Golgi-associated retrograde protein (GARP) complex-dependent endosomes to trans Golgi network retrograde trafficking is controlled by Rab4b.

BACKGROUND: The trafficking of cargoes from endosomes to the trans-Golgi network requires numerous sequential and coordinated steps. Cargoes are sorted into endosomal-derived carriers that are transported, tethered, and fused to the trans-Golgi network. The tethering step requires several complexes, including the Golgi-associated retrograde protein complex, whose localization at the trans-Golgi network is determined by the activity of small GTPases of the Arl and Rab family. However, how the Golgi-associated retrograde protein complex recognizes the endosome-derived carriers that will fuse with the trans-Golgi network is still unknown. METHODS: We studied the retrograde trafficking to the trans-Golgi network by using fluorescent cargoes in cells overexpressing Rab4b or after Rab4b knocked-down by small interfering RNA in combination with the downregulation of subunits of the Golgi-associated retrograde protein complex. We used immunofluorescence and image processing (Super Resolution Radial Fluctuation and 3D reconstruction) as well as biochemical approaches to characterize the consequences of these interventions on cargo carriers trafficking. RESULTS: We reported that the VPS52 subunit of the Golgi-associated retrograde protein complex is an effector of Rab4b. We found that overexpression of wild type or active Rab4b increased early endosomal to trans-Golgi network retrograde trafficking of the cation-independent mannose-6-phosphate receptor in a Golgi-associated retrograde protein complex-dependent manner. Conversely, overexpression of an inactive Rab4b or Rab4b knockdown attenuated this trafficking. In the absence of Rab4b, the internalized cation-independent mannose 6 phosphate receptor did not have access to VPS52-labeled structures that look like endosomal subdomains and/or endosome-derived carriers, and whose subcellular distribution is Rab4b-independent. Consequently, the cation-independent mannose-6-phosphate receptor was blocked in early endosomes and no longer had access to the trans-Golgi network. CONCLUSION: Our results support that Rab4b, by controlling the sorting of the cation-independent mannose-6-phosphate receptor towards VPS52 microdomains, confers a directional specificity for cargo carriers en route to the trans-Golgi network. Given the importance of the endocytic recycling in cell homeostasis, disruption of the Rab4b/Golgi-associated retrograde protein complex-dependent step could have serious consequences in pathologies.

Periprostatic Adipose Tissue Displays a Chronic Hypoxic State that Limits Its Expandability.

White adipose tissue accumulates at various sites throughout the body, some adipose tissue depots exist near organs whose function they influence in a paracrine manner. Prostate gland is surrounded by a poorly characterized adipose depot called periprostatic adipose tissue (PPAT), which plays emerging roles in prostate-related disorders. Unlike all other adipose depots, PPAT secretes proinflammatory cytokines even in lean individuals and does not increase in volume during obesity. These unique features remain unexplained because of the poor structural and functional characterization of this tissue. This study characterized the structural organization of PPAT in patients compared with abdominopelvic adipose tissue (APAT), an extraperitoneal adipose depot, the accumulation of which is correlated to body mass index. Confocal microscopy followed by three-dimensional reconstructions showed a sparse vascular network in PPAT when compared with that in APAT, suggesting that this tissue is hypoxic. Unbiased comparisons of PPAT and APAT transcriptomes found that most differentially expressed genes were related to the hypoxia response. High levels of the hypoxia-inducible factor 2α confirmed the presence of an adaptive response to hypoxia in PPAT. This chronic hypoxic state was associated with inflammation and fibrosis, which were not further up-regulated by obesity. This fibrosis and inflammation explain the failure of PPAT to expand in obesity and open new mechanistic avenues to explain its role in prostate-related disorders, including cancer.

REDD1 deficiency protects against nonalcoholic hepatic steatosis induced by high-fat diet.

Nonalcoholic fatty liver disease is a chronic liver disease which is associated with obesity and insulin resistance. We investigated the implication of REDD1 (Regulated in development and DNA damage response-1), a stress-induced protein in the development of hepatic steatosis. REDD1 expression was increased in the liver of obese mice and morbidly obese patients, and its expression correlated with hepatic steatosis and insulin resistance in obese patients. REDD1 deficiency protected mice from the development of hepatic steatosis induced by high-fat diet (HFD) without affecting body weight gain and glucose intolerance. This protection was associated with a decrease in the expression of lipogenic genes, SREBP1c, FASN, and SCD-1 in liver of HFD-fed REDD1-KO mice. Healthy mitochondria are crucial for the adequate control of lipid metabolism and failure to remove damaged mitochondria is correlated with liver steatosis. Expression of markers of autophagy and mitophagy, Beclin, LC3-II, Parkin, BNIP3L, was enhanced in liver of HFD-fed REDD1-KO mice. The number of mitochondria showing colocalization between LAMP2 and AIF was increased in liver of HFD-fed REDD1-KO mice. Moreover, mitochondria in liver of REDD1-KO mice were smaller than in WT. These results are correlated with an increase in PGC-1α and CPT-1 expression, involved in fatty acid oxidation. In conclusion, loss of REDD1 protects mice from the development of hepatic steatosis.

Transfer of dysbiotic gut microbiota has beneficial effects on host liver metabolism.

Gut microbiota dysbiosis has been implicated in a variety of systemic disorders, notably metabolic diseases including obesity and impaired liver function, but the underlying mechanisms are uncertain. To investigate this question, we transferred caecal microbiota from either obese or lean mice to antibiotic-free, conventional wild-type mice. We found that transferring obese-mouse gut microbiota to mice on normal chow (NC) acutely reduces markers of hepatic gluconeogenesis with decreased hepatic PEPCK activity, compared to non-inoculated mice, a phenotypic trait blunted in conventional NOD2 KO mice. Furthermore, transferring of obese-mouse microbiota changes both the gut microbiota and the microbiome of recipient mice. We also found that transferring obese gut microbiota to NC-fed mice then fed with a high-fat diet (HFD) acutely impacts hepatic metabolism and prevents HFD-increased hepatic gluconeogenesis compared to non-inoculated mice. Moreover, the recipient mice exhibit reduced hepatic PEPCK and G6Pase activity, fed glycaemia and adiposity. Conversely, transfer of lean-mouse microbiota does not affect markers of hepatic gluconeogenesis. Our findings provide a new perspective on gut microbiota dysbiosis, potentially useful to better understand the aetiology of metabolic diseases.

ERK1 is dispensable for mouse pancreatic beta cell function but is necessary for glucose-induced full activation of MSK1 and CREB.

AIMS/HYPOTHESIS: Insufficient insulin secretion from pancreatic beta cells, which is associated with a decrease in beta cell mass, is a characteristic of type 2 diabetes. Extracellular signal-related kinase 1 and 2 (ERK1/2) inhibition in beta cells has been reported to affect insulin secretion, gene transcription and survival, although whether ERK1 and ERK2 play distinct roles is unknown. The aim of this study was to assess the individual roles of ERK1 and ERK2 in beta cells using ERK1 (also known as Mapk3)-knockout mice (Erk1 -/- mice) and pharmacological approaches. METHODS: NAD(P)H, free cytosolic Ca2+ concentration and insulin secretion were determined in islets. ERK1 and ERK2 subplasmalemmal translocation and activity was monitored using total internal reflection fluorescence microscopy. ERK1/2, mitogen and stress-activated kinase1 (MSK1) and cAMP-responsive element-binding protein (CREB) activation were evaluated by western blot and/or immunocytochemistry. The islet mass was determined from pancreatic sections. RESULTS: Glucose induced rapid subplasmalemmal recruitment of ERK1 and ERK2. When both ERK1 and ERK2 were inhibited simultaneously, the rapid transient peak of the first phase of glucose-induced insulin secretion was reduced by 40% (p < 0.01), although ERK1 did not appear to be involved in this process. By contrast, ERK1 was required for glucose-induced full activation of several targets involved in beta cell survival; MSK1 and CREB were less active in Erk1 -/- mouse beta cells (p < 0.01) compared with Erk1 +/+ mouse beta cells, and their phosphorylation could only be restored when ERK1 was re-expressed and not when ERK2 was overexpressed. Finally, the islet mass of Erk1 -/- mice was slightly increased in young animals (4-month-old mice) vs Erk1 +/+ mice (section occupied by islets [mean ± SEM]: 0.74% ± 0.03% vs 0.62% ± 0.04%; p < 0.05), while older mice (10 months old) were less prone to age-associated pancreatic peri-insulitis (infiltrated islets [mean ± SEM]: 7.51% ± 1.34% vs 2.03% ± 0.51%; p < 0.001). CONCLUSIONS/INTERPRETATION: ERK1 and ERK2 play specific roles in beta cells. ERK2 cannot always compensate for the lack of ERK1 but the absence of a clear-cut phenotype in Erk1 -/- mice shows that ERK1 is dispensable in normal conditions.

Rab4b controls an early endosome sorting event by interacting with the γ-subunit of the clathrin adaptor complex 1.

The endocytic pathway is essential for cell homeostasis and numerous small Rab GTPases are involved in its control. The endocytic trafficking step controlled by Rab4b has not been elucidated, although recent data suggested it could be important for glucose homeostasis, synaptic homeostasis or adaptive immunity. Here, we show that Rab4b is required for early endosome sorting of transferrin receptors (TfRs) to the recycling endosomes, and we identified the AP1γ subunit of the clathrin adaptor AP-1 as a Rab4b effector and key component of the machinery of early endosome sorting. We show that internalised transferrin (Tf) does not reach Vamp3/Rab11 recycling endosomes in the absence of Rab4b, whereas it is rapidly recycled back to the plasma membrane. By contrast, overexpression of Rab4b leads to the accumulation of internalised Tf within AP-1- and clathrin-coated vesicles. These vesicles are poor in early and recycling endocytic markers except for TfR and require AP1γ for their formation. Furthermore, the targeted overexpression of the Rab4b-binding domain of AP1γ to early endosome upon its fusion with FYVE domains inhibited the interaction between Rab4b and endogenous AP1γ, and perturbed Tf traffic. We thus proposed that the interaction between early endocytic Rab4b and AP1γ could allow the budding of clathrin-coated vesicles for subsequent traffic to recycling endosomes. The data also uncover a novel type of endosomes, characterised by low abundance of either early or recycling endocytic markers, which could potentially be generated in cell types that naturally express high level of Rab4b.

Caloric restriction modulates Mcl-1 expression and sensitizes lymphomas to BH3 mimetic in mice.

Caloric restriction (CR) is proposed to decrease tumorigenesis through a variety of mechanisms including effects on glycolysis. However, the understanding of how CR affects the response to cancer therapy is still rudimentary. Here, using the Eµ-Myc transgenic mouse model of B-cell lymphoma, we report that by reducing protein translation, CR can reduce expression of the prosurvival Bcl-2 family member Mcl-1 and sensitize lymphomas to ABT-737-induced death in vivo. By using Eµ-Myc lymphoma cells lacking p53, we showed that CR mimetics such as 2-deoxyglucose led to a decrease in Mcl-1 expression and sensitized lymphoma cells to ABT-737-induced death independently of p53. In keeping with this, Eµ-Myc lymphoma cells lacking the BH3-only proapoptotic members Noxa, Puma, or Bim were also sensitized by CR mimetics to ABT-737-induced death. Remarkably, neither the loss of both Puma and Noxa, the loss of both Puma and Bim, nor the loss of all three BH3-only proteins prevented sensitization to ABT-737 induced by CR mimetics. Thus, CR can influence Mcl-1 expression and sensitize cells to BH3 mimetic-induced apoptosis, independently of the main BH3-only proteins and of p53. Exploiting this may improve the efficiency of, or prevent resistance to, cancer therapy.

Adipocyte Glucocorticoid Receptor Activation With High Glucocorticoid Doses Impairs Healthy Adipose Tissue Expansion by Repressing Angiogenesis.

In humans, glucocorticoids (GCs) are commonly prescribed because of their anti-inflammatory and immunosuppressive properties. However, high doses of GCs often lead to side effects, including diabetes and lipodystrophy. We recently reported that adipocyte glucocorticoid receptor (GR)-deficient (AdipoGR-KO) mice under corticosterone (CORT) treatment exhibited a massive adipose tissue (AT) expansion associated with a paradoxical improvement of metabolic health compared with control mice. However, whether GR may control adipose development remains unclear. Here, we show a specific induction of hypoxia-inducible factor 1α (HIF-1α) and proangiogenic vascular endothelial growth factor A (VEGFA) expression in GR-deficient adipocytes of AdipoGR-KO mice compared with control mice, together with an increased adipose vascular network, as assessed by three-dimensional imaging. GR activation reduced HIF-1α recruitment to the Vegfa promoter resulting from Hif-1α downregulation at the transcriptional and posttranslational levels. Importantly, in CORT-treated AdipoGR-KO mice, the blockade of VEGFA by a soluble decoy receptor prevented AT expansion and the healthy metabolic phenotype. Finally, in subcutaneous AT from patients with Cushing syndrome, higher VEGFA expression was associated with a better metabolic profile. Collectively, these results highlight that adipocyte GR negatively controls AT expansion and metabolic health through the downregulation of the major angiogenic effector VEGFA and inhibition of vascular network development.

GIPR expression is induced by thiazolidinediones in a PPARγ-independent manner and repressed by obesogenic stimuli.

Adipose tissue (AT) dysfunctions are associated with the onset of insulin resistance (IR) and type 2 diabetes mellitus (T2DM). Targeting glucose-dependent insulinotropic peptide receptor (GIPR) is a valid option to increase the efficacy of glucagon-like peptide 1 (GLP-1) receptor agonists in T2DM treatment. Nevertheless, the therapeutic potential of targeting the GIP/GIPR axis and its effect on the AT are controversial. In this work, we explored the expression and regulation of GIPR in precursor cells and mature adipocytes, investigating if and how obesogenic stimuli and thiazolidinediones perturb GIPR expression. Using publicly available gene expression datasets, we assessed that, among white adipose tissue (WAT) cells, adipocytes express lower levels of GIPR compared to cells of mesothelial origin, pericytes, dendritic and NK/T cells. However, we report that GIPR levels markedly increase during the in vitro differentiation of both murine and human adipocytes, from 3T3-L1 and human mesenchymal precursor cells (MSCs), respectively. Notably, we demonstrated that thiazolidinediones - ie. synthetic PPARγ agonists widely used as anti-diabetic drugs and contained in the adipogenic mix - markedly induce GIPR expression. Moreover, using multiple in vitro systems, we assessed that thiazolidinediones induce GIPR in a PPARγ-independent manner. Our results support the hypothesis that PPARγ synthetic agonists may be used to increase GIPR levels in AT, potentially affecting in turn the targeting of GIP system in patients with metabolic dysfunctions. Furthermore, we demonstrate in vitro and in vivo that proinflammatory stimuli, and especially the TNFα, represses GIPR both in human and murine adipocytes, even though discordant results were obtained between human and murine cellular systems for other cytokines. Finally, we demonstrated that GIPR is negatively affected also by the excessive lipid engulfment. Overall, we report that obesogenic stimuli - ie. pro-inflammatory cytokines and the increased lipid accumulation - and PPARγ synthetic ligands oppositely modulate GIPR expression, possibly influencing the effectiveness of GIP agonists.

Metformin and cancer therapy.

PURPOSE OF REVIEW: To focus on the potential role of metformin, a widely used antidiabetic drug, in cancer treatment. RECENT FINDINGS: Epidemiological, preclinical and cellular studies have shown in the last 6 years that metformin exerts antitumoral properties. Here, we review the very last findings concerning metformin action in cancer. The results of the first clinical trials as well as the combined action of metformin and chemotherapeutics agents in vitro and in vivo will be discussed. Recent studies show that metformin could also regulate inflammation and, therefore, may play a role in tumor microenvironment. Finally, we will present the latest publications concerning the molecular mechanisms implicated in metformin action, especially the AMP-activated kinase-independent pathways. SUMMARY: The numerous in-vitro and in-vivo studies warrant the ongoing clinical trials, which should definitively help us to determine if metformin could be used in cancer therapy.

The Stress-Responsive microRNA-34a Alters Insulin Signaling and Actions in Adipocytes through Induction of the Tyrosine Phosphatase PTP1B.

Metabolic stresses alter the signaling and actions of insulin in adipocytes during obesity, but the molecular links remain incompletely understood. Members of the microRNA-34 (miR-34 family play a pivotal role in stress response, and previous studies showed an upregulation of miR-34a in adipose tissue during obesity. Here, we identified miR-34a as a new mediator of adipocyte insulin resistance. We confirmed the upregulation of miR-34a in adipose tissues of obese mice, which was observed in the adipocyte fraction exclusively. Overexpression of miR-34a in 3T3-L1 adipocytes or in fat pads of lean mice markedly reduced Akt activation by insulin and the insulin-induced glucose transport. This was accompanied by a decreased expression of VAMP2, a target of miR-34a, and an increased expression of the tyrosine phosphatase PTP1B. Importantly, PTP1B silencing prevented the inhibitory effect of miR-34a on insulin signaling. Mechanistically, miR-34a decreased the NAD+ level through inhibition of Naprt and Nampt, resulting in an inhibition of Sirtuin-1, which promoted an upregulation of PTP1B. Furthermore, the mRNA expression of Nampt and Naprt was decreased in adipose tissue of obese mice. Collectively, our results identify miR-34a as a new inhibitor of insulin signaling in adipocytes, providing a potential pathway to target to fight insulin resistance.

Insulin induces REDD1 expression through hypoxia-inducible factor 1 activation in adipocytes.

REDD1 (regulated in development and DNA damage responses) is essential for the inhibition of mTORC1 (mammalian target of rapamycin complex) signaling pathway in response to hypoxia. REDD1 expression is regulated by many stresses such as hypoxia, oxidative stress, and energy depletion. However, the regulation of REDD1 expression in response to insulin remains unknown. In the present study, we demonstrate that in murine and in human adipocytes, insulin stimulates REDD1 expression. Insulin-induced REDD1 expression occurs through phosphoinositide 3-kinase/mTOR-dependent pathways. Moreover, using echinomycin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, and HIF-1alpha small interfering RNA, we demonstrate that insulin stimulates REDD1 expression only through the transcription factor HIF-1. In conclusion, our study shows that insulin stimulates REDD1 expression in adipocytes.

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function.

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

A Citrullus colocynthis fruit extract acutely enhances insulin-induced GLUT4 translocation and glucose uptake in adipocytes by increasing PKB phosphorylation.

ETHNOPHARMACOLOGICAL RELEVANCE: Citrullus colocynthis (L.) Schrad is a common fruit in traditional medicine and used as remedy against various diseases, especially diabetes. Up to now, its anti-diabetic effects have been fully attributed to its enhancement of pancreatic insulin secretion. Whether C. colocynthis also ameliorates insulin action in peripheral tissues has not been investigated. AIM OF THE STUDY: In the present study, using 3T3-L1 adipocytes as cell model, we have investigated whether colocynth fruit extracts affect insulin action. MATERIALS AND METHODS: Various extracts were prepared from the C. colocynthis fruit and screened using a cell-based 96 well plate GLUT4 translocation assay. Promising extracts were further studied for their effects on glucose uptake and cell viability. The effect on insulin signal transduction was determined by Western blot and the molecular composition was established by LC-MS. RESULTS: The ethyl acetate fractions of aqueous non-defatted extracts of seed and pulp, designated Sna1 and Pna1, acutely enhanced insulin-induced GLUT4 translocation. In accordance, both extracts increased insulin-stimulated cellular glucose uptake. Pna1, which displayed greater effects on GLUT4 and glucose uptake than Sna1, was further investigated and was demonstrated to increase GLUT4 translocation without changing the half-maximum dose (ED50) of insulin, nor changing GLUT4 translocation kinetics. At the molecular level, Pna1 was found to enhance insulin-induced PKB phosphorylation without changing phosphorylation of the insulin receptor. Pna1 appeared not to be toxic to cells and, like insulin, restored cell viability during serum starvation. By investigating the molecular composition of Pna1, nine compounds were identified that made up 87% of the mass of the extract, one of which is likely to be responsible for the insulin-enhancing effects of Pna1. CONCLUSIONS: The C. colocynthis fruit possesses insulin-enhancing activity. This activity may explain in part its anti-diabetic effects in traditional medicine. It also identifies the C. colocynthis as a source of a potential novel insulin enhancer that may prove to be useful to reduce hyperglycemia in type 2 diabetes.

TNFα Mediates Inflammation-Induced Effects on PPARG Splicing in Adipose Tissue and Mesenchymal Precursor Cells.

Low-grade chronic inflammation and reduced differentiation capacity are hallmarks of hypertrophic adipose tissue (AT) and key contributors of insulin resistance. We identified PPARGΔ5 as a dominant-negative splicing isoform overexpressed in the AT of obese/diabetic patients able to impair adipocyte differentiation and PPARγ activity in hypertrophic adipocytes. Herein, we investigate the impact of macrophage-secreted pro-inflammatory factors on PPARG splicing, focusing on PPARGΔ5. We report that the epididymal AT of LPS-treated mice displays increased PpargΔ5/cPparg ratio and reduced expression of Pparg-regulated genes. Interestingly, pro-inflammatory factors secreted from murine and human pro-inflammatory macrophages enhance the PPARGΔ5/cPPARG ratio in exposed adipogenic precursors. TNFα is identified herein as factor able to alter PPARG splicing-increasing PPARGΔ5/cPPARG ratio-through PI3K/Akt signaling and SRp40 splicing factor. In line with in vitro data, TNFA expression is higher in the SAT of obese (vs. lean) patients and positively correlates with PPARGΔ5 levels. In conclusion, our results indicate that inflammatory factors secreted by metabolically-activated macrophages are potent stimuli that modulate the expression and splicing of PPARG. The resulting imbalance between canonical and dominant negative isoforms may crucially contribute to impair PPARγ activity in hypertrophic AT, exacerbating the defective adipogenic capacity of precursor cells.

Inhibition of eIF5A hypusination reprogrammes metabolism and glucose handling in mouse kidney.

Inhibition of the eukaryotic initiation factor 5A activation by the spermidine analogue GC7 has been shown to protect proximal cells and whole kidneys against an acute episode of ischaemia. The highlighted mechanism involves a metabolic switch from oxidative phosphorylation toward glycolysis allowing cells to be transiently independent of oxygen supply. Here we show that GC7 decreases protein expression of the renal GLUT1 glucose transporter leading to a decrease in transcellular glucose flux. At the same time, GC7 modifies the native energy source of the proximal cells from glutamine toward glucose use. Thus, GC7 acutely and reversibly reprogrammes function and metabolism of kidney cells to make glucose its single substrate, and thus allowing cells to be oxygen independent through anaerobic glycolysis. The physiological consequences are an increase in the renal excretion of glucose and lactate reflecting a decrease in glucose reabsorption and an increased glycolysis. Such a reversible reprogramming of glucose handling and oxygen dependence of kidney cells by GC7 represents a pharmacological opportunity in ischaemic as well as hyperglycaemia-associated pathologies from renal origin.

TAXOMET: A French Prospective Multicentric Randomized Phase II Study of Docetaxel Plus Metformin Versus Docetaxel Plus Placebo in Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND: Docetaxel (DOCE) is a standard of care in metastatic castration-resistant prostate cancer (mCRPC). Several retrospective studies suggested a decrease in Prostate Cancer incidence and mortality with metformin (MET). MET has also demonstrated anti-tumor activity in Prostate Cancer preclinical models, with increased apoptosis when added to DOCE. We aimed at exploring the role of MET in combination with DOCE in mCRPC. PATIENTS AND METHODS: Non-diabetic mCRPC patients were randomly assigned to receive DOCE 75 mg/m2 every 21 days + prednisone (5 mg. BID) with either MET 850 mg BID (D+M) or placebo (D+P) up to 10 cycles. Prostate-Specific Antigen (PSA) response ≥50% from baseline was the primary end point. Secondary end points included objective response rate (ORR), progression-free survival (PFS), overall survival (OS), toxicity and quality of life (QoL). RESULTS: Out of 99 patients were randomized (D+M = 50; D+P = 49) in 10 French centers. The median follow-up was 86 (IQR 73-88) months. The PSA-response rate reached 66% in the D+M arm, but was not different from that observed in the D+P arm (63%, P = 0,94). In the D+M and D+P arms, the ORR was 28% and 24%, the median PFS was 7.8 and 6.0 months and the median OS was 27 and 20 months (ns), respectively. Diarrhea grade I to II was more frequent in the MET arm (66% vs. 43%). No impairment of QoL was observed. CONCLUSION: MET addition failed to improve the standard DOCE regimen in mCRPC. Further research targeting tumor cell metabolism should be performed.

Apelin and APJ regulation in adipose tissue and skeletal muscle of type 2 diabetic mice and humans.

Apelin, an adipocyte-secreted factor upregulated by insulin, is increased in adipose tissue (AT) and plasma with obesity. Apelin was recently identified as a new player in the control of glucose homeostasis. However, the regulation of apelin and APJ (apelin receptor) expression in skeletal muscle in relation to insulin resistance or type 2 diabetes is not known. Thus we studied apelin and APJ expression in AT and muscle in different mice models of obesity and in type 2 diabetic patients. In insulin-resistant high-fat (HF)-fed mice, apelin and APJ expression were increased in AT compared with control. This was not the case in AT of highly insulin-resistant db/db mice. In skeletal muscle, apelin expression was similar in control and HF-fed mice and decreased in db/db mice. APJ expression was decreased in both HF-fed and db/db mice. Control subjects and type 2 diabetic patients were subjected to a hyperinsulinemic-euglycemic clamp, and tissues biopsies were obtained before and at the end of the clamp. There was no significant difference in basal apelin and APJ expression in AT and muscle between control and diabetic patients. However, apelin plasma levels were significantly increased in diabetic patients. During the clamp, hyperinsulinemia increased apelin and APJ expression in AT of control but not in diabetic subjects. In muscle, only APJ mRNA levels were increased in control but also in diabetic patients. Taken together, these data show that apelin and APJ expression in mice and humans is regulated in a tissue-dependent manner and according to the severity of insulin resistance.

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Circulating Levels of Soluble Dipeptidyl Peptidase-4 Are Dissociated from Inflammation and Induced by Enzymatic DPP4 Inhibition.

Dipeptidyl peptidase-4 (DPP-4) controls glucose homeostasis through enzymatic termination of incretin action. We report that plasma DPP-4 activity correlates with body weight and fat mass, but not glucose control, in mice. Genetic disruption of adipocyte Dpp4 expression reduced plasma DPP-4 activity in older mice but did not perturb incretin levels or glucose homeostasis. Knockdown of hepatocyte Dpp4 completely abrogated the obesity-associated increase in plasma DPP-4 activity, reduced liver cytokine expression, and partially attenuated inflammation in adipose tissue without changes in incretin levels or glucose homeostasis. In contrast, circulating levels of soluble DPP4 (sDPP4) were dissociated from inflammation in mice with endothelial-selective or global genetic inactivation of Dpp4. Remarkably, inhibition of DPP-4 enzymatic activity upregulated circulating levels of sDPP4 originating from endothelial or hematopoietic cells without inducing systemic or localized inflammation. Collectively, these findings reveal unexpected complexity in regulation of soluble versus enzymatic DPP-4 and control of inflammation and glucose homeostasis.