Special Issue: "Anti-inflammatory Effects of Glucagon-like Peptide-1".

From the failure of gut extracts in diabetic patients' therapy to the effective action in cardiovascular outcomes [...].

Caveolin-1 and Atherosclerosis: Regulation of LDLs Fate in Endothelial Cells.

Caveolae are 50-100 nm cell surface plasma membrane invaginations observed in terminally differentiated cells. They are characterized by the presence of the protein marker caveolin-1. Caveolae and caveolin-1 are involved in regulating several signal transduction pathways and processes. It is well recognized that they have a central role as regulators of atherosclerosis. Caveolin-1 and caveolae are present in most of the cells involved in the development of atherosclerosis, including endothelial cells, macrophages, and smooth muscle cells, with evidence of either pro- or anti-atherogenic functions depending on the cell type examined. Here, we focused on the role of caveolin-1 in the regulation of the LDLs' fate in endothelial cells.

Transitions Between Highly Automated and Longitudinally Assisted Driving: The Role of the Initiator in the Fight for Authority.

OBJECTIVE: A driving simulator study explored how drivers behaved depending on their initial role during transitions between highly automated driving (HAD) and longitudinally assisted driving (via adaptive cruise control). BACKGROUND: During HAD, drivers might issue a take-over request (TOR), initiating a transition of control that was not planned. Understanding how drivers behave in this situation and, ultimately, the implications on road safety is of paramount importance. METHOD: Sixteen participants were recruited for this study and performed transitions of control between HAD and longitudinally assisted driving in a driving simulator. While comparing how drivers behaved depending on whether or not they were the initiators, different handover strategies were presented to analyze how drivers adapted to variations in the authority level they were granted at various stages of the transitions. RESULTS: Whenever they initiated the transition, drivers were more engaged with the driving task and less prone to follow the guidance of the proposed strategies. Moreover, initiating a transition and having the highest authority share during the handover made the drivers more engaged with the driving task and attentive toward the road. CONCLUSION: Handover strategies that retained a larger authority share were more effective whenever the automation initiated the transition. Under driver-initiated transitions, reducing drivers' authority was detrimental for both performance and comfort. APPLICATION: As the operational design domain of automated vehicles (Society of Automotive Engineers [SAE] Level 3/4) expands, the drivers might very well fight boredom by taking over spontaneously, introducing safety issues so far not considered but nevertheless very important.

Anti-Inflammatory Effects of GLP-1R Activation in the Retina.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone, mainly produced by enteroendocrine L cells, which participates in the regulation of glucose homeostasis, and in reduction in body weight by promoting satiety. Actions of GLP-1 are mediated by activation of its receptor GLP-1R, which is widely expressed in several tissues including the retina. The effects of GLP-1R activation are useful in the management of type 2 diabetes mellitus (T2DM). In addition, the activation of GLP-1R has anti-inflammatory effects in several organs, suggesting that it may be also useful in the treatment of inflammatory diseases. Inflammation is a common element in the pathogenesis of several ocular diseases, and the protective effects of treatment with GLP-1 emerged also in retinal diseases. In this review we highlight the anti-inflammatory effects of GLP-1R activation in the retina. Firstly, we summarized the pathogenic role of inflammation in ocular diseases. Then, we described the pleiotropic effects of GLP-1R activation on the cellular components of the retina which are mainly involved in the pathogenesis of inflammatory retinal diseases: the retinal ganglion cells, retinal pigment epithelial cells and endothelial cells.

High Glucose Impairs Expression and Activation of MerTK in ARPE-19 Cells.

MerTK (Mer Tyrosine Kinase) is a cell surface receptor that regulates phagocytosis of photoreceptor outer segments (POS) in retinal pigment epithelial (RPE) cells. POS phagocytosis is impaired in several pathologies, including diabetes. In this study, we investigate whether hyperglycemic conditions may affect MerTK expression and activation in ARPE-19 cells, a retinal pigment epithelial cellular model. ARPE-19 cells were cultured in standard (CTR) or high-glucose (HG) medium for 24 h. Then, we analyzed: mRNA levels and protein expression of MerTK and ADAM9, a protease that cleaves the extracellular region of MerTK; the amount of cleaved Mer (sMer); and the ability of GAS6, a MerTK ligand, to induce MerTK phosphorylation. Since HG reduces miR-126 levels, and ADAM9 is a target of miR-126, ARPE-19 cells were transfected with miR-126 inhibitor or mimic; then, we evaluated ADAM9 expression, sMer, and POS phagocytosis. We found that HG reduced expression and activation of MerTK. Contextually, HG increased expression of ADAM9 and the amount of sMer. Overexpression of miR-126 reduced levels of sMer and improved phagocytosis in ARPE-19 cells cultured with HG. In this study, we demonstrate that HG compromises MerTK expression and activation in ARPE-19 cells. Our results suggest that HG up-regulates ADAM9 expression, leading to increased shedding of MerTK. The consequent rise in sMer coupled to reduced expression of MerTK impairs binding and internalization of POS in ARPE-19 cells.

The Hormetic Effect of Metformin: "Less Is More"?

Metformin (MTF) is the first-line therapy for type 2 diabetes (T2DM). The euglycemic effect of MTF is due to the inhibition of hepatic glucose production. Literature reports that the principal molecular mechanism of MTF is the activation of 5'-AMP-activated protein kinase (AMPK) due to the decrement of ATP intracellular content consequent to the inhibition of Complex I, although this effect is obtained only at millimolar concentrations. Conversely, micromolar MTF seems to activate the mitochondrial electron transport chain, increasing ATP production and limiting oxidative stress. This evidence sustains the idea that MTF exerts a hormetic effect based on its concentration in the target tissue. Therefore, in this review we describe the effects of MTF on T2DM on the principal target organs, such as liver, gut, adipose tissue, endothelium, heart, and skeletal muscle. In particular, data indicate that all organs, except the gut, accumulate MTF in the micromolar range when administered in therapeutic doses, unmasking molecular mechanisms that do not depend on Complex I inhibition.

Glibenclamide Mimics Metabolic Effects of Metformin in H9c2 Cells.

BACKGROUND: Sulfonylureas, such as glibenclamide, are antidiabetic drugs that stimulate beta-cell insulin secretion by binding to the sulfonylureas receptors (SURs) of adenosine triphosphate-sensitive potassium channels (KATP). Glibenclamide may be also cardiotoxic, this effect being ascribed to interference with the protective function of cardiac KATP channels for which glibenclamide has high affinity. Prompted by recent evidence that glibenclamide impairs energy metabolism of renal cells, we investigated whether this drug also affects the metabolism of cardiac cells. METHODS: The cardiomyoblast cell line H9c2 was treated for 24 h with glibenclamide or metformin, a known inhibitor of the mitochondrial respiratory chain. Cell viability was evaluated by sulforodhamine B assay. ATP and AMP were measured according to the enzyme coupling method and oxygen consumption by using an amperometric electrode, while Fo-F1 ATP synthase activity assay was evaluated by chemiluminescent method. Protein expression was measured by western blot. RESULTS: Glibenclamide deregulated energy balance of H9c2 cardiomyoblasts in a way similar to that of metformin. It inhibited mitochondrial complexes I, II and III with ensuing impairment of oxygen consumption and ATP synthase activity, ATP depletion and increased AMPK phosphorylation. Furthermore, glibenclamide disrupted mitochondrial subcellular organization. The perturbation of mitochondrial energy balance was associated with enhanced anaerobic glycolysis, with increased activity of phosphofructo kinase, pyruvate kinase and lactic dehydrogenase. Interestingly, some additive effects of glibenclamide and metformin were observed. CONCLUSIONS: Glibenclamide deeply alters cell metabolism in cardiac cells by impairing mitochondrial organization and function. This may further explain the risk of cardiovascular events associated with the use of this drug, alone or in combination with metformin.

Cavin-1 and Caveolin-1 are both required to support cell proliferation, migration and anchorage-independent cell growth in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a childhood soft tissue tumor with broad expression of markers that are typically found in skeletal muscle. Cavin-1 is a recently discovered protein actively cooperating with Caveolin-1 (Cav-1) in the morphogenesis of caveolae and whose role in cancer is drawing increasing attention. Using a combined in silico and in vitro analysis here we show that Cavin-1 is expressed in myogenic RMS tumors as well as in human and primary mouse RMS cultures, exhibiting a broad subcellular localization, ranging from nuclei and cytosol to plasma membrane. In particular, the coexpression and plasma membrane interaction between Cavin-1 and Cav-1 characterized the proliferation of human and mouse RMS cell cultures, while a downregulation of their expression levels was observed during the myogenic differentiation. Knockdown of Cavin-1 or Cav-1 in the human RD and RH30 cells led to impairment of cell proliferation and migration. Moreover, loss of Cavin-1 in RD cells impaired the anchorage-independent cell growth in soft agar. While the loss of Cavin-1 did not affect the Cav-1 protein levels in RMS cells, Cav-1 overexpression and knockdown triggered a rise or depletion of Cavin-1 protein levels in RD cells, respectively, in turn reflecting on increased or decreased cell proliferation, migration and anchorage-independent cell growth. Collectively, these data indicate that the interaction between Cavin-1 and Cav-1 underlies the cell growth and migration in myogenic tumors.

Autonomous artificial intelligence versus teleophthalmology for diabetic retinopathy.

Purpose: To assess the role of artificial intelligence (AI) based automated software for detection of Diabetic Retinopathy (DR) compared with the evaluation of digital retinography by two double masked retina specialists. Methods: Two-hundred one patients (mean age 65 ± 13 years) with type 1 diabetes mellitus or type 2 diabetes mellitus were included. All patients were undergoing a retinography and spectral domain optical coherence tomography (SD-OCT, DRI 3D OCT-2000, Topcon) of the macula. The retinal photographs were graded using two validated AI DR screening software (Eye Art TM and IDx-DR) designed to identify more than mild DR. Results: Retinal images of 201 patients were graded. DR (more than mild DR) was detected by the ophthalmologists in 38 (18.9%) patients and by the AI-algorithms in 36 patients (with 30 eyes diagnosed by both algorithms). Ungradable patients by the AI software were 13 (6.5%) and 16 (8%) for the Eye Art and IDx-DR, respectively. Both AI software strategies showed a high sensitivity and specificity for detecting any more than mild DR without showing any statistically significant difference between them. Conclusions: The comparison between the diagnosis provided by artificial intelligence based automated software and the reference clinical diagnosis showed that they can work at a level of sensitivity that is similar to that achieved by experts.

Caveolin-1 is essential for metformin inhibitory effect on IGF1 action in non-small-cell lung cancer cells.

Metformin causes an AMP/ATP ratio increase and AMP-activated protein kinase (AMPK) activation. Since caveolin-1 (Cav-1) plays a role in AMPK activation and energy balance, we investigated whether Cav-1 could participate in metformin's inhibitory effect on IGF1 signaling. The effect of metformin was studied in two non-small-cell lung cancer (NSCLC) cell lines, Calu-1 and Calu-6, expressing higher and lower amounts of Cav-1, respectively. In Calu-1, but not in Calu-6 cells, metformin reduced phosphorylation of type 1 insulin-like growth factor receptor (IGF-IR) substrates Akt and Forkhead transcription factor 3a (FOXO3a), inhibited IGF1-dependent FOXO3a nuclear exit, and decreased IGF1-dependent cell proliferation. Here, we show that sensitivity of NSCLC cells to metformin was dependent on Cav-1 expression and that metformin required Cav-1 to induce AMPK phosphorylation and AMP/ATP ratio increase. Cav-1 silencing in Calu-1 and overexpression in Calu-6 reduced and improved, respectively, the inhibitory effect of metformin on IGF1-dependent Akt phosphorylation. Prolonged metformin treatment in Calu-6 cells induced a dose-dependent expression increase of Cav-1 and OCT1, a metformin transporter. Cav-1 and OCT1 expression was associated with the antiproliferative effect of metformin in Calu-6 cells (IC(50)=18 mM). In summary, these data suggest that Cav-1 is required for metformin action in NSCLC cells.

The plant hormone abscisic acid increases in human plasma after hyperglycemia and stimulates glucose consumption by adipocytes and myoblasts.

The plant hormone abscisic acid (ABA) is released from glucose-challenged human pancreatic ß cells and stimulates insulin secretion. We investigated whether plasma ABA increased during oral and intravenous glucose tolerance tests (OGTTs and IVGTTs) in healthy human subjects. In all subjects undergoing OGTTs (n=8), plasma ABA increased over basal values (in a range from 2- to 9-fold). A positive correlation was found between the ABA area under the curve (AUC) and the glucose AUC. In 4 out of 6 IVGTTs, little or no increase of ABA levels was observed. In the remaining subjects, the ABA increase was similar to that recorded during OGTTs. GLP-1 stimulated ABA release from an insulinoma cell line and from human islets, by ∼10- and 2-fold in low and high glucose, respectively. Human adipose tissue also released ABA in response to high glucose. Nanomolar ABA stimulated glucose uptake, similarly to insulin, in rat L6 myoblasts and in murine 3T3-L1 cells differentiated to adipocytes, by increasing GLUT-4 translocation to the plasma membrane. Demonstration that a glucose load in humans is followed by a physiological rise of plasma ABA, which can enhance glucose uptake by adipose tissues and muscle cells, identifies ABA as a new mammalian hormone involved in glucose metabolism.

Klotho: A new therapeutic target in diabetic retinopathy?

Klotho (Kl) is considered an antiaging gene, mainly for the inhibition of the insulin-like growth factor-1 signaling. Kl exists as full-length transmembrane, which acts as co-receptor for fibroblast growth factor receptor, and in soluble forms (sKl). The sKl may exert pleiotropic effects on organs and tissues by regulating several pathways involved in the pathogenesis of diseases associated with oxidative and inflammatory state. In diabetic Patients, serum levels of Kl are significantly decreased compared to healthy subjects, and are related to duration of diabetes. In diabetic retinopathy (DR), one of the most common microvascular complications of type 2 diabetes, serum Kl levels are negatively correlated with progression of the disease. A lot of evidences showed that Kl regulates several mechanisms involved in maintaining homeostasis and functions of retinal cells, including phagocytosis, calcium signaling, secretion of vascular endothelial growth factor A (VEGF-A), maintenance of redox status, and melanin biosynthesis. Experimental data have been shown that Kl exerts positive effects on several mechanisms involved in onset and progression of DR. In particular, treatment with Kl: (1) Prevents apoptosis induced by oxidative stress in human retinal endothelial cells and in retinal pigment epithelium (RPE) cells; (2) reduces secretion of VEGF-A by RPE cells; and (3) decreases subretinal fibrosis and preserves autophagic activity. Therefore, Kl may become a novel biomarker and a good candidate for the treatment of DR.

Crosstalk between the Rod Outer Segments and Retinal Pigmented Epithelium in the Generation of Oxidative Stress in an In Vitro Model.

Dysfunction of the retinal pigment epithelium (RPE) is associated with several diseases characterized by retinal degeneration, such as diabetic retinopathy (DR). However, it has recently been proposed that outer retinal neurons also participate in the damage triggering. Therefore, we have evaluated the possible crosstalk between RPE and photoreceptors in priming and maintaining oxidative damage of the RPE. For this purpose, we used ARPE-19 cells as a model of human RPE, grown in normal (NG, 5.6 mM) or high glucose (HG, 25 mM) and unoxidized (UOx) or oxidized (Ox) mammalian retinal rod outer segments (OSs). ARPE-19 cells were efficient at phagocytizing rod OSs in both NG and HG settings. However, in HG, ARPE-19 cells treated with Ox-rod OSs accumulated MDA and lipofuscins and displayed altered LC3, GRP78, and caspase 8 expression compared to untreated and UOx-rod-OS-treated cells. Data suggest that early oxidative damage may originate from the photoreceptors and subsequently extend to the RPE, providing a new perspective to the idea that retinal degeneration depends solely on a redox alteration of the RPE.

One-year follow-up comparison of two hybrid closed-loop systems in Italian children and adults with type 1 diabetes.

Background and aims: Tandem Control-IQ and MiniMed 780G are the main Advanced Hybrid Closed Loop (AHCL) systems currently available in pediatric and adult patients with Type 1 Diabetes (T1D). The aim of our study was to evaluate glycemic control after 1-year of follow-up extending our previous study of 1-month comparison between the two systems. Methods: We retrospectively compared clinical and continuous glucose monitoring (CGM) data from the patients included in the previous study which have completed 1-year observation period. The study population consisted of 74 patients, 42 Minimed 780G users and 32 Tandem Control-IQ users. Linear mixed models with random intercept were performed to study the variations over time and the interaction between time and system; Mann-Whitney or T-test were used to compare systems at 1-year. Results: Both systems have been shown to be effective in maintaining the glycemic improvement achieved one month after starting AHCL. Significant changes over time were observed for TIR, TAR, TAR>250mg/dl, average glucose levels and SD (p<0.001). At 1-year follow-up Minimed 780G obtained better improvement in TIR (p<0.001), TAR (p=0.002), TAR>250mg/dl (p=0.001), average glucose levels (p<0.001). The comparison of the glycemic parameters at 1-year showed a significant superiority of Minimed 780G in terms of TIR (71% vs 68%; p=0.001), TAR (p=0.001), TAR>250 (p=0.009), average glucose levels(p=0.001) and SD (p=0.031). Conclusions: The use of AHCL systems led to a significant improvement of glycemic control at 1-month, which is maintained at 1-year follow-up. MiniMed is more effective than Tandem in reaching the International recommended glycemic targets. Continuous training and education in the use of technology is essential to get the best out of the most advanced technological tools.

The effect of inconsistent steering guidance during transitions from Highly Automated Driving.

This driving simulator study investigated the effect of inconsistent steering guidance during system and user-initiated transitions from Highly Automated Driving (HAD). In particular, the aim of the study was to understand if steering conflicts could be achieved by adopting inconsistent steering guidance and whether these conflicts could be exploited to accelerate drivers' steering engagement within a limited time. Inconsistent steering guidance was generated by switching the guidance on and off at 3 different frequencies (0.1, 0.2 and 0.3 Hz). Results revealed that steering engagement has more to do with the initiation rather than the quality of the steering guidance. In fact, drivers were more engaged with the steering task when they initiated the transition themselves. Compared to system-initiated transitions, in user-initiated ones, drivers exerted stronger steering inputs throughout the transition, which allowed them to maintain larger Time To Lane Crossing (TTLC) values with fewer steering corrections. During system-initiated transitions, drivers started to actively engage with the steering activity only after more than 5 s from the start of the transition but were able to achieve a steering behaviour close to the one shown during user-initiated transitions at 10 s.

Emerging Role of Caveolin-1 in GLP-1 Action.

Glucagon-like peptide-1 (GLP-1) is a gut hormone mainly produced in the intestinal epithelial endocrine L cells, involved in maintaining glucose homeostasis. The use of GLP-1 analogous and dipeptidyl peptidase-IV (DPP-IV) inhibitors is well-established in Type 2 Diabetes. The efficacy of these therapies is related to the activation of GLP-1 receptor (GLP-1R), which is widely expressed in several tissues. Therefore, GLP-1 is of great clinical interest not only for its actions at the level of the beta cells, but also for the extra-pancreatic effects. Activation of GLP-1R results in intracellular signaling that is regulated by availability of downstream molecules and receptor internalization. It has been shown that GLP-1R co-localizes with caveolin-1, the main component of caveolae, small invagination of the plasma membrane, which are involved in controlling receptor activity by assembling signaling complexes and regulating receptor trafficking. The aim of this review is to outline the important role of caveolin-1 in mediating biological effects of GLP-1 and its analogous.

Caveolin-1 Down-Regulation Reduces VEGF-A Secretion Induced by IGF-1 in ARPE-19 Cells.

The insulin-like growth factor 1 (IGF-1) stimulates expression and secretion of vascular endothelial growth factor-A (VEGF-A), the main actor in ocular neovascularization, by RPE cells. Activity of IGF-1 is regulated by interaction between its receptor and Caveolin-1 (Cav-1), the main component of caveolae. The aim of this study was to investigate whether modulation of Cav-1 expression affects synthesis and secretion of VEGF-A. ARPE-19 cells were transfected with small interfering RNA for Cav-1 (si-Cav-1) and with control siRNA (si-CTR) and stimulated with IGF-1. We found that down-regulation of Cav-1 did not affect activation of IGF-1R but regulated in an opposite manner the phosphorylation of Akt and Erk1/2. Moreover, we found that IGF-1 increased mRNA levels of VEGF-A in both si-CTR and in si-Cav-1 ARPE-19 cells and that Cav-1 silencing significantly reduced basal and IGF-1-stimulated VEGF-A release. Then we investigated the response of the microvascular endothelial cell line HMEC-1 to secretory products of ARPE-19 cells by evaluating wound healing closure, finding that conditioned media from si-Cav-1-ARPE-19 cells reduced endothelial cell migration rate. These data demonstrate that Cav-1 regulates secretion of VEGF-A, and that the depletion of Cav-1 reduces IGF-1 induced VEGF-A secretion in ARPE-19 cells and the migratory potential of their secretory products.

A Comparison of Two Hybrid Closed-Loop Systems in Italian Children and Adults With Type 1 Diabetes.

Tandem Control-IQ and Minimed 780G represent the most Advanced Hybrid Closed Loop (AHCL) systems currently available in pediatric and adult subjects with Type 1 Diabetes (T1D). We retrospectively compared clinical and continuous glucose monitoring data from 51 patients who upgraded to Minimed 780G system and have completed 1-month observation period with data from 39 patients who upgraded to Tandem Control-IQ. Inverse probability weighting was used to minimize the basal characteristics imbalances. Both AHCL systems showed a significant improvement in glycemic parameters. Minimed 780G group achieved higher TIR increase (p= 0.004) and greater reduction of blood glucose average (p= 0.001). Tandem Control-IQ system significantly reduced the occurrence of TBR (p= 0.010) and the Coefficient of Variation of glucose levels (p= 0.005). The use of ACHL systems led to a significant improvement of glycemic control substantially reaching the International recommended glycemic targets. Minimed 780G appears to be more effective in managing hyperglycemia, while Tandem Control-IQ seems to be more effective in reducing time in hypoglycemia.