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Background Empagliflozin can curb inflammation and oxidative stress, through sodium-proton exchanger (NHE) inhibition, in a model of lipotoxicity in human myeloid angiogenic cells (MAC), which mediate endothelial repairing processes. Aim of this study is to assess in human MAC whether: (1) Stearic acid (SA) induced inflammation and increase in oxidant stress is accompanied by bioenergetic alterations; (2) empagliflozin anti-lipotoxic action is concomitant with coherent changes in bioenergetic metabolism, possibly via NHE blockade. Methods MAC were isolated from peripheral blood of healthy volunteers and incubated in the presence/absence of SA (100 μM for 3 h) with/without empagliflozin (EMPA 100 μM) or amiloride (Ami 100 μM) for 1 h. Cell respiration (oxygen consumption rate OCR) and anaerobic glycolysis (measured as proton production rate) were recorded in real-time by Seahorse technology, and ATP production (anaerobic glycolysis- and oxphos-derived) rates were calculated. Results SA, at the concentration causing inflammation and increased oxidant stress, altered cell bioenergetics of human MAC, with overall reductions in basal OCR and oxphos-derived ATP production (all p Conclusions In human MAC: (1) SA altered cell bioenergetics, concomitantly with inflammation and oxidant stress; (2) EMPA possibly inhibited mitochondrial respiration, (3) the protective effect of EMPA against SA-induced lipotoxicity was unlikely to be mediated through bioenergetic metabolism.

SGLT-2 inhibitors (SGLT2i) reduce cardiovascular (CV) risk in people with type 2 diabetes and atherosclerotic CV diseases. The inflammatory function of macrophages (Mφ) - which play a pivotal role in atherosclerosis - is strictly dependent on their metabolism. Aim: To determine whether empagliflozin (EMPA) reduces inflammation and modifies bioenergetic metabolism in proinflammatory human Mφ (M1). Methods: M1 were differentiated from healthy donors’ monocytes and incubated with/without EMPA 100 µM for 16h. Biomarkers of inflammation and oxidative stress were quantified by qPCR. Steady-state energetic metabolism (fluxes: pmol/min for 5x104 cells) was measured by indirect microcalorimetry [oxidations of lipids (LipOX,) glucose and amino acids; anaerobic glycolysis (AnaerGlyc); tricarboxylic acid cycle (TCA); energy expenditure (EPR); maximum estimate of ATP production (ATPflux)] which is based on the integration of measured O2 and H+ (Agilent Seahorse), lactate and NH4 (enzymatic method) fluxes with the stoichiometric equations of metabolic pathways. Results: In M1, EMPA reduces (≈50%) inflammation (IL1β, IL8, TNFα and MCP1; p Conclusions: EMPA alters M1 inflammation along with cytosolic (AnaerGlyc) and - to a greater extent - mitochondrial (TCA) bioenergetics. EMPA-induced metabolic dysfunction of M1 in vivo might result into reduced metaflammation and contribute to SGLT2i-dependent CV protection. Disclosure V. Spigoni: None. G. Cinquegrani: None. F. Fantuzzi: None. S. Lorusso: None. A. Dei cas: Other Relationship; Self; Novo Nordisk. R. C. Bonadonna: Advisory Panel; Self; Sanofi, Speaker’s Bureau; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company. Funding Italian Diabete Ricerca Foundation; Eli Lilly Italy

Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression.We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-βH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| We identified a total of 264 genes stably expressed in EndoC-βH1 cells and human islets following cytokine- or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells.

SUMMARYCellular identity during development is under the control of transcription factors that form gene regulatory networks. However, the transcription factors and gene regulatory networks underlying cellular identity in the human adult pancreas remain largely unexplored. Here, we integrate multiple single-cell RNA-sequencing datasets of the human adult pancreas, totaling 7393 cells, and comprehensively reconstruct gene regulatory networks. We show that a network of 142 transcription factors forms distinct regulatory modules that characterize pancreatic cell types. We present evidence that our approach identifies regulators of cell identity in the human adult pancreas. We predict that HEYL, BHLHE41 and JUND are active in acinar, beta and alpha cells, respectively, and show that these proteins are present in the human adult pancreas as well as in human induced pluripotent stem cell (hiPSC)-derived islet cells. Using single-cell transcriptomics, we found that JUND represses beta cell genes in hiPSC-alpha cells. Both BHLHE41 and JUND depletion seemed to increase the number of sc-enterochromaffin cells in hiPSC-derived islets. The comprehensive gene regulatory network atlas can be explored interactively online. We anticipate our analysis to be the starting point for a more sophisticated dissection of how transcription factors regulate cell identity in the human adult pancreas. Furthermore, given that transcription factors are major regulators of embryo development and are often perturbed in diseases, a comprehensive understanding of how transcription factors work will be relevant in development and disease.HIGHLIGHTSReconstruction of gene regulatory networks for human adult pancreatic cell typesAn interactive resource to explore and visualize gene expression and regulatory statesPrediction of putative transcription factors that drive pancreatic cell identityBHLHE41 depletion in primary islets induces apoptosis

DNAJC3, also known as P58IPK, is an Hsp40 family member that interacts with and inhibits PKR-like ER-localized eIF2α kinase (PERK). Dnajc3 deficiency in mice causes pancreatic β-cell loss and diabetes. Loss-of-function mutations in DNAJC3 cause early-onset diabetes and multisystemic neurodegeneration. The aim of our study was to investigate the genetic cause of early-onset syndromic diabetes in two unrelated patients, and elucidate the mechanisms of β-cell failure in this syndrome., info:eu-repo/semantics/published

β dedifferentiation is recognized as one of the main mechanisms responsible for the disappearance of β cells from pancreatic islets of diabetic subjects, but the underlying process is still unclear. Noradrenergic innervation is known to inhibit insulin secretion, and in pancreas of diabetic subjects a 3-fold increase of noradrenergic fibers was observed compared to nondiabetic subjects, directly related to the % of dedifferentiated cells within the islets. The increase in fibers has been associated with worsening β cell function, suggesting a role in the pathogenesis of the disease and perhaps in the process of dedifferentiation. The aim of our study was to use in vitro human β cell models to explore a direct role of innervation in the process of noradrenergic dedifferentiation. The human β cell line EndoC-βH1 was exposed to different concentrations of noradrenaline (NA) (0.4 μM, 4 μM and 40 μM) for 24 and 72 h. The expression of β cell dedifferentiation markers was assessed before and after stimulation of the α2A adrenergic receptor, by morphology (immunofluorescence (IF)), qPCR and ELISA. FGF2 (100 ng/mL), a known inducer of β dedifferentiation in vitro, was used as a positive control. NA treatment did not have any impact on β cell viability, while, as expected, it slightly reduced insulin secretion after 24 h. The expression of β cell dedifferentiation markers (ALDH1a3) was increased by approximately 30% after stimulation with NA, while mature β cell marker were reduced (NKX6.1) up to 60% after 72 h (by IF detection). Moreover, the mRNA expression of FOXO1, the main transcription factor involved in the dedifferentiation process, was reduced up to 35.4% after 72 h stimulation with NA. We observed a dose and time dependent pattern in all the experiments. In conclusion, the data suggest a direct role of noradrenergic fibers in the dedifferentiation process, potentially identifying a new target for the prevention and therapy of type 2 diabetes. Disclosure F. Cinti: None. F. Fantuzzi: None. A. Carfí: None. T. Mezza: None. C. Cefalo: None. S. Moffa: None. G. Sorice: None. A. Giaccari: Speaker’s Bureau; Self; Amgen, AstraZeneca, Eli Lilly and Company, Merck Sharp & Dohme Corp., Mundipharma, Novo Nordisk A/S, Sanofi-Aventis. M. Cnop: None. Funding European Foundation for the Study of Diabetes

The clear evidence of cardiovascular benefits in cardiovascular outcome trials of sodium-glucose cotransporter 2 inhibitors (SGLT2i) in type 2 diabetes might suggest an effect on atherosclerotic plaque vulnerability and/or thrombosis, in which myeloid angiogenic cells (MAC) and platelets (PLT) are implicated. We tested the effects of SGLT2i on inflammation and oxidant stress in a model of stearic acid (SA)-induced lipotoxicity in MAC and on PLT activation. The possible involvement of the Na+/H+ exchanger (NHE) was also explored., info:eu-repo/semantics/published

Coffee is an important source of bioactive compounds, including caffeine, trigonelline, and phenolic compounds. Several studies have highlighted the preventive effects of coffee consumption on major cardiometabolic (CM) diseases, but the impact of different coffee dosages on markers of CM risk in a real-life setting has not been fully understood. This study aimed to investigate the effect of coffee and cocoa-based confectionery containing coffee consumption on several CM risk factors in healthy subjects., info:eu-repo/semantics/published

Coffee is an important source of bioactive compounds, including caffeine, trigonelline, and phenolic compounds. Several studies have highlighted the preventive effects of coffee consumption on major cardiometabolic diseases, but the impact of coffee dosage on markers of cardiometabolic risk is not well understood. Moreover, the pool of coffee-derived circulating metabolites in real-life settings is unknown. This study evaluated the bioavailability and effects on recognised cardiometabolic markers of coffee bioactives, considering different levels of consumption. An innovative experimental design, including both a chronic and an acute sub-study, and a comprehensive analytical approach were used.A 3-arm, randomised, crossover trial was conducted in 21 healthy volunteers (age, 23 ± 2 y; BMI, 22.3 ± 2.5 kg/m2) (Mena et al., Trials 2017, 18, 527). Volunteers were assigned to consume 3 treatments for 4 weeks, including 1 cup of espresso coffee/day, 3 cups of espresso coffee/day, and 1 cup of espresso coffee plus 2 cocoa-based confectionary products containing-coffee twice per day. The last day of each treatment, blood and urine samples were collected at specific time points for 24 hours. Dietary intake, body weight, BMI, waist circumference, blood pressure, fasting glucose, insulin, LDL- and HDL-cholesterol, triglycerides, nitric oxide, inflammatory markers (IL-8, TNFα, VEGF), trimethylamine-N-oxide (TMAO), DNA damage, DNA catabolites, and eicosanoids were assessed. The pool of coffee-derived circulating metabolites was also assessed in acute conditions. Untargeted metabolomics was performed.Energy intake did not change among treatments after 4 weeks, while significant differences were observed in the intake of saturated fatty acids and carbohydrates. The effect of different coffee dosages on the set of cardiometabolic markers assessed was negligible. Plasma and urinary pharmacokinetic profiles were evaluated for 6 caffeine metabolites, 3 trigonelline derivatives, and up to 40 phenolic metabolites. Pharmacokinetics highlighted the different “waves” of circulating metabolites occurring upon repeated coffee consumption. Differences in several pharmacokinetic parameters were observed among treatments, which may support the long-term cardiometabolic benefits of certain patterns of coffee consumption. Multivariate analyses clearly differentiated treatments on the basis of the urinary metabolome.This work provided a comprehensive picture of the impact of different coffee dosages on the pool of coffee-derived circulating metabolites, the urinary metabolome, and a wide number of cardiometabolic markers. Multivariate analyses focused on inter-individual differences are ongoing to better understand the effect of coffee on cardiometabolic health.

Friedreich ataxia is an autosomal recessive neurodegenerative disease associated with a high diabetes prevalence. No treatment is available to prevent or delay disease progression. Friedreich ataxia is caused by intronic GAA trinucleotide repeat expansions in the frataxin-encoding FXN gene that reduce frataxin expression, impair iron-sulfur cluster biogenesis, cause oxidative stress, and result in mitochondrial dysfunction and apoptosis. Here we examined the metabolic, neuroprotective and frataxin-inducing effects of glucagon-like-peptide 1 (GLP-1) analogs in in vivo and in vitro models and in Friedreich ataxia patients. The GLP-1 analog exenatide improved glucose homeostasis of frataxin-deficient mice through enhanced insulin content and secretion in pancreatic β-cells. Exenatide induced frataxin and iron-sulfur cluster-containing proteins in β-cells and brain, and was protective to sensory neurons in dorsal root ganglia. GLP-1 analogs also induced frataxin expression, reduced oxidative stress and improved mitochondrial function in Friedreich ataxia patients' induced pluripotent stem cell-derived β-cells and sensory neurons. The frataxin-inducing effect of exenatide was confirmed in a pilot trial in Friedreich ataxia patients, showing modest frataxin induction in platelets over a 5-week treatment course. Taken together, GLP-1 analogs improve mitochondrial function in frataxin-deficient cells and induce frataxin expression. Our findings identify incretin receptors as a therapeutic target in Friedreich ataxia., info:eu-repo/semantics/published

Increased non high-density lipoprotein (HDL)/low-density lipoprotein (LDL) cholesterol levels are independent risk factors for cardiovascular (CV) mortality with no documented threshold. A new combination of nutraceuticals (berberine 200 mg, monacolin K 3 mg, chitosan 10 mg and coenzyme Q 10 mg) with additive lipid-lowering properties has become available. The aim of the study is to test the efficacy of the nutraceutical formulation (one daily) in lowering non-HDL cholesterol vs. placebo at 12 weeks in individuals with non-HDL-cholesterol levels ≥160 mg/dL. 39 subjects (age 52 ± 11 years; 54% females; body mass index 27 ± 4 kg/m²) were randomized (3:1) in a double blind phase II placebo-controlled study. At baseline, 4 and 12 weeks main clinical/biohumoral parameters, pro-inflammatory cytokines, (gut)-hormones, proprotein convertase subtilisin/kexin type 9 (PCSK9) levels and endothelial progenitor cell (EPC) number were assessed. Baseline characteristics were comparable in the two groups. The intervention significantly decreased non-HDL cholesterol (-30 ± 20 mg/dL; p = 0.012), LDL cholesterol (-31 ± 18 mg/dL, p = 0.011) and apolipoprotein (Apo) B (-14 ± 12 mg/dL, p = 0.030) levels compared to the placebo. Pro-inflammatory, hormonal, PCSK9 and EPC levels remained stable throughout the study in both groups. The intervention was well tolerated. Three adverse events occurred: Epstein Barr virus infection, duodenitis and asymptomatic but significant increase in creatine phosphokinase (following intense physical exercise) which required hospitalization. The tested nutraceutical formulation may represent a possible therapeutic strategy in dyslipidemic individuals in primary prevention., info:eu-repo/semantics/published

Background and aims Saturated free fatty acids (SFAs) can induce lipotoxicity in different cells. No studies have investigated the effects of SFA in circulating angiogenic cells (CACs), which play a key role in endothelial repair processes. The aim of the study was to assess the effects of SFAs, specifically stearic acid (SA), on viability and function of CACs and to investigate potential underlying molecular mechanisms. Methods CACs were isolated from healthy subjects by established methods. CACs were incubated with BSA-complexed stearate (100 μM) to assess the time course (from 8 to 24 h exposure) of the effects on viability and apoptosis (activation of caspases 3/7), angiogenic function (tube formation assay), pro-inflammatory cytokine (IL-1β, IL-6, IL-8, MCP-1 and TNFα) gene expression (qPCR) and secretion (ELISA), activation of MAPK (JNK, p38 and Erk1/2) by Western blot and endoplasmic reticulum (ER) stress marker (CHOP, BIP, ATF4, XBP-1 and sXBP-1) gene expression by qPCR. Results Stearic acid activates effector caspases in CACs in a dose- and time-dependent manner. SA also impairs CAC function and increases pro-inflammatory molecule (IL-1β, IL-6, IL-8, MCP-1 and TNFα) gene expression and secretion in CACs starting from 3 h of incubation. The activation of JNK by SA mediates pro-inflammatory response, but it may be not necessary for apoptosis. Moreover, SA induces the expression of ER stress markers across the three branches of the ER stress response. Conclusions In humans, both function and viability of CACs are exquisitely vulnerable to physiologic concentrations of stearate; lipotoxic impairment of endothelial repair processes may be implicated in vascular damage caused by SFAs.

The consumption of foodstuffs yielding circulating compounds able to maintain endothelial function by improving nitric oxide (NO) bioavailability can be considered as an effective strategy for cardiovascular disease prevention. This work assessed the in vitro effects of urolithin A, urolithin B, and urolithin B-glucuronide, ellagitannin-derived metabolites of colonic origin, on NO release and endothelial NO synthase (eNOS) activation in primary human aortic endothelial cells (HAECs). Urolithins were tested both individually at 15 μM and as a mixture of 5 μM each, at different time points. The biotransformation of these molecules in cell media due to cell metabolism was also evaluated by UHPLC-MS(n). The mix of urolithins at 5 μM significantly increased nitrite/nitrate levels following 24 h of incubation, while single urolithins at 15 μM did not modify NO bioavailability. Both the mix of urolithins at 5 μM and urolithin B-glucuronide at 15 μM activated eNOS expression. All urolithins underwent metabolic reactions, but these were limited to conjugation with sulfate moieties. This study represents a step forward in the understanding of cardiovascular health benefits of ellagitannin-rich foodstuffs and backs the idea that peripheral cells may contribute to urolithin metabolism., info:eu-repo/semantics/published

Myeloid angiogenic cells (MACs) play a key role in endothelial repairing processes and functionality but their activity may be impaired by the lipotoxic effects of some molecules like stearic acid (SA). Among the dietary components potentially able to modulate endothelial function in vivo, (poly)phenolic compounds represent serious candidates. Here, we apply a comprehensive multidisciplinary approach to shed light on the prospects of Bergamot (Citrus bergamia), a citrus fruit rich in flavanones and other phenolic compounds, in the framework of lipotoxicity-induced MACs impairment. The flavanone profile of bergamot juice was characterized and 16 compounds were identified, with a new 3-hydroxy-3-methylglutaryl (HMG) flavanone, isosakuranetin-7-O-neohesperidoside-6″-O-HMG, described for the first time. Then, a pilot bioavailability study was conducted in healthy volunteers to assess the circulating flavanone metabolites in plasma and urine after consumption of bergamot juice. Up to 12 flavanone phase II conjugates (sulfates and glucuronides of hesperetin, naringenin and eriodyctiol) were detected and quantified. Finally, the effect of some of the metabolites identified in vivo, namely hesperetin-7-O-glucuronide, hesperetin-3'-O-glucuronide, naringenin-7-O-glucuronide and naringenin-4'-O-glucuronide, was tested, at physiological concentrations, on gene expression of inflammatory markers and apoptosis in MACs exposed to SA. Under these conditions, naringenin-4'-O-glucuronide and hesperetin-7-O-glucuronide were able to modulate inflammation, while no flavanone glucuronide was effective in curbing stearate-induced lipoapoptosis. These results demonstrate that some flavanone metabolites, derived from the in vivo transformation of bergamot juice phenolics in humans, may mitigate stearate-induced inflammation in MACs., info:eu-repo/semantics/published

Neonatal diabetes is caused by single gene mutations reducing pancreatic β cell number or impairing β cell function. Understanding the genetic basis of rare diabetes subtypes highlights fundamental biological processes in β cells. We identified 6 patients from 5 families with homozygous mutations in the YIPF5 gene, which is involved in trafficking between the endoplasmic reticulum (ER) and the Golgi. All patients had neonatal/early-onset diabetes, severe microcephaly, and epilepsy. YIPF5 is expressed during human brain development, in adult brain and pancreatic islets. We used 3 human β cell models (YIPF5 silencing in EndoC-βH1 cells, YIPF5 knockout and mutation knockin in embryonic stem cells, and patient-derived induced pluripotent stem cells) to investigate the mechanism through which YIPF5 loss of function affects β cells. Loss of YIPF5 function in stem cell-derived islet cells resulted in proinsulin retention in the ER, marked ER stress, and β cell failure. Partial YIPF5 silencing in EndoC-βH1 cells and a patient mutation in stem cells increased the β cell sensitivity to ER stress-induced apoptosis. We report recessive YIPF5 mutations as the genetic cause of a congenital syndrome of microcephaly, epilepsy, and neonatal/early-onset diabetes, highlighting a critical role of YIPF5 in β cells and neurons. We believe this is the first report of mutations disrupting the ER-to-Golgi trafficking, resulting in diabetes., info:eu-repo/semantics/published