Activation of G protein-coupled receptors by ketone bodies: Clinical implication of the ketogenic diet in metabolic disorders

Ketogenesis takes place in hepatocyte mitochondria where acetyl-CoA derived from fatty acid catabolism is converted to ketone bodies (KB), namely β-hydroxybutyrate (β-OHB), acetoacetate and acetone. KB represent important alternative energy sources under metabolic stress conditions. Ketogenic diets (KDs) are low-carbohydrate, fat-rich eating strategies which have been widely proposed as valid nutritional interventions in several metabolic disorders due to its substantial efficacy in weight loss achievement. Carbohydrate restriction during KD forces the use of FFA, which are subsequently transformed into KB in hepatocytes to provide energy, leading to a significant increase in ketone levels known as "nutritional ketosis". The recent discovery of KB as ligands of G protein-coupled receptors (GPCR) - cellular transducers implicated in a wide range of body functions - has aroused a great interest in understanding whether some of the clinical effects associated to KD consumption might be mediated by the ketone/GPCR axis. Specifically, anti-inflammatory effects associated to KD regimen are presumably due to GPR109A-mediated inhibition of NLRP3 inflammasome by β-OHB, whilst lipid profile amelioration by KDs could be ascribed to the actions of acetoacetate via GPR43 and of β-OHB via GPR109A on lipolysis. Thus, this review will focus on the effects of KD-induced nutritional ketosis potentially mediated by specific GPCRs in metabolic and endocrinological disorders. To discriminate the effects of ketone bodies per se, independently of weight loss, only studies comparing ketogenic vs isocaloric non-ketogenic diets will be considered as well as short-term tolerability and safety of KDs

Endothelial progenitor cell number and telomere lenght in circulating leukocytes: new early markers of cardiovascular risk

Il numero di Cellule Progenitrici Endoteliali (EPCs) circolanti e la Lunghezza Telomerica Leucocitaria (LTL) sono considerati marcatori emergenti di rischio cardiovascolare (CV), in quanto ridotti in presenza di fattori di rischio e patologie CV. Non esistono, ad oggi, studi riguardanti il loro possibile ruolo quali marcatori di stratificazione precoce di rischio CV in una popolazione di soggetti giovani adulti sani. Obiettivi: Il progetto di ricerca si divide in due parti. STUDIO 1 Valutare il numero di EPCs in una popolazione di giovani adulti sani, in relazione alla presenza di fattori di rischio CV noti con particolare riferimento all’insulinoresistenza (IR). STUDIO 2 Valutare la LTL in giovani adulti sani, in relazione alla presenza di fattori di rischio CV, di aterosclerosi subclinica e di indice cumulativo di rischio CV. Materiali e metodi: E’ stato valutato il numero di EPCs circolanti in 122 soggetti adulti sani (73M/49F; età 37±8 anni), normotesi ed in assenza di alcuna terapia farmacologica. In tutti i soggetti sono state raccolte le principali variabili cliniche ed antropometriche. E’ stato effettuato un prelievo a digiuno per la determinazione di glicemia, insulinemia (IRI), profilo lipidico, uricemia, inibitore dell’attivatore del plasminogeno (PAI-1), proteina C reattiva ad alta sensibilità (hsPCR) ed emocromo. L’IR è stata definita in base ai valori di IRI a digiuno. E’ stato effettuato un esame ultrasonografico per la determinazione dello spessore medio intimale (IMT) che rileva l’ispessimento della parete dell’arteria carotidea, e quindi indice di aterosclerosi subclinica, e calcolato il Framingham Risk score (FRs), un indice cumulativo di rischio CV. Il numero di EPCs è stato valutato mediante tecnica citofluorimetrica misurando l’espressione di specifici antigeni di superficie (CD133, CD34, e KDR) nella popolazione linfomonocitaria. Si è proceduto dapprima alla ottimizzazione delle fasi del protocollo d'analisi (lisi dei globuli rossi, settaggio delle condizioni di analisi FACS). Per ogni soggetto sono stati allestiti due campioni: il primo marcato con gli Abs specifici per la valutazione del numero di EPCs il secondo marcato con immunoglobuline IgG1 aspecifiche (controllo negativo). All'interno della stessa popolazione, in un sottogruppo di 82 soggetti, è stata misurata anche la lunghezza dei telomeri in cellule mononucleate isolate da sangue periferico. E' stato dapprima estratto il DNA con metodica standard e quindi valutata la lunghezza dei telomeri tramite PCR quantitativa. Per normalizzare i campioni in contenuto di DNA è stato valutato in parallelo un gene presente in singola copia per ottenere una T/S ratio tra Ct telomerico e Ct del singolo gene ulteriormente normalizzato per la T/S ratio di un DNA controllo (standard interno). Risultati: STUDIO 1 Il numero di EPCs è risultato inversamente correlato con l’indice di massa corporea (rho=-0.18;p<0.05), circonferenza vita (-0.2;<0.05), pressione sistolica (-0.23;<0.01) e diastolica (-0.21;<0.05), uricemia (-0.24;<0.005), PAI-1 (-0.19;<0.05) ed IRI (-0.2;<0.05) e direttamente correlato con i valori di colesterolo HDL (0.18;<0.05). Il genere maschile è risultato indipendentemente associato ad un ridotto numero di EPCs rispetto alle altre covariate(p<0.05). Dividendo la popolazione in sottogruppi in base al genere e alla presenza di IR, il numero di EPCs risultava minore nei maschi insulinoresistenti rispetto ai maschi insulinosensibili (p<0.05), questa differenza non si evidenziava nel genere femminile. STUDIO 2 La LTL è risultata inversamente correlata con i valori di età (rho=-0.24; p<0.05), circonferenza vita (-0.29;<0.01), trigliceridi (-0.25;<0.05), PAI-1 (-0.28;=0.01), hsPCR (-0.23;<0.05), IMT (-0.24;<0.05) e FRs (-0.28;<0.01) e direttamente correlata con colesterolo HDL (0.35;=0.001) ed EPCs (0.26;<0.05). I valori di IMT, di colesterolo HDL ed il numero di EPCs, sono risultati indipendentemente correlati alla LTL (p<0.05). Conclusioni: STUDIO 1 Il ridotto numero di EPCs nei maschi potrebbe contribuire a spiegare il maggiore rischio CV nel genere maschile rispetto a quello femminile in premenopausa di pari età. Inoltre, il ridotto numero di EPCs nei maschi insulinoresistenti potrebbe rappresentare uno dei possibili meccanismi tramite cui la IR si accompagna ad un aumentato rischio CV nel genere maschile. STUDIO 2 I risultati suggeriscono che la LTL possa essere considerata un marcatore precoce di rischio CV e di aterosclerosi subclinica, anche in una popolazione di soggetti giovani adulti sani a basso rischio CV. La correlazione indipendente tra LTL e numero di EPCs e l’origine midollare comune di queste due popolazioni cellulari, suggerisce l’esistenza di una meccanismo fisiopatologico condiviso alla base di queste alterazioni implicate nello sviluppo e progressione della patologia CV. Numero di EPCs e LTL si confermano possibili marcatori di stratificazione precoce di rischio CV anche in soggetti giovani adulti sani, seppure ulteriori studi siano necessari per confermarne un ruolo prognostico.Circulating Endothelial Progenitor cell (EPC) number and Leukocyte Telomere Length (LTL) are considered new markers of cardiovascular (CV) risk, since they are both reduced in the presence of CV risk factors and diseases. No studies are available to date investigating EPC number and LTL as early markers in the stratification of CV risk in a population of healthy young adults. Aim of the study: the project consists of two sub-studies STUDY 1 To evaluate EPC number in a population of healthy young subjects, in relation to the presence of the traditional CV risk factors particularly insulin resistance (IR) STUDY 2 To evaluate LTL in healthy young adults, in relation to the presence of CV risk factors, subclinical atherosclerosis and the cumulative index of CV risk. Methods: EPC number was evaluated in 122 young healthy subjects (73M/49F; 37±8yrs), normotensive and not taking any medication. The main clinical and anthropometric variables were evaluated in all subjects. Fasting blood samples were drawn in each subject for the determination of plasma glucose, insulin (IRI), lipid profile, uric acid, high sensitive C-reactive protein (hsCRP), PAI-1 and EPC number. IR was defined on the basis of fasting IRI values. A B-mode ultrasound examination was performed to determine the Intima Media Thickness (IMT), as a validate marker of subclinical atherosclerosis, and the Framingham Risk score (FRs) was calculated, as a cumulative index of CV risk. EPCs were identified as cells positive for CD34, CD133 and Kinase insert Domain Receptor (KDR) cell-surface antigens within the lymphomonocytes, by flow cytometry analysis. We firstly optimised the experimental conditions: red cells lysis and FACS parameters. Non-specific binding of IgG1 was used as negative control (isotype). LTL was measured in a subgroup of 82 subjects starting from peripheral blood mononuclear cells (PBMCs). DNA isolated from PBMCs was amplified by a specific quantitative real-time PCR reaction to evaluate telomere length. In parallel a single copy gene amplification was performed to obtain normalized data. LTL is expressed as T/S ratio values. Risults: STUDY 1 EPC number resulted inversely correlated with body mass index (rho=-0.18;p<0.05), waist (-0.2;<0.05), systolic (-0.23;<0.01) and diastolic blood pressure (-0.21;<0.05), uric acid (-0.24;<0.005), PAI-1 (-0.19;<0.05) and IRI (-0.2;<0.05) and directly correlated with HDL-cholesterol (0.18;<0.05). EPC number was lower in males and the male gender was the only independent predictor of low EPC count (p<0.05). By dividing the population in four subgroups based on gender and insulin resistance, EPC levels were lower in insulin resistant compared to insulin sensitive males (p<0.05) with no differences in females. STUDY 2 LTL resulted inversely correlated with age (rho=-0.24; p<0.05), waist (-0.29;<0.01), triglycerides (-0.25;<0.05), PAI-1 (-0.28;=0.01), hsPCR (-0.23;<0.05), IMT (-0.24;<0.05) and FRs (-0.28;<0.01) and directly correlated with HDL-cholesterol (0.35;=0.001) and EPC count (0.26;<0.05). IMT, HDL-cholesterol, and EPC number resulted independently correlated to LTL (p<0.05). Conclusions: STUDY 1 The male gender is an independent predictor of low EPC levels in healthy subjects. This might contribute to explaining the higher CV risk in males compared to pre-menopausal age-matched females. In this study a reduced EPC number in insulin resistant males represents one of the putative mechanisms by wich IR is paralleled by a worsening of the CV risk. STUDY 2 These data suggest that LTL can be considered a new early marker of CV risk and subclinical atherosclerosis also in healthy young adults with low CV risk. The independent correlation between LTL and EPC number and the common bone marrow origin, suggest a shared pathophysiological mechanism of impairment leading to the progression of CV disease. EPC number and LTL can be considered new markers for the early stratification of CV risk also in a population of healthy young adults. Longitudinal prospective studies are warranted to validate their prognostic value

Empagliflozin does not reverse lipotoxicity-induced impairment in human myeloid angiogenic cell bioenergetics.

info:eu-repo/semantics/publishe

Pioglitazone Improves In Vitro Viability and Function of Endothelial Progenitor Cells from Individuals with Impaired Glucose Tolerance

BACKGROUND: Evidence suggests that the PPARγ-agonist insulin sensitizer pioglitazone, may provide potential beneficial cardiovascular (CV) effects beyond its anti-hyperglycaemic function. A reduced endothelial progenitor cell (EPC) number is associated with impaired glucose tolerance (IGT) or diabetes, conditions characterised by increased CV risk. AIM: To evaluate whether pioglitazone can provide benefit in vitro in EPCs obtained from IGT subjects. MATERIALS AND METHODS: Early and late-outgrowth EPCs were obtained from peripheral blood mononuclear cells of 14 IGT subjects. The in vitro effect of pioglitazone (10 µM) with/without PPARγ-antagonist GW9662 (1 µM) was assessed on EPC viability, apoptosis, ability to form tubular-like structures and pro-inflammatory molecule expression. RESULTS: Pioglitazone increased early and late-outgrowth EPC viability, with negligible effects on apoptosis. The capacity of EPCs to form tubular-like structures was improved by pioglitazone in early (mean increase 28%; p=0.005) and late-outgrowth (mean increase 30%; p=0.037) EPCs. Pioglitazone reduced ICAM-1 and VCAM-1 adhesion molecule expression in both early (p=0.001 and p=0.012 respectively) and late-outgrowth (p=0.047 and p=0.048, respectively) EPCs. Similarly, pioglitazone reduced TNFα gene and protein expression in both early (p=0.034;p=0.022) and late-outgrowth (p=0.026;p=0.017) EPCs compared to control. These effects were prevented by incubation with the PPARγ-antagonist GW9662. CONCLUSION: Pioglitazone exerts beneficial effects in vitro on EPCs isolated from IGT subjects, supporting the potential implication of pioglitazone as a CV protective agents

Vildagliptin, but not glibenclamide, increases circulating endothelial progenitor cell number: A 12-month randomized controlled trial in patients with type 2 diabetes

Background: Fewer circulating endothelial progenitor cells (EPCs) and increased plasma (C-term) stromal cell-derived factor 1α (SDF-1α), a substrate of DPP-4, are biomarkers, and perhaps mediators, of cardiovascular risk and mortality. Short-term/acute treatment with DPP-4 inhibitors improve EPC bioavailability; however, long-term effects of DPP-4i on EPCs bioavailability/plasma (C-term) SDF-1α are unknown. Methods: Randomized (2:1) open-label trial to compare the effects of vildagliptin (V) (100 mg/day) vs glibenclamide (G) (2.5 mg bid to a maximal dose of 5 mg bid) on circulating EPC levels at 4 and 12 months of treatment in 64 patients with type 2 diabetes in metformin failure. At baseline, and after 4 and 12 months, main clinical/biohumoral parameters, inflammatory biomarkers, concomitant therapies, EPC number (CD34+/CD133+/KDR+/106 cytometric events) and plasma (C-term) SDF-1α (R&D system) were assessed. Results: Baseline characteristics were comparable in the two groups. V and G similarly and significantly (p < 0.0001) improved glucose control. At 12 months, V significantly increased EPC number (p < 0.05) and significantly reduced (C-term) SDF-1α plasma levels (p < 0.01) compared to G, with no differences in inflammatory biomarkers. Conclusions: V exerts a long-term favorable effect on EPC and (C-term) SDF-1α levels at glucose equipoise, thereby implying a putative beneficial effect on vascular integrity. Trial registration Clinical Trials number: NCT01822548; name: Effect of Vildagliptin vs. Glibenclamide on Circulating Endothelial Progenitor Cell Number Type 2 Diabetes. Registered 28 March, 2013

Effects on nitric oxide production of urolithins, gut-derived ellagitannin metabolites, in human aortic endothelial cells

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-MSn . 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

N-3 PUFA increase bioavailability and function of endothelial progenitor cells

BACKGROUND AND AIMS: Recent data suggest that n-3 PUFA exert beneficial effects on endothelial progenitor cell (EPC) biology. We sought to investigate whether these effects might be mediated by enhanced EPC in vitro function and/or in vivo bioavailability. METHODS AND RESULTS: CACs and late-outgrowth EPCs were isolated from peripheral blood mononuclear cells obtained from 12 donor buffy-coats. The effect of n-3 PUFA (EPA : DHA = 0.9 : 1.5; 9 μM EPA plus 15 μM DHA) was tested on CAC/EPC viability, function (tube-formation) and pro-inflammatory molecule expression. Circulating EPC (cells positive for CD34, CD133 and kinase insert domain receptor - KDR cell-surface antigens by flow cytometry) number was evaluated in 20 healthy subjects (10 F/10 M, 32 ± 5 years), randomized to receive 4 mackerel or sardine portions per week for 6 weeks followed by a 6 week free-diet period. N-3 PUFA improved CAC and late-outgrowth EPC viability (p < 0.05) and the capacity to form tube-like structures in CACs (+38%; p < 0.05) and late-outgrowth EPCs (+15%; p < 0.05). ICAM-1 expression was reduced in both CACs (p < 0.05) and late-outgrowth EPCs (p < 0.05) and VCAM-1 in late-outgrowth EPCs (p < 0.005). N-3 PUFA significantly decreased TNF-α and MCP-1 expression in CACs and IL-8, TNF-α and MCP-1 in late-outgrowth EPCs (p < 0.05). Circulating EPC number significantly improved after 6 weeks of a fish-enriched diet (p < 0.01) and returned to baseline levels after a 6 week free-diet period (p < 0.01). Plasma EPA levels were independently and positively associated with EPC levels (p < 0.005). CONCLUSION: Our findings support the case of a beneficiary role played by n-3 PUFA in EPC function and bioavailability

Bioavailability of Bergamot (Citrus bergamia) Flavanones and Biological Activity of Their Circulating Metabolites in Human Pro-Angiogenic Cells

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

Bioavailability of Bergamot (Citrus bergamia) Flavanones and Biological Activity of Their Circulating Metabolites in Human Pro-Angiogenic Cells

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

Stearic acid at physiologic concentrations induces in vitro lipotoxicity in circulating angiogenic cells

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.SCOPUS: ar.jinfo:eu-repo/semantics/publishe