Human blood vessel organoids reveal a critical role for CTGF in maintaining microvascular integrity.

The microvasculature plays a key role in tissue perfusion and exchange of gases and metabolites. In this study we use human blood vessel organoids (BVOs) as a model of the microvasculature. BVOs fully recapitulate key features of the human microvasculature, including the reliance of mature endothelial cells on glycolytic metabolism, as concluded from metabolic flux assays and mass spectrometry-based metabolomics using stable tracing of 13C-glucose. Pharmacological targeting of PFKFB3, an activator of glycolysis, using two chemical inhibitors results in rapid BVO restructuring, vessel regression with reduced pericyte coverage. PFKFB3 mutant BVOs also display similar structural remodelling. Proteomic analysis of the BVO secretome reveal remodelling of the extracellular matrix and differential expression of paracrine mediators such as CTGF. Treatment with recombinant CTGF recovers microvessel structure. In this work we demonstrate that BVOs rapidly undergo restructuring in response to metabolic changes and identify CTGF as a critical paracrine regulator of microvascular integrity.

Ocular application of electrospun materials for drug delivery and cellular therapies.

The constraints of delivering conventional drugs, biologics and cell-based therapeutics to target ocular sites necessitate the fabrication of novel drug delivery systems to treat diverse ocular diseases. Conventional ocular drug delivery approaches are prone to low bioavailability, poor penetration and degradation of therapeutics, including cell-based therapies, leading to the need for frequent topical applications or intraocular injections. However, owing to their exceptional structural properties, nanofibrous and microfibrous electrospun materials have gained significant interest in ocular drug delivery and biomaterial applications. This review covers the recent developments of electrospun fibers for the delivery of drugs, biologics, cells, growth factors and tissue regeneration in treating ocular diseases. The insights from this review can provide a thorough understanding of the selection of materials for the fabrication of nano- and/or micro-fibrous systems for ocular applications, with a particular interest in achieving controlled drug release and cell therapy. A detailed modality for fabricating different types of nano- and micro-fibers produced from electrospinning and factors influencing generation are also discussed.

Retinal pigment epithelium extracellular vesicles are potent inducers of age-related macular degeneration disease phenotype in the outer retina.

Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors atrophy and/or retinal and choroidal angiogenesis. Here we use AMD patient-specific RPE cells with the Complement Factor H Y402H high-risk polymorphism to perform a comprehensive analysis of extracellular vesicles (EVs), their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced polarised EV secretion. Multi-omics analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids, which mediate key AMD features including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. Moreover, AMD RPE EVs induce amyloid fibril formation, revealing their role in drusen formation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the acquisition of AMD features such as stress vacuoles, cytoskeletal destabilization and abnormalities in the morphology of the nucleus. Retinal organoid treatment with apical AMD RPE EVs leads to disrupted neuroepithelium and the appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6/Vsx2, suggesting injury-induced regenerative pathways activation. These findings indicate that AMD RPE EVs are potent inducers of AMD phenotype in the neighbouring RPE and retinal cells.

Google Classroom as a Teaching Tool for Undergraduate Embryology.

Context and aim Modern teaching of medicine has evolved into a beyond-the-classroom experience. Learning management systems (LMSs) have made this possible because of easy accessibility and user-friendliness. The COVID-19 lockdown further accentuated the need for this mode of education delivery. General embryology (GE) is a subject under human anatomy that does not rely on "touch-and-feel" as much as other medical subjects. Assess Google Classroom (GC) as a teaching tool to deliver an online undergraduate-level general embryology (GE) course. Settings and design A cross-sectional study involving 211 undergraduate medical students across India. Methods and material A pre-and post-quiz model was adopted to evaluate the efficiency of a five-lecture course on GE. The course content was delivered via 20-minute YouTube video lectures, uploaded on GC. Lastly, student feedback regarding gadget preferences and the overall learning experience was collected. Statistical Analysis: The confidence interval was set at 95%, and a p-value

Long term high glucose exposure induces premature senescence in retinal endothelial cells.

Purpose: Features of cellular senescence have been described in diabetic retinal vasculature. The aim of this study was to investigate how the high glucose microenvironment impacts on the senescence program of retinal endothelial cells. Methods: Human retinal microvascular endothelial cells were cultured under control and high glucose conditions of 5 mM and 25 mM D-glucose, respectively. Isomeric l-glucose was used as the osmotic control. Cells were counted using CASY technology until they reached their Hayflick limit. Senescence-associated ß-Galactosidase was used to identify senescent cells. Endothelial cell functionality was evaluated by the clonogenic, 3D tube formation, and barrier formation assays. Cell metabolism was characterized using the Seahorse Bioanalyzer. Gene expression analysis was performed by bulk RNA sequencing. Retinal tissues from db/db and db/+ mice were evaluated for the presence of senescent cells. Publicly available scRNA-sequencing data for retinas from Akimba and control mice was used for gene set enrichment analysis. Results: Long term exposure to 25 mM D-Glucose accelerated the establishment of cellular senescence in human retinal endothelial cells when compared to 5 mM D-glucose and osmotic controls. This was shown from 4 weeks, by a significant slower growth, higher percentages of cells positive for senescence-associated ß-galactosidase, an increase in cell size, and lower expression of pRb and HMGB2. These senescence features were associated with decreased clonogenic capacity, diminished tubulogenicity, and impaired barrier function. Long term high glucose-cultured cells exhibited diminished glycolysis, with lower protein expression of GLUT1, GLUT3, and PFKFB3. Transcriptomic analysis, after 4 weeks of culture, identified downregulation of ALDOC, PFKL, and TPI1, in cells cultured with 25 mM D-glucose when compared to controls. The retina from db/db mice showed a significant increase in acellular capillaries associated with a significant decrease in vascular density in the intermediate and deep retinal plexuses, when compared to db/+ mice. Senescent endothelial cells within the db/db retinal vasculature were identified by senescence-associated ß-galactosidase staining. Analysis of single cell transcriptomics data for the Akimba mouse retina highlighted an enrichment of senescence and senescence-associated secretory phenotype gene signatures when compared to control mice. Conclusion: A diabetic-like microenvironment of 25 mM D-glucose was sufficient to accelerate the establishment of cellular senescence in human retinal microvascular endothelial cells.

A Novel Online Dissection Course on Lower Limb Anatomy During the COVID-19 Pandemic.

INTRODUCTION: The teaching of human anatomy, a medical subject that relies heavily on live teaching, teacher-student interactivity, and visuospatial skills, has suffered tremendously since the COVID-19 pandemic mandated the shutting down of medical institutions. The medical education fraternity was compelled to replace the traditional teaching method of hands-on cadaveric dissections (HOCDs) with online education to overcome this new challenge, but it came at the cost of reduced student engagement and lesser spatial orientation. METHOD: In this cross-sectional, questionnaire-based study, we designed a novel online dissection course on lower limb anatomy and collected student feedback on the same from consenting Phase I Bachelor of Medicine, Bachelor of Surgery (MBBS) students of Symbiosis Medical College for Women, Pune, India. The course design consisted of three different modes: a live Zoom session using a handheld camera phone, a pre-recorded video dissection uploaded on the institute learning management system, and a Powerpoint presentation with high-resolution photographs of each dissected layer; and the feedback intended to find out what works best for the students. Overall feedback regarding their preferences in terms of presentation design, use of background music in pre-recorded videos, and overall learning experience was also collected. The course consisted of six two-hour teaching sessions. The first three sessions each used a different mode of teaching, repeating the same pattern in the next three sessions. The first mode of teaching implemented was a live Zoom session where instructors used a hand-held cell phone camera to show specimens that had been dissected a day prior. The second mode involved a pre-recorded video showing step-by-step dissection performed by the instructor which was then uploaded on the Institute Learning Management System. Of the two pre-recorded videos, background music consisting of a low-volume instrumental track was added to the second video. The third mode utilized Powerpoint presentations containing high-resolution photographs of each dissected layer on a separate slide along with labeling. The presentations were shown to the students over a Zoom call. A Google Form (GF) questionnaire was created after validation by subject experts to gather the students' feedback on the teaching and learning of anatomy via these sessions. The GF responses were collected and analyzed using Microsoft Excel. RESULTS: 41.7% of students recommended the use of a combination of all three modes in the same session, while 36.7% favored pre-recorded videos. 86.7% of students said that a good quality presentation design helps in keeping them engaged and only 23% of students favored the use of background music for increasing their ability to concentrate. 63.3% of students found the learning experience highly satisfactory. CONCLUSION: Although virtual dissection teaching methods may not be able to completely replace HOCDs, a well-planned online dissection course incorporating multiple modes of online dissections with an emphasis on good quality presentation design and frequent teacher-student interactivity can provide a strong impetus for learning in the absence of live teaching methods.

Current concepts on endothelial stem cells definition, location, and markers.

Ischemic vascular disease is a major cause of mortality and morbidity worldwide, and regeneration of blood vessels in perfusion-deficient tissues is a worthwhile therapeutic goal. The idea of delivering endothelial stem/progenitor cells to repair damaged vasculature, reperfuse hypoxic tissue, prevent cell death, and consequently diminish tissue inflammation and fibrosis has a strong scientific basis and clinical value. Various labs have proposed endothelial stem/progenitor cell candidates. This has created confusion, as there are profound differences between these cell definitions based on isolation methodology, characterization, and reparative biology. Here, a stricter definition based on stem cell biology principles is proposed. Although preclinical studies have often been promising, results from clinical trials have been highly contradictory and served to highlight multiple challenges associated with disappointing therapeutic benefit. This article reviews recent accomplishments in the field and discusses current difficulties when developing endothelial stem cell therapies. Emerging evidence that disputes the classic view of the bone marrow as the source for these cells and supports the vascular wall as the niche for these tissue-resident endothelial stem cells is considered. In addition, novel markers to identify endothelial stem cells, including CD157, EPCR, and CD31low VEGFR2low IL33+ Sox9+ , are described.

miR-130a activates the VEGFR2/STAT3/HIF1α axis to potentiate the vasoregenerative capacity of endothelial colony-forming cells in hypoxia.

Hypoxia modulates reparative angiogenesis, which is a tightly regulated pathophysiological process. MicroRNAs (miRNAs) are important regulators of gene expression in hypoxia and angiogenesis. However, we do not yet have a clear understanding of how hypoxia-induced miRNAs fine-tune vasoreparative processes. Here, we identify miR-130a as a mediator of the hypoxic response in human primary endothelial colony-forming cells (ECFCs), a well-characterized subtype of endothelial progenitors. Under hypoxic conditions of 1% O2, miR-130a gain-of-function enhances ECFC pro-angiogenic capacity in vitro and potentiates their vasoreparative properties in vivo. Mechanistically, miR-130a orchestrates upregulation of VEGFR2, activation of STAT3, and accumulation of HIF1α via translational inhibition of Ddx6. These findings unveil a new role for miR-130a in hypoxia, whereby it activates the VEGFR2/STAT3/HIF1α axis to enhance the vasoregenerative capacity of ECFCs.

The Long Pentraxin PTX3 as a New Biomarker and Pharmacological Target in Age-Related Macular Degeneration and Diabetic Retinopathy.

Age related macular degeneration (AMD) and diabetic retinopathy (DR) are multifactorial, neurodegenerative and inflammatory diseases of the eye primarily involving cellular and molecular components of the outer and inner blood-retina barriers (BRB), respectively. Largely contributed by genetic factors, particularly polymorphisms in complement genes, AMD is a paradigm of retinal immune dysregulation. DR, a major complication of diabetes mellitus, typically presents with increased vascular permeability and occlusion of the retinal vasculature that leads, in the proliferative form of the disease, to neovascularization, a pathogenic trait shared with advanced AMD. In spite of distinct etiology and clinical manifestations, both pathologies share common drivers, such as chronic inflammation, either of immune (in AMD) or metabolic (in DR) origin, which initiates and propagates degeneration of the neural retina, yet the underlying mechanisms are still unclear. As a soluble pattern recognition molecule with complement regulatory functions and a marker of vascular damage, long pentraxin 3 (PTX3) is emerging as a novel player in ocular homeostasis and a potential pharmacological target in neurodegenerative disorders of the retina. Physiologically present in the human eye and induced in inflammatory conditions, this protein is strategically positioned at the BRB interface, where it acts as a "molecular trap" for complement, and modulates inflammation both in homeostatic and pathological conditions. Here, we discuss current viewpoints on PTX3 and retinal diseases, with a focus on AMD and DR, the roles therein proposed for this pentraxin, and their implications for the development of new therapeutic strategies.

Vascular Regeneration for Ischemic Retinopathies: Hope from Cell Therapies.

Retinal vascular diseases, such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, ocular ischemic syndrome and ischemic optic neuropathy, are leading causes of vision impairment and blindness. Whilst drug, laser or surgery-based treatments for the late stage complications of many of these diseases are available, interventions that target the early vasodegenerative stages are lacking. Progressive vasculopathy and ensuing ischemia is an underpinning pathology in many of these diseases, leading to hypoperfusion, hypoxia, and ultimately pathological neovascularization and/or edema in the retina and other ocular tissues, such as the optic nerve and iris. Therefore, repairing the retinal vasculature may prevent progression of ischemic retinopathies into late stage vascular complications. Various cell types have been explored for their vascular repair potential. Endothelial progenitor cells, mesenchymal stem cells and induced pluripotent stem cells are studied for their potential to integrate with the damaged retinal vasculature and limit ischemic injury. Clinical trials for some of these cell types have confirmed safety and feasibility in the treatment of ischemic diseases, including some retinopathies. Another promising avenue is mobilization of endogenous endothelial progenitors, whereby reparative cells are moved from their niche to circulating blood to target and home into ischemic tissues. Several aspects and properties of these cell types have yet to be elucidated. Nevertheless, we foresee that cell therapy, whether through delivery of exogenous or enhancement of endogenous reparative cells, will become a valuable and beneficial treatment for ischemic retinopathies.

Therapies for Type 1 Diabetes: Current Scenario and Future Perspectives.

Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing ß cells located in the endocrine pancreas in areas known as islets of Langerhans. The current standard-of-care for T1D is exogenous insulin replacement therapy. Recent developments in this field include the hybrid closed-loop system for regulated insulin delivery and long-acting insulins. Clinical studies on prediction and prevention of diabetes-associated complications have demonstrated the importance of early treatment and glucose control for reducing the risk of developing diabetic complications. Transplantation of primary islets offers an effective approach for treating patients with T1D. However, this strategy is hampered by challenges such as the limited availability of islets, extensive death of islet cells, and poor vascular engraftment of islets post-transplantation. Accordingly, there are considerable efforts currently underway for enhancing islet transplantation efficiency by harnessing the beneficial actions of stem cells. This review will provide an overview of currently available therapeutic options for T1D, and discuss the growing evidence that supports the use of stem cell approaches to enhance therapeutic outcomes.

Biomechanical comparison of 2 augmented glenoid designs: an integrated kinematic finite element analysis.

BACKGROUND: Augmented glenoid implants are available to help restore the biomechanics of the glenohumeral joint with excessive retroversion. It is imperative to understand their behavior to make a knowledgeable preoperative decision. Therefore, our goal was to identify an optimal augmented glenoid design based on finite element analysis (FEA) under maximum physiological loading. METHODS: FEA models of 2 augmented glenoid designs-wedge and step-were created per the manufacturers' specifications and virtually implanted in a scapula model to correct 20° of retroversion. Simulation of shoulder abduction was performed using the FEA shoulder model. The glenohumeral force ratio, relative micromotion, and stress levels on the cement mantle, glenoid vault, and backside of the implants were compared between the 2 designs. RESULTS: The force ratio was 0.56 for the wedge design and 0.87 for the step design. Micromotion (combination of distraction, translation, and compression) was greater for the step design than the wedge design. Distraction measured 0.05 mm for the wedge design and 0.14 mm for the step component. Both implants showed a similar pattern for translation; however, compression was almost 3 times greater for the step component. Both implants showed high stress levels on the cement mantle. At the glenoid vault and on the implants, the stress levels were 1.65 MPa and 6.62 MPa, respectively, for the wedge design and 3.78 MPa and 13.25 MPa, respectively, for the step design. CONCLUSION: Implant design slightly affects joint stability; however, it plays a major role regarding long-term survival. Overall, the augmented wedge design provides better implant fixation and stress profiles with less micromotion.

The effect of glenohumeral radial mismatch on different augmented total shoulder arthroplasty glenoid designs: a finite element analysis.

BACKGROUND: Augmented glenoid implants to correct bone loss can possibly reconcile current prosthetic failures and improve long-term performance for total shoulder arthroplasty. Biomechanical implant studies have suggested benefits from augmented glenoid components, but limited evidence exists on optimal design. METHODS: An integrated kinematic finite element analysis (FEA) model was used to evaluate optimal augmented glenoid design based on biomechanical performance in translation in the anteroposterior plane similar to clinical loading and failure mechanisms with osteoarthritis. Computer-aided design software models of 2 different commercially available augmented glenoid designs-wedge (Equinox; Exactech, Inc., Gainesville, FL, USA) and step (STEPTECH; DePuy Synthes, Warsaw, IN, USA) were created according to precise manufacturer's dimensions of the implants. Using FEA, they were virtually implanted to correct 20° of retroversion. Two glenohumeral radial mismatches, 3.5/4 mm and 10 mm, were evaluated for joint stability and implant fixation simulating high-risk conditions for failure. RESULTS: The wedged and step designs showed similar glenohumeral joint stability under both radial mismatches. Surrogate for micromotion was a combination of distraction, translation, and compression. With similar behavior and measurements for distraction and translation, compression dictated micromotion (wedge: 3.5 mm = 0.18 mm and 10 mm = 0.10 mm; step: 3.5 mm = 0.19 mm and 10 mm = 0.25 mm). Stress levels on the backside of the implant and on the cement mantle were higher using a step design. DISCUSSION: Greater radial mismatch has the advantage of providing higher glenohumeral stability with tradeoffs, such as higher implant and cement mantle stress levels, and micromotion worse when using a step design.

Cholecystokinin (CCK) and related adjunct peptide therapies for the treatment of obesity and type 2 diabetes.

Cholecystokinin (CCK) is a hormone secreted from I-cells of the gut, as well as neurons in the enteric and central nervous system, that binds and activates CCK-1 and CCK-2 receptors to mediate its biological actions. To date knowledge relating to the physiological significance of CCK has predominantly focused around induction of short-term satiety. However, CCK has also been highlighted to possess important actions in relation to the regulation of insulin secretion, as well as overall beta-cell function and survival. Consequently, this has led to the development of enzymatically stable, biologically active, CCK peptide analogues with proposed therapeutic promise for both obesity and type 2 diabetes. In addition, several studies have demonstrated metabolic, and therapeutically relevant, complementary biological actions of CCK with those of the incretin hormones GIP and GLP-1, as well as with amylin and leptin. Thus, stable CCK derivatives not only offer promise as potential independent weight-reducing and glucose-lowering drugs, but also as effective adjunctive therapies. This review focuses on the recent and ongoing developments of CCK in the context of new therapies for obesity and type 2 diabetes.

Beneficial metabolic effects of dietary epigallocatechin gallate alone and in combination with exendin-4 in high fat diabetic mice.

OBJECTIVE: Significant attempts are being made to generate multifunctional, hybrid or peptide combinations as novel therapeutic strategies for type 2 diabetes, however this presents key challenges including design and pharmaceutical development. In this study, we evaluated metabolic properties of oral nutritional supplement epigallocatechin gallate (EGCG) in combination with GLP-1 agonist exendin-4 in a mouse model of dietary-induced diabetes and obesity. METHODS: EGCG, exendin-4 or combination of both were administered twice-daily over 28 days to high fat (HF) mice on background of low-dose streptozotocin. Energy intake, body weight, fat mass, glucose tolerance, insulin sensitivity, lipid profile, biochemical and hormone markers, and islet histology were examined. RESULTS: All treatment groups exhibited significantly reduced body weight, fat mass, circulating glucose and insulin concentrations, and HbA1c levels which were independent of changes in energy intake. Similarly, there was marked improvement in glycaemic control, glucose-stimulated insulin release, insulin sensitivity, total cholesterol and triglycerides, with most prominent effects observed following combination therapy. Circulating corticosterone concentrations and 11beta-hydroxysteroid dehydrogenase type1 (11ß-HSD1) staining (in pancreas) were beneficially decreased without changes in circulating interleukin 6 (IL-6), alanine transaminase (ALT) and glutathione reductase. Combination therapy resulted in increased islet area and number, beta cell area, and pancreatic insulin content. Generally, metabolic effects were much more pronounced in mice which received combination therapy. CONCLUSIONS: EGCG alone and particularly in combination with exendin-4 exerts positive metabolic properties in HF mice. EGCG may be useful dietary adjunct alongside GLP-1 mimetics in treatment of diabetes and related disorders.

Metabolic and neuroprotective effects of dapagliflozin and liraglutide in diabetic mice.

This study assessed the metabolic and neuroprotective actions of the sodium glucose cotransporter-2 inhibitor dapagliflozin in combination with the GLP-1 agonist liraglutide in dietary-induced diabetic mice. Mice administered low-dose streptozotocin (STZ) on a high-fat diet received dapagliflozin, liraglutide, dapagliflozin-plus-liraglutide (DAPA-Lira) or vehicle once-daily over 28 days. Energy intake, body weight, glucose and insulin concentrations were measured at regular intervals. Glucose tolerance, insulin sensitivity, hormone and biochemical analysis, dual-energy X-ray absorptiometry densitometry, novel object recognition, islet and brain histology were examined. Once-daily administration of DAPA-Lira resulted in significant decreases in body weight, fat mass, glucose and insulin concentrations, despite no change in energy intake. Similar beneficial metabolic improvements were observed regarding glucose tolerance, insulin sensitivity, HOMA-IR, HOMA-ß, HbA1c and triglycerides. Plasma glucagon, GLP-1 and IL-6 levels were increased and corticosterone concentrations decreased. DAPA-Lira treatment decreased alpha cell area and increased insulin content compared to dapagliflozin monotherapy. Recognition memory was significantly improved in all treatment groups. Brain histology demonstrated increased staining for doublecortin (number of immature neurons) in dentate gyrus and synaptophysin (synaptic density) in stratum oriens and stratum pyramidale. These data demonstrate that combination therapy of dapagliflozin and liraglutide exerts beneficial metabolic and neuroprotective effects in diet-induced diabetic mice. Our results highlight important personalised approach in utilising liraglutide in combination with dapagliflozin, instead of either agent alone, for further clinical evaluation in treatment of diabetes and associated neurodegenerative disorders.

Sequential induction of beta cell rest and stimulation using stable GIP inhibitor and GLP-1 mimetic peptides improves metabolic control in C57BL/KsJ db/db mice.

AIMS/HYPOTHESIS: GIP(6-30)Cex-K(40)[Pal] has been characterised as a fatty-acid-derived gastric inhibitory polypeptide (GIP) inhibitor that can induce pancreatic beta cell rest by diminishing the incretin effect. We investigated its therapeutic efficacy with and without the glucagon-like peptide-1 (GLP-1) beta cell cytotropic agent liraglutide. METHODS: The therapeutic efficacy of GIP(6-30)Cex-K(40)[Pal] alone, and in combination with liraglutide, was determined in C57BL/KsJ db/db mice using a sequential 12 h administration schedule. RESULTS: GIP(6-30)Cex-K(40)[Pal] was devoid of cAMP-generating or insulin-secretory activity, and inhibited GIP-induced cAMP production and insulin secretion. GIP(6-30)Cex-K(40)[Pal] also inhibited GIP-induced glucose-lowering and insulin-releasing actions in mice. Dose- and time-dependent studies in mice revealed that 2.5 nmol/kg GIP(6-30)Cex-K(40)[Pal], and 0.25 nmol/kg liraglutide, imparted distinct biological effects for 8-12 h post administration. When GIP(6-30)Cex-K(40)[Pal] (2.5 nmol/kg) and liraglutide (0.25 nmol/kg) were administered sequentially at 12 h intervals (at 08:00 and 20:00 hours) to db/db mice for 28 days, mice treated with GIP(6-30)Cex-K(40)[Pal] (08:00 hours) and liraglutide (20:00 hours) displayed pronounced reductions in circulating glucose and insulin. Both oral and intraperitoneal glucose tolerance and glucose-stimulated plasma insulin concentrations were improved together with enhanced insulin sensitivity. The expression of genes involved in adipocyte lipid deposition was generally decreased. The other treatment modalities, including GIP(6-30)Cex-K(40)[Pal] (08:00 and 20:00 hours), liraglutide (08:00 and 20:00 hours) and liraglutide (08:00 hours) combined with GIP(6-30)Cex-K(40)[Pal] (20:00 hours), also imparted beneficial effects but these were not as prominent as those of GIP(6-30)Cex-K(40)[Pal] (08:00 hours) and liraglutide (20:00 hours). CONCLUSION/INTERPRETATION: These data demonstrate that periods of beta cell rest combined with intervals of beta cell stimulation benefit diabetes control and should be further evaluated as a potential treatment option for type 2 diabetes.

A Novel CCK-8/GLP-1 Hybrid Peptide Exhibiting Prominent Insulinotropic, Glucose-Lowering, and Satiety Actions With Significant Therapeutic Potential in High-Fat-Fed Mice.

Glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) exert important complementary beneficial metabolic effects. This study assessed the biological actions and therapeutic utility of a novel (pGlu-Gln)-CCK-8/exendin-4 hybrid peptide compared with the stable GLP-1 and CCK mimetics exendin-4 and (pGlu-Gln)-CCK-8, respectively. All peptides significantly enhanced in vitro insulin secretion. Administration of the peptides, except (pGlu-Gln)-CCK-8 alone, in combination with glucose significantly lowered plasma glucose and increased plasma insulin in mice. All treatments elicited appetite-suppressive effects. Twice-daily administration of the novel (pGlu-Gln)-CCK-8/exendin-4 hybrid, (pGlu-Gln)-CCK-8 alone, or (pGlu-Gln)-CCK-8 in combination with exendin-4 for 21 days to high-fat-fed mice significantly decreased energy intake, body weight, and circulating plasma glucose. HbA1c was reduced in the (pGlu-Gln)-CCK-8/exendin-4 hybrid and combined parent peptide treatment groups. Glucose tolerance and insulin sensitivity also were improved by all treatment modalities. Interestingly, locomotor activity was decreased in the hybrid peptide group, and these mice also exhibited reductions in circulating triglyceride and cholesterol levels. Pancreatic islet number and area, as well ß-cell area and insulinotropic responsiveness, were dramatically improved by all treatments. These studies highlight the clear potential of dual activation of GLP-1 and CCK1 receptors for the treatment of type 2 diabetes.

A novel DPP IV-resistant C-terminally extended glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through glucagon and GLP-1 receptor activation.

AIMS/HYPOTHESIS: Modification of the structure of glucagon could provide useful compounds for the potential treatment of obesity-related diabetes. METHODS: This study evaluated N-acetyl-glucagon, (D-Ser(2))glucagon and an analogue of (D-Ser(2))glucagon with the addition of nine amino acids from the C-terminal of exendin(1-39), namely (D-Ser(2))glucagon-exe. RESULTS: All analogues were resistant to dipeptidyl peptidase IV degradation. N-Acetyl-glucagon lacked acute insulinotropic effects in BRIN BD11 cells, whereas (D-Ser(2))glucagon and (D-Ser(2))glucagon-exe evoked significant (p < 0.001) insulin release. (D-Ser(2))glucagon-exe stimulated cAMP production (p < 0.001) in glucagon- and GLP-1-receptor (GLP-1R)-transfected cells but not in glucose-dependent insulinotropic polypeptide-receptor-transfected cells. In normal mice, N-acetyl-glucagon and (D-Ser(2))glucagon retained glucagon-like effects of increasing (p < 0.001) plasma glucose and insulin levels. (D-Ser(2))glucagon-exe was devoid of hyperglycaemic actions but substantially (p < 0.001) increased plasma insulin levels. (D-Ser(2))glucagon-exe reduced the glycaemic excursion (p < 0.01) and increased the insulin secretory (p < 0.01) response following a glucose challenge 12 h after administration. Studies in GLP-1R knockout mice confirmed involvement of the GLP-1R pathway in the biological actions of (D-Ser(2))glucagon-exe. Twice-daily administration of (D-Ser(2))glucagon-exe to high-fat-fed mice for 28 days significantly (p < 0.05 to p < 0.001) reduced body weight, energy intake and non-fasting glucose levels, as well as increasing insulin concentrations. Glucose tolerance and insulin sensitivity were significantly (p < 0.01) improved and energy expenditure, O2 consumption and locomotor activity were (p < 0.05 to p < 0.001) augmented. The metabolic benefits were accompanied by increases in pancreatic islet number (p < 0.001) and area (p < 0.05), as well as beta cell area (p < 0.05). Beneficial effects were largely retained for 14 days following cessation of treatment. CONCLUSIONS/INTERPRETATION: This study emphasises the potential of (D-Ser(2))glucagon-exe for the treatment of obesity-related diabetes.