging showed complete remission of all KS-related pulmonary nodules solely after resolution of hypercortisolism. CONCLUSION: KS may occur in the setting of endogenous CS and may go into remission after cure of hypercortisolism without further specific treatment.

CYP2W1 is highly expressed in adrenal glands and is positively associated with the response to mitotane in adrenocortical carcinoma.

BACKGROUND: Adrenocortical tumors comprise frequent adenomas (ACA) and rare carcinomas (ACC). Human cytochrome P450 2W1 (CYP2W1) is highly expressed in some cancers holding the potential to activate certain drugs into tumor cytotoxins. OBJECTIVE: To investigate the CYP2W1 expression in adrenal samples and its relationship with clinical outcome in ACC. MATERIAL AND METHODS: CYP2W1 expression was investigated by qRT-PCR in 13 normal adrenal glands, 32 ACA, 25 ACC, and 9 different non-adrenal normal tissue samples and by immunohistochemistry in 352 specimens (23 normal adrenal glands, 33 ACA, 239 ACC, 67 non-adrenal normal or neoplastic samples). RESULTS: CYP2W1 mRNA expression was absent/low in normal non-adrenal tissues, but high in normal and neoplastic adrenal glands (all P<0.01 vs non-adrenal normal tissues). Accordingly, CYP2W1 immunoreactivity was absent/low (H-score 0-1) in 72% of non-adrenal normal tissues, but high (H-score 2-3) in 44% of non-adrenal cancers, in 65% of normal adrenal glands, in 62% of ACAs and in 50% of ACCs (all P<0.001 vs non-adrenal normal tissues), being significantly increased in steroid-secreting compared to non-secreting tumors. In ACC patients treated with mitotane only, high CYP2W1 immunoreactivity adjusted for ENSAT stage was associated with longer overall survival and time to progression (P<0.05 and P<0.01, respectively), and with a better response to therapy both as palliative (response/stable disease in 42% vs 6%, P<0.01) or adjuvant option (absence of disease recurrence in 69% vs 45%, P<0.01). CONCLUSION: CYP2W1 is highly expressed in both normal and neoplastic adrenal glands making it a promising tool for targeted therapy in ACC. Furthermore, CYP2W1 may represent a new predictive marker for the response to mitotane treatment.

Adipocyte chemerin release is induced by insulin without being translated to higher levels in vivo.

BACKGROUND: Chemerin is an adipokine that regulates insulin sensitivity and insulin secretion. Prolonged hyperinsulinaemia is associated with higher systemic chemerin, and insulin induces adipose tissue chemerin release. These findings led us to hypothesize that systemic chemerin may be associated with post-prandial glucose metabolism and/or may even be induced after oral glucose load. Therefore, the effect of insulin on adipocyte chemerin levels and systemic chemerin in mice was analysed. Further, systemic levels of chemerin after oral glucose load in nondiabetic individuals were studied. DESIGN AND METHODS: Chemerin levels were determined in adipocytes after short-term and long-term treatment with insulin. Effects of acute hyperinsulinaemia were studied in mice. Chemerin was measured during oral glucose tolerance test in 66 healthy, nondiabetic individuals stratified for established body mass index categories. RESULTS: Insulin induces chemerin release from adipocytes within 24 h, while cellular levels are not affected. Short-term hyperinsulinaemia also upregulates adipocyte chemerin in vitro but has no effect on adipose tissue and chemerin serum levels of mice. Systemic chemerin is higher in overweight/obese than normal-weight controls and positively correlates with total cholesterol. Chemerin is not associated with markers of insulin sensitivity like fasting glucose or insulin. Fasting chemerin levels are similar to concentrations measured 1 and 2 h after oral glucose uptake in overweight and obese donors. CONCLUSIONS: Post-prandial hyperinsulinaemia does not contribute to higher chemerin levels in nondiabetic individuals.

Intracellular survival of Staphylococcus aureus in adipocyte-like differentiated 3T3-L1 cells is glucose dependent and alters cytokine, chemokine, and adipokine secretion.

Although obesity and type 2 diabetes mellitus are associated with Gram-positive infections and a worse clinical outcome, it is unknown whether adipocytes can be infected by Gram-positive bacteria. Adipocyte-like differentiated 3T3-L1 cells and Staphylococcus aureus were used for infection experiments under normoglycemic (100 mg/dl) and hyperglycemic (450 mg/dl) conditions in the presence/absence of insulin (1 µm). Intracellular presence and survival of S. aureus was investigated quantitatively. Supernatant cytokines, chemokines, and adipokines were measured by ELISA. Lipid metabolism and cellular morphology of infected adipocytes were investigated by different techniques. The present study provides the proof of principle that adipocyte-like cells can be infected by S. aureus dose dependently for up to 5 d. Importantly, low bacterial inocula did not affect cell viability. Intracellular survival of S. aureus was glucose dependent but not insulin dependent, and insulin receptor expression and insulin receptor signaling were not altered. Infection increased macrophage chemoattractant protein-1, visfatin, and IL-6 secretion, whereas resistin and adiponectin were decreased. Infected adipocytes had higher intracellular triacylglycerol concentrations and larger lipid droplets because of a decreased lipolysis. Taken together, infection of adipocytes by S. aureus is glucose dependent, inhibits cellular lipolysis, and affects the secretion of immunomodulating adipokines differentially. Because cell viability is not affected during infection, adipose tissue might function as a host for chronic infection by bacteria-causing metabolic, proinflammatory, and prodiabetic disturbances.

Systemic resistin is increased in type 2 diabetic patients treated with loop diuretics.

Increased serum resistin was found in rodent models of obesity and insulin resistance, whereas contradictory results have been obtained in human studies. In humans, resistin is primarily released by monocytes/macrophages, suggesting that soluble levels may be associated with macrophage activation. Here, systemic and monocyte-released resistin levels were found to be similar in type 2 diabetic (T2D) patients, overweight controls and normal-weight controls. When adjusted for body mass index and age, serum resistin modestly correlated with gamma-glutamyltransferase levels, fasting glucose and interleukin-6. Systemic resistin was marginally increased in T2D patients treated with beta-blockers or urate-lowering drugs and was considerably higher in patients treated with loop diuretics. Monocyte-released resistin was even reduced by the loop diuretic furosemide, excluding the possibility that this drug may directly stimulate resistin synthesis. In summary, the current data indicate that changes accompanying renal dysfunction but not obesity or type 2 diabetes are associated with increased serum resistin.

In vivo suppression of visfatin by oral glucose uptake: evidence for a novel incretin-like effect by glucagon-like peptide-1 (GLP-1).

BACKGROUND: Visfatin represents an adipokine of the visceral adipose tissue exerting pleiotropic effects. The aim of the study was to characterize the physiological regulation of visfatin release in vivo in healthy, nondiabetic probands and in adipocytes in vitro. SUBJECTS AND METHODS: One hundred healthy subjects (64 females and 36 males) underwent an oral glucose tolerance test (75 g). Probands were characterized by anthropometric and laboratory parameters. Fasting and postprandial (1 and 2 h) serum concentrations of glucose, insulin, and visfatin were measured. Stimulation experiments using glucose, insulin, mannitol, and glucagon-like peptide-1 (GLP-1) were performed on 3T3-L1 adipocytes including Western blot analysis. RESULTS: Fasting visfatin levels were not different between males/females or lean/obese individuals but were negatively (r = -0.5; P < 0.001) correlated with fasting glucose levels. Visfatin levels were rapidly and significantly suppressed (P < 0.001) upon an oral glucose intake, and this suppression was more pronounced in overweight and female probands. In vitro experiments demonstrated that hyperglycemia, osmotic stress, and sex steroids did not influence visfatin release. In contrast, insulin strongly (P = 0.002) inhibited visfatin release in vitro by approximately 50%, and this suppression was more pronounced under hyperglycemia. Importantly, GLP-1 strongly (P < 0.001) inhibited adipocytic visfatin release by approximately 50%. CONCLUSIONS: Insulin and GLP-1 are responsible for the rapid suppression of visfatin levels upon an oral glucose uptake in healthy probands. The inhibitory effect of GLP-1 on adipocytic visfatin release together with the absence of direct glucose effects on visfatin release suggests the existence of a novel incretin-like effect represented by a GLP-1/visfatin/axis.

Bile acid signaling after an oral glucose tolerance test.

Monitoring bile acids as signal molecules in combination with a bile acid synthesis marker and the FXR regulator fibroblast growth factor 19 (FGF19), this study addresses significant postprandial changes. The efficacy of the different pathways to regulate bile acid synthesis through short heterodimer partner (SHP) dependent FXR modulation in liver, and SHP independent activation via FGF19 is demonstrated. Characteristic changes of the bile acid profile during an oral glucose tolerance test (oGTT) were investigated in 73 individuals. 15 bile acid species including conjugated and unconjugated forms were quantitatively determined with LC-MS/MS in serum samples collected at three time points during the oGTT. All conjugated bile acid species showed the same time course, a significant increase at 60 min after the glucose intake and an incline at 120 min. In contrast, a consistent decline of all unconjugated bile acids was monitored. 7α-Hydroxy-4-cholesten-3-one, an early bile acid synthesis marker, showed an inverse response with a significant decrease at 60 min which proves the efficient and rapid downregulation of CYP7A1 via FXR activation through bile acid signaling. Significantly higher levels of FGF19 were observed 120 min after glucose intake and 60 min after bile acid excursion.

Elevated free fatty acids and impaired adiponectin bioactivity contribute to reduced SOD2 protein in monocytes of type 2 diabetes patients.

Type 2 diabetes (T2D) is characterized by increased oxidative stress contributing to the development of cardiovascular disease (CVD). Monocytes are critically important in the pathogenesis of CVD and antioxidant enzymes like superoxide dismutase (SOD2) protect these cells from excessive reactive oxygen species (ROS). Adiponectin is an adipocyte-derived protein with atheroprotective function and the effect of adiponectin on monocyte SOD2 was analyzed herein. Adiponectin upregulated SOD2 mRNA and dose- and time-dependently induced SOD2 protein in primary human monocytes. Elevated systemic free fatty acids (FFA) are commonly found in T2D patients and palmitic acid as well as oleic acid reduced monocyte SOD2 protein. Adiponectin mediated upregulation of SOD2, however, was not affected by FFA incubation. SOD2 protein was reduced in T2D monocytes compared to monocytes of age- and body mass index-matched healthy controls. Adiponectin still induced SOD2 in T2D monocytes but efficiency tended to be reduced. In summary this study indicates that elevated systemic free fatty acids and impaired adiponectin activity contribute to reduced SOD2 and most likely increased oxidative stress in T2D monocytes.

C1q/TNF-related protein-3 represents a novel and endogenous lipopolysaccharide antagonist of the adipose tissue.

Proteins secreted by adipocytes (adipokines) play an important role in the pathophysiology of type 2 diabetes mellitus and the associated chronic and low-grade state of inflammation. It was the aim to characterize the antiinflammatory potential of the new adipocytokine, C1q/TNF-related protein-3 (CTRP-3), which shows structural homologies to the pleiotropic adipocytokine adiponectin. mRNA and protein expression of CTRP-3 was analyzed by RT-PCR and Western blot. Recombinant CTRP-3 and small interfering RNA-based strategies were used to investigate the effect of CTRP-3 on toll-like receptor (TLR) ligand, lipopolysaccharide (LPS)-, and lauric acid-induced chemokine release of monocytes and adipocytes. Together with complex ELISA-based techniques, a designed TLR4/myeloid differentiation protein-2 fusion molecule shown to bind LPS was used to prove the ability of CTRP-3 to act as endogenous LPS antagonist. CTRP-3 is synthesized in monocytes and adipocytes. The recombinant protein dose-dependently inhibits the release of chemokines in monocytes and adipocytes that were induced by lauric acid, LPS, and other TLR ligands in vitro and ex vivo. CTRP-3 inhibits monocyte chemoattractant protein-1 release in adipocytes, whereas small interfering RNA-mediated knockdown of CTRP-3 up-regulates monocyte chemoattractant protein-1 release, reduces lipid droplet size, and decreases intracellular triglyceride concentration in adipocytes, causing a dedifferentiation into a more proinflammatory and immature phenotype. By using a designed TLR4/MD-2 fusion molecule, it is shown by different techniques that CTRP-3 specifically and effectively inhibits the binding of LPS to its receptor, TLR4/MD-2. CTRP-3 inhibits three basic and common proinflammatory pathways involved in obesity and type 2 diabetes mellitus (adipo-inflammation) by acting as an endogenous LPS antagonist of the adipose tissue.

Toll-like receptor ligands cause proinflammatory and prodiabetic activation of adipocytes via phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase but not interferon regulatory factor-3.

Here, we aim to investigate the mechanisms of Toll-like receptor (TLR)-induced prodiabetic and proinflammatory activation of adipocytes and to detect differences in the responsiveness of TLRs to their respective ligands between adipocytes isolated from inflamed vs. noninflamed adipose tissue. Experiments using specific ligands for all known TLRs were performed in murine 3T3-L1 adipocytes and in human adipocytes isolated from noninflamed and inflamed adipose tissue. IL-6 and monocyte chemoattractant protein-1 (MCP-1) release were measured by ELISA. The expression of the signal transduction proteins phospho-extracellular signal-regulated kinase (P-Erk), P-c-Jun N-terminal kinase (JNK), and P-interferon regulatory factor-3 was investigated by Western blot analysis. Additionally, functional inhibitors of MAPK kinase-1/-2 and JNK-1/-2 were used in the stimulation experiments. Activation of TRL4 by lipopolysaccharide (LPS) and TLR1/2 by Pam(3)Cys up-regulates IL-6 and MCP-1 release in adipocytes via specific activation of Erk. Stimulation of adipocytes by macrophage activating lipopeptide-2 (MALP-2) induces MCP-1 but has no effect on IL-6 release. This stimulatory effect on MCP-1 release is antagonized by inhibition of both mitogen-activated protein kinase-1/-2 and JNK-1/-2. Phosphorylation of Erk and JNK is up-regulated after stimulation by MALP-2. In human adipocytes isolated from noninflamed adipose tissue, LPS and Pam(3)Cys, but not MALP-2, are potent inducers of IL-6 and MCP-1. MALP-2 is able to induce IL-6 and MCP-1 release in adipocytes isolated from inflamed adipose tissue, whereas these adipocytes lost their ability to respond to LPS. The present results point to a role of the adipose tissue in innate immunity. TLR-ligand-induced proinflammatory and prodiabetic activation of adipocytes might couple visceral adipose tissue dysfunction with insulin resistance and type 2 diabetes mellitus.

Effects of the new adiponectin paralogous protein CTRP-3 and of LPS on cytokine release from monocytes of patients with type 2 diabetes mellitus.

AIMS/HYPOTHESIS: It was the aim to investigate the hypothesis that the new C1q/TNF-family member CTRP-3 (C1q/TNF-related protein-3) acts anti-inflammatory in human monocytes from healthy controls and patients with type 2 diabetes mellitus (T2D). METHODS: Monocytes were isolated from 20 healthy controls and 30 patients with T2D. IL-6 and TNF concentrations were measured by ELISA. CTRP-3 was expressed in insect cells and used for stimulation experiments. RESULTS: Basal IL-6 and TNF were not different in control and in T2D monocytes. LPS-stimulation (1 microg/ml) significantly (p<0.001) increased IL-6 and TNF in the supernatants of control and in T2D monocytes to a similar extent. CTRP-3 (1 microg/ml) significantly (p=0.03) inhibited LPS-induced IL-6 in control monocytes but not in T2D monocytes. TNF upon co-stimulation with LPS and CTRP-3 was significantly (p=0.012) lower in control than in T2D monocytes. LPS-induced TNF concentration was significantly and positively correlated with serum total cholesterol and LDL cholesterol in T2D patients. CONCLUSIONS: CTRP-3 inhibits LPS-induced IL-6 and TNF release. This anti-inflammatory effect is lost in T2D. Serum cholesterol concentration affects the pro-inflammatory potential of LPS to induce TNF release from T2D monocytes in the presence or absence of CTRP-3. CTRP-3 might partly account for the pro-inflammatory state in T2D.

Adiponectin downregulates galectin-3 whose cellular form is elevated whereas its soluble form is reduced in type 2 diabetic monocytes.

Galectin-3 plays a role in atherosclerotic diseases, and the effect of adiponectin that protects from atherosclerotic diseases on monocytic galectin-3 was analysed. Adiponectin reduced galectin-3 mRNA, its cellular and soluble form, and this effect was impaired in T2D cells. Cellular galectin-3 was higher in monocytes of overweight than normal-weight donors and was highest in T2D cells. Cellular galectin-3 positively correlated with the BMI of the donors and negatively with soluble monocyte galectin-3. Circulating levels of total adiponectin did not correlate with cellular or soluble galectin-3 indicating that additional factors contribute to higher cellular monocytic galectin-3 in obesity and T2D.

[Hormone-induced spontaneous femoral neck fracture in a 28-year-old female patient]. / Hormonell bedingte Schenkelhalsspontanfraktur bei einer 28-jährigen Patientin.

CASE REPORT: A 28-year-old woman was referred to hospital with a spontaneous femoral neck fracture. Dual energy X-ray absorptiometry showed severe osteoporosis without apparent cause or risk factors. The basic endocrinologic evaluation revealed hypercortisolism, and the diagnosis of Cushing's disease could be made following biochemical test and magnetic resonance imaging. A transsphenoidal resection of the pituitary gland tumor was performed and led to a cure of the disease. The patient received calcium and vitamin D substitution as well as biphosphonates. CONCLUSION: Glucocorticoid-induced osteoporosis is the most frequent cause of secondary osteoporosis. Only few cases with occult endogenous Cushing's syndrome and osteoporosis as the main manifestation of the disease have been reported. Nevertheless, hypercortisolism should be excluded whenever osteoporosis without apparent cause is diagnosed.

[Reversible metabolic syndrome]. / Reversibles metabolisches Syndrom hormoneller Ursache.

A 52-year-old male patient was admitted to the emergency department with dyspnea and hypertensive urgency. During the previous 6 months, the patient had noticed leg edema, weight gain (particularly in the face and abdomen), and impotence. 1.5 years ago, he was diagnosed with hypertension resistant to medication. After an accident at work 1 year ago, osteoporosis was diagnosed with vertebral and rib fractures. Measurement of sleeping midnight salivary cortisol levels together with 24-h urine free cortisol excretion and an overnight low-dose 1-mg dexamethasone suppression test proved overt hypercortisolism. The high-dose 8-mg dexamethasone suppression suggested an adrenal or ectopic source of hypercortisolism. By contrast, elevated adreno-corticotropic hormone (ACTH) levels and a corticotropin-releasing hormone stimulation test gave evidence for a pituitary source of hypercortisolism. However, pituitary magnetic resonance imaging failed to reveal a pituitary adenoma. Moreover, computed tomography scans of thorax and abdomen were negative. In this situation, an inferior petrosal vein sampling was performed and revealed an ACTH gradient (central-systemic) >3 with lateralization to the right side. The patient underwent a selective, partial, transsphenoidal resection and was cured from clinical signs and symptoms caused by hypercortisolism. Subsequent hormonal replacement therapy of postoperative pituitary insufficiency was necessary.