Metformin to reduce metabolic complications and inflammation in patients on systemic glucocorticoid therapy: a randomised, double-blind, placebo-controlled, proof-of-concept, phase 2 trial.

BACKGROUND: An urgent need to reduce the metabolic side-effects of glucocorticoid overexposure has been recognised, as glucocorticoid excess can lead to Cushing's syndrome, which is associated with high morbidity. We aimed to evaluate the potential of metformin to reverse such effects while sparing the anti-inflammatory benefits of glucocorticoids. METHODS: We did a randomised, double-blind, placebo-controlled, proof-of-concept, phase 2 trial involving four hospitals in the UK. Patients without diabetes were eligible if they were between the ages of 18 and 75 years with an inflammatory disease treated with continuous prednisolone (≥20 mg/day for ≥4 weeks and remaining on ≥10 mg/day for the subsequent 12 weeks, or its cumulative dose-equivalent). Eligible patients were randomly allocated (1:1) to either the metformin or placebo groups, using a computer-generated randomisation table stratified according to age and BMI. Metformin and placebo were administered orally for 12 weeks in escalating doses: 850 mg/day for the first 5 days, 850 mg twice a day for the next 5 days, and 850 mg three times a day subsequently. The primary outcome was the between-group difference in visceral-to-subcutaneous fat area ratio over 12 weeks, assessed by CT. Secondary outcomes included changes in metabolic, bone, cardiovascular, and inflammatory parameters over 12 weeks. Our analysis followed a modified intention-to-treat principle for the primary outcome. This study is registered with ClinicalTrials.gov, NCT01319994. FINDINGS: Between July 17, 2012, and Jan 14, 2014, 849 patients were assessed for study eligibility, of which 53 were randomly assigned to receive either metformin (n=26) or placebo (n=27) for 12 weeks. 19 patients in the metformin group and 21 in the placebo group were eligible for the primary outcome analysis. Both groups received an equivalent cumulative dose of glucocorticoids (1860 mg prednisolone-equivalent [IQR 1060-2810] in the metformin group vs 1770 mg [1020-2356] in the placebo group); p=0·76). No change in the visceral-to-subcutaneous fat area ratio between the treatment groups was observed (0·11, 95% CI -0·02 to 0·24; p=0·09), but patients in the metformin group lost truncal subcutaneous fat compared with the placebo group (-3835 mm2, 95% CI -6781 to -888; p=0·01). Improvements in markers of carbohydrate, lipid, liver, and bone metabolism were observed in the metformin group compared with the placebo group. Additionally, those in the metformin group had improved fibrinolysis, carotid intima-media thickness, inflammatory parameters, and clinical markers of disease activity. The frequency of pneumonia (one event in the metformin group vs seven in the placebo group; p=0·01), overall rate of moderate-to-severe infections (two vs 11; p=0·001), and all-cause hospital admissions due to adverse events (one vs nine; p=0·001) were lower in the metformin group than in the placebo group. Patients in the metformin group had more events of diarrhoea than the placebo group (18 events vs eight; p=0·01). INTERPRETATION: No significant changes in the visceral-to-subcutaneous fat area ratio between the treatment groups were observed; however, metformin administration did improve some of the metabolic profile and clinical outcomes for glucocorticoid-treated patients with inflammatory disease, which warrants further investigation. FUNDING: Barts Charity and Merck Serono.

Efficacy of Monotherapy with Biologics and JAK Inhibitors for the Treatment of Rheumatoid Arthritis: A Systematic Review.

Despite recommendations suggesting that biological and targeted synthetic disease-modifying antirheumatic drugs (b/tsDMARDs) should be used in combination with methotrexate in the treatment of rheumatoid arthritis (RA), up to one-third of patients with RA are treated with monotherapy. The objective of the systematic literature review reported here was to evaluate the clinical evidence regarding the efficacy of b/tsDMARDs as monotherapy in the treatment of RA. MEDLINE®, Embase®, and the Cochrane Central Trials Register (to April 11, 2017) and the American College of Rheumatology and European League Against Rheumatism conference proceedings (2010-2016) were searched for randomized controlled trials evaluating the efficacy of b/tsDMARDs as monotherapy for RA in adults. Forty-four monotherapy studies of abatacept, adalimumab, baricitinib, certolizumab pegol, etanercept, sarilumab, sirukumab, tocilizumab, and tofacitinib reported in 71 publications were identified. Tocilizumab had the most studies (14), followed by etanercept (10) and adalimumab (9). These b/tsDMARDs were consistently shown to be efficacious treatments, regardless of whether patients were intolerant of or had never used conventional synthetic (cs) DMARDs. However, better treatment outcomes were usually achieved with combination therapy, and this was observed for all b/tsDMARDs assessed by this review. Only a few studies provided a head-to-head comparison between b/tsDMARD treatments or between b/tsDMARD monotherapy and combination therapy, and as many were initial RA treatments they were not generalizable to usual care. In conclusion, evidence from randomized trials suggests that the b/tsDMARDs studied are effective as monotherapy. In general, some patient responses seem better with combination therapy and the durability of monotherapy is less than combination therapy. There is, however, a need for longer-term head-to-head trials to establish positioning of these interventions in the treatment algorithm for RA. FUNDING: Pfizer.Plain Language Summary: Plain language summary available on the journal website.

The Impact of Biologics and Tofacitinib on Cardiovascular Risk Factors and Outcomes in Patients with Rheumatic Disease: A Systematic Literature Review.

INTRODUCTION: Rheumatic diseases are autoimmune, inflammatory diseases often associated with cardiovascular (CV) disease, a major cause of mortality in these patients. In recent years, treatment with biologic and targeted synthetic disease-modifying anti-rheumatic drugs (DMARDs), either as monotherapy or in combination with other drugs, have become the standard of treatment. In this systematic literature review, we evaluated the effect of treatment with biologic or tofacitinib on the CV risk and outcomes in these patients. METHODS: A systematic search was performed in MEDLINE, Embase, the Cochrane Central Register of Controlled Trials, and Cochrane Database of Systematic Reviews for articles reporting on CV risk and events in patients with rheumatic disease treated with a biologic agent or tofacitinib. Articles identified were subjected to two levels of screening. Articles that passed the first level based on title and abstract were assessed on full-text evaluation. The quality of randomized clinical trials was assessed by Jadad scoring system and the quality of the other studies and abstracts was assessed using the Downs and Black instrument. The data extracted included study design, baseline patient characteristics, and measurements of CV risk and events. RESULTS: Of the 5722 articles identified in the initial search, screening yielded 105 unique publications from 90 unique studies (33 clinical trials, 39 prospective cohort studies, and an additional 18 retrospective studies) that reported CV risk outcomes. A risk of bias analysis for each type of report indicated that they were of good or excellent quality. Importantly, despite some limitations in data reported, there were no indications of significant increase in adverse CV events or risk in response to treatment with the agents evaluated. CONCLUSIONS: Treatment with biologic or tofacitinib appears to be well-tolerated with respect to CV outcomes in these patients.

Immunogenicity of Biologics in Chronic Inflammatory Diseases: A Systematic Review.

OBJECTIVES: A systematic review was conducted to explore the immunogenicity of biologic agents across inflammatory diseases and its potential impact on efficacy/safety. METHODS: Literature searches were conducted through November 2016 to identify controlled and observational studies of biologics/biosimilars administered for treatment of rheumatoid arthritis (RA), psoriatic arthritis (PsA), juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS), non-radiographic axial spondyloarthritis (nr-axSpA), psoriasis (Ps), Crohn's disease, and ulcerative colitis. RESULTS: Of >21,000 screened publications, 443 were included. Anti-drug antibody (ADAb) rates varied widely among biologics across diseases (and are not directly comparable because of immunoassay heterogeneity); the highest overall rates were reported with infliximab (0-83%), adalimumab (0-54%), and infliximab biosimilar CT-P13 (21-52%), and the lowest with secukinumab (0-1%), ustekinumab (1-11%), etanercept (0-13%), and golimumab (0-19%). Most ADAbs were neutralizing, except those to abatacept and etanercept. ADAb+ versus ADAb- patients had lower rates of clinical response to adalimumab (RA, PsA, JIA, AS, Ps), golimumab (RA), infliximab (RA, PsA, AS, Ps), rituximab (RA), ustekinumab (Ps), and CT-P13 (RA, AS). Higher rates of infusion-related reactions were reported in infliximab- and CT-P13-treated ADAb+ patients. Background immunosuppressives/anti-proliferatives reduced biologic immunogenicity across diseases. CONCLUSIONS: Based on reviewed reports, biologic/biosimilar immunogenicity differs among agents, with the highest rates observed with infliximab and adalimumab. As ADAb formation in biologic-/biosimilar-treated patients may increase the risk of lost response, the immunogenicity of these agents is an important (albeit not the only) consideration in the treatment decision-making process.

Comparison of Etanercept Monotherapy and Combination Therapy with Methotrexate in Psoriatic Arthritis: Results from 2 Clinical Trials.

OBJECTIVE: To evaluate the clinical/functional outcomes associated with etanercept (ETN) monotherapy versus combination therapy in psoriatic arthritis (PsA). METHODS: Data from patients with PsA who received ETN alone (n = 322) or combined with methotrexate (MTX; n = 152) for 24 weeks in 2 placebo-controlled clinical trials were summarized across studies. RESULTS: Similar proportions of patients in the monotherapy and combination therapy groups achieved the PsA Response Criteria (80% and 83%) and the American College of Rheumatology improvements of 20% (ACR20; both 70%); numerically higher proportions receiving monotherapy achieved ACR50 (55% vs 48%) and ACR70 (35% vs 27%). Little between-group difference was observed in the 28-joint Disease Activity Score with C-reactive protein, the Psoriasis Area and Severity Index, and the Health Assessment Questionnaire-Disability Index improvement. CONCLUSION: ETN with and without MTX provided similar benefits in active PsA.

The effects of chronic candesartan treatment on cardiac and hepatic adenosine monophosphate-activated protein kinase in rats submitted to surgical stress.

INTRODUCTION: adenosine monophosphate-activated protein kinase (AMPK) plays a prominent role as a metabolic stress sensor, and it has recently been suggested that the renin-angiotensin system, in addition to its role in stress regulation, may play a significant role in regulating the AMPK system. This study aimed to evaluate the effects of candesartan, an angiotensin II receptor blocker, on cardiac and hepatic AMPK activity basally as well as after surgical stress under general anesthesia. MATERIALS AND METHODS: Male Wistar rats were treated with 5 mg/kg/day candesartan in their drinking water for two weeks. Levels of cardiac and hepatic AMPK activity were determined, using a kinase activity assay, basally and after surgical stress under general anesthesia. RESULTS: Chronic administration of candesartan increased hepatic AMPK activity approximately 4 times (p<0.05) while no significant change was demonstrated in cardiac AMPK. Cardiac and hepatic AMPK activities were not significantly increased by surgical stress alone performed under anesthesia. However, chronic treatment with candesartan decreased AMPK activity in both liver and heart after surgical stress under anesthesia (p<0.01 for both comparisons). CONCLUSIONS: While chronic candesartan treatment may stimulate AMPK activity in certain organs such as the liver, when combined with surgical stress under anesthesia it inhibits pathways regulating AMPK activity.

The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release.

This study aimed to investigate whether the growth hormone release and metabolic effects of ghrelin on AMPK activity of peripheral tissues are mediated by cannabinoid receptor type 1 (CB1) and the central nervous system. CB1-knockout (KO) and/or wild-type mice were injected peripherally or intracerebroventricularly with ghrelin and CB1 antagonist rimonabant to study tissue AMPK activity and gene expression (transcription factors SREBP1c, transmembrane protein FAS, enzyme PEPCK, and protein HSL). Growth hormone levels were studied both in vivo and in vitro. Peripherally administered ghrelin in liver, heart, and adipose tissue AMPK activity cannot be observed in CB1-KO or CB1 antagonist-treated mice. Intracerebroventricular ghrelin treatment can influence peripheral AMPK activity. This effect is abolished in CB1-KO mice and by intracerebroventricular rimonabant treatment, suggesting that central CB1 receptors also participate in the signaling pathway that mediates the effects of ghrelin on peripheral tissues. Interestingly, in vivo or in vitro growth hormone release is intact in response to ghrelin in CB1-KO animals. Our data suggest that the metabolic effects of ghrelin on AMPK in peripheral tissues are abolished by the lack of functional CB1 receptor via direct peripheral effect and partially through the central nervous system, thus supporting the existence of a possible ghrelin-cannabinoid-CB1-AMPK pathway.

CB1 receptor mediates the effects of glucocorticoids on AMPK activity in the hypothalamus.

AMP-activated protein kinase (AMPK), a regulator of cellular and systemic energy homeostasis, can be influenced by several hormones. Tissue-specific alteration of AMPK activity by glucocorticoids may explain the increase in appetite, the accumulation of lipids in adipose tissues, and the detrimental cardiac effects of Cushing's syndrome. Endocannabinoids are known to mediate the effects of various hormones and to influence AMPK activity. Cannabinoids have central orexigenic and direct peripheral metabolic effects via the cannabinoid receptor type 1 (CB1). In our preliminary experiments, WT mice received implants of a corticosterone-containing pellet to establish a mouse model of Cushing's syndrome. Subsequently, WT and Cb1 (Cnr1)-knockout (CB1-KO) littermates were treated with corticosterone and AMPK activity in the hypothalamus, various adipose tissues, liver and cardiac tissue was measured. Corticosterone-treated CB1-KO mice showed a lack of weight gain and of increase in hypothalamic and hepatic AMPK activity. In adipose tissues, baseline AMPK activity was higher in CB1-KO mice, but a glucocorticoid-induced drop was observed, similar to that observed in WT mice. Cardiac AMPK levels were reduced in CB1-KO mice, but while WT mice showed significantly reduced AMPK activity following glucocorticoid treatment, CB1-KO mice showed a paradoxical increase. Our findings indicate the importance of the CB1 receptor in the central orexigenic effect of glucocorticoid-induced activation of hypothalamic AMPK activity. In the periphery adipose tissues, changes may occur independently of the CB1 receptor, but the receptor appears to alter the responsiveness of the liver and myocardial tissues to glucocorticoids. In conclusion, our data suggest that an intact cannabinoid pathway is required for the full metabolic effects of chronic glucocorticoid excess.

Ghrelin and cannabinoids require the ghrelin receptor to affect cellular energy metabolism.

INTRODUCTION: Ghrelin is a potent orexigenic brain-gut peptide with lipogenic and diabetogenic effects, possibly mediated by growth hormone secretagogue receptor (GHS-R1a). Cannabinoids also have orexigenic and lipogenic effects. AMPK is a regulator of energy homeostasis and we have previously shown that ghrelin and cannabinoids stimulate hypothalamic AMPK activity while inhibiting it in the liver and adipose tissue, suggesting that AMPK mediates both the central appetite-inducing and peripheral effects of ghrelin and cannabinoids. AIMS: Using GHS-R KO mice, we investigated whether the known ghrelin receptor GHS-R1a is required for the tissue-specific effects of ghrelin on AMPK activity, and if an intact ghrelin signalling pathway is necessary for the effects of cannabinoids on AMPK activity. METHODS: Wild-type and GHS-R KO mice were treated intraperitoneally with ghrelin 500 ng/g bodyweight or CB1 agonist HU210 20 ng/g and hypothalamic, hepatic and adipose AMPK activity was studied using a functional kinase assay. RESULTS: Ghrelin and HU210 significantly stimulated hypothalamic AMPK activity in wild-type animals (mean±SEM, 122.5±5.2% and 128±11.6% of control, p<0.05) and inhibited it in liver (55.1±4.8% and 62.2±14.5%, p<0.01) and visceral fat (mesenteric fat (MF): 54.6±16% and 52.0±9.3%, p<0.05; epididymal fat (EF): 47.9±12.1% and 45.6±1.7%, p<0.05). The effects of ghrelin, and interestingly also HU210, on hypothalamic, visceral fat and liver AMPK activity were abolished in the GHS-R KO mice (hypothalamus: 107.9±7.7% and 87.4±13.3%, liver: 100.5±11.6% and 116.7±5.4%, MF: 132.1±29.9% and 107.1±32.7%, EF: 89.8±7.3% and 91.7±18.3%, p>0.05). CONCLUSIONS: Ghrelin requires GHS-R1a for its effect on hypothalamic, liver and adipose tissue AMPK activity. An intact ghrelin signalling pathway is necessary for the effects of cannabinoids on AMPK activity.

Measurement of AMP-activated protein kinase activity and expression in response to ghrelin.

Ghrelin is a circulating brain-gut peptide that is known to exert several metabolic effects such as stimulating appetite, inducing adiposity, increasing bone formation, and influencing the cardiovascular functions. AMP-activated protein kinase (AMPK), a highly conserved heterotrimeric protein that plays a key role in energy homeostasis, has been shown to mediate many of these metabolic effects of ghrelin. Ghrelin is shown to stimulate hypothalamic AMPK activity and inhibit liver and adipose tissue AMPK activity. The effects of ghrelin on AMPK activity can be studied using an elegant kinase assay, which involves immunoprecipitating AMPK protein from the tissue of interest followed by quantifying its enzymatic activity using radiolabeled adenosine triphosphate (ATP) in the presence of a suitable substrate. As a surrogate marker of AMPK activity, AMPK Thr(172) phosphorylation can be measured by Western blotting. Information about the AMPK pathway can also be gained by studying the mRNA expression of various AMPK subunits and by Western blotting for phosphorylated acetyl-CoA carboxylase, a key AMPK target. These methods have been widely used and published for investigating the effects of ghrelin on AMPK activity. In this chapter, we look into these experiments' methodology in detail.

Modifications in basal and stress-induced hypothalamic AMP-activated protein kinase (AMPK) activity in rats chronically treated with an angiotensin II receptor blocker.

5' adenosine monophosphate-activated protein kinase (AMPK) plays a prominent role as a metabolic stress sensor. The role of hypothalamic AMPK in response to restraint and surgical stress has not been previously investigated. It has been recently suggested that the renin-angiotensin system, in addition to its role in stress regulation, may play a significant role in regulating metabolic pathways including the regulation of the AMPK system. This study was thus aimed to evaluate the effects of candesartan, an angiotensin II AT1 receptor blocker drug, on hypothalamic AMPK activity under basal conditions and after restraint in conscious rats or after surgical stress under general anesthesia. Male Wistar rats were treated with 5 mg/kg/day candesartan in the drinking water for 2 weeks. The hypothalamic AMPK activity was determined under basal and stress conditions, using a kinase activity assay. Chronic administration of candesartan significantly increased hypothalamic AMPK activity. Hypothalamic AMPK activity was also increased by restraint stress whereas no change was observed during surgical stress under anesthesia. The high levels of hypothalamic AMPK activation observed in candesartan-treated rats were not changed by restraint stress but were reduced to control levels by anesthesia and surgery. In conclusion, chronic candesartan treatment and restraint stress in conscious rats stimulate the hypothalamic AMPK activity, whereas surgical stress under anesthesia inhibits pathways regulating the AMPK activity even in candesartan-treated rats.

The expression of ghrelin O-acyltransferase (GOAT) in human tissues.

Ghrelin is a circulating growth hormone-releasing and appetite-inducing brain-gut peptide. It needs to be acylated on its serine-3 with octanoate for its endocrine actions. The acyl-transferase that catalyses ghrelin octanoylation has recently been identified and named as GOAT (ghrelin O-acyltransferase); GOAT enzyme is coded by the MBOAT4 gene. This study aimed to investigate GOAT expression in the human. The distribution of GOAT mRNA expression was studied in various human tissues using classical and real-time reverse transcription and polymerase chain reaction. GOAT expression was found in all tissues studied (stomach, adrenal cortex, breast, right and left colon, duodenum, jejunum, ileum, fat, Fallopian tube, gallbladder, lymph node, lymphocyte cell line, kidney, liver, lung, muscle, myocardium, pituitary, oesophagus, pancreas, ovary, placenta, prostate, testis, spleen and thyroid). The widespread expression of GOAT corresponds to the widespread distribution of ghrelin expression. GOAT expression was high in stomach and gut, the major ghrelin-secreting tissues, and in the pituitary, in which ghrelin is known to show autocrine and paracrine effects. Identification of GOAT expression in various tissues support the concept that in addition to the important endocrine effect of acylated ghrelin, the paracrine effects of locally synthetised and acylated ghrelin may be important.

The ghrelin/GOAT/GHS-R system and energy metabolism.

Ghrelin is a brain-gut peptide that was discovered through reverse pharmacology and was first isolated from extracts of porcine stomach. Ghrelin binds to growth hormone secretagogue receptor (GHS-R) and is acylated on its serine 3 residue by ghrelin O-acyltransferase (GOAT). Several important biological functions of ghrelin have been identified, which include its growth hormone-releasing and appetite-inducing effects. Ghrelin exerts its central orexigenic effect mainly by acting on the hypothalamic arcuate nucleus via the activation of the GHS-R. Peripherally ghrelin has multiple metabolic effects which include promoting gluconeogenesis and fat deposition. These effects together with the increased food intake lead to an overall body weight gain. AMP-activated protein kinase, which is a key enzyme in energy homeostasis, has been shown to mediate the central and peripheral metabolic effects of ghrelin. The hypothalamic fatty acid pathway, hypothalamic mitochondrial respiration and uncoupling protein 2 have all been shown to act as the downstream targets of AMPK in mediating the orexigenic effects of ghrelin. Abnormal levels of ghrelin are associated with several metabolic conditions such as obesity, type 2 diabetes, Prader-Willi syndrome and anorexia nervosa. The ghrelin/GOAT/GHS-R system is now recognised as a potential target for the development of anti-obesity treatment.

Mechanisms of metformin action on glucose transport and metabolism in human adipocytes.

The mechanisms of metformin effects on glucose transport and metabolism were investigated in human adipocytes. Human preadipocytes obtained from surgical biopsies were differentiated in vitro into adipocytes and the effects of metformin on glucose uptake, glucose oxidation and the involved signaling pathways were analyzed. Metformin (1mM, 24h) increased glucose uptake 2.3±0.2-fold (p<0.001 vs. basal) in human adipocytes, without altering cell viability and oxygen consumption. Metformin did not alter GLUT-1 mRNA expression and protein content but increased GLUT-4 mRNA expression and cellular protein content, leading to increased GLUT-4 protein content in the plasma membrane. Neither basal nor insulin-induced phosphorylation of Akt at Ser-473 and AS160 (Akt substrate of 160kDa) at Thr-642 were enhanced by metformin. Suppression of metformin-induced AMP-activated protein kinase (AMPK) activity by AMPKα1 silencing, however, reduced metformin-associated GLUT-4 expression and stimulation of glucose uptake. In addition, metformin induced glucose oxidation. In conclusion, activation of AMPKα1 without impairment of cell respiration is crucial for metformin-mediated increase in GLUT-4 protein content and glucose uptake in human adipocytes.

Ghrelin's role as a major regulator of appetite and its other functions in neuroendocrinology.

Ghrelin is a circulating growth-hormone-releasing and appetite-inducing brain-gut peptide. It is a known natural ligand of the growth hormone secretagogue receptor (GHS-R). Ghrelin is acylated on its serine 3 residue by ghrelin O-acyltransferase (GOAT). The acylation is essential for its orexigenic and adipogenic effects. Ghrelin exerts its central orexigenic effect through activation of various hypothalamic and brain stem neurons. Several new intracellular targets/mediators of the appetite-inducing effect of ghrelin in the hypothalamus have recently been identified, including the AMP-activated protein kinase, its upstream kinase calmodulin kinase kinase 2, components of the fatty acid pathway and the uncoupling protein 2. The ghrelin/GOAT/GHS-R system is now recognised as a potential target for the development of anti-obesity treatment. Ghrelin regulates the function of the anterior pituitary through stimulation of secretion not only of growth hormone, but also of adrenocorticotrophin and prolactin. The implication of ghrelin and its receptor in the pathogenesis of the neuroendocrine tumors will also be discussed in this review.

Alterations in adipose tissue during critical illness: An adaptive and protective response?

RATIONALE: Critical illness is characterized by lean tissue wasting, whereas adipose tissue is preserved. Overweight and obese critically ill patients may have a lower risk of death than lean patients, suggestive of a protective role for adipose tissue during illness. OBJECTIVES: To investigate whether adipose tissue could protectively respond to critical illness by storing potentially toxic metabolites, such as excess circulating glucose and triglycerides. METHODS: We studied adipose tissue morphology and metabolic activity markers in postmortem biopsies of 61 critically ill patients and 20 matched control subjects. Adipose morphology was also studied in in vivo biopsies of 27 patients and in a rabbit model of critical illness (n = 22). MEASUREMENTS AND MAIN RESULTS: Adipose tissue from critically ill patients revealed a higher number and a smaller size of adipocytes and increased preadipocyte marker levels as compared with control subjects. Virtually all adipose biopsies from critically ill patients displayed positive macrophage staining. The animal model demonstrated similar changes. Glucose transporter levels and glucose content were increased. Glucokinase expression was up-regulated, whereas glycogen and glucose-6-phosphate levels were low. Acetyl CoA carboxylase protein and fatty acid synthase activity were increased. Hormone-sensitive lipase activity was not altered, whereas lipoprotein lipase activity was increased. A substantially increased AMP-activated protein kinase activity may play a crucial role. CONCLUSIONS: Postmortem adipose tissue biopsies from critically ill patients displayed a larger number of small adipocytes in response to critical illness, revealing an increased ability to take up circulating glucose and triglycerides. Similar morphologic changes were present in vivo. Such changes may render adipose tissue biologically active as a functional storage depot for potentially toxic metabolites, thereby contributing to survival.

AMPK as a mediator of hormonal signalling.

AMP-activated protein kinase (AMPK) is a key molecular player in energy homeostasis at both cellular and whole-body levels. AMPK has been shown to mediate the metabolic effects of hormones such as leptin, ghrelin, adiponectin, glucocorticoids and insulin as well as cannabinoids. Generally, activated AMPK stimulates catabolic pathways (glycolysis, fatty acid oxidation and mitochondrial biogenesis) and inhibits anabolic pathways (gluconeogenesis, glycogen, fatty acid and protein synthesis), and has a direct appetite-regulating effect in the hypothalamus. Drugs that activate AMPK, namely metformin and thiazolidinediones, are often used to treat metabolic disorders. Thus, AMPK is now recognised as a potential target for the treatment of obesity and associated co-morbidities.

Shedding light on the intricate puzzle of ghrelin's effects on appetite regulation.

Ghrelin, a hormone primarily produced by the stomach, has a wide range of metabolic and non-metabolic effects. It also stimulates food intake through activation of various hypothalamic and brain stem neurons. A series of recent studies have explored the intracellular mechanisms of the appetite-inducing effect of ghrelin in the hypothalamus, shedding light on the intricate mechanisms of appetite regulation. AMP-activated protein kinase (AMPK) is a key metabolic enzyme involved in appetite regulation. Calmodulin kinase kinase 2 (CaMKK2) has been identified as an upstream kinase of AMPK and a key mediator in the effect of ghrelin on AMPK activity. The fatty acid pathway, hypothalamic mitochondrial respiration, and uncoupling protein 2 have been outlined as downstream targets of AMPK and mediators of ghrelin's appetite stimulating effect. This short overview summarises the present data in this field.

Changes in adenosine 5'-monophosphate-activated protein kinase as a mechanism of visceral obesity in Cushing's syndrome.

OBJECTIVE: Features of the metabolic syndrome such as central obesity with insulin resistance and dyslipidemia are typical signs of Cushing's syndrome and common side effects of prolonged glucocorticoid treatment. AMP-activated protein kinase (AMPK), a key regulatory enzyme of lipid and carbohydrate metabolism as well as appetite, is involved in the development of the deleterious metabolic effects of excess glucocorticoids, but no data are available in humans. In the current study, we demonstrate the effect of high glucocorticoid levels on AMPK activity of human adipose tissue samples from patients with Cushing's syndrome. METHODS: AMPK activity and mRNA expression of genes involved in lipid metabolism were assessed in visceral adipose tissue removed at abdominal surgery of 11 patients with Cushing's syndrome, nine sex-, age-, and weight-matched patients with adrenal incidentalomas, and in visceral adipose tissue from four patients with non-endocrine-related abdominal surgery. RESULTS: The patients with Cushing's syndrome exhibited a 70% lower AMPK activity in visceral adipose tissue as compared with both incidentalomas and control patients (P = 0.007 and P < 0.001, respectively). Downstream targets of AMPK fatty acid synthase and phosphoenol-pyruvate carboxykinase were up-regulated in patients with Cushing's syndrome. AMPK activity was inversely correlated with 0900 h serum cortisol and with urinary free cortisol. CONCLUSIONS: Our data suggest that glucocorticoids inhibit AMPK activity in adipose tissue, suggesting a novel mechanism to explain the deposition of visceral adipose tissue and the consequent central obesity observed in patients with iatrogenic or endogenous Cushing's syndrome.

Protein western array analysis in human pituitary tumours: insights and limitations.

The molecular analysis of pituitary tumours has received a great deal of attention, although the majority of studies have concentrated on the genome and the transcriptome. We aimed to study the proteome of human pituitary adenomas. A protein array using 1005 monoclonal antibodies was used to study GH-, corticotrophin- and prolactin-secreting as well as non-functioning pituitary adenomas (NFPAs). Individual protein expression levels in the tumours were compared with the expression profile of normal pituitary tissue. Out of 316 proteins that were detected in the pituitary tissue samples, 116 proteins had not previously been described in human pituitary tissue. Four prominent differentially expressed proteins with potential importance to tumorigenesis were chosen for validation by immunohistochemistry and western blotting. In the protein array analysis heat shock protein 110 (HSP110), a chaperone associated with protein folding, and B2 bradykinin receptor, a potential regulator of prolactin secretion, were significantly overexpressed in all adenoma subtypes, while C-terminal Src kinase (CSK), an inhibitor of proto-oncogenic enzymes, and annexin II, a calcium-dependent binding protein, were significantly underexpressed in all adenoma subtypes. The immunohistochemical analysis confirmed the overexpression of HSP110 and B2 bradykinin receptor and underexpression of CSK and annexin II in pituitary adenoma cells when compared with their corresponding normal pituitary cells. Western blotting only partially confirmed the proteomics data: HSP110 was significantly overexpressed in prolactinomas and NFPAs, the B2 bradykinin receptor was significantly overexpressed in prolactinomas, annexin II was significantly underexpressed in somatotrophinomas, while CSK did not show significant underexpression in any tumour. Protein expression analysis of pituitary samples disclosed both novel proteins and putative protein candidates for pituitary tumorigenesis, though validation using conventional techniques are necessary to confirm the protein array data.