Pathophysiology and Clinical Management of Bile Acid Diarrhea

Bile acid malabsorption (BAM) represents a common cause of chronic diarrhea whose prevalence is under-investigated. We reviewed the evidence available regarding the pathophysiology and clinical management of bile acid diarrhea (BAD). BAD results from dysregulation of the enterohepatic recirculation of bile acids. It has been estimated that 25-33% of patients with functional diarrhea and irritable bowel syndrome with diarrhea have BAM. Currently, the selenium homotaurocholic acid test is the gold standard for BAD diagnosis and severity assessment. However, it is an expensive method and not widely available. The validation of the utility in the clinical practice of several other serum markers, such as 7 alpha-hydroxy-4-cholesten-3-one (C4) and the fibroblast growth factor 19 (FGF19) is ongoing. The first-line treatment of patients with BAD is bile acid sequestrants. Patients that are refractory to first-line therapy should undergo further diagnostics to confirm the diagnosis and to treat the underlying cause of BAD. An early and correct diagnosis of BAD would improve patient's quality of life, avoiding additional diagnostic tests that burden health care systems. Considering the limited availability and tolerability of specific medications for BAD treatment, future research is awaited to identify other therapeutic approaches, such as gut microbiota modulating therapies

FGF-2b and h-PL transform duct and non-endocrine human pancreatic cells into endocrine insulin secreting cells by modulating differentiating genes

Background: Diabetes mellitus (DM) is a multifactorial disease orphan of a cure. Regenerative medicine has been proposed as novel strategy for DM therapy. Human fibroblast growth factor (FGF)-2b controls β-cell clusters via autocrine action, and human placental lactogen (hPL)-A increases functional β-cells. We hypothesized whether FGF-2b/hPL-A treatment induces β-cell differentiation from ductal/non-endocrine precursor(s) by modulating specific genes expression. Methods: Human pancreatic ductal-cells (PANC-1) and non-endocrine pancreatic cells were treated with FGF-2b plus hPL-A at 500 ng/mL. Cytofluorimetry and Immunofluorescence have been performed to detect expression of endocrine, ductal and acinar markers. Bromodeoxyuridine incorporation and annexin-V quantified cells proliferation and apoptosis. Insulin secretion was assessed by RIA kit, and electron microscopy analyzed islet-like clusters. Results: Increase in PANC-1 duct cells de-differentiation into islet-like aggregates was observed after FGF-2b/hPL-A treatment showing ultrastructure typical of islets-aggregates. These clusters, after stimulation with FGF-2b/hPL-A, had significant (p < 0.05) increase in insulin, C-peptide, pancreatic and duodenal homeobox 1 (PDX-1), Nkx2.2, Nkx6.1, somatostatin, glucagon, and glucose transporter 2 (Glut-2), compared with control cells. Markers of PANC-1 (Cytokeratin-19, MUC-1, CA19-9) were decreased (p < 0.05). These aggregates after treatment with FGF-2b/hPL-A significantly reduced levels of apoptosis. Conclusions: FGF-2b and hPL-A are promising candidates for regenerative therapy in DM by inducing de-differentiation of stem cells modulating pivotal endocrine genes

Enhancement of anti-leukemia activity of NK cells in vitro and in vivo by inhibition of leukemia cell-induced NK cell damage

Acute myeloid leukemia (AML) cells induce, in vitro, NK cell abnormalities (NKCAs) including apoptosis and activating receptor down-regulation. The potential negative impact of AML cells on the therapeutic efficacy of NK cell-based strategies prompted us to analyze the mechanisms underlying NKCAs and to develop approaches to protect NK cells from NKCAs. NKCA induction by the AML leukemia cells target a subpopulation of peripheral blood NK cells and is interleukin-2 independent but is abrogated by a long-term culture of NK (LTNK) cells at 37°C. LTNK cells displayed a significantly enhanced ability to damage AML cells in vitro and inhibited the subcutaneous growth of ML-2 cells grafted into CB17 SCID mice. Actinomycin D restored the susceptibility of LTNK cells to NKCAs while TAPI-0, a functional analog of the tissue inhibitor of metalloproteinase (TIMP) 3, inhibits ML-2 cell-induced NKCAs suggesting that the generation of NK cell resistance to NKCAs involves RNA transcription and metalloproteinase (MPP) inactivation. This conclusion is supported by the reduced susceptibility to AML cell-induced NKCAs of LTNK cells in which TIMP3 gene and protein are over-expressed. This information may contribute to the rational design of targeted strategies to enhance the efficacy of NK cell-based-immunotherapy of AML with haploidentical NK cells

Regolazione epatica del segnale insulinico

Il diabete mellito di tipo 2 (DMT2) è caratterizzato da elevati livelli di insulino resistenza (IR) e da un difetto della cellula beta pancreatica. L’iperglicemia insorge principalmente a causa di un aumento della produzione di glucosio a livello epatico che induce uno stato di iperinsulinemia. Lo scopo di questo lavoro è studiare la fisiopatologia della disfunzione epatica nel DMT2. Nel nostro studio abbiamo utilizzato due modelli sperimentali: 1) Linee cellulari epatiche immortalizzate derivate da topi knockout (KO) (IR-/-) e “wild type” (WT) (IR+/+) per il recettore insulinico (IR); 2) Tessuto epatico estratto da topi IR+/+, eterozigoti con un singolo allele knockout per IR+/-, e IR-/-. L’analisi proteomica, condotta su triplicati sperimentali biologici, in elettroforesi bidimensionale (2D-Gel), evidenziava diverse proteine di interesse: sono state trovate più di 20 proteine differentemente sintetizzate nei fenotipi studiati, sia nei tessuti che in vitro. Cinque proteine sono state identificate mediante spettrometria di massa (MALDI-TOF): Peroxiredoxin-6, XRN2, Annessina 1, Calreticulina, HMGB1. Lo studio dei livelli di sintesi e della funzione delle cinque proteine ha rivelato il loro coinvolgimento nella regolazione del segnale insulinico, della sintesi proteica, del metabolismo e dello stress ossidativo. Il confronto di questi dati con quelli ottenuti dall’analisi trascrittomica (Real-Time PCR) e l’analisi Western Blot, ha dimostrato che la mancanza del recettore insulinico sia in vitro che in vivo induce un’alterazione del processo di traduzione.In the western society and developing countries the incidence of insulin resistance, type-2 diabetes mellitus (T2DM) and augmented metabolic risk for cardiovascular disease and T2DM, are increasing exponentially. This raise is due mainly to an urbanized life style and to age of population. The shared feature of these related diseases is an impaired response to insulin target tissues, insulin resistance, and/or reduction of insulin secretion in pancreatic beta cells. Studies of these aspects are performed: in vitro, culture cell lines of fibroblasts/hepatocytes derived from insulin receptor knockout (IRKO) mice transformed by adenovirus SV40; ex vivo, in genetically modified animal (mouse) models for insulin receptor knockout (IRKO). It has been analyzed the differences in proteins expression profile and insulin signaling transmission in IRKO hepatocytes and IRKO mouse liver tissues compared with the wild type and heterozygous phenotypes. The aims of this study are to detect differences in proteins expression that could be modulated by insulin and could be involved in cell survival, cell cycle and oxidative stress. In these conditions it is possible to study the specific role of IRKO in hepatic cell lines and analyze the different modulation in activity and expression of insulin related substrates (basal conditions). On the other hand, in mouse liver tissue (ex-vivo) it is possible to detect differences in insulin substrates expression and activation in diabetic ketoacidotic conditions (pathophysiological conditions). To isolate the differentially expressed proteins we used 2D-PAGE analysis of protein extracts of knockout, wild type and heterozygous mouse liver tissues and murine hepatocytes phenotypes (wild type and knockout). Five proteins at present, of the 22 proteins differentially expressed, have been identified by mass spectrometry. It has also been discovered a defect in post transcription proteins production that it is associated with a related increases in mRNA levels and again it has been described a defect in myotubules proteins expression but differently not in albumin production

Regolazione epatica del segnale insulinico

Il diabete mellito di tipo 2 (DMT2) è caratterizzato da elevati livelli di insulino resistenza (IR) e da un difetto della cellula beta pancreatica. L’iperglicemia insorge principalmente a causa di un aumento della produzione di glucosio a livello epatico che induce uno stato di iperinsulinemia. Lo scopo di questo lavoro è studiare la fisiopatologia della disfunzione epatica nel DMT2. Nel nostro studio abbiamo utilizzato due modelli sperimentali: 1) Linee cellulari epatiche immortalizzate derivate da topi knockout (KO) (IR-/-) e “wild type” (WT) (IR+/+) per il recettore insulinico (IR); 2) Tessuto epatico estratto da topi IR+/+, eterozigoti con un singolo allele knockout per IR+/-, e IR-/-. L’analisi proteomica, condotta su triplicati sperimentali biologici, in elettroforesi bidimensionale (2D-Gel), evidenziava diverse proteine di interesse: sono state trovate più di 20 proteine differentemente sintetizzate nei fenotipi studiati, sia nei tessuti che in vitro. Cinque proteine sono state identificate mediante spettrometria di massa (MALDI-TOF): Peroxiredoxin-6, XRN2, Annessina 1, Calreticulina, HMGB1. Lo studio dei livelli di sintesi e della funzione delle cinque proteine ha rivelato il loro coinvolgimento nella regolazione del segnale insulinico, della sintesi proteica, del metabolismo e dello stress ossidativo. Il confronto di questi dati con quelli ottenuti dall’analisi trascrittomica (Real-Time PCR) e l’analisi Western Blot, ha dimostrato che la mancanza del recettore insulinico sia in vitro che in vivo induce un’alterazione del processo di traduzione.In the western society and developing countries the incidence of insulin resistance, type-2 diabetes mellitus (T2DM) and augmented metabolic risk for cardiovascular disease and T2DM, are increasing exponentially. This raise is due mainly to an urbanized life style and to age of population. The shared feature of these related diseases is an impaired response to insulin target tissues, insulin resistance, and/or reduction of insulin secretion in pancreatic beta cells. Studies of these aspects are performed: in vitro, culture cell lines of fibroblasts/hepatocytes derived from insulin receptor knockout (IRKO) mice transformed by adenovirus SV40; ex vivo, in genetically modified animal (mouse) models for insulin receptor knockout (IRKO). It has been analyzed the differences in proteins expression profile and insulin signaling transmission in IRKO hepatocytes and IRKO mouse liver tissues compared with the wild type and heterozygous phenotypes. The aims of this study are to detect differences in proteins expression that could be modulated by insulin and could be involved in cell survival, cell cycle and oxidative stress. In these conditions it is possible to study the specific role of IRKO in hepatic cell lines and analyze the different modulation in activity and expression of insulin related substrates (basal conditions). On the other hand, in mouse liver tissue (ex-vivo) it is possible to detect differences in insulin substrates expression and activation in diabetic ketoacidotic conditions (pathophysiological conditions). To isolate the differentially expressed proteins we used 2D-PAGE analysis of protein extracts of knockout, wild type and heterozygous mouse liver tissues and murine hepatocytes phenotypes (wild type and knockout). Five proteins at present, of the 22 proteins differentially expressed, have been identified by mass spectrometry. It has also been discovered a defect in post transcription proteins production that it is associated with a related increases in mRNA levels and again it has been described a defect in myotubules proteins expression but differently not in albumin production

Glucagon Like Peptide 1 and MicroRNA in Metabolic Diseases: Focusing on GLP1 Action on miRNAs

Glucagon like peptide 1 (GLP1) is an incretin hormone released from the enteroendocrine L-type cells of the lower gastrointestinal tract. The active isoforms of GLP1 are rapidly degraded (<2 min) by protease dipeptidyl peptidase-4 (DPP-4) after their release. Among its functions, GLP1 exerts a pivotal role in regulating glucose and lipid metabolism. In particular, GLP1 increases glucose stimulated insulin secretion, functional pancreatic β-cell mass and decreases glucagon secretion from pancreatic α-cells. GLP1 can also be a regulator of lipid and lipoprotein metabolism ameliorating diabetic dyslipidemia, liver steatosis, and promoting satiety. Interestingly, it has been found that GLP1 and GLP1 agonists can modulate the expression of different microRNAs (miRNAs), a ~22 nucleotides small non-coding RNAs, key modulators of protein expression. In particular, in pancreas, GLP1 increases the expression levels of miRNA-212 and miRNA-132, stimulating insulin secretion. Similarly, GLP1 decreases miRNA-338 levels, leading to an increase of pancreatic β-cell function, followed by an improvement of diabetic conditions. Moreover, GLP1 modulation of miRNAs expression in the liver regulates hepatic lipid storage. Indeed, GLP1 down-regulates miRNA-34a and miRNA-21 and up-regulates miRNA-200b and miRNA-200c expression in liver, reducing intra hepatic lipid accumulation and liver steatosis. Clinical and pre-clinical studies, discussed in this review, suggest that modulation of GLP1/miRNAs pathway may be a useful and innovative therapeutic strategy for prevention and treatment of metabolic disorders, such as diabetes mellitus and liver steatosis

GLP1-R: a Novel Pharmacological Target for Treating Human Bronchial Hyperresponsiveness

Asthma is associated with several comorbidities such as Type 2 Diabetes Mellitus (T2DM) which may lead to bronchial hyperersponsiveness (BHR). Since glucagon-like peptide 1 regulates glucose homeostasis, we pharmacologically investigated the influence of the glucagon-like peptide 1 receptor (GLP1-R) agonist exendin-4 on the bronchial BHR induced in human isolated airways. The effect of exendin-4 was assessed in human isolated airways undergoing overnight passive sensitization and high glucose stimulation, two conditions mimicking ex vivo the BHR typical of asthmatic and diabetic patients, respectively. GLP1-R activation modulated the bronchial contractile tone induced by transmural stimulation (Emax -56.7±3.6%, onset of action 28.2±4.4 min). Exendin-4 prevented the BHR induced by both high glucose stimulation and passive sensitization (-37.8±7.5% and -74.9±3.9%, P0.05 vs. passively sensitized tissues). The GLP1-R stimulation by overnight incubation with exendin-4 induced the over-expression of adenylyl cyclase isoform V (+48.4±1.3%, P<0.05 vs. passively sensitized tissues) and restored the cAMP levels depleted by this procedure (+330.8±63.3%, P<0.05 vs. passively sensitized tissues). In conclusion, GLP1-R may represent a novel target for treating BHR by activating the cAMP-dependent PKA pathway in human airways, and GLP1-R agonists could be used as a 'new' class to treat asthmatic patients and T2DM patients with BHR

Prdx6 Prevents Diabetic Myopathy by Improving Skeletal Muscle Cell Differentiation

Diabetes mellitus is characterized by a state of hyperglycemia resulting from altered insulin secretion by pancreatic beta cell, insulin action or both. This pathological condition is frequently associated with muscle mass loss, a condition defined as sarcopenia, and diabetic myopathy, represented by an impairment of the regenerative power of muscle fiber and by an altered differentiation of progenitor cells. Oxidative stress has been identified as the main cause of muscular alterations typical of diabetic patients. We showed that Peroxiredoxin 6 (Prdx6), a relatively new antioxidant enzyme belonging from the Peroxiredoxin family, has a central role in glucose homeostasis by exerting a potent antioxidant role. Based on these results, in the present study we aimed to verify whether Prdx6 modulates the association between diabetes and the progression of myopathy and sarcopenia. Firstly, we evaluated the gene expression of the main factors involved in the differentiation of myogenic muscle cells such as MyoD and Myogenin in murine knockout models for Prdx6 (Prdx6-/-). We observed significant decreased levels of both genes in Prdx6-/- mice compared to controls, suggesting an impairment of the regenerative potential of muscle fibers. These data were, also, confirmed by using in vitro cell model of murine myoblasts (C2C7) knockdown for Prdx6. Moreover, in the murine models, the process of muscle atrophy was studied by evaluating the gene expression of MuRF1 and Atrogin-1 that finely regulate protein degradation at skeletal muscle level. According to our hypothesis, the expression levels of both enzymes were significantly increased confirming the presence of muscle atrophy. Our study, for the first time, highlights a fundamental role of Prdx6 in the preservation of muscle mass, suggesting how Prdx6 can be considered a potential therapeutic target for diabetic myopathy and sarcopenia. However, further studies are needed in order to understand the molecular mechanism underlying this phenomenon. Disclosure F. Pacifici: None. B. Capuani: None. F. Piermarini: None. D. Pastore: None. R. Arriga: None. A. Coppola: None. S. Rea: None. G. Donadel: None. A. Bellia: None. D. Della-Morte: None. D. Lauro: None