Attilio Pingitore

Identification of 9-cis-retinoic acid as a pancreas-specific autacoid that attenuates glucose-stimulated insulin secretion

The all-trans-retinoic acid (atRA) isomer, 9-cis-retinoic acid (9cRA), activates retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in vitro. RARs control multiple genes, whereas RXRs serve as partners for RARs and other nuclear receptors that regulate metabolism. Physiological function has not been determined for 9cRA, because it has not been detected in serum or multiple tissues with analytically validated assays. Here, we identify 9cRA in mouse pancreas by liquid chromatography/tandem mass spectrometry (LC/MS/MS), and show that 9cRA decreases with feeding and after glucose dosing and varies inversely with serum insulin. 9cRA reduces glucose-stimulated insulin secretion (GSIS) in mouse islets and in the rat β-cell line 832/13 within 15 min by reducing glucose transporter type 2 (Glut2) and glucokinase (GK) activities. 9cRA also reduces Pdx-1 and HNF4α mRNA expression, ∼8- and 80-fold, respectively: defects in Pdx-1 or HNF4α cause maturity onset diabetes of the young (MODY4 and 1, respectively), as does a defective GK gene (MODY2). Pancreas β-cells generate 9cRA, and mouse models of reduced β-cell number, heterozygous Akita mice, and streptozotocin-treated mice have reduced 9cRA. 9cRA is abnormally high in glucose-intolerant mice, which have β-cell hypertropy, including mice with diet-induced obesity (DIO) and ob/ob and db/db mice. These data establish 9cRA as a pancreas-specific autacoid with multiple mechanisms of action and provide unique insight into GSIS.

The diet-derived short chain fatty acid propionate improves beta-cell function in humans and stimulates insulin secretion from human islets in vitro.

Diet‐derived short chain fatty acids (SCFAs) improve glucose homeostasis in vivo, but the role of individual SCFAs and their mechanisms of action have not been defined. This study evaluated the effects of increasing colonic delivery of the SCFA propionate on β‐cell function in humans and the direct effects of propionate on isolated human islets in vitro.

Bid as a potential target of apoptotic effects exerted by low doses of PPARγ and RXR ligands in breast cancer cells.

The combined treatment with nanomolar doses of the PPARγ ligand Rosiglitazone (BRL) and the RXR ligand 9-cis‑retinoic acid (9RA) induces a p53-dependent apoptosis in MCF7, SKBR3 and T47D human breast cancer cells. Since MCF7 cells express a wild-type p53 protein, while SKBR3 and T47D cells harbor endogenous mutant p53, we elucidated the mechanism through which PPARγ and RXR ligands triggered apoptotic processes independently of p53 transcriptional activity. We showed an upregulation of Bid expression enhancing the association between Bid/p53 in both cytosol and mitochondria after the ligand treatment. Particularly in the mitochondria, the complex involves the truncated Bid that plays a key role in the apoptotic process induced by BRL and 9RA, since the disruption of mitochondrial membrane potential, the induction of PARP cleavage and the percentage of TUNEL-positive cells were reversed after knocking down Bid. Moreover, PPARγ and RXR ligands were able to reduce mitochondrial GST activity, which was no longer noticeable silencing Bid expression, suggesting the potential of Bid in the regulation of mitochondrial intracellular reactive oxygen species scavenger activity. Our data, providing new insight into the role of p53/Bid complex at the mitochondria in promoting breast cancer cell apoptosis upon low doses of PPARγ and RXR ligands, address Bid as a potential target in the novel therapeutical strategies for breast cancer.

GPR55‐dependent stimulation of insulin secretion from isolated mouse and human islets of Langerhans

The novel cannabinoid receptor GPR55 is expressed by rodent islets and it has been implicated in β‐cell function in response to a range of ligands. This study evaluated the effects of GPR55 ligands on intracellular calcium ([Ca2+]i) levels and insulin secretion from islets isolated from GPR55 knockout (GPR55 −/−) mice, age‐matched wildtype (WT) mice and human pancreas.

Proliferative and anti-proliferative effects of retinoic acid at doses similar to endogenous levels in Leydig MLTC-1/R2C/TM-3 cells

BACKGROUND Vitamin A is suggested to be protective against oxidative stress. However, different authors observed pro-oxidant effects of retinoids both in experimental works and clinical trials. These discordances are the bases for the investigation of the proliferative and anti-proliferative properties of retinoic acid (RA) in biological systems. METHODS Cell viability is determined with the MTT assay. Oxidative stress parameters are detected measuring catalase (CAT) and glutathione S-transferase (GST) enzymatic activities. FABP5 mRNA levels are measured by RT-PCR. Autophagy and apoptosis are analyzed by Monodansylcadaverine (MDC) staining and TUNEL assay, respectively. RESULTS AND CONCLUSIONS RA, at nutraceutic/endogenous doses (10-200 nM), increases cell viability of testes tumor Leydig cell lines (MLTC-1 and R2C) and modulates antioxidant enzyme activities, as CAT and GST. RA is able to induce proliferation through non-classical and redox-dependent mechanisms accompanied by increased levels of FABP5 mRNA. The redox environment of the cell is currently thought to be extremely important for controlling either apoptosis or autophagy. Apoptosis occurs at pharmacological doses, while autophagy, which plays a critical role in removing damaged or surplus organelles in order to maintain cellular homeostasis, is triggered at the critical concentration of 500 nM RA, both in normal and tumoral cells. Slight variations of RA concentrations are evaluated as a threshold value to distinguish between the proliferative or anti-proliferative effects. GENERAL SIGNIFICANCE Although retinoids have a promising role as antineoplastic agents, physiological levels of RA play a key role in Leydig cancer progression, fostering proliferation and growth of testicular tumoral mass.

APT070 (mirococept), a membrane‐localizing C3 convertase inhibitor, attenuates early human islet allograft damage in vitro and in vivo in a humanized mouse model

A major obstacle to islet cell transplantation is the early loss of transplanted islets resulting from the instant blood‐mediated inflammation reaction (IBMIR). The activation of complement pathways plays a central role in IBMIR. The aim of this study was to test the inhibitory effect of “painting” human islets with APT070, a membrane‐localizing C3 convertase inhibitor, on inflammation evoked by exposure to human serum in vitro and by transplantation in vivo in a humanized diabetic mouse model.

Crystallographic Study and Biological Evaluation of 1,4-dimethyl- N -alkylcarbazoles†

The 9-(bromoalkyl)-1,4-dimethyl-9H-carbazole (2a-d) derivatives, characterized by the presence of five or seven methylenic spacer groups bonded to the carbazole nitrogen, have been synthesized from the corresponding 1,4- dimethyl-9H-carbazole and appropriate dibromoalkane following a general synthetic method. All the prepared species have been fully characterized by means of IR, and (1)H and (13)C NMR spectroscopy, GC-MS and Elemental analysis. Good crystals of the 2c have been obtained and the crystal structure has been solved by means of X-ray diffractometry. In order to study the cytotoxic effect of 2a, 2b, 2c, 2d carbazole derivatives on A2780 ovarian cancer cells, we performed MTT assay after exposure of this cell population to those compounds in a concentration range from 1 to 10μM. Finally, we want to verify whether the cytotoxic effect of the 2c carbazole is mediated by apoptotic mechanisms, by performing chromatin condensation assay on the A2780 cell cultures upon the carbazole treatment at concentration of 10 μM for 72h. All together our data demonstrate that carbazole derivatives exert inhibitory effects on ovarian cancer cell growth, highlighting a stronger and a dose-dependent anti proliferative activity displayed by 2c carbazole, designating this compound, as a better candidate in the treatment of human ovarian cancer.

Fine tuning of insulin secretion by release of nerve growth factor from mouse and human islet β-cells

Nerve growth factor (NGF) is a protein required for neuronal development that also has regulatory functions in non-neuronal cells. Both NGF and its membrane receptors trkA and p75(NTR) are expressed by islet β-cells. In this study we dynamically profiled NGF secretion from islets and used selective trkA and p75(NTR) inhibitors to identify the role of endogenous NGF in β-cell stimulus-secretion coupling. NGF secretion from mouse islets was transient and did not accompany the sustained second phase of glucose-induced insulin secretion. Despite being present in human islets, NGF was not released at sufficient levels to be quantified. Inhibition of NGF signaling through trkA and p75(NTR) increased basal insulin secretion from both human and mouse islets, but impaired glucose-stimulated insulin secretion. These data support a role for islet NGF in fine-tuning insulin secretion, to both maintain a low basal level of insulin output and contribute to the biphasic secretory response to glucose.

Vitamin A and Diabesity: New Insight for Drug Discovery

Obesity, insulin resistance, metabolic syndrome and type 2 diabetes have reached epidemic proportions, from the term: diabesity. Vitamin A is delivered by a specific binding protein called retinol-binding protein 4 (RBP4) a soluble protein, emerging to have a role in insulin resistance, the major cause of diabetes is highly associated with adipose tissue inflammation and obesity with action. RBP4, interacts with two receptors, the Toll-like receptor 4 (TLR4) and the plasma membrane protein are stimulated by retinoic acid 6 (STRA6), leading to the activation of c-Jun N-terminal protein kinase (JNK) pathways and JAK2/STAT5 cascade, respectively. Both mechanisms sustain insulin resistance. Therefore, ablation of STRA6 protects mice from RBP4-induced suppression of insulin signaling. In addition, mice harboring deletion of a specific chaperon for retinol, show infiltration of α-cells in the core of pancreatic islets, where usually only β-cells reside, showing a pre-diabetic like phenotype. Not only proteins in vitamin A shuttle and signaling are emerging in diabesity, recently, the discovery of 9cis retinoic acid (9cRA) with effects on controlling glucose levels have opened a new scenario. So far, only pancreas β-cells have been shown to synthesize it, and high levels of 9cRA correlate with obesity mice models. In this article, we summarize the recent literature present on this topic raising the hypothesis.

The adhesion receptor GPR56 is activated by extracellular matrix collagen III to improve β-cell function

AimsG-protein coupled receptor 56 (GPR56) is the most abundant islet-expressed G-protein coupled receptor, suggesting a potential role in islet function. This study evaluated islet expression of GPR56 and its endogenous ligand collagen III, and their effects on β-cell function.MethodsGPR56 and collagen III expression in mouse and human pancreas sections was determined by fluorescence immunohistochemistry. Effects of collagen III on β-cell proliferation, apoptosis, intracellular calcium ([Ca2+]i) and insulin secretion were determined by cellular BrdU incorporation, caspase 3/7 activities, microfluorimetry and radioimmunoassay, respectively. The role of GPR56 in islet vascularisation and innervation was evaluated by immunohistochemical staining for CD31 and TUJ1, respectively, in pancreases from wildtype (WT) and Gpr56−/− mice, and the requirement of GPR56 for normal glucose homeostasis was determined by glucose tolerance tests in WT and Gpr56−/− mice.ResultsImmunostaining of mouse and human pancreases revealed that GPR56 was expressed by islet β-cells while collagen III was confined to the peri-islet basement membrane and islet capillaries. Collagen III protected β-cells from cytokine-induced apoptosis, triggered increases in [Ca2+]i and potentiated glucose-induced insulin secretion from WT islets but not from Gpr56−/− islets. Deletion of GPR56 did not affect glucose-induced insulin secretion in vitro and it did not impair glucose tolerance in adult mice. GPR56 was not required for normal islet vascularisation or innervation.ConclusionWe have demonstrated that collagen III improves islet function by increasing insulin secretion and protecting against apoptosis. Our data suggest that collagen III may be effective in optimising islet function to improve islet transplantation outcomes, and GPR56 may be a target for the treatment of type 2 diabetes.