Nicoleta Cristina Olarescu

Adipocytes as a Source of Increased Circulating Levels of Nicotinamide Phosphoribosyltransferase/Visfatin in Active Acromegaly

BACKGROUND Nicotinamide phosphoribosyltransferase (NAMPT)/visfatin is a widely expressed protein with various effects on glucose and lipid metabolism, cell survival, and inflammation. AIM We hypothesized that NAMPT was related to metabolic disturbances in active acromegaly. METHODS Body composition, glucose metabolism, and NAMPT levels were measured in 47 patients with active, untreated acromegaly and 24 age-, sex-, and body mass index-matched controls. The in vitro effects of GH/IGF-I on NAMPT expression in human sc adipocytes (SCA), visceral adipocytes, osteoblasts, and hepatocytes were studied. The effects of overnight incubation with the highly specific NAMPT inhibitor FK866 on the GH-stimulated monocyte chemotactic protein-1 and IL-6 expression in mature SCA were evaluated. RESULTS NAMPT was increased in active acromegaly (P = 0.004) and correlated negatively with limb (arms + legs) fat percentage (% fat, r = -0.32; P = 0.032). After adjusting for age, gender, leptin, and GH, the circulating NAMPT correlated negatively with limb and total body fat percentage (% fat limbs, r = -0.43, P = 0.006; % fat total body, r = -0.36, P = 0.022) and correlated positively with limb and total body lean percentage (% lean limbs, r = 0.31, P = 0.047; % lean total body, r = 0.33, P = 0.034). No correlation between NAMPT and glucose metabolic parameters was found. In vitro studies revealed that GH increased NAMPT expression in adipocytes. The inhibition of NAMPT enzymatic activity attenuated GH-induced monocyte chemotactic protein-1 expression in SCA. CONCLUSIONS NAMPT is increased in active acromegaly and may be an inflammatory mediator that causes monocyte infiltration in adipose tissue.

The Impact of Adipose Tissue on Insulin Resistance in Acromegaly

Adipose tissue (AT) is recognized as key contributor to the systemic insulin resistance and overt diabetes seen in metabolic syndrome. Acromegaly is a disease characterized by excessive secretion of growth hormone (GH) and insulin-like growth factor I (IGF-I). GH is known both for its action on AT and for its detrimental effect on glucose metabolism and insulin signaling. In active acromegaly, while body fat deports are diminished, insulin resistance is increased. Early studies have demonstrated defects in insulin action, both at the hepatic and extrahepatic (i.e., muscle and fat) levels, in active disease. This review discusses recent data suggesting that AT inflammation, altered AT distribution, and impaired adipogenesis are potential mechanisms contributing to systemic insulin resistance in acromegaly.

Dual-Energy X-Ray Absorptiometry Is a Valid Method to Estimatimate Visceral Adipose Tissue in Adult Patients With Prader-Willi Syndrome During Treatment With Growth Hormone

CONTEXT Visceral adipose tissue (VAT) is established as a risk factor for type 2 diabetes and cardiovascular disease, but the radiation exposure and cost of computed tomography (CT) measurements limits its daily clinical use. OBJECTIVE The main objective of this study was to compare the degree of agreement between VAT measurements by a new dual-energy X-ray absorptiometry (DXA) application and one of the standard methods, CT, in a population of patients with Prader-Willi syndrome (PWS) before and after GH treatment. Furthermore, we tested whether VAT estimations by these two methods are equivalent in assessing the metabolic risk in this population. DESIGN AND PATIENTS Data from the Norwegian population of a multicenter study in adults with genetically proven PWS were used. Subjects with complete anthropometry, biochemical, and imagistic measurements at all study visits (baseline and after 12 and 24 months of GH treatment) (n = 14, six men) were included. VAT was quantified both using CT scans (GE Lightspeed 16 Pro) of the abdomen at L2-L3 level and a total body DXA scan (GE Healthcare Lunar Prodigy). RESULTS VAT DXA was strongly associated with VAT CT at baseline (r = 0.97) and after 12 (r = 0.90) and 24 months (r = 0.89) of GH treatment (all P < .001). We found moderate to strong positive correlations between VAT by both methods, and blood pressure, weight, body mass index, waist circumference, glucose metabolism, and other fat depots (arms, legs, android, trunk, total body) but no association with age, gender, blood lipids, and IGF-I. Adiponectin was negatively associated with the amount of VAT. At baseline, the highest correlation with homeostasis model assessment of insulin resistance (HOMA-IR) was found for VAT DXA (r = 0.76, P = .001) and VAT CT (r = 0.75, P = .002), respectively. CONCLUSION VAT can be accurately estimated by DXA, in patients with PWS, and might contribute to the assessment of the metabolic risk.

GH action influences adipogenesis of mouse adipose tissue-derived mesenchymal stem cells

GH influences adipocyte differentiation, but both stimulatory and inhibitory effects have been described. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are multipotent and are able to differentiate into adipocytes, among other cells. Canonical Wnt/β-catenin signaling activation impairs adipogenesis. The aim of the present study was to elucidate the role of GH on AT-MSC adipogenesis using cells isolated from male GH receptor knockout (GHRKO), bovine GH transgenic (bGH) mice, and wild-type littermate control (WT) mice. AT-MSCs from subcutaneous (sc), epididiymal (epi), and mesenteric (mes) AT depots were identified and isolated by flow cytometry (Pdgfrα+ Sca1+ Cd45- Ter119- cells). Their in vitro adipogenic differentiation capacity was determined by cell morphology and real-time RT-PCR. Using identical in vitro conditions, adipogenic differentiation of AT-MSCs was only achieved in the sc depot, and not in epi and mes depots. Notably, we observed an increased differentiation in cells isolated from sc-GHRKO and an impaired differentiation of sc-bGH cells as compared to sc-WT cells. Axin2, a marker of Wnt/β-catenin activation, was increased in mature sc-bGH adipocytes, which suggests that activation of this pathway may be responsible for the decreased adipogenesis. Thus, the present study demonstrates that (i) adipose tissue in mice has a well-defined population of Pdgfrα+ Sca1+ MSCs; (ii) the differentiation capacity of AT-MSCs varies from depot to depot regardless of GH genotype; (iii) the lack of GH action increases adipogenesis in the sc depot; and iv) activation of the Wnt/β-catenin pathway might mediate the GH effect on AT-MSCs. Taken together, the present results suggest that GH diminishes fat mass in part by altering adipogenesis of MSCs.

The Metabolic Risk in Patients Newly Diagnosed with Acromegaly Is Related to Fat Distribution and Circulating Adipokines and Improves after Treatment

Background/Aims: Adipose tissue (AT) distribution is closely related to metabolic disease risk. Growth hormone (GH) reduces visceral and total body fat mass and induces whole-body insulin resistance. Our aim was to assess the effects of total and visceral AT (VAT) distribution and derived adipokines on systemic insulin resistance and lipid metabolism in acromegaly. Methods: Seventy adult patients with active acromegaly (43 males, age 49 ± 14 years) were evaluated before treatment, and a subset (n = 30, 20 males) was evaluated after treatment for acromegaly. Body composition and VAT, glucose metabolism parameters, lipids, C-reactive protein, and selected adipokines (vaspin, omentin, adiponectin, and leptin) were measured. Results: At baseline, VAT was positively associated with glucose metabolism parameters and with lipids. GH, but not IGF-I, was negatively associated with all AT depots (visceral, trunk, limbs, and total; 0.41 ≤ r ≤ 0.61, p < 0.001 for all) and positively associated with vaspin (r = 0.31, p = 0.013). The fat deposition after treatment was predominantly located on trunk and visceral depots. The lipid profile partially improved, with increases in HDL and apolipoprotein A-I and a decrease in lipoprotein(a). Vaspin decreased and omentin increased. Adiponectin and leptin did not change significantly. The improvement in homeostasis model assessment for insulin resistance (HOMA-IR) was best predicted by the decreases in IGF-I and vaspin and the lack of an increase in trunk fat (R2 = 0.59, p = 0.001). Conclusions: (1) VAT is a metabolic risk factor for patients with active acromegaly; (2) vaspin and omentin levels are influenced by the disease activity but are not associated with VAT mass; (3) fat deposition after treatment occurs predominantly on the trunk and in visceral depots, and (4) insulin resistance decreases and the lipid profile partially improves with treatment.

Inflammatory adipokines contribute to insulin resistance in active acromegaly and respond differently to different treatment modalities.

BACKGROUND Active acromegaly is associated with insulin resistance, but it is uncertain whether inflammation in adipose tissue is a contributing factor. AIM To test if GH/IGF1 promotes inflammation in adipocytes, and if this is relevant for systemic insulin resistance in acromegaly. Furthermore, to investigate the effect of treatment modalities (transsphenoidal surgery (TS), somatostatin analogs (SAs), and pegvisomant (PGV)) on glucose metabolism and inflammatory biomarkers in acromegaly. METHODS The in vitro effects of GH/IGF1 on gene expression of adipokines in human adipocytes were investigated. Body composition, glucose metabolism, and circulating adipokines (adiponectin (AD), high-molecular weight AD (HMWAD), leptin, vascular endothelial growth factor-A (VEGF-A), monocyte chemotactic protein 1 (MCP1), and thioredoxin (TRX)) were measured in 37 patients with active acromegaly before and after treatment. RESULTS In vitro GH, but not IGF1, increased VEGF and MCP1 in human adipocytes. In all treatment groups, body fat increased and IGF1 decreased to the same extent. Fasting glucose decreased in the TS (P=0.016) and PGV (P=0.042) groups, but tended to increase in the SA group (P=0.078). Insulin and HOMA-IR decreased in both TS and SA groups, while the PGV group showed no changes. Serum VEGF and MCP1 decreased significantly in the TS group only (P=0.010, P=0.002), while HMWAD increased with PGV treatment only (P=0.018). A multivariate analysis model identified the changes in GH and VEGF as predictors of improvement in HOMA-IR after treatment (R²=0.39, P=0.002). CONCLUSIONS i) GH directly promotes inflammation of human adipocytes by increasing VEGF and MCP1 levels; ii) glucose metabolism and inflammation (VEGF and MCP1) improve to some extent after treatment, despite an increase in adipose tissue mass; and iii) the decrease in insulin resistance after therapy in acromegaly depends, to some extent, on treatment modalities.

Treatment of acromegaly increases BMD but reduces Trabecular Bone Score - a longitudinal study

CONTEXT Bone turnover is increased in acromegaly. Despite normalization of bone turnover after treatment, the risk for vertebral fractures remains increased. Gonadal status, but not BMD, is correlated with vertebral fractures. Trabecular bone score (TBS) is related to bone microarchitecture. OBJECTIVE The aim of this study is to assess the longitudinal change in TBS and BMD following treatment for acromegaly. DESIGN, SETTING, PATIENTS, INTERVENTIONS, AND MAIN OUTCOME MEASURES: This longitudinal study included 48 patients with acromegaly between 2005 and 2015. BMD, TBS, and markers for bone turnover (P1NP and CTX-1) were measured at baseline and following treatment. RESULTS Following treatment, the mean TBS decreased by 3.0 (±7.0) %, whereas the BMD at the lumbar spine (LS) increased by 3.2 (±4.9) % (both P<0.01). The changes in BMD LS and TBS were not correlated (P=0.87). The TBS change was found to be -4.5 % (±6.7; P=0.003) in men and -0.3 % (±6.8; P=0.85) in women (P=0.063 for interaction men vs women). The mean BMD LS increased in men +4.2 g/cm(2) (±4.3; P<0.001), but not in women +1.5 g/cm(2) (±5.6; P=0.36); (P=0.073 for interaction). BMD increased in the ultradistal radius and total body (both P<0.01). The increase in BMD LS was associated with a decrease in P1NP and CTX-1 (P<0.001) and with lower P1NP and CTX-1 at the follow-up (P<0.02). CONCLUSION Treatment of acromegaly affects TBS and BMD at LS in different manners. The reduction of bone turnover markers predicts the increase in BMD but not the decrease in TBS. The DXA changes were more pronounced in men. Alterations in trabecular bone architecture may explain the persistent fracture risk despite the increase in BMD after disease control.

Increased Serum and Bone Matrix Levels of the Secreted Wnt Antagonist DKK-1 in Patients With Growth Hormone Deficiency in Response to Growth Hormone Treatment

CONTEXT Growth hormone (GH) substitution of adult-onset growth hormone deficiency (aoGHD) patients partially reverses unfavorable body composition profile. Wnt signaling pathway has being acknowledged as an important modulator of bone mass and of energy metabolism in adipose tissue and in β-cells. OBJECTIVE To assess the role of selected Wnt antagonists in bone and glucose metabolism before and after GH replacement in aoGHD. PATIENTS AND METHODS Patients from two randomized placebo-controlled studies of GH replacement in aoGHD were used. In study 1, 39 patients received GH or placebo for 9 months with 4 months wash-out. In study 2, iliac bone biopsies were obtained before and after GH or placebo (n = 10 each) for 12 months. Body composition and serum (study 1) and bone matrix (study 2) levels of Wnt antagonists (DKK-1, sFRP-3, WIF-1, and SOST) were quantified before and after GH. In vitro effect of GH and IGF-1 on DKK-1 secretion and expression of Wnt signaling modulators was assessed in human osteoblasts and mature adipocytes. RESULTS GH replacement increased circulating and bone matrix levels of DKK-1, but not sFRP-3, WIF-1, and SOST. Furthermore, DKK-1 secretion increased in human osteoblasts stimulated by GH in vitro, with no effects on other cells. At baseline and after treatment, circulating DKK-1 was negatively associated with bone mass, but not fat mass or measures of insulin resistance, in aoGHD patients. CONCLUSIONS An increase in DKK-1 may limit the effects of GH on bone mass, but does not seem to impact the increase in insulin resistance following GH substitution.

Increased serum and bone matrix levels of transforming growth factor β1 in patients with GH deficiency in response to GH treatment

OBJECTIVE Patients with adult onset GH deficiency (aoGHD) have secondary osteoporosis, which is reversed by long-term GH substitution. Transforming growth factor β1 (TGFβ1 or TGFB1) is abundant in bone tissue and could mediate some effects of GH/IGFs on bone. We investigated its regulation by GH/IGF1 in vivo and in vitro. DESIGN AND METHODS The effects of GH substitution (9-12 months, placebo controlled) on circulating and cortical bone matrix contents of TGFβ1 were investigated in patients with aoGHD. The effects of GH/IGF1 on TGFβ1 secretion in osteoblasts (hFOB), adipocytes, and THP-1 macrophages as well as the effects on release from platelets were investigated in vitro. RESULTS In vivo GH substitution increased TGFβ1 protein levels in cortical bone and serum. In vitro, GH/IGF1 stimulation induced a significant increase in TGFβ1 secretion in hFOB. In contrast, no major effect of GH/IGF1 on TGFβ1 was found in adipocytes and THP-1 macrophages. Finally, a minor modifying effect on SFLLRN-stimulated platelet release of TGFβ1 was observed in the presence of IGF1. CONCLUSION GH substitution increases TGFβ1 in vivo and in vitro, and this effect could contribute to improved bone metabolism during such therapy, potentially reflecting direct effect of GH/IGF1 on bone cells.

Gene expression profiling of fast- and slow- growing gonadotroph non-functioning pituitary adenomas

OBJECTIVE Reliable biomarkers associated with aggressiveness of non-functioning gonadotroph adenomas (GAs) are lacking. As the growth of tumor remnants is highly variable, molecular markers for growth potential prediction are necessary. We hypothesized that fast- and slow-growing GAs present different gene expression profiles and reliable biomarkers for tumor growth potential could be identified, focusing on the specific role of epithelial-mesenchymal transition (EMT). DESIGN AND METHODS Eight GAs selected for RNA sequencing were equally divided into fast- and slow-growing group by the tumor volume doubling time (TVDT) median (27.75 months). Data were analyzed by tophat2, cufflinks and cummeRbund pipeline. 40 genes were selected for RT-qPCR validation in 20 GAs based on significance, fold-change and pathway analyses. The effect of silencing MTDH (metadherin) and EMCN (endomucin) on in vitro migration of human adenoma cells was evaluated. RESULTS 350 genes were significantly differentially expressed (282 genes upregulated and 68 downregulated in the fast group, P-adjusted <0.05). Among 40 selected genes, 11 showed associations with TVDT (-0.669<R<-0.46, P < 0.05). These were PCDH18, UNC5D, EMCN, MYO1B, GPM6A and six EMT-related genes (SPAG9, SKIL, MTDH, HOOK1, CNOT6L and PRKACB). MTDH, but not EMCN, demonstrated involvement in cell migration and association with EMT markers. CONCLUSIONS Fast- and slow-growing GAs present different gene expression profiles, and genes related to EMT have higher expression in fast-growing tumors. In addition to MTDH, identified as an important contributor to aggressiveness, the other genes might represent markers for tumor growth potential and possible targets for drug therapy.