Animal Models Of Diabetes Research Articles

Diabetes-Induced Amplification of Nociceptive DRG Neuron Output by Upregulation of Somatic T-Type Ca2+ Channels.

The development of pain symptoms in peripheral diabetic neuropathy (PDN) is associated with the upregulation of T-type Ca2+ channels (T-channels) in the soma of nociceptive DRG neurons. Moreover, a block of these channels in DRG neurons effectively reversed mechanical and thermal hyperalgesia in animal diabetic models, indicating that T-channel functioning in these neurons is causally linked to PDN. However, no particular mechanisms relating the upregulation of T-channels in the soma of nociceptive DRG neurons to the pathological pain processing in PDN have been suggested. Here we have electrophysiologically identified voltage-gated currents expressed in nociceptive DRG neurons and developed a computation model of the neurons, including peripheral and central axons. Simulations showed substantially stronger sensitivity of neuronal excitability to diabetes-induced T-channel upregulation at the normal body temperature compared to the ambient one. We also found that upregulation of somatic T-channels, observed in these neurons under diabetic conditions, amplifies a single action potential invading the soma from the periphery into a burst of multiple action potentials further propagated to the end of the central axon. We have concluded that the somatic T-channel-dependent amplification of the peripheral nociceptive input to the spinal cord demonstrated in this work may underlie abnormal nociception at different stages of diabetes development.

A Universal Hydrogel Microneedle for on-Site Detection of Small Molecules, Proteins, and Ribonucleic Acids

Point-of-care testing (POCT) of clinical biomarkers is critical to health monitoring and timely treatment, yet biosensing assays capable of detecting biomarkers without the need for costly external equipment and reagents are limited. Blood-based assays are, specifically, challenging as blood collection is invasive, and several processing steps are required. A promising alternative to blood is interstitial fluid (ISF), which originates from blood and fills the extracellular space; thus, it has common biomarkers with plasma/serum while also containing biomarkers unique to the local cells. A wide range of metabolites, including amino acids, lipids, nucleotides, and protein biomarkers, can be detected in ISF, emphasizing the potential of ISF for health monitoring. Microneedle (MN)-based biosensors have recently emerged as a promising approach for investigating the ISF. They enable minimally invasive penetration through the skin for ISF access. Specifically, Hydrogel-based MNs have two main advantages; i) their fabrication is cost-effective and simple, ii) they are highly biocompatible. Therefore, they overcome the challenges with conventional solid MNs. However, Hydrogel Microneedles (HMN) capable of both extracting ISF and sensing an analyte on-microneedle (without any need for post-sampling processing) have been limited. Here, we report a versatile assay that employs HMNs to both extract (ISF) and perform on-MN sensing of a specific analyte/biomarker in a minimally invasive manner. Our assay incorporates graphene oxide-nucleic acid (GO.NA) optical sensors for sensing. The GO.NA optical sensor consists of GO nanosheets conjugated to fluorophore-modified nucleic acid (NA), in which GO acts as a quencher. Single-stranded NAs have high affinity for binding to GO, therefore, in the absence of the biomarker of interest the NAs are tightly bound to GO; in turn, their fluorophore tag is quenched. However, in the presence of a specific biomarker, the NAs bind to their target, inducing a conformational change that distances the fluorophore tag from GO, leading to fluorescence recovery and generation of an optical signal. GO also acts as a substrate for NA immobilization; therefore, it protects the NA from degradation and prevents any potential NA release from HMN-GO.NA. To enable microscope-free patch visualization and optical measurements for POCT, we developed a miniaturized smartphone-based system that captures fluorescence images of the HMN-GO.NA patches (Scheme 1A and 1B), which are then analyzed using freely available software (ImageJ). Our system could successfully measure six clinically important biomarkers (glucose, uric acid, insulin, and serotonin as well as microribonucleic acid 210 and 21) in-vitro (using phantom gel) and ex-vivo (using porcine skin). Further, we have been able to accurately detect glucose and uric acid in live diabetic animal models, proving the efficacy of our system. Figure 1

Updates on RPE cell damage in diabetic retinopathy (Review).

Diabetic retinopathy (DR) is a microvascular complication of diabetes. The retinal pigment epithelium (RPE) forms the outer layer of the blood‑retinal barrier and serves a role in maintaining retinal function. RPE cell injury has been revealed in diabetic animal models, and high glucose (HG) levels may cause damage to RPE cells by increasing the levels of oxidative stress, promoting pro‑inflammatory gene expression, disrupting cell proliferation, inducing the endothelial‑mesenchymal transition, weakening tight conjunctions and elevating cell death mechanisms, such as apoptosis, ferroptosis and pyroptosis. Non‑coding RNAs including microRNAs, long non‑coding RNAs and circular RNAs participate in RPE cell damage caused by HG levels, which may provide targeted therapeutic strategies for the treatment of DR. Plant extracts such as citrusin and hesperidin, and a number of hypoglycemic drugs, such as sodium‑glucose co‑transporter 2 inhibitors, metformin and glucagon‑like peptide‑1 receptor agonists, exhibit potential RPE protective effects; however, the detailed mechanisms behind these effects remain to be fully elucidated. An in‑depth understanding of the contribution of the RPE to DR may provide novel perspectives and therapeutic targets for DR.

ANIMAL MODELS OF DIABETIC NEPHROPATHY

Diabetic nephropathy (DN) is the most common complication of diabetes mellitus (DM). It is characterized by high blood glucose levels or hyperglycemia and is accompanied by changes in lipid, carbohydrate, and protein metabolism which can lead to an increased risk of complications due to vascular disease. DN is probably the most insidious among these complications, causing substantial morbidity and mortality. In this article, we will review the literature on animal models of diabetes. We will discuss several species as animal models for Type 1 and 2 diabetes, including zebrafish, rabbits, mice, rats, and rat models. This article also provides various methods used in research with model animals and presents the required result for studying diabetic DN.

A critical review on therapeutic approaches of CRISPR-Cas9 in diabetes mellitus.

Diabetes mellitus (D.M.) is a common metabolic disorder caused mainly by combining two primary factors, which are (1) defects in insulin production by the pancreatic β-cells and (2) responsiveness of insulin-sensitive tissues towards insulin. Despite the rapid advancement in medicine to suppress elevated blood glucose levels (hyperglycemia) and insulin resistance associated with this hazard, a demand has undoubtedly emerged to find more effective and curative dimensions in therapeutic approaches against D.M. The administration of diabetes treatment that emphasizes insulin production and sensitivity may result in unfavorable side effects, reduced adherence, and potential treatment ineffectiveness. Recent progressions in genome editing technologies, for instance, in zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR-Cas)-associated nucleases, have greatly influenced the gene editing technology from concepts to clinical practices. Improvements in genome editing technologies have also opened up the possibility to target and modify specific genome sequences in a cell directly. CRISPR/Cas9 has proven effective in utilizing ex vivo gene editing in embryonic stem cells and stem cells derived from patients. This application has facilitated the exploration of pancreatic beta-cell development and function. Furthermore, CRISPR/Cas9 enables the creation of innovative animal models for diabetes and assesses the effectiveness of different therapeutic strategies in treating the condition. We, therefore, present a critical review of the therapeutic approaches of the genome editing tool CRISPR-Cas9 in treating D.M., discussing the challenges and limitations of implementing this technology.

Biological Evidence of Improved Wound Healing Using Autologous Micrografts in a Diabetic Animal Model

Background: Tissue healing consists of four main phases: coagulation, inflammation, proliferation, and remodeling. In diabetic patients, this process is stagnant in the inflammatory stage, leading to chronic wounds. The aim of this study is to evaluate in an animal model the biological evidence related to the use of the Rigenera® technology (Turin Italy), an innovative mechanical procedure to isolate autologous micrografts (AMG). Methods: Fifty male Wistar rats were divided into four groups: control (C), control treated with micrografts (CM), diabetic (DB), and diabetic treated with micrografts (DBM). The experimental setup involved: the quantification of the total collagen and elastic fibers; histopathological analysis; immunohistochemical analysis for collagen type I (COL1), collagen type III (COL3), vascular endothelial growth factor (VEGF-A), and interleukin 4 (IL4) and 10 (IL10); evaluation of the oxidative stress; measurement of gluthatione (GSH); and, finally, an enzyme-linked immunosorbent assay (ELISA) on tumor necrosis factor-α (TNF-α). Results: The AMG technology induces a faster healing process: VEGF-A, IL4, IL10, and GSH increased, while TNF-α and oxidative stress decreased. Conclusions: Animals treated with micrografts showed more favorable results for healing compared to those that did not receive treatment, demonstrating a positive participation of the micrografts in the treatment of difficult-to-heal wounds.

Integrin β5 subunit regulates hyperglycemia-induced vascular endothelial cell apoptosis through FoxO1-mediated macroautophagy.

Hyperglycemia frequently induces apoptosis in endothelial cells and ultimately contributes to microvascular dysfunction in patients with diabetes mellitus (DM). Previous research reported that the expression of integrins as well as their ligands was elevated in the diseased vessels of DM patients. However, the association between integrins and hyperglycemia-induced cell death is still unclear. This research was designed to investigate the role played by integrin subunit β5 (ITGB5) in hyperglycemia-induced endothelial cell apoptosis. We used leptin receptor knockout (Lepr-KO) ( db / db ) mice as spontaneous diabetes animal model. Selective deletion of ITGB5 in endothelial cell was achieved by injecting vascular targeted adeno-associated virus via tail vein. Besides, we also applied small interfering RNA in vitro to study the mechanism of ITGB5 in regulating high glucose-induced cell apoptosis. ITGB5 and its ligand, fibronectin, were both upregulated after exposure to high glucose in vivo and in vitro . ITGB5 knockdown alleviated hyperglycemia-induced vascular endothelial cell apoptosis and microvascular rarefaction in vivo.In vitro analysis revealed that knockdown of either ITGB5 or fibronectin ameliorated high glucose-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). In addition, knockdown of ITGB5 inhibited fibronectin-induced HUVEC apoptosis, which indicated that the fibronectin-ITGB5 interaction participated in high glucose-induced endothelial cell apoptosis. By using RNA-sequencing technology and bioinformatic analysis, we identified Forkhead Box Protein O1 (FoxO1) as an important downstream target regulated by ITGB5. Moreover, we demonstrated that the excessive macroautophagy induced by high glucose can contribute to HUVEC apoptosis, which was regulated by the ITGB5-FoxO1 axis. The study revealed that high glucose-induced endothelial cell apoptosis was positively regulated by ITGB5, which suggested that ITGB5 could potentially be used to predict and treat DM-related vascular complications.

Metabolite profiling and biochemical investigation of the antidiabetic potential of Loranthus pulverulentus Wall n-butanol fraction in diabetic animal models

Ethnopharmacological relevanceGlobally, 537 million individuals are estimated to have diabetes. The traditional use of herbs for ameliorating diabetes symptoms is a common practice in Pakistan and use of Loranthus pulverulentus Wall (L. pulverulentus) by local people in Azad Jammu and Kashmir has been reported. Aim of the studyIn the present study, the antidiabetic potential of standardized n-butanol fraction of leaves of L. pulverulentus Wall, which is a parasite of Dalbergia sisso Roxb was assessed in both alloxan (ALX) and streptozotocin (STZ) diabetic animal models. Materials and methodsChemical characterization of BF was performed using HPLC, GCMS and UHPLC-MS. The effect of the fraction (250 mg/kg) on insulin, plasma free fatty acids, L-lactate, pyruvate, MDA, HbA1c and glycogen levels in ALX and STZ animal models was determined. Liver and renal profiles were analyzed in the STZ model. Toxicological studies were performed by hemolytic, Ames mutagenicity, DNA protection, and thrombolytic assays. Histopathological analysis of the pancreas, liver, and kidney was performed. ResultsBF demonstrated highly significant (p < 0.001) antidiabetic potential in both diabetic models. BF significantly (p < 0.05) improved OGTT results in alloxanized diabetic mice and blocked the absorption of glucose from the gut. A significant (p < 0.001) increase in insulin levels and glycogen content in liver tissue and a decrease in plasma FFA, MDA, HbA1c, L-lactate, and pyruvate levels in STZ-diabetic mice were recorded. GC-MS and chromatographic analysis showed the presence of catechin, eugenol, longifolene, caryophyllene, Ar-tumerone and Geranyl-alpha-terpinene. Various metabolites with antidiabetic potential, including 4-hydroxycinnamyl alcohol 4-D-glucoside, zingerone glucoside, trans-trismethoxy resveratrol-d4, epigallocatechin 3-O-cinnamate, and β-glucogallin, were identified using UHPLC-MS. Animals treated with BF showed marked improvements in cellular structures of the pancreas, liver and kidneys. This fraction is non-mutagenic and protects the DNA. ConclusionThe experimental fraction contained potential antidiabetic bioactive compounds responsible for alleviating diabetes-associated biochemical dysregulation. The fraction increased insulin levels and enhanced glycogen storage in muscles and the liver. It blocked glucose absorption from the intestine and substantially decreased HbA1c, lactate, pyruvate, free fatty acids, lipid, liver and kidney damage. Therefore, the use of BF for the treatment of type-2 diabetes may be beneficial.

Phosphorylation of αB-Crystallin Involves Interleukin-1β-Mediated Intracellular Retention in Retinal Müller Cells: A New Mechanism Underlying Fibrovascular Membrane Formation.

Chronic inflammation plays a pivotal role in the pathology of proliferative diabetic retinopathy (PDR), in which biological alterations of retinal glial cells are one of the key elements. The phosphorylation of αB-crystallin/CRYAB modulates its molecular dynamics and chaperone activity, and attenuates αB-crystallin secretion via exosomes. In this study, we investigated the effect of phosphorylated αB-crystallin in retinal Müller cells on diabetic mimicking conditions, including interleukin (IL)-1β stimuli. Human retinal Müller cells (MIO-M1) were used to examine gene and protein expressions with real-time quantitative PCR, enzyme linked immunosorbent assay (ELISA), and immunoblot analyses. Cell apoptosis was assessed by Caspase-3/7 assay and TdT-mediated dUTP nick-end labeling staining. Retinal tissues isolated from the Spontaneously Diabetic Torii (SDT) fatty rat, a type 2 diabetic animal model with obesity, and fibrovascular membranes from patients with PDR were examined by double-staining immunofluorescence. CRYAB mRNA was downregulated in MIO-M1 cells with the addition of 10ng/mL IL-1β; however, intracellular αB-crystallin protein levels were maintained. The αB-crystallin serine 59 (Ser59) residue was phosphorylated with IL-1β application in MIO-M1 cells. Cell apoptosis in MIO-M1 cells was induced by CRYAB knockdown. Immunoreactivity for Ser59-phosphorylated αB-crystallin and glial fibrillary acidic protein was colocalized in glial cells of SDT fatty rats and fibrovascular membranes. The Ser59 phosphorylation of αB-crystallin was modulated by IL-1β in Müller cells under diabetic mimicking inflammatory conditions, suggesting that αB-crystallin contributes to the pathogenesis of PDR through an anti-apoptotic effect.

CXCL5 suppression recovers neovascularization and accelerates wound healing in diabetes mellitus

BackgroundHigher chemokine C-X-C motif ligand 5 (CXCL5) level was observed in type 2 diabetes mellitus (DM) patients; however, its role in diabetic vasculopathy was not clarified. This study aimed to explore the impacts and mechanistic insights of CXCL5 in neovasculogenesis and wound healing in DM.MethodsEndothelial progenitor cells (EPCs) and human aortic endothelial cells (HAECs) were used in vitro. Streptozotocin-induced diabetic mice and Leprdb/JNarl mice were used as type 1 and type 2 DM models. Moreover, CXCL5 knockout mice were used to generate diabetic mice. Hindlimb ischemia surgery, aortic ring assays, matrigel plug assay, and wound healing assay were conducted.ResultsCXCL5 concentrations were increased in plasma and EPCs culture medium from type 2 DM patients. CXCL5 neutralizing antibody upregulated vascular endothelial growth factor (VEGF)/stromal cell-derived factor-1 (SDF-1) and promoted cell function in EPCs from type 2 DM patients and high glucose-treated EPCs from non-DM subjects as well as HAECs. CXCL5 directly up-regulated interleukin (IL)-1β/IL-6/tumor necrosis factor-α and down-regulated VEGF/SDF-1 via ERK/p65 activation through chemokine C-X-C motif receptor 2 (CXCR2). CXCL5 neutralizing antibody recovered the blood flow after hindlimb ischemia, increased circulating EPC number, and enhanced VEGF and SDF-1 expression in ischemic muscle. CXCL5 suppression promoted neovascularization and wound healing in different diabetic animal models. The above observation could also be seen in streptozotocin-induced CXCL5 knockout diabetic mice.ConclusionsCXCL5 suppression could improve neovascularization and wound healing through CXCR2 in DM. CXCL5 may be regarded as a potential therapeutic target for vascular complications of DM.

Therapeutic potential of elema-1,3,7(11),8-tetraen-8,12-lactam from Curcuma wenyujin on diabetic retinopathy via anti-inflammatory and anti-angiogenic pathways

Ethnopharmacological relevanceIn traditional Chinese medicine, the causes of diabetic retinopathy (DR) are blood stasis and heat. Curcuma wenyujin Y. H. Chen & C. Ling and its extracts have the effects of promoting blood circulation to remove blood stasis, clearing the heart, and cooling the blood, and have been used in the treatment of DR. Elema-1,3,7 (11),8-tetraen-8,12-lactam (Ele), an N-containing sesquiterpene isolated from this plant. However, the anti-inflammatory and anti-angiogenic effects of Ele and its therapeutic potential in DR are still unknown. Aim of the studyTo evaluate the anti-inflammatory and anti-angiogenic effects of Ele and its therapeutic potential in DR. Materials and methodsIn vitro, anti-inflammatory and anti-angiogenic effects were assessed using TNF-α or VEGF-stimulated HUVECs. Protein expression was analyzed using Western blotting. ICAM-1 and TNF-α mRNA expressions were analyzed using real-time quantitative RT-PCR. The therapeutic potential in DR was assessed using both animal models of STZ-induced diabetes and oxygen-induced retinopathy. The retinal vascular permeability was measured using Evans blue, and the quantitation of retinal leukostasis using FITC-coupled Con A. The retinal neovascular tufts were analyzed using fluorescein angiography and counting pre-retinal vascular lumens. ResultsEle inhibited NF-κB pathway, and ICAM-1, TNF-α mRNA expression in TNF-α- stimulated HUVECs. It also inhibits the multistep process of angiogenesis by inhibiting the phosphorylation of VEGFR2 and its downstream signaling kinases Src, Erk1/2, Akt, and mTOR in VEGF-stimulated HUVECs. Intravitreal injection of Ele can significantly reduce retinal microvascular leakage, leukostasis, and expression of ICAM-1, TNF-α in diabetic rats and inhibits oxygen-induced retinal neovascularization and VEGFR2 phosphorylation in OIR mice. ConclusionsEle has anti-inflammatory and anti-angiogenic effects through inhibiting NF-κB and VEGFR2 signaling pathways, and it may be a potential drug candidate for DR.

Contribution of animal models to diabetes research: Its history, significance, and translation to humans.

Diabetes mellitus is still expanding globally and is epidemic in developing countries. The combat of this plague has caused enormous economic and social burdens related to a lowered quality of life in people with diabetes. Despite recent significant improvements of life expectancy in patients with diabetes, there is still a need for efforts to elucidate the complexities and mechanisms of the disease processes to overcome this difficult disorder. To this end, the use of appropriate animal models in diabetes studies is invaluable for translation to humans and for the development of effective treatment. In this review, a variety of animal models of diabetes with spontaneous onset in particular will be introduced and discussed for their implication in diabetes research.

Characterization of a S1PR2 specific 11C-labeled radiotracer in streptozotocin-induced diabetic murine model

BackgroundDiabetes mellitus is a chronic progressive metabolic disorder that affects millions of people worldwide. Emerging evidence suggests the important roles of sphingolipid metabolism in diabetes. In particular, sphingosine-1-phosphate (S1P) and S1P receptor 2 (S1PR2) have important metabolic functions and are involved in several metabolic diseases. In diabetes, S1PR2 can effectively preserve β cells and improve glucose/insulin tolerance in high-fat diet induced and streptozotocin (STZ)-induced diabetic mouse models. We previously developed a group of potent and selective S1PR2 ligands and radioligands. MethodsIn this study, we continued our efforts and characterized our leading S1PR2 radioligand, [11C]TZ34125, in a STZ-induced diabetic mouse model. [11C]TZ34125 was radiosynthesized in an automated synthesis module and in vitro saturation binding assay was performed using recombinant human S1PR2 membrane. In vitro saturation autoradiography analysis was also performed to determine the binding affinity of [11C]TZ34125 against mouse tissues. Type-1 diabetic mouse model was developed following a single high dose of STZ in C57BL/6 mice. Ex vivo biodistribution was performed to evaluate the distribution and amount of [11C]TZ34125 in tissues. In vitro autoradiography analysis was performed to compare the uptake of [11C]TZ34125 between diabetic and control animals in mouse spleen and pancreas. ResultsOur in vitro saturation binding assay using [11C]TZ34125 confirmed [11C]TZ34125 is a potent radioligand to recombinant human S1PR2 membrane with a Kd value of 0.9 nM. Saturation autoradiographic analysis showed [11C]TZ34125 has a Kd of 67.5, 45.9, and 25.0 nM to mouse kidney, spleen, and liver tissues respectively. Biodistribution study in STZ-induced diabetic mice showed the uptake of [11C]TZ34125 was significantly elevated in the spleen (~2 fold higher) and pancreas (~1.4 fold higher) compared to normal controls. The increased uptake of [11C]TZ34125 was further confirmed using autoradiographic analysis in the spleen and pancreases of STZ-induced diabetic mice, indicating S1PR2 can potentially act as a biomarker of diabetes in pancreases and inflammation in spleen. Future mechanistic analysis and in vivo quantitative assessment using non-invasive PET imaging in large animal model of diabetes is worthwhile. ConclusionsOverall, our data showed an increased uptake of our lead S1PR2-specific radioligand, [11C]TZ34125, in the spleen and pancreases of STZ-induced diabetic mice, and demonstrated [11C]TZ34125 has a great potential for preclinical and clinical usage for assessment of S1PR2 in diabetes and inflammation.

Reactive oxygen species linked diabetes mellitus management by natural products: A Pre-clinical study in rat

The environment has a great impact on health maintenance. Environmental stress factors generate reactive oxygen species (ROS) inour bodies. As a result, pro-oxidant status of our body is dominated over the antioxidant status that induces health disorders. Diabetesmellitus (DM), a common metabolic cum lifestyle disease, is now considered as X syndrome. Environmental stressors through ROSgeneration can impose point mutation for the production of mutant enzymes, leading to cellular metabolic disorders, especiallycarbohydrate metabolism, an important cause of diabetes. Moreover, free radicals through cellular signaling system down-regulategene expression of antioxidant enzymes that produce high levels of cellular free radicals in metabolic organs, which may lead to DM.Natural products with strong antioxidant activities have a major role in managing diabetes. Nutraceuticals in E. jumbulana, Camelliasinensis, H. antidysenterica majorly contribute to such management by increasing gene expression of antioxidant enzymes andcarbohydrate metabolic enzymes that favor glucose utilization in cell. It has also been established that such nutraceuticals can generate β cells from hepatic stem cells, so plasma insulin and c-peptide levels are elevated. Insulin receptor gene expression is also corrected by such nutraceuticals in diabetic model animals. The efficacy of such nutraceuticals is comparable with antidiabetic gold standard drugs. Such studies have been confirmed by genomics and proteomics studies using real-time PCR followed by western blotting studies. This field of research unfolds a new domain known as ‘Neutrogenomics’ in health science.It is concluded that natural products may be the parallel management process of modern therapy as per the guideline of WHO.

SAR and lead optimization of (Z)-5-(4-hydroxy-3-methoxybenzylidene)-3-(2-morpholinoacetyl)thiazolidine-2,4-dione as a potential multi-target antidiabetic agent

In case of metabolic disorder like Diabetes mellitus (DM), a number of key enzymes are abnormally expressed and hence they might be excellent targets for antidiabetic drug design. Multi-target design strategy has recently attracted great attention to treat challenging diseases. We have previously reported a vanillin-thiazolidine-2,4-dione hybrid 3 as multitarget inhibitor of α-glucosidase, α-amylase, PTP-1B and DPP-4. The reported compound predominantly exhibited good in-vitro DPP-4 inhibition only. Current research describes the goal to optimize an early lead compound. The efforts were focused on enhancing the capability of manipulating multiple pathways at the same time for the treatment of diabetes. The central 5-benzylidinethiazolidine-2,4-dione for Lead compound (Z)-5-(4-hydroxy-3-methoxybenzylidene)-3-(2-morpholinoacetyl)thiazolidine-2,4-dione (Z-HMMTD) was left unchanged. While East and West moieties were altered by the introduction of different building blocks conceived by using a number of rounds of predictive docking studies performed on X-ray crystal structures of four target enzymes. This systematic SAR led to the syntheses of new potent multi-target antidiabetic compounds 47–49 and 55–57 with many fold increase in the in-vitro potency compared to Z-HMMTD. The potent compounds showed good in-vitro and in-vivo safety profile. Compound 56 emerged excellent as glucose-uptake promotor via hemi diaphragm of the rat. Moreover, the compounds demonstrated antidiabetic activity in STZ-induced diabetic animal model.

Protein Extract of a Probiotic Strain of Hafnia alvei and Bacterial ClpB Protein Improve Glucose Tolerance in Mice

A commercial strain of Hafnia alvei (H. alvei) 4597 bacteria was shown to reduce food intake and promote weight loss, effects possibly induced by the bacterial protein ClpB, an antigen-mimetic of the anorexigenic α-melanocyte-stimulating hormone. A decrease in the basal plasma glucose levels was also observed in overweight fasted humans and mice receiving H. alvei. However, it is not known whether H. alvei influences sweet taste preference and whether its protein extract or ClpB are sufficient to increase glucose tolerance; these are the objectives tested in the present study. C57BL/6J male mice were kept under standard diet and were gavaged daily for 17 days with a suspension of H. alvei (4.5 × 107 CFU/animal) or with H. alvei total protein extract (5 μg/animal) or saline as a control. Sweet taste preference was analyzed via a brief-access licking test with sucrose solution. Glucose tolerance tests (GTT) were performed after the intraperitoneal (IP) or intragastric (IG) glucose administration at the 9th and 15th days of gavage, respectively. The expression of regulatory peptides’ mRNA levels was assayed in the hypothalamus. In another experiment performed in non-treated C57BL/6J male mice, effects of acute IP administration of recombinant ClpB protein on glucose tolerance were studied by both IP- and IG-GTT. Mice treated with the H. alvei protein extract showed an improved glucose tolerance in IP-GTT but not in IG-GTT. Both groups treated with H. alvei bacteria or protein extract showed a reduction of pancreatic tissue weight but without significant changes to basal plasma insulin. No significant effects of H. alvei bacteria or its total protein extract administration were observed on the sweet taste preference, insulin tolerance and expression of regulatory peptides’ mRNA in the hypothalamus. Acute administration of ClpB in non-treated mice increased glucose tolerance during the IP-GTT but not the IG-GTT, and reduced basal plasma glucose levels. We conclude that both the H. alvei protein extract introduced orally and the ClpB protein administered via IP improve glucose tolerance probably by acting at the glucose postabsorptive level. Moreover, H. alvei probiotic does not seem to influence the sweet taste preference. These results justify future testing of both the H. alvei protein extract and ClpB protein in animal models of diabetes.

Effects of aerobic training and vitamin D supplementation on glycemic indices and adipose tissue gene expression in type 2 diabetic rats

Type 2 diabetes mellitus (T2DM) is a progressive metabolic disorder mainly caused by overweight and obesity that accumulates pro-inflammatory factors in adipose tissue. Studies have confirmed the efficacy of exercise and vitamin D supplementation in preventing, controlling, and treating diabetes. While, reduced physical activity and vitamin D deficiency are related to increased adiposity, blood glucose level, insulin concentration, and insulin resistance. This study purposed to investigate the effect of 8-week aerobic training with vitamin D supplementation on the expression of AMPK, PGC-1α, and UCP-1 genes expression in the visceral adipose tissue of obese rats with T2DM. In this experimental study, fifty male Wistar rats were divided into 5 groups (n = 10): aerobic training and vitamin D supplementation (AT + Vit D), aerobic training (5 days/week for 8 weeks; AT), vitamin D supplementation (Vit D), diabetic control (C) and NC (Non-Diabetic Control). AT + Vit D and AT groups practiced an 8-week aerobic training, 5 days a week. Vit D and AT + Vit D groups receive 5000 IU of vitamin D by injection once a week while AT and C received sesame oil. After blood sampling, visceral fat was taken to measure AMPK, PGC-1α, and UCP1 gene expression. Data were statistically analyzed by One-way ANOVA and paired sample t-test at a significance level of p < 0.05. Based on our results BW, BMI, WC, visceral fat, insulin, glucose, and HOMA-IR were significantly lower in the AT + Vit D, AT, and Vit D groups compared with the C group (p < 0.01). Furthermore, AT + Vit D, AT, and Vit D upregulated AMPK, PGC-1α, and UCP1 gene expression compared to the C. Based on the results compared to AT and Vit D, AT + Vit D significantly upregulated AMPK (p = 0.004; p = 0.001), PGC-1α (p = 0.010; p = 0.001), and UCP1 (p = 0.032; p = 0.001) gene expression, respectively. Also, AT induced more significant upregulations in the AMPK (p = 0.001), PGC-1α (p = 0.001), and UCP1 gene expression (p = 0.001) than Vit D. Vitamin D supplementation enhanced the beneficial effects of aerobic training on BW, BMI, WC, visceral fat, insulin, glucose, and HOMA-IR in diabetic rats. We also observed that separate AT or Vit D upregulated the gene expression of AMPK, PGC-1α, and UCP1 however, combined AT + Vit D upregulated AMPK, PGC-1α, and UCP1 more significantly. These results suggested that combining aerobic training and vitamin D supplementation exerted incremental effects on the gene expressions related to adipose tissue in animal models of diabetes.

1734-P: Polyhormonal Cells of the Pancreatic Islets in Congenital Hyperinsulinism

One of the possible causes of insulin deficiency in diabetes is dedifferentiation of β-cells into immature phenotypes with decreased insulin content or into the polyhormonal cells containing both insulin and glucagon. Polyhormonal cells have been detected in the pancreas in diabetic animal models as well as in humans with type 2 diabetes and longstanding type 1 diabetes, suggesting this phenomenon is a consequence of hyperglycemia. However, there is no information about the presence of polyhormonal cells in opposite metabolic conditions such as hypoglycemia in infants with congenital hyperinsulinism (CHI). We examined pancreatic biopsies from 6 infants with diffuse CHI and 5 infants with focal CHI using double immunofluorescence with antibodies to insulin and glucagon. In focal CHI, we found a significant proportion of α-cells containing glucagon and insulin within the focal lesion and in the rest of the tissue. Similarly, in diffuse CHI, we found the majority of α-cells contained glucagon and insulin in all pancreatic islets. The proportion of insulin and glucagon varied in the polyhormonal cells - from cells with equal expression of these hormones to cells with the prevalence of insulin or glucagon. In one diffuse case and in two focal lesions, we observed polyhormonal cells with significantly decreased glucagon expression and normal insulin expression. Outside the latter focal lesions, cells had normal level of glucagon expression. Our results indicate that polyhormonal cells are not exclusive for diabetes and hyperglycemia. We found polyhormonal cells in the pancreas of infants with CHI under hypoglycemic conditions. We suggest that this indicates high degree of the plasticity of the pancreatic cells. The role of plasticity in infants with CHI is yet to be established. Disclosure Y.Krivova: None. A.E.Proshchina: None. D.Gubaeva: None. M.Melikyan: None. D.Otlyga: None.

92-LB: Safety, Tolerability, and Clinical Effects of ZP8396, a Novel Long-Acting Amylin Analog—A Single Ascending Dose Trial

ZP8396 is an amylin analog designed for once weekly dosing that has demonstrated the potential to reduce body weight and improve glycemia in animal models of obesity and diabetes. We performed a randomized, double-blind, placebo-controlled trial to assess safety, pharmacokinetics, and pharmacodynamics of a single subcutaneous injection of ZP8396 in healthy lean and overweight male subjects. A total of 56 subjects (mean age was 38.1 years; mean BMI of 25.6 kg/m2) were randomized to ZP8396 or placebo (6:2) within seven dose cohorts ranging from 0.04 mg to 2.4 mg. The mean half-life of ZP8396 was approximately 10 days. After 1 week of observation, mean body weight decreased by -0.6%, 2.6%, 3.6%, and 4.2% from baseline following a single dose of placebo, 0.7 mg, 1.4 mg and 2.4 mg, respectively (Figure 1). ZP8396 was well tolerated, with no serious or severe adverse events (AEs) and no withdrawals. The most frequent related AEs were decreased appetite, nausea and vomiting, most events were mild and transient. No anti-drug antibodies were detected. In conclusion, administration of a single dose of ZP8396 resulted in dose-dependent and consistent reductions in body weight supporting the potential of ZP8396 as a treatment for obesity. Additional clinical studies of longer duration will be necessary to fully assess the clinical potential of ZP8396. Disclosure M. B. Olsen: Stock/Shareholder; Novo Nordisk A/S, Zealand Pharma A/S. U. Hövelmann: None. J. Griffin: Employee; Zealand Pharma A/S. K. M. Knudsen: Employee; Zealand Pharma A/S. T. Johansen: Employee; Zealand Pharma A/S. D. Kendall: Employee; Zealand Pharma A/S. Stock/Shareholder; Zealand Pharma A/S, MannKind Corporation. T. Heise: Research Support; Adocia, AstraZeneca, Biocon, Crinetics Pharmaceuticals, Inc., Eli Lilly and Company. Speaker's Bureau; Eli Lilly and Company. Consultant; Gan &amp; Lee Pharmaceuticals. Research Support; Genova. Advisory Panel; Novo Nordisk. Research Support; Novo Nordisk. Speaker's Bureau; Novo Nordisk. Research Support; Sanofi, Zealand Pharma A/S.

#3482 IN VIVO EVALUATIONS OF THE PROTECTIVE EFFECTS OF NEW FORMULATIONS FOR PERITONEAL DIALYSIS ADOPTING A POWERFUL MULTI-PHOTON MICROSCOPY BASED APPROACH

Abstract Background and Aims Peritoneal dialysis (PD) is a renal replacement therapy that enables metabolic waste products and excess fluids to be eliminated through the peritoneal membrane. Exposure to conventional dialysates at high glucose concentrations is considered critical for the pathogenesis of peritoneal fibrosis, angiogenesis, and epithelial-mesenchymal transition (EMT). These events largely contribute to peritoneal membrane aging, resulting in ultrafiltration and clearance failure. Pre-clinical research in this field suffers from the lack of valid and reproducible in vivo systems and so far has been limited to in vitro systems of mesothelial or endothelial cell lines. In vitro studies on mesothelial cell monolayers have validated the significant protective effects of dialysates with alternative osmo-metabolic agents. With their better biocompatibility these novel formulations could provide a valid substitute for conventional solutions (1,2). The method proposed, based on multi-photon microscopy, aims to study the physiology of the peritoneal membrane during dialysis exchange and to validate the effects of biocompatible dialysates in animal models of fibrosis as observed in the course of dialysis treatment. Method We have implemented a surgical procedure to optimize the stability of a flap of parietal peritoneum to allow direct microscope observation. The innovative technology of multi-photon microscopy enables one to observe the vessels of microcirculation in vivo and provides a 3D rendering of the peritoneal membrane, giving an overview of all the single layers without the use of specific markers (Figure 1). One may also assess specific phenomena induced by the fibrotic process, such as the thickening of the sub-mesothelial interstitium, exploiting the autofluorescence signal from excited collagen fibers caused by the physical phenomenon of Second Harmonic generation. In vivo microscopy evaluation of peritoneal membrane senescence parameters was conducted in rats subjected to one daily intraperitoneal injection (10 mL/day) for 15 days with one of the following PD solutions: a commercially available glucose-based neutral-pH, low-GDP, PD solution (Physioneal 3.86%, Baxter Healthcare, Italy), and a new single-chamber low glucose PD solution containing osmo-metabolic agents such as L-carnitine and xylitol (XyloCore HS, Galenica Senese, Italy). XyloCore HS is a lactate-buffered PD solution with glucose (1.5%), L-carnitine (0.02%) and xylitol (2.0%). The osmotic strength of the two PD solutions tested was comparable. Results Treatment with XyloCore HS was associated with a significant reduction in thickness of the sub-mesothelial interstitium (p = 0.013), the density of collagen fibers (p = 0.012) and the vascular composition (p = 0.006), as well as the number of branch points (p = 0.0335), when compared to rats treated with a commercial glucose-based PD solution, Physioneal. Figure 2 shows the different thicknesses of sub-mesothelial stroma upon treatment with the PD solutions under investigation. Conclusion Previous in vitro studies have shown that XyloCore (1,2), a novel glucose-sparing PD solution currently in Phase III clinical development (ELIXIR - NCT03994471), is able to counteract the glucotoxic effects on the peritoneal membrane induced by conventional dialysates. Our pre-clinical in vivo methodology not only confirms our previous in vitro findings, but also suggests that long-term protective effects may be achieved with XyloCore. Further studies are in progress in a diabetic animal model to extend the favorable peritoneal in vivo findings of XyloCore treatment to a systemic level, possibly by improving glycemic control.