The entero-insular axis: a journey in the physiopathology of diabetes

Glycemic homeostasis is an essential mechanism for the proper working of an organism. However, balance in blood lipid and protein levels also plays an important role. The discovery of the hormone insulin and the description of its function for glycemic control made fundamental scientific progress in this field. However, since then our view of the problem has been deeply influenced only in terms of glucose and insulin (in an insulin-centric and glucose-centric way). Based on recent scientific discoveries, a fine and sophisticated network of hormonal and metabolic interactions, involving almost every apparatus and tissue of the human body, has been theorized. Efficient metabolic homeostasis is founded on these intricate interactions. Although it is still not fully defined, this complex network can undergo alterations that lead to metabolic disorders such as diabetes mellitus (DM). The endocrine pancreas plays a crucial role in the metabolic balance of an organism, but insulin is just one of the elements involved and each single pancreatic islet hormone is worthy of our concern. Moreover, pancreatic hormones need to be considered in a general view, concerning both their systemic function as direct mediators and as hormones, which, in turn, are regulated by other hormones or other substances. This more complex scenario should be taken into account for a better understanding of the pathophysiology and the therapeutic algorithms of DM. As a consequence, improvements in modern medicine could help to contemplate this new perspective. This review is focused on some aspects of gut-pancreas interaction, aiming to integrate this synergy into a wider context involving other organs and tissues

Coffee Restores Expression of lncRNAs Involved in Steatosis and Fibrosis in a Mouse Model of NAFLD

Background and aim: Coffee intake exerts protective effects against non-alcoholic fatty liver disease (NAFLD), although without fully cleared mechanisms. In this study we aimed to assess whether coffee consumption may influence the expression of long non-coding RNAs (lncRNAs) in the liver. Methods: C57BL/6J mice were fed a 12-week standard diet (SD), high-fat diet (HFD) or HFD plus decaffeinated coffee solution (HFD + coffee). Expression of specific lncRNAs involved in NAFLD was analyzed by real-time PCR. For the most differentially expressed lncRNAs, the analysis was also extended to their mRNA targets. Results: Decaffeinated coffee intake reduced body weight gain, prevented NAFLD, lowered hyperglycemia and hypercholesterolemia. NAFLD was associated with lower hepatic expression of Gm16551, a lncRNA inhibiting de novo lipogenesis, and higher expression of H19, a lncRNA promoting fibrogenesis. Coffee intake restored Gm16551 to levels observed in lean mice and downregulated gene expression of its targets acetyl coenzyme A carboxylase 1 and stearoyl coenzyme A desaturase 1. Furthermore, coffee consumption markedly decreased hepatic expression of H19 and of its target gene collagen alpha-1(I) chain; consistently, in mice fed HFD + coffee liver expression of αSMA protein returned to levels of mice fed SD. Expression of lncRNA involved in circadian clock such as fatty liver-related lncRNA 1 (FLRL1) and fatty liver-related lncRNA 2 (FLRL2) were upregulated by HFD and were also modulated by coffee intake. Conclusion: Hepatoprotective effects of coffee may be depending on the modulation of lncRNAs involved in key pathways of NAFLD onset and progression

High glomerular filtration rate is associated with impaired arterial stiffness and subendocardial viability ratio in prediabetic subjects.

BACKGROUND AND AIMS High glomerular filtration rate (HGFR) is associated with cardiovascular damage in the setting of various conditions such as obesity and diabetes. Prediabetes was also associated with increased GFR, however, the association between prediabetes, HGFR and cardiovascular damage has not been investigated. In this study, we investigated the association between HGFR and early markers of cardiovascular disease in subjects with prediabetes. METHODS AND RESULTS Augmentation pressure (Aug), augmentation index (AIx), subendocardial viability ratio (SEVR), pulse wave velocity (PWV), intima-media thickness (IMT) and estimated GFR (eGFR) were evaluated in 230 subjects with prediabetes. The eGFR was assessed using the Chronic Kidney Disease Epidemiology Collaboration formula. HGFR was defined as an eGFR above the 75th percentile. Prediabetic subjects were divided into two groups according to presence/absence of HGFR: 61 subjects with HGFR and 169 subjects without HGFR. Subjects with HGFR showed higher Aug, AIx and lower SEVR compared with prediabetic subjects with lower eGFR (14.1 ± 7.2 vs 10.8 ± 6.2, 32.9 ± 12.7 vs 27.6 ± 11.7, 153.5 ± 27.8 vs 162 ± 30.2, p < 0.05). No differences were found in PWV and IMT values between the two groups. Then, we performed multiple regression analysis to test the relationship between Aug, SEVR and several cardiovascular risk factors. In multiple regression analysis Aug was associated with age, systolic blood pressure (BP), HOMA-IR and eGFR; the major determinants of SEVR were systolic BP, HOMA-IR and eGFR. CONCLUSION Subjects with prediabetes and HGFR exhibited an increased Aug, AIx and a reduced SEVR. These alterations are associated with eGFR, insulin resistance and systolic BP

Atorvastatin but not pravastatin impairs mitochondrial function in human pancreatic islets and rat β-cells. Direct effect of oxidative stress

Statins are a class of drugs widely prescribed as frontline therapy for lowering plasma LDL-cholesterol in cardiovascular risk prevention. Several clinical reports have recently suggested an increased risk of type 2 diabetes associated with chronic use of these drugs. The pathophysiology of this effect remains to be fully elucidated but impaired β-cell function constitutes a potential mechanism. The aim of this study was to explore the effect of a chronic treatment with lipophilic and hydrophilic statins on β-cell function, using human pancreatic islets and rat insulin-secreting INS-1 cells; we particularly focused on the role of mitochondria and oxidative stress. The present study demonstrates, for the first time, that atorvastatin (lipophilic) but not pravastatin (hydrophilic) affected insulin release and mitochondrial metabolism due to the suppression of antioxidant defense system and induction of ROS production in pancreatic β-cell models. Mevalonate addition and treatment with a specific antioxidant (N-AcetylCysteine) effectively reversed the observed defects. These data demonstrate that mitochondrial oxidative stress is a key element in the pathogenesis of statin-related diabetes and may have clinical relevance to design strategies for prevention or reduction of statin induced β-cell dysfunction and diabetes in patients treated with lipophilic statins

MOLECULAR CLONING, CHARACTERIZATION AND EXPRESSION ANALYSIS OF THE RBM20, A NOVEL RIBONUCLEOPROTEIN GENE ASSOCIATED TO FAMILIAL DILATED CARDIOMYOPATHY.

Lo splicing alternativo del pre-RNA messaggero (pre-mRNA) \ue8 un processo strettamente regolato che coinvolge lo spliceosoma e proteine di legame all\u2019RNA che possono reprimere o attivare i siti di splicing selezionati. Oltre il 15% delle malattie genetiche umane sono state associate a mutazioni negli elementi di splicing agenti in cis, ma poche tra queste sono attribuibili a fattori agenti in trans che controllano lo splicing alternativo. Gli effetti di queste mutazioni che coinvolgono il macchinario di splicing basale e i regolatori dello splicing alternativo sono stati associati all\u2019autismo, alla sclerosi laterale amiotrofica e al cancro. Nel cuore e nel muscolo scheletrico, lo splicing alternativo svolge un ruolo critico per la funzione muscolare. In entrambi i sistemi, le transizioni di splicing avvengono secondo delle vie temporali strettamente raggruppate, in cui sono coinvolte le stesse proteine. Un ruolo diretto dei regolatori dello splicing nelle patologie cardiache \ue8 stato dimostrato recentemente per due proteine con un motivo di legame all\u2019RNA (RBM), RBM25 e RBM20. Lo studio sperimentale presentato in questa tesi \ue8 focalizzato sulla proteina RBM20. RBM20, proteina con un motivo di legame all\u2019RNA di tipo 20, \ue8 un fattore agente in trans espresso preferenzialmente nel tessuto cardiaco, che regola lo splicing alternativo di geni aventi un ruolo chiave nella funzionalit\ue0 cardiaca, compresi quelli che regolano l\u2019omeostasi ionica, la biologia del sarcomero e la trasduzione del segnale. Mutazioni del gene RBM20 sono associate a cardiomiopatia dilatativa familiare e la maggior parte di queste altera i residui amminoacidici presenti nel dominio ricco in arginine e serine della proteina. I motivi funzionali della proteina RBM20 sono stati predetti attraverso l\u2019omologia di sequenza, ma sono stati poco caratterizzati a livello funzionale. In questo studio \ue8 stato clonato il cDNA della isoforma cardiaca della proteina RBM20 umana e della corrispondente proteina murina. L\u2019utilizzo di vettori di espressione della proteina RBM20 prodotti nel corso dello studio ha contribuito alla caratterizzazione funzionale dei domini richiesti per la localizzazione nucleare di RBM20. Analisi di microscopia confocale hanno mostrato che RBM20 ha una localizzazione nucleare con distribuzione punteggiata a livello di strutture denominate \u201cnuclear speckles\u201d, questa distribuzione \ue8 tipica dei fattori di splicing con un dominio ricco in arginine e serine (RS). Attraverso la produzione di vettori che esprimono forme tronche della proteina e comparando la loro distribuzione subcellulare abbiamo identificato le sequenze necessarie alla localizzazione nucleare di RBM20. Questa regione comprende il dominio di legame all\u2019RNA e il dominio ricco in arginine e serine. La sequenza \ue8 conservata in molte specie che appartengono solo alle proteine ortologhe di RBM20. Questi studi dimostrano l\u2019esistenza di una selezione specifica durante l\u2019evoluzione nella regolazione post-trascrizionale del cuore, indicando RBM20 come un fattore chiave negli eventi di regolazione dello splicing richiesti per la funzione cardiaca.Alternative splicing of pre-messenger RNA is a tightly regulated process that involves the spliceosome and additional RNA binding proteins that can repress or activate splice site selection. More than 15% of human genetic diseases have been associated to mutations in cis-acting splice elements, but very few of them are attributable to trans-factors that control alternative splicing. The effects of these mutations that involve the basal splicing machinery and regulators of alternative splicing have been associated with autism spectrum disorders, amyotrophic lateral sclerosis and cancer. In heart and skeletal muscle, alternative splicing plays a critical role in muscle function. In both systems, splicing transition occurs in tightly grouped temporal ways, partially involving the same proteins. A direct involvement of splicing regulators to cardiac diseases has been proven recently for two RNA binding motif (RBM) protein, the RBM25 and the RBM20. This experimental study is focused on the RBM20 protein. RNA binding motif protein type 20 (RBM20) is a trans-acting factor expressed preferentially in heart tissue, which regulates alternative splicing of gene that have a key role in cardiac function, including ion homeostasis, sarcomere biology and signal transduction. Mutations in the RBM20 gene are linked to familial dilated cardiomyopathy and the most of them alters residues in the arginine-serine domain of the protein. The functional motifs of the RBM20 protein have been predicted by sequence homology, but poorly functionally characterized. In the present study by cloning the cardiac isoform of human RBM20 and the mouse RBM20 full length cDNA I provided functional annotations of structural domains required for the RBM20 nuclear localization. Confocal microscopy analysis showed that RBM20 had a punctuate localization in nuclear speckles, which is typical of RS splicing factors. By producing expressing vectors for truncated proteins and comparing their subcellular distribution we identified the sequences necessary for RBM20 full nuclear retention. The region overlaps both RNA binding motif and arginine-serine domain. The sequence is conserved in many species but belongs only to RBM20 protein orthologs. These studies demonstrate a specific evolutionary selection for post-transcriptional regulation in heart, highlighting RBM20 as a key factor for regulation of splicing events required for cardiac function

Metformin: When Should We Fear Lactic Acidosis?

Metformin, a molecule belonging to the biguanide family, represents one of the most commonly prescribed medications for the treatment of diabetes mellitus in the world. Over the sixty years during which it has been used, many benefits have been described, which are not limited to the treatment of diabetes mellitus. However, since metformin is similar to other members of the same drug family, there is still much concern regarding the risk of lactic acidosis. This article aims to highlight the correlation between the use of metformin and the onset of renal damage or lactic acidosis. Metformin-associated lactic acidosis exists; however, it is rare. The appropriate use of the drug, under safe conditions, induces benefits without risks

Clinical and Molecular Biomarkers for Diagnosis and Staging of NAFLD

Non-alcoholic fatty liver disease (NAFLD) is the most common hepatic pathology in industrialized countries, affecting about 25% of the general population. NAFLD is a benign condition, however, it could evolve toward more serious diseases, including non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and finally, hepatocellular carcinoma (HCC). Liver biopsy is still the gold standard for NAFLD diagnosis. Due to the risks associated with liver biopsy and the impossibility to apply it on a large scale, it is now necessary to identify non-invasive biomarkers, which may reliably identify patients at higher risk of progression. Therefore, several lines of research have tried to address this issue by identifying novel biomarkers using omics approaches, including lipidomics, metabolomics and RNA molecules’ profiling. Thus, in this review, we firstly report the conventional biomarkers used in clinical practice for NAFL and NASH diagnosis as well as fibrosis staging, and secondly, we pay attention to novel biomarkers discovered through omics approaches with a particular focus on RNA biomarkers (microRNAs, long-noncoding RNAs), showing promising diagnostic performance for NAFL/NASH diagnosis and fibrosis staging

Chronic exposure to GLP-1 increases GLP-1 synthesis and release in a pancreatic alpha cell line (α-TC1): evidence of a direct effect of GLP-1 on pancreatic alpha cells.

Incretin therapies, which are used to treat diabetic patients, cause a chronic supra-physiological increase in GLP-1 circulating levels. It is still unclear how the resulting high hormone concentrations may affect pancreatic alpha cells. The present study was designed to investigate the effects of chronic exposure to high GLP-1 levels on a cultured pancreatic alpha cell line.α-TC1-6 cell line was cultured in the presence or absence of GLP-1 (100 nmol/l) for up to 72 h. In our model GLP-1 receptor (GLP-1R) was measured. After the cells were exposed to GLP-1 the levels of glucagon secretion were measured. Because GLP-1 acts on intracellular cAMP production, the function of GLP-1R was studied. We also investigated the effects of chronic GLP-1 exposure on the cAMP/MAPK pathway, Pax6 levels, the expression of prohormone convertases (PCs), glucagon gene (Gcg) and protein expression, glucagon and GLP-1 production.In our model, we were able to detect GLP-1R. After GLP-1 exposure we found a reduction in glucagon secretion. During further investigation of the function of GLP-1R, we found an activation of the cAMP/MAPK/Pax6 pathway and an increase of Gcg gene and protein expression. Furthermore we observed a significant increase in PC1/3 protein expression, GLP-1 intracellular content and GLP-1 secretion.Our data indicate that the chronic exposure of pancreatic alpha cells to GLP-1 increases the ability of these cells to produce and release GLP-1. This phenomenon occurs through the stimulation of the transcription factor Pax6 and the increased expression of the protein convertase PC1/3

1 h Postload Glycemia Is Associated with Low Endogenous Secretory Receptor for Advanced Glycation End Product Levels and Early Markers of Cardiovascular Disease

We investigated the correlation of the soluble receptor for advanced glycation end products (sRAGE) and endogenous secretory RAGE (esRAGE) with markers of cardiovascular disease in subjects with normal glucose tolerance (NGT) and 1 h postload glucose &ge;155 mg/dL after an oral glucose tolerance test. We stratified 282 subjects without a previous diagnosis of diabetes into three groups: 123 controls (NGT and 1 h postload glycemia &lt;155 mg/dL), 84 NGT and 1 h postload glycemia &ge;155 mg/dL (NGT 1 h high), and 75 subjects with impaired fasting glucose and/or impaired glucose tolerance (IFG/IGT). NGT 1 h high subjects exhibited lower esRAGE (0.36 &plusmn; 0.18 vs. 0.4 5 &plusmn; 0.2, p &lt; 0.05) and higher S100A12 levels than controls (5684 (3193.2&ndash;8295.6) vs. 3960.1 (2101.8&ndash;7419), p &lt; 0.05). Furthermore, they showed an increased pulse wave velocity (PWV) and intima&ndash;media thickness (IMT). No differences were found between the NGT 1 h high group and the IFG/IGT group regarding cardiometabolic profiles. After multiple regression analyses, esRAGE was associated with glycated hemoglobin (HbA1c) and high-sensitivity C-reactive protein (hs-CRP). Age, HbA1c, and esRAGE were the determinants of IMT, whereas S100A12 and systolic pressure were the determinants of PWV. The NGT 1 h high group exhibited low esRAGE levels and an altered cardiometabolic profile. HbA1c, S100A12, and hs-CRP were associated with these alterations. In conclusion, subjects with NGT are not a homogeneous population, and they present different cardiovascular and glycometabolic risks

Thyroid Cancer and Circadian Clock Disruption

Thyroid cancer (TC) represents the most common malignancy of the endocrine system, with an increased incidence across continents attributable to both improvement of diagnostic procedures and environmental factors. Among the modifiable risk factors, insulin resistance might influence the development of TC. A relationship between circadian clock machinery disfunction and TC has recently been proposed. The circadian clock machinery comprises a set of rhythmically expressed genes responsible for circadian rhythms. Perturbation of this system contributes to the development of pathological states such as cancer. Several clock genes have been found deregulated upon thyroid nodule malignant transformation. The molecular mechanisms linking circadian clock disruption and TC are still unknown but could include insulin resistance. Circadian misalignment occurring during shift work, jet lag, high fat food intake, is associated with increased insulin resistance. This metabolic alteration, in turn, is associated with a well-known risk factor for TC i.e., hyperthyrotropinemia, which could also be induced by sleep disturbances. In this review, we describe the mechanisms controlling the circadian clock function and its involvement in the cell cycle, stemness and cancer. Moreover, we discuss the evidence supporting the link between circadian clockwork disruption and TC development/progression, highlighting its potential implications for TC prevention, diagnosis and therapy