Alessandra Puddu

Evidence for the Gut Microbiota Short-Chain Fatty Acids as Key Pathophysiological Molecules Improving Diabetes

In type 2 diabetes, hyperglycemia, insulin resistance, increased inflammation, and oxidative stress were shown to be associated with the progressive deterioration of beta-cell function and mass. Short-chain fatty acids (SCFAs) are organic fatty acids produced in the distal gut by bacterial fermentation of macrofibrous material that might improve type 2 diabetes features. Their main beneficial activities were identified in the decrease of serum levels of glucose, insulin resistance as well as inflammation, and increase in protective Glucagon-like peptide-1 (GLP-1) secretion. In this review, we updated evidence on the effects of SCFAs potentially improving metabolic control in type 2 diabetes.

Glycated fetal calf serum affects the viability of an insulin-secreting cell line in vitro.

The purpose of the present study was to evaluate the direct effects of advanced glycation end products (AGEs) on beta-cells by their exposure to a glycated serum to estimate the cellular viability and the related insulin secretion. Glycation of fetal calf serum was obtained by incubation with 50 mol/L ribose at 37 degrees C for 7 days; at the end of this incubation period, the pentosidine content ranged between 15 and 16 x 10(5) pmol/L. HIT-T15 cells, a pancreatic islet cell line, were grown and cultured for 5 days in Roswell Park Memorial Institute (RPMI) medium containing either not glycated (NGS) or glycated (GS) fetal calf serum. Cellular oxidative stress (ie, thiobarbituric acid-reactive substances) was assessed by high-performance liquid chromatography. Cellular viability was evaluated by detection of proliferation, cell necrosis, and cell apoptosis rate. The insulin secretion and the related intracellular content were evaluated by enzyme-linked immunosorbent assay. The present study reported, after 5 days of exposure to the glycation environment, a moderately reduced cellular proliferation (-20.44% +/- 2.92%) with a corresponding increase of cell necrosis (+67.7% +/- 1.56%) and cell apoptosis (+39.83% +/- 2.92%) rate in comparison with the untreated cells. Oxidative intracellular stress was higher in GS conditions compared with the NGS ones (+293.3% +/- 87.53%). Insulin release from GS-treated HIT-T15 cells was lower than that of NGS-treated cells both when cells were stimulated with low glucose concentration (2.8 mmol/L, -30.3% +/- 4.91%) or when they were challenged with high glucose concentration (16.7 mmol/L, -29.2% +/- 5.82%). Incubation of HIT-T15 cells with glycated serum also caused a significant decrease of insulin intracellular content (-44.47% +/- 9.98%). Thus, AGEs were shown to exert toxic effects on insulin-secreting cells. Chronically high intracellular oxidative stress, due to accumulation of AGEs, affects the insulin secretion machinery. The present data suggest a pivotal role of the non-enzymatic glycation process in the onset and progression of diabetes during aging and a direct adverse effect of a glycated environment on the pancreatic islet cells.

Advanced glycation endproducts and diabetes. Beyond vascular complications.

Advanced Glycation Endproducts (AGEs) are a group of heterogeneous compounds formed by the non enzymatic reactions between aldehydic group of reducing sugars with proteins, lipids or nucleic acids. Formation and accumulation of AGEs is related with the aging process and is accelerated in diabetes. Type 2 diabetes, the most common form of diabetes, is characterized by hyperglycaemia and insulin resistance associated to a progressive deterioration of beta cell function and mass. The pathogenic role of AGEs in vascular diabetic complications is widely recognised. Recently other aspects of the detrimental effects of AGEs in type 2 diabetes are emerged: AGEs interfere with the complex molecular pathway of insulin signaling, leading to insulin resistance; AGEs modify the insulin molecule, and, consequently, its function; AGEs decrease insulin secretion and insulin content. In this article we review the role of AGEs in type 2 diabetes, beyond their involvement in vascular complications.

Advanced Glycation End Products Play Adverse Proinflammatory Activities in Osteoporosis

Osteoporosis is a major public health burden that is expected to further increase as the global population ages. In the last twenty years, advanced glycation end products (AGEs) have been shown to be critical mediators both in the pathogenesis and development of osteoporosis and other chronic degenerative diseases related to aging. The accumulation of AGEs within the bone induces the formation of covalent cross-links with collagen and other bone proteins which affects the mechanical properties of tissue and disturbs bone remodelling and deterioration, underlying osteoporosis. On the other hand, the gradual deterioration of the immune system during aging (defined as immunosenescence) is also characterized by the generation of a high level of oxidants and AGEs. The synthesis and accumulation of AGEs (both localized within the bone or in the systemic circulation) might trigger a vicious circle (in which inflammation and aging merged in the word “Inflammaging”) which can establish and sustain the development of osteoporosis. This narrative review will update the molecular mechanisms/pathways by which AGEs induce the functional and structural bone impairment typical of osteoporosis.

Quinazolinedione SIRT6 inhibitors sensitize cancer cells to chemotherapeutics

The NAD(+)-dependent sirtuin SIRT6 is highly expressed in human breast, prostate, and skin cancer where it mediates resistance to cytotoxic agents and prevents differentiation. Thus, SIRT6 is an attractive target for the development of new anticancer agents to be used alone or in combination with chemo- or radiotherapy. Here we report on the identification of novel quinazolinedione compounds with inhibitory activity on SIRT6. As predicted based on SIRT6s biological functions, the identified new SIRT6 inhibitors increase histone H3 lysine 9 acetylation, reduce TNF-α production and increase glucose uptake in cultured cells. In addition, these compounds exacerbate DNA damage and cell death in response to the PARP inhibitor olaparib in BRCA2-deficient Capan-1 cells and cooperate with gemcitabine to the killing of pancreatic cancer cells. In conclusion, new SIRT6 inhibitors with a quinazolinedione-based structure have been identified which are active in cells and could potentially find applications in cancer treatment.

Update on the Protective Molecular Pathways Improving Pancreatic Beta-Cell Dysfunction

The primary function of pancreatic beta-cells is to produce and release insulin in response to increment in extracellular glucose concentrations, thus maintaining glucose homeostasis. Deficient beta-cell function can have profound metabolic consequences, leading to the development of hyperglycemia and, ultimately, diabetes mellitus. Therefore, strategies targeting the maintenance of the normal function and protecting pancreatic beta-cells from injury or death might be crucial in the treatment of diabetes. This narrative review will update evidence from the recently identified molecular regulators preserving beta-cell mass and function recovery in order to suggest potential therapeutic targets against diabetes. This review will also highlight the relevance for novel molecular pathways potentially improving beta-cell dysfunction.

Retinal Pigment Epithelial Cells Express a Functional Receptor for Glucagon-Like Peptide-1 (GLP-1)

Glucagon-like peptide-1 (GLP-1) is a gut-derived incretin hormone that has been shown to improve glucose homeostasis in type 2 diabetes. The biological effects of GLP-1 are mediated by its specific receptor GLP-1R that is expressed in a wide range of tissues, where it is responsible of the extra-pancreatic effects of GLP-1. Since the retinal pigment epithelium (RPE), that forms the outer retinal barrier, has a key role in protecting from diabetic retinopathy (DR), we investigated the potential expression and function of GLP-1R in a RPE cell line. ARPE-19 cells were cultured in DMEM/F12 supplemented with 10% FBS. The expression of GLP-1R was evaluated at both mRNA and protein levels. Then, the activation postreceptor intracellular signal transduction pathways (extracellular signal-regulated kinases 1 and 2 [ERK1/2] and protein kinase B [PKB]) were assessed by western blot in normal cells or silenced for GLP-1R in the presence or absence of 10 nmol/L GLP-1. The potential connections between intracellular signalling pathways triggered by GLP-1 stimulation were performed before incubating cells with kinase pharmacological inhibitors of mitogen-activated protein kinase (MEK)1/2, phosphatydilinositol-3kinase (PI3K), or epidermal growth factor receptor (EGFR). The results showed that GLP1R is expressed at both mRNA and protein level in ARPE-19 cells. Stimulation with GLP-1 strongly activated PKB and ERK1/2 phosphorylation till 40 min of exposure. GLP-1-mediated activation of both kinases was dependent on the upstream activation of PI3K and EGFR. Finally, treatment with GLP-1 did not affect the spontaneous release of VEGF-A from ARPE-19 cells. In conclusion, this paper showed that the presence of functional GLP-1R is expressed in RPE cells. These data might represent the rationale to further investigate the potential direct beneficial effects of GLP-1 treatment against DR.

Acipimox reduces circulating levels of insulin and associated neutrophilic inflammation in metabolic syndrome

Metabolic syndrome is a proatherosclerotic condition clustering cardiovascular risk factors, including glucose and lipid profile alterations. The pathophysiological mechanisms favoring atherosclerotic inflammation in the metabolic syndrome remain elusive. Here, we investigated the potential role of the antilipolytic drug acipimox on neutrophil- and monocyte-mediated inflammation in the metabolic syndrome. Acipimox (500 mg) was orally administered to metabolic syndrome patients (n = 11) or healthy controls (n = 8). Serum and plasma was collected before acipimox administration (time 0) as well as 2-5 h afterward to assess metabolic and hematologic parameters. In vitro, the effects of the incubation with metabolic syndrome serum were assessed on human neutrophil and monocyte migration toward the proatherosclerotic chemokine CCL3. Two to five hours after acipimox administration, a significant reduction in circulating levels of insulin and nonesterified fatty acid (NEFA) was shown in metabolic syndrome patients. At time 0 and 2 h after acipimox administration, metabolic syndrome serum increased neutrophil migration to CCL3 compared with healthy controls. No effect was shown in human monocytes. At these time points, serum-induced neutrophil migration positively correlated with serum levels of insulin and NEFA. Metabolic syndrome serum or recombinant insulin did not upregulate CCR5 expression on neutrophil surface membrane, but it increased intracellular JNK1/2 phosphorylation. Insulin immunodepletion blocked serum-induced neutrophil migration and associated JNK1/2 phosphorylation. Although mRNA expression of acipimox receptor (GPR109) was shown in human neutrophils, 5-500 μM acipimox did not affect insulin-induced neutrophil migration. In conclusion, results suggest that acipimox inhibited neutrophil proatherosclerotic functions in the metabolic syndrome through the reduction in circulating levels of insulin.

An emerging role of glucagon-like peptide-1 in preventing advanced-glycation-end-product-mediated damages in diabetes.

Glucagon-like peptide-1 (GLP-1) is a gut hormone produced in the intestinal epithelial endocrine L cells by differential processing of the proglucagon gene. Released in response to the nutrient ingestion, GLP-1 plays an important role in maintaining glucose homeostasis. GLP-1 has been shown to regulate blood glucose levels by stimulating glucose-dependent insulin secretion and inhibiting glucagon secretion, gastric emptying, and food intake. These antidiabetic activities highlight GLP-1 as a potential therapeutic molecule in the clinical management of type 2 diabetes, (a disease characterized by progressive decline of beta-cell function and mass, increased insulin resistance, and final hyperglycemia). Since chronic hyperglycemia contributed to the acceleration of the formation of Advanced Glycation End-Products (AGEs, a heterogeneous group of compounds derived from the nonenzymatic reaction of reducing sugars with free amino groups of proteins implicated in vascular diabetic complications), the administration of GLP-1 might directly counteract diabetes pathophysiological processes (such as pancreatic β-cell dysfunction). This paper outlines evidence on the protective role of GLP-1 in preventing the deleterious effects mediated by AGEs in type 2 diabetes.

Glucagon-like peptide-1 triggers protective pathways in pancreatic beta-cells exposed to glycated serum

Advanced glycation end products (AGEs) might play a pathophysiological role in the development of diabetes and its complications. AGEs negatively affect pancreatic beta-cell function and the expression of transcriptional factors regulating insulin gene. Glucagon-like peptide-1 (GLP-1), an incretin hormone that regulates glucose homeostasis, might counteract the harmful effects of AGEs on the beta cells in culture. The aim of this study was to identify the intracellular mechanisms underlying GLP-1-mediated protection from AGE-induced detrimental activities in pancreatic beta cells. HIT-T15 cells were cultured for 5 days with glycated serum (GS, consisting in a pool of AGEs), in the presence or absence of 10 nmol/L GLP-1. After evaluation of oxidative stress, we determined the expression and subcellular localization of proteins involved in maintaining redox balance and insulin gene expression, such as nuclear factor erythroid-derived 2 (Nrf2), glutathione reductase, PDX-1, and MafA. Then, we investigated proinsulin production. The results showed that GS increased oxidative stress, reduced protein expression of all investigated factors through proteasome activation, and decreased proinsulin content. Furthermore, GS reduced ability of PDX-1 and MafA to bind DNA. Coincubation with GLP-1 reversed these GS-mediated detrimental effects. In conclusion, GLP-1, protecting cells against oxidants, triggers protective intercellular pathways in HIT-T15 cells exposed to GS.