Glucagon induces the hepatic expression of inflammatory markers in vitro and in vivo.

Glucagon exerts multiple hepatic actions, including stimulation of glycogenolysis/gluconeogenesis. The liver plays a crucial role in chronic inflammation by synthesizing proinflammatory molecules, which are thought to contribute to insulin resistance and hyperglycaemia. Whether glucagon affects hepatic expression of proinflammatory cytokines and acute-phase reactants is unknown. Herein, we report a positive relationship between fasting glucagon levels and circulating interleukin (IL)-1ß (r = 0.252, p = .042), IL-6 (r = 0.230, p = .026), fibrinogen (r = 0.193, p = .031), complement component 3 (r = 0.227, p = .024) and high sensitivity C-reactive protein (r = 0.230, p = .012) in individuals without diabetes. In CD1 mice, 4-week continuous treatment with glucagon induced a significant increase in circulating IL-1ß (p = .02), and IL-6 (p = .001), which was countered by the contingent administration of the glucagon receptor antagonist, GRA-II. Consistent with these results, we detected a significant increase in the hepatic activation of inflammatory pathways, such as expression of NLRP3 (p < .02), and the phosphorylation of nuclear factor kappaB (NF-κB; p < .02) and STAT3 (p < .01). In HepG2 cells, we found that glucagon dose-dependently stimulated the expression of IL-1ß (p < .002), IL-6 (p < .002), fibrinogen (p < .01), complement component 3 (p < .01) and C-reactive protein (p < .01), stimulated the activation of NLRP3 inflammasome (p < .01) and caspase-1 (p < .05), induced the phosphorylation of TRAF2 (p < .01), NF-κB (p < .01) and STAT3 (p < .01). Preincubating cells with GRA-II inhibited the ability of glucagon to induce an inflammatory response. Using HepaRG cells, we confirmed the dose-dependent ability of glucagon to stimulate the expression of NLRP3, the phosphorylation of NF-κB and STAT3, in the absence of GRA-II. These results suggest that glucagon has proinflammatory effects that may participate in the pathogenesis of hyperglycaemia and unfavourable cardiometabolic risk profile.

Metabolites Profiling of Melanoma Interstitial Fluids Reveals Uridine Diphosphate as Potent Immune Modulator Capable of Limiting Tumor Growth.

Tumor interstitial fluid (TIF) surrounds and perfuses tumors and collects ions, metabolites, proteins, and extracellular vesicles secreted by tumor and stromal cells. Specific metabolites, accumulated within the TIF, could induce metabolic alterations of immune cells and shape the tumor microenvironment. We deployed a metabolomic approach to analyze the composition of melanoma TIF and compared it to the plasma of C57BL6 mice, engrafted or not with B16-melanoma cells. Among the classes of metabolites analyzed, monophosphate and diphosphate nucleotides resulted enriched in TIF compared to plasma samples. The analysis of the effects exerted by guanosine diphosphate (GDP) and uridine diphosphate (UDP) on immune response revealed that GDP and UDP increased the percentage of CD4+CD25+FoxP3- and, on isolated CD4+ T-cells, induced the phosphorylation of ERK, STAT1, and STAT3; increased the activity of NF-κB subunits p65, p50, RelB, and p52; increased the expression of Th1/Th17 markers including IFNγ, IL17, T-bet, and RORγt; and reduced the expression of IL13, a Th2 marker. Finally, we observed that local administrations of UDP in B16-engrafted C57BL6 mice reduced tumor growth and necrotic areas. In addition, UDP-treated tumors showed a higher presence of MHCIIhi tumor-associated macrophage (TAM) and of CD3+CD8+ and CD3+CD4+ tumor-infiltrating T-lymphocytes (TILs), both markers of anti-tumor immune response. Consistent with this, intra-tumoral gene expression analysis revealed in UDP-treated tumors an increase in the expression of genes functionally linked to anti-tumor immune response. Our analysis revealed an important metabolite acting as mediator of immune response, which could potentially represent an additional tool to be used as an adjuvant in cancer immunotherapy.

The TRIB3 R84 variant is associated with increased left ventricular mass in a sample of 2426 White individuals.

BACKGROUND: Prior studies in animal models showed that increased cardiac expression of TRIB3 has a pathogenic role in inducing left ventricular mass (LVM). Whether alterations in TRIB3 expression or function have a pathogenic role in inducing LVM increase also in humans is still unsettled. In order to address this issue, we took advantage of a nonsynonymous TRIB3 Q84R polymorphism (rs2295490), a gain-of-function amino acid substitution impairing insulin signalling, and action in primary human endothelial cells which has been associated with insulin resistance, and early vascular atherosclerosis. METHODS: SNP rs2295490 was genotyped in 2426 White adults in whom LVM index (LVMI) was assessed by validated echocardiography-derived measures. RESULTS: After adjusting for age and sex, LVMI progressively and significantly increased from 108 to 113, to 125 g/m2 in Q84Q, Q84R, and R84R individuals, respectively (Q84R vs. Q84Q, P = 0.03; R84R vs. Q84Q, P < 0.0001). The association between LVMI and the Q84R and R84R genotype remained significant after adjusting for blood pressure, smoking habit, fasting glucose levels, glucose tolerance status, anti-hypertensive treatments, and lipid-lowering therapy (Q84R vs. Q84Q, P = 0.01; R84R vs. Q84Q, P < 0.0001). CONCLUSIONS: We found that the gain-of-function TRIB3 Q84R variant is significantly associated with left ventricular mass in a large sample of White nondiabetic individual of European ancestry.

Effects of Alpha-2-HS-glycoprotein on cognitive and emotional assessment in prediabetic and diabetic subjects.

BACKGROUND: High concentrations of Alpha-2-HS-glycoprotein, also called Fetuin-A (Fet-A), are associated with insulin resistance, obesity, non-alcoholic fatty liver disease, type 2 diabetes and polycystic ovary syndrome. Moreover, Fet-A is able to cross the bloodbrain barrier into ischemic brain tissue in adult humans. Although the brain is an important target of insulin action, there is little evidence associating serum levels of Fet-A with psychiatric conditions such as depression and cognitive decline, and no reports about the presence and degree of anxiety disorders. METHODS: We have examined cognitive and emotional alterations in a Caucasian population of 94 subjects. RESULTS: Our data confirmed that, irrespective of insulin sensitivity status, circulating Fet-A levels are positively associated with an increased risk of showing signs of depression according to the BDI-II test, and have reported new evidences of a positive association between Fet-A and state- and trait- anxiety, as measured by the STAI questionnaires. In contrast, no association was observed between Fet-A levels and cognitive performance on the MMSE. LIMITATIONS: Although the study includes a well-characterized population, the small sample size and cross sectional nature are important limitations, and this results should not be considered definitive. The data are based only on Caucasian subjects and their generalizability to other ethnic groups should be done with caution. CONCLUSION: Overall, these data suggest for the first time a role of Fet-A as an independent risk factor in the development of symptoms of anxiety and depression in prediabetic and diabetic subjects.

The Functional Polymorphism of DDAH2 rs9267551 Is an Independent Determinant of Arterial Stiffness.

Background: The association of circulating asymmetric dimethylarginine (ADMA) levels with cardiovascular risk and arterial stiffness has been reportedly demonstrated, although the causal involvement of ADMA in the pathogenesis of these conditions is still debated. Dimethylaminohydrolase 2 (DDAH2) is the enzyme responsible for ADMA hydrolysis in the vasculature, and carriers of the polymorphism rs9267551 C in the 5'-UTR of DDAH2 have been reported to have higher DDAH2 expression and reduced levels of serum ADMA. Approach and Results: We genotyped rs9267551 in 633 adults of European ancestry and measured their carotid-femoral pulse wave velocity (cfPWV), the gold-standard method to estimate arterial stiffness. cfPWV resulted significantly lower in rs9267551 C allele carriers (Δ = -1.12 m/s, P < 0.01) after correction for age, sex and BMI, and a univariate regression showed that the presence of rs9267551 C variant was negatively associated with cfPWV (ß = -0.110, P < 0.01). In a multivariable regression model, subjects carrying the rs9267551 C allele manifested significantly lower cfPWV than GG carriers (ß = -0.098, P = 0.01) independently from several potential confounders. We measured circulating ADMA levels in a subset of 344 subjects. A mediation analysis revealed that the effect of DDAH2 rs9267551 genotype on cfPWV was mediated by the variation in ADMA levels. Conclusions: These evidences hint that the presence of rs9267551 C allele may explain, at least in part, a reduction in vessel rigidity as measured by cfPWV, and support the attribution of a causative role to ADMA in the pathogenesis of arterial stiffness.

CD31+ Extracellular Vesicles From Patients With Type 2 Diabetes Shuttle a miRNA Signature Associated With Cardiovascular Complications.

Innovative biomarkers are needed to improve the management of patients with type 2 diabetes mellitus (T2DM). Blood circulating miRNAs have been proposed as a potential tool to detect T2DM complications, but the lack of tissue specificity, among other reasons, has hampered their translation to clinical settings. Extracellular vesicle (EV)-shuttled miRNAs have been proposed as an alternative approach. Here, we adapted an immunomagnetic bead-based method to isolate plasma CD31+ EVs to harvest vesicles deriving from tissues relevant for T2DM complications. Surface marker characterization showed that CD31+ EVs were also positive for a range of markers typical of both platelets and activated endothelial cells. After characterization, we quantified 11 candidate miRNAs associated with vascular performance and shuttled by CD31+ EVs in a large (n = 218) cross-sectional cohort of patients categorized as having T2DM without complications, having T2DM with complications, and control subjects. We found that 10 of the tested miRNAs are affected by T2DM, while the signature composed by miR-146a, -320a, -422a, and -451a efficiently identified T2DM patients with complications. Furthermore, another CD31+ EV-shuttled miRNA signature, i.e., miR-155, -320a, -342-3p, -376, and -422a, detected T2DM patients with a previous major adverse cardiovascular event. Many of these miRNAs significantly correlate with clinical variables held to play a key role in the development of complications. In addition, we show that CD31+ EVs from patients with T2DM are able to promote the expression of selected inflammatory mRNAs, i.e., CCL2, IL-1α, and TNFα, when administered to endothelial cells in vitro. Overall, these data suggest that the miRNA cargo of plasma CD31+ EVs is largely affected by T2DM and related complications, encouraging further research to explore the diagnostic potential and the functional role of these alterations.

HDL (High-Density Lipoprotein) and ApoA-1 (Apolipoprotein A-1) Potentially Modulate Pancreatic α-Cell Glucagon Secretion.

OBJECTIVE: Subjects with low levels of HDL (high-density lipoprotein) and ApoA-1 (apolipoprotein A-1) have increased risk to develop type 2 diabetes. HDL levels are an independent predictor of ß-cell function and positively modulate it. Type 2 diabetes is characterized by defects in both ß and α-cell function, but the effect of HDL and ApoA1 on α-cell function is unknown. Approach and Results: We observed a significant negative correlation (r=-0.422, P<0.0001) between HDL levels and fasting glucagon in a cohort of 132 Italian subjects. In a multivariable regression analysis including potential confounders such as age, sex, BMI, triglycerides, total cholesterol, fasting and 2-hour postload glucose, and fasting insulin, the association between HDL and fasting glucagon remained statistically significant (ß=-0.318, P=0.006). CD1 mice treated with HDL or ApoA-1 for 3 consecutive days showed a 32% (P<0.001) and 23% (P<0.05) reduction, respectively, in glucagon levels following insulin-induced hypoglycemia, compared with controls. Treatment of pancreatic αTC1 clone 6 cells with HDL or ApoA-1 for 24 hours resulted in a significant reduction of glucagon expression (P<0.04) and secretion (P<0.01) after an hypoglycemic stimulus and increased Akt (RAC-alpha serine/threonine-protein kinase) and FoxO1 (forkhead/winged helix box gene, group O-1) phosphorylation. Pretreatment with Akt inhibitor VIII, PI3K (phosphatidylinositol 3-kinase) inhibitor LY294002, and HDL receptor SCARB-1 (scavenger receptor class B type 1) inhibitor BLT-1 (block lipid transport-1) restored αTC1 cell response to low glucose levels. CONCLUSIONS: These results support the notion that HDL and ApoA-1 modulate glucagon expression and secretion by binding their cognate receptor SCARB-1, and activating the PI3K/Akt/FoxO1 signaling cascade in an in vitro α-cell model. Overall, these results raise the hypothesis that HDL and ApoA-1 may have a role in modulating glucagon secretion.

Association between Serum Mg2+ Concentrations and Cardiovascular Organ Damage in a Cohort of Adult Subjects.

Magnesium (Mg2+) levels are associated with insulin resistance, hypertension, atherosclerosis, and type 2 diabetes (T2DM). We evaluated the clinical utility of physiological Mg2+ in assessing subclinical cardiovascular organ damage including increased carotid artery intima- media thickness (c-IMT) and left ventricular mass index (LVMI) in a cohort of well-characterized adult non-diabetic individuals. Age- and gender-adjusted correlations between Mg2+ and metabolic parameters showed that Mg2+ circulating levels were correlated negatively with body mass index (BMI), fasting glucose, and 2h-oral glucose tolerance test (OGTT) glucose. Similarly, Mg2+ levels were significantly and negatively related to c-IMT and LVMI. A multivariate regression analysis revealed that age (ß = 0.440; p < 0.0001), BMI (ß = 0.225; p < 0.0001), and Mg2+ concentration (ß = -0.122; p < 0.01) were independently associated with c-IMT. Age (ß = 0.244; p = 0.012), Mg2+ (ß = -0.177; p = 0.019), and diastolic blood pressure (ß = 0.184; p = 0.038) were significantly associated with LVMI in women, while age (ß = 0.211; p = 0.019), Mg2+ (ß = -0.171; p = 0.038) and the homeostasis model assessment index of insulin resistance (HOMA-IR) (ß = -0.211; p = 0.041) were the sole variables associated with LVMI in men. In conclusion, our data support the hypothesis that the assessment of Mg2+ as part of the initial work-up might help unravel the presence of subclinical organ damage in subjects at increased risk of cardiovascular complications.

Are Circulating Mg2+ Levels Associated with Glucose Tolerance Profiles and Incident Type 2 Diabetes?

Magnesium (Mg2+) is an enzyme co-factor that plays a key role in many biochemical reactions, as well as in glucose metabolism. Clinical evidences have demonstrated that depletion of serum Mg2+ increases exponentially with the duration of type 2 diabetes mellitus (T2DM). Diabetes is associated with low Mg2+, and hypomagnesemia is associated with insulin resistance, inflammation, and increased risk for cardiovascular disease. In subjects at high risk of inflammation and insulin resistance, supplementation of Mg2+ alone ameliorates both phenotypes, slowing the development and progression of hepatic steatosis. We analyze the relationship between serum Mg2+ levels and the onset of T2DM in a large cohort of well-characterized adult white individuals participating in the CATAMERI study, who were reexamined after a mean follow-up of 5.6 ± 0.9 years. In our analysis we acquired a significant negative correlation between Mg2+ levels, fasting glucose, and 2h-post load glucose in subjects who underwent an OGTT. Moreover, Mg2+ levels correlated negatively with fasting insulin levels, and positively with the lipid profile. As for the detrimental effect of lower circulating Mg2+ levels, our data revealed a significant reduction of T2DM risk of about 20% for each 1 mg/dL increase of circulating Mg2+. The present results are consistent with the theory that Mg2+ supplementation could ameliorate insulin sensitivity reducing the risk to develop T2DM.

A functional variant of the dimethylarginine dimethylaminohydrolase-2 gene is associated with myocardial infarction in type 2 diabetic patients.

BACKGROUND: Myocardial infarction is the main mortality cause in patients with type 2 diabetes (T2DM). Endothelial dysfunction due to reduced bioavailability of nitric oxide (NO) is an early step of atherogenesis. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthesis, and it is metabolized by the enzymes dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2. The functional variant rs9267551 C, in the promoter region of DDAH2, has been linked to increased DDAH2 expression, and lower ADMA plasma levels, and was associated with lower risk of coronary artery disease in large-scale genome-wide association studies (GWAS) performed in the general population. However, it is unknown whether this association holds true in T2DM patients. To address this issue, we investigated whether rs9267551 is associated with risk of myocardial infarction in two cohorts of T2DM patients. METHODS: SNP rs9267551 was genotyped in 1839 White T2DM patients from the Catanzaro Study (CZ, n = 1060) and the Gargano Heart Study-cross sectional design (GHS, n = 779). Cases were patients with a previous myocardial infarction, controls were asymptomatic patients with neither previous myocardial ischemia nor signs of it at resting and during a maximal symptom limited stress electrocardiogram. RESULTS: Carriers of allele rs9267551 C showed a dose dependent reduction in the risk of myocardial infarction [(CZ = OR 0.380, 95% CI 0.175-0.823, p = 0.014), (GHS = 0.497, 0.267-0.923, p = 0.027), (Pooled = 0.458, 0.283-0.739, p = 0.001)] which remained significant after adjusting for sex, age, BMI, smoking, HbA1c, total cholesterol HDL, and triglyceride levels [(CZ = 0.307, 0.106-0.885, p = 0.029), (GHS = 0.512, 0.270-0.970, p = 0.040), (Pooled = 0.458, 0.266-0.787, p = 0.005)]. CONCLUSIONS: We found that rs9267551 polymorphism is significantly associated with myocardial infarction in T2DM patients of European ancestry from two independent cohorts. It is possible that in subjects carrying the protective C allele less ADMA accumulates in endothelial cells causing vascular protection as a consequence of higher nitric oxide availability.

The polymorphism rs35767 at IGF1 locus is associated with serum urate levels.

Previous studies suggested that the IGF-1/IGF-1 receptor signaling pathway may contribute to regulate uric acid levels. To confirm this hypothesis, we assessed the effects of the IGF-1-raising genetic variant rs35767 on urate levels in serum and urine, and we investigated IGF-1 ability to modulate the expression of transporters involved in reabsorption and secretion of uric acid in the kidney. The study population included 2794 adult Whites. 24-hour urinary uric acid concentration was available for 229 subjects. rs35767 polymorphism was screened using TaqMan genotyping assays. HEK293 (human embryonic kidney-293) cell line was treated with IGF-1 (1, 5, 10, 50 nM) for 24-hours, and differences in the expression of urate transporters were evaluated via Western Blot and real time rtPCR. Individuals carrying the IGF-1-raising allele (rs35767 T) exhibited significantly lower levels of serum urate according to both additive and recessive models, after correction for gender, age, BMI, glucose tolerance, glomerular filtration rate, and anti-hypertensive treatment. TT genotype carriers displayed higher uricosuria than C allele carriers did, after adjusting for confounders. Exposure of HEK293 cells to IGF-1 resulted in a dose-dependent increase of uric acid transporters deputed to uric acid excretion (MRP4, NPT1 and BCRP), and reduction of GLUT9 expression, the major mediator of uric acid reabsorption, both at mRNA and protein level. We observed a significant association between the functional polymorphism rs35767 near IGF1 with serum urate concentrations and we provide a mechanistic explanation supporting a causal role for IGF-1 in the regulation of uric acid homeostasis.

Teneligliptin enhances the beneficial effects of GLP-1 in endothelial cells exposed to hyperglycemic conditions.

High-glucose-induced oxidative stress contributes to cardiovascular endothelial damage in diabetes. Glucagon-like peptide 1 (GLP-1) is beneficial to endothelial cells, but its effects are diminished when cells are continuously exposed to high glucose. Teneligliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor that prevents oxidative stress, apoptosis and the metabolic memory effect. We explored the potential additive effects of Teneligliptin and GLP-1 in hyperglycemia-damaged endothelial cells. Human umbilical vein endothelial cells (HUVECs) were exposed to normal-glucose (5 mmol/L) or high-glucose (HG, 25 mmol/L) for 21 days, or to HG for 14 days followed by normal-glucose for 7 days (HM). These cells were continually treated with Teneligliptin 3.0 µmol/L, alone or in combination with an acute dose of GLP-1 50 nmol/L. DPP-4 was upregulated under hyperglycemic conditions, but Teneligliptin reduced DPP-4 expression and activity. Simultaneous Teneligliptin and GLP-1 synergistically increased the antioxidant response and reduced ROS levels in HG- and HM-exposed HUVECs. Concurrent treatment also enhanced cell proliferation, reduced apoptotic gene expression and ameliorated endoplasmic reticulum stress in HG- and HM-exposed HUVECs. Thus, long-term Teneligliptin treatment reduced DPP-4 levels and activity in HUVECs exposed to chronic hyperglycemia. Moreover, Teneligliptin enhanced the beneficial effects of GLP-1 on oxidative stress, proliferation, apoptosis and endoplasmic reticulum homeostasis.

Inflammageing and metaflammation: The yin and yang of type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is characterised by chronic low-grade inflammation, recently referred to as 'metaflammation', a relevant factor contributing to the development of both diabetes and its complications. Nonetheless, 'canonical' anti-inflammatory drugs do not yield satisfactory results in terms of prevention of diabetes progression and of cardiovascular events, suggesting that the causal mechanisms fostering metaflammation deserve further research to identify new druggable targets. Metaflammation resembles ageing-induced low-grade inflammation, previously referred to as inflammageing, in terms of clinical presentation and the molecular profile, pointing to a common aetiology for both conditions. Along with the mechanisms proposed to fuel inflammageing, here we dissect a plethora of pathological cascades triggered by gluco- and lipotoxicity, converging on candidate phenomena possibly explaining the enduring pro-inflammatory program observed in diabetic tissues, i.e. persistent immune-system stimulation, accumulation of senescent cells, epigenetic rearrangements, and alterations in microbiota composition. We discuss the possibility of harnessing these recent discoveries in future therapies for T2DM. Moreover, we review recent evidence regarding the ability of diets and physical exercise to modulate selected inflammatory pathways relevant for the diabetic pathology. Finally, we examine the latest findings showing putative anti-inflammatory mechanisms of anti-hyperglycaemic agents with proven efficacy against T2DM-induced cardiovascular complications, in order to gain insights into quickly translatable therapeutic approaches.

Short-term sustained hyperglycaemia fosters an archetypal senescence-associated secretory phenotype in endothelial cells and macrophages.

Diabetic status is characterized by chronic low-grade inflammation and an increased burden of senescent cells. Recently, the senescence-associated secretory phenotype (SASP) has been suggested as a possible source of inflammatory factors in obesity-induced type 2 diabetes. However, while senescence is a known consequence of hyperglycaemia, evidences of SASP as a result of the glycaemic insult are missing. In addition, few data are available regarding which cell types are the main SASP-spreading cells in vivo. Adopting a four-pronged approach we demonstrated that: i) an archetypal SASP response that was at least partly attributable to endothelial cells and macrophages is induced in mouse kidney after in vivo exposure to sustained hyperglycaemia; ii) reproducing a similar condition in vitro in endothelial cells and macrophages, hyperglycaemic stimulus largely phenocopies the SASP acquired during replicative senescence; iii) in endothelial cells, hyperglycaemia-induced senescence and SASP could be prevented by SOD-1 overexpression; and iiii) ex vivo circulating angiogenic cells derived from peripheral blood mononuclear cells from diabetic patients displayed features consistent with the SASP. Overall, the present findings document a direct link between hyperglycaemia and the SASP in endothelial cells and macrophages, making the SASP a highly likely contributor to the fuelling of low-grade inflammation in diabetes.

Insulin-like growth factor-1 is a negative modulator of glucagon secretion.

Glucagon secretion involves a combination of paracrine, autocrine, hormonal, and autonomic neural mechanisms. Type 2 diabetes often presents impaired glucagon suppression by insulin and glucose. Insulin-like growth factor-I (IGF-1) has elevated homology with insulin, and regulates pancreatic ß-cells insulin secretion. Insulin and IGF-1 receptors share considerable structure homology and function. We hypothesized the existence of a mechanism linking the inhibition of α-cells glucagon secretion to IGF-1. Herein, we evaluated the association between plasma IGF-1 and glucagon levels in 116 nondiabetic adults. After adjusting for age gender and BMI, fasting glucagon levels were positively correlated with 2-h post-load glycaemia, HOMA index and fasting insulin, and were negatively correlated with IGF-1 levels. In a multivariable regression, the variables independently associated to fasting glucagon were circulating IGF-1 levels, HOMA index and BMI, explaining 20.7% variation. To unravel the molecular mechanisms beneath IGF-1 and glucagon association, we investigated whether IGF-1 directly modulates glucagon expression and secretion in an in vitro model of α-cells. Our data showed that IGF-1 inhibits the ability of low glucose concentration to stimulate glucagon expression and secretion via activation of the phosphatidylinositol-3-kinase/Akt/FoxO1 pathway. Collectively, our results suggest a new regulatory role of IGF-1 on α-cells biological function.

Plasma kisspeptin levels are associated with insulin secretion in nondiabetic individuals.

To evaluate if plasma kisspeptin concentrations are associated with insulin secretion, as suggested by recent in vitro studies, independently of confounders. 261 nondiabetic subjects were stratified into tertiles according to kisspeptin values. Insulin secretion was assessed using indexes derived from oral glucose tolerance test (OGTT). After adjusting for age, gender, and BMI, subjects in the highest (tertile 3) kisspeptin group exhibited significantly lower values of insulinogenic index, corrected insulin response (CIR30), and Stumvoll indexes for first-phase and second-phase insulin release as compared with low (tertile 1) or intermediate (tertile 2) kisspeptin groups. Univariate correlations between kisspeptin concentration and metabolic variables showed that kisspeptin concentration was significantly and positively correlated with age, blood pressure, and 2-h post-load glucose, and inversely correlated with BMI, and waist circumference. There was an inverse relationship between kisspeptin levels and OGTT-derived indexes of glucose-stimulated insulin secretion. A multivariable regression analysis in a model including all the variables significantly correlated with kisspeptin concentration showed thar age (ß = -0.338, P<0.0001), BMI (ß = 0.272, P<0.0001), 2-h post-load glucose (ß = -0.229, P<0.0001), and kisspeptin (ß = -0.105, P = 0.03) remained associated with insulinogenic index. These factors explained 34.6% of the variance of the insulinogenic index. In conclusion, kisspeptin concentrations are associated with insulin secretion independently of important determinants of glucose homeostasis such as gender, age, adiposity, 2-h post-load glucose, and insulin sensitivity.

Uric Acid Is Associated With Inflammatory Biomarkers and Induces Inflammation Via Activating the NF-κB Signaling Pathway in HepG2 Cells.

OBJECTIVE: Serum uric acid (UA) has been associated with increased risk of cardiovascular and metabolic diseases. However, the causal mechanisms linking elevated UA levels to cardio-metabolic diseases are still unsettled. One potential explanation for how UA might contribute to cardio-metabolic disease might be its ability to induce systemic inflammation. APPROACH AND RESULTS: Herein, we report a positive relationship between serum UA and acute-phase reactants, such as high-sensitivity C-reactive protein, fibrinogen, ferritin, complement C3, and erythrocyte sedimentation rate, in a cohort of 2731 nondiabetic adults. The relationship remains significant after adjustment for several confounders, including age, sex, adiposity, anti-hypertensive treatments or diuretics use. To confirm the existence of a causal relationship, we examined the effect of UA on the expression of inflammatory biomarkers in human hepatoma HepG2 cells and characterized the signaling pathway by which UA acts. We show that UA stimulates the expression of C-reactive protein, fibrinogen, ferritin, and complement C3 in a dose-dependent fashion. The proinflammatory effects of UA were abrogated by benzbromarone, a specific inhibitor of UA transporters. Exposure of cells to UA resulted in activation of the IκB kinase/IκBα/NF-κB signaling pathway that was attenuated by benzbromarone. The effect of UA was completely blocked by the antioxidant N-acetylcysteine. CONCLUSIONS: These in vivo and in vitro data suggest that hyperuricemia might induce the expression of hepatic inflammatory molecules by activating the proinflammatory NF-κB signaling cascade. Because inflammation has an important pathogenetic role in metabolic and cardiovascular disease, our study may help understanding the mechanism by which hyperuricemia may contribute to organ damage.

Liraglutide prevents cognitive decline in a rat model of streptozotocin-induced diabetes independently from its peripheral metabolic effects.

Diabetes has been identified as a risk factor for cognitive dysfunctions. Glucagone like peptide 1 (GLP-1) receptor agonists have neuroprotective effects in preclinical animal models. We evaluated the effects of GLP-1 receptor agonist, liraglutide (LIR), on cognitive decline associated with diabetes. Furthermore, we studied LIR effects against hippocampal neurodegeneration induced by streptozotocin (STZ), a well-validated animal model of diabetes and neurodegeneration associated with cognitive decline. Diabetes and/or cognitive decline were induced in Wistar rats by intraperitoneal or intracerebroventricular injection of STZ and then rats were treated with LIR (300µg/kg daily subcutaneously) for 6 weeks. Rats underwent behavioral tests: Morris water maze, passive avoidance, forced swimming (FST), open field, elevated plus maze, rotarod tests. Furthermore, LIR effects on hippocampal neurodegeneration and mTOR pathway (AKT, AMPK, ERK and p70S6K) were assessed. LIR improved learning and memory only in STZ-treated animals. Anxiolytic effects were observed in all LIR-treated groups but pro-depressant effects in CTRL rats were observed. At a cellular/molecular level, intracerebroventricular STZ induced hippocampal neurodegeneration accompanied by decreased phosphorylation of AMPK, AKT, ERK and p70S6K. LIR reduced hippocampal neuronal death and prevented the decreased phosphorylation of AKT and p70S6K; AMPK was hyper-phosphorylated in comparison to CTRL group, while LIR had no effects on ERK. LIR reduced animal endurance in the rotarod test and this effect might be also linked to a reduction in locomotor activity during only the last two minutes of the FST. LIR had protective effects on cognitive functions in addition to its effects on blood glucose levels. LIR effects in the brain also comprised anxiolytic and pro-depressant actions (although influenced by reduced endurance). Finally, LIR protected from diabetes-dependent hippocampal neurodegeneration likely through an effect on mTOR pathway.

Everolimus improves memory and learning while worsening depressive- and anxiety-like behavior in an animal model of depression.

Everolimus (EVR) is an orally-administered rapamycin analog that selectively inhibits the mammalian target of rapamycin (mTOR) kinase (mainly mTORC1 and likely mTORC2) and the related signaling pathway. mTOR is a serine/threonine protein kinase regulating multiple important cellular functions; dysfunction of mTOR signaling has also been implicated in the pathophysiology of several neurological, neurodegenerative, developmental and cognitive disorders. EVR is widely used as an anti-neoplastic therapy and more recently in children with tuberous sclerosis complex (TSC). However, no clear correlation exists between EVR use and development of central side effects e.g. depression, anxiety or cognitive impairment. We studied the effects of a 3 weeks administration of EVR in mice chronically treated with betamethasone 21-phosphate disodium (BTM) as a model of depression and cognitive decline. EVR treatment had detrimental effects on depressive- and anxiety-like behavior while improving cognitive performance in both control (untreated) and BTM-treated mice. Such effects were accompanied by an increased hippocampal neurogenesis and synaptogenesis. Our results therefore might support the proposed pathological role of mTOR dysregulation in depressive disorders and confirm some previous data on the positive effects of mTOR inhibition in cognitive decline. We also show that EVR, possibly through mTOR inhibition, may be linked to the development of anxiety. The increased hippocampal neurogenesis by EVR might explain its ability to improve cognitive function or protect from cognitive decline. Our findings suggest some caution in the use of EVR, particularly in the developing brain; patients should be carefully monitored for their psychiatric/neurological profiles in any clinical situation where an mTOR inhibitor and in particular EVR is used e.g. cancer treatment, TSC or immunosuppression.

PHLPP phosphatases as a therapeutic target in insulin resistance-related diseases.

INTRODUCTION: Pleckstrin homology domain leucine-rich repeat protein phosphatases (PHLPPs), originally identified as Akt kinase hydrophobic motif specific phosphatases, have subsequently been shown to regulate several molecules recurring within the insulin signaling pathway. This observation suggests that PHLPP phosphatases may have a clinically relevant role in the pathogenesis of insulin resistance-related diseases and may thus represent suitable targets for the treatment of these conditions. AREAS COVERED: The literature pertaining to PHLPPs substrates is reviewed herein, along with information on the molecular players involved in regulating the activity and expression of PHLPP phosphatases. In the present review, knowledge of genetic variants in the genes that encode for PHLPP isozymes and the surrounding regulatory regions is also summarized. In addition, data from the studies addressing the role of PHLPPs in insulin resistance-related disorders and from those investigating the possibility to manipulate these phosphatases for therapeutic purposes are presented. EXPERT OPINION: A number of issues should be resolved before PHLPPs are pursued as therapeutic targets including: the mechanisms regulating the specificity of PHLPP isozymes; the possibility of differentially regulating PHLPP family members and the possible impact of PHLPPs modulation on the risk of cancer.