Cell death induced in L-cells by treatment with thymidine: Staging of the process and relationship to apoptosis

Summary The purpose of this study was to characterize the stages in the development of thymidine-induced cell death. L-cells were characterized by both morphologic and quantitative techniques and evaluated at 24, 48, and 72 h of treatment. Cells first enlarged (stage I); about 50% of these enlarged cells then decreased in size with blebbing and compacting (stage II). This residual cell body transformed into a smooth eosinophilic hyaline body (stage III) by 72 h, many of which could be identified within the vacuolar system of viable cells. These changes were reflected in morphologic counts and Coulter sizing. Cell death (loss of labeled DNA) began in stage II and was most prominent in stage III. No cleavage of DNA into oligonucleosomal fragments was detected by agarose gel electrophoresis at any stage. The similarity of these changes to the complete spectrum of apoptosis in vivo is discussed.     

Signalling pathways involved in the detection of peptones by murine small intestinal enteroendocrine L-cells

Glucagon like peptide-1 is an insulinotropic hormone released from intestinal L-cells in response to food ingestion. Here, we investigated mechanisms underlying the sensing of peptones by primary small intestinal L-cells. Meat, casein and vegetable-derived peptones (5 mg/ml), the L-amino acids Phe, Trp, Gln and Ala (20 mM each), and the dipeptide glycine-sarcosine (20 mM) stimulated GLP-1 secretion from primary cultures prepared from the small intestine. Further mechanistic studies were performed with meat peptone, and revealed the elevation of intracellular calcium in L-cells. Inhibition of the calcium sensing receptor (CaSR), transient receptor potential (TRP) channels and Q-type voltage gated calcium channels (VGCC) significantly attenuated peptone-stimulated GLP-1 release and reduced intracellular Ca²⁺ responses. CaSR inhibition also attenuated the GLP-1 secretory response to Gln. Targeting these pathways in L-cells could be used to increase endogenous production of GLP-1 and offer exploitable avenues for the development of therapeutics to treat diabetes and obesity.     

Effect of kolaviron on islet dynamics in diabetic rats

Kolaviron, a biflavonoid isolated from the edible seeds of Garcinia kola, lowers blood glucose in experimental models of diabetes; however, the underlying mechanisms are not yet fully elucidated. The objective of the current study was to assess the effects of kolaviron on islet dynamics in streptozotocin-induced diabetic rats. Using double immunolabeling of glucagon and insulin, we identified insulin-producing β- and glucagon-producing α-cells in the islets of diabetic and control rats and determined the fractional β-cell area, α-cell area and islet number. STZ challenged rats presented with islet hypoplasia and reduced β-cell area concomitant with an increase in α-cell area. Kolaviron restored some islet architecture in diabetic rats through the increased β-cell area. Overall, kolaviron-treated diabetic rats presented a significant (p < 0.05) increase in the number of large and very large islets compared to diabetic control but no difference in islet number and α-cell area. The β-cell replenishment potential of kolaviron and its overall positive effects on glycemic control suggest that it may be a viable target for diabetes treatment.     

Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer

Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include intestinal enteroendocrine cells genetically engineered to secrete insulin in response to physiologic stimuli; responsiveness occurs at the exocytosis level to regulate the acute release of recombinant insulin. In this work, we established a human cellular model to demonstrate that meat hydrolysate can simultaneously stimulate glucagon-like peptide-1 (GLP-1, an enteroendocrine cell-derived incretin hormone) and recombinant insulin secretion from the engineered human NCI-H716 intestinal cell line. Cells were genetically modified using the recombinant adeno-associated virus (rAAV)-mediated insulin gene transfer. Recombinant cells were then differentiated to display endocrine features, in particular the formation of granule-like compartments. A fusion protein of insulin and enhanced green fluorescence protein (EGFP) was designed to reveal the compartments of localization of the fusion protein and assess its co-localization with endogenous GLP-1. Our work provides a unique human cellular model for regulated insulin release through genetic engineering of GLP-1-secreting intestinal cells, which is expected to be useful for cell-based therapies of IDD.     

Synthesis,characterization and evaluation of the suppression of insulin resistance in Type-II diabetes mellitus animals by treatment with metal complex

The present study is characterized toward thespesone isolation from Thespesia populnea (Malvaceae). Subsequently it was modified and characterized to study its effect on diabetes related symptoms. The complex is administered to diabetes induced mice with the doses of 5, 10 and 20 mg/kg, p.o. and the effect of complex on the level of body weight, lipid profile and blood glucose was studied after 22 days. The results have indicated that diabetic mice show a significant (p < 0.01) decrease in the level of serum triglyceride, plasma glucose and increase in body weight. Hence the present investigation reveals that newly synthesized complex is useful in the management of Type-II diabetes mellitus because of its ability to reduce insulin resistance.     

Heat-regulated production and secretion of insulin from a human artificial chromosome vector

Human artificial chromosomes (HACs) behave as independent minichromosomes and are potentially useful as a way to achieve safe, long-term expression of a transgene. In this study, we sought to elucidate the potential of HAC vectors carrying the human proinsulin transgene for gene therapy of insulin-dependent diabetes mellitus (IDDM) using non-beta-cells as a host for the vector. To facilitate the production of mature insulin in non-beta-cells and to safely regulate the level of transgene expression, we introduced furin-cleavable sites into the proinsulin coding region and utilized the heat shock protein 70 (Hsp70) promoter. We used Cre-loxP-mediated recombination to introduce the gene cassettes onto 21DeltapqHAC, a HAC vector whose structure is completely defined, present in human fibrosarcoma HT1080 cells. We observed long-term expression and stable retention of the transgene without aberrant translocation of the HAC constructs. As expected, the Hsp70 promoter allowed us to regulate gene expression with temperature, and the production and secretion of intermediates of mature insulin were made possible by the furin-cleavable sites we had introduced into proinsulin. This study can be an initial step on the application of HAC vectors on the gene delivery to non-beta-cells, which might provide a direction for future treatment for diabetes.     

Effects of subchronic acrylamide treatment on the endocrine pancreas of juvenile male Wistar rats

Acrylamide (AA) is a well-known industrial monomer with carcinogenic, mutagenic, neurotoxic and endocrine disruptive effects on living organisms. AA has been the subject of renewed interest owing to its presence in various food products. We investigated the potential adverse effects of oral AA treatment on the endocrine pancreas of juvenile rats using histochemical, immunohistochemical, stereological and biochemical methods. Thirty juvenile male Wistar rats were divided into one control and two AA treatment groups: one treated with 25 mg/kg AA and the other treated with 50 mg/kg AA for 21 days. We found a significant decrease in β-cell mass. The significant decrease in β-cell optical density and unchanged blood glucose levels indicate that normoglycemia in AA treated rats may result from intensive exocytosis of insulin-containing secretory granules. By contrast with β-cells, we observed increased α-cell mass. The slight increase in α-cell cytoplasmic volume suggests retention of glucagon in α-cells, which is consistent with the significant increase in α-cell optical density for AA treated animals. The number of islets of Langerhans did not change significantly in AA treated groups. Our findings suggest that AA treatment causes decreased β-cell mass and moderate α-cell mass increase in the islets of Langerhans of juvenile male Wistar rats.     

Insulin therapy and its consequences for the mother,foetus, and newborn in gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is a disease characterised by glucose intolerance and first diagnosed in pregnancy. This condition relates to an anomalous placental environment and aberrant placental vascular function. GDM-associated hyperglycaemia changes the placenta structure leading to abnormal development and functionality of this vital organ. Aiming to avoid the GDM-hyperglycaemia and its deleterious consequences in the mother, the foetus and newborn, women with GDM are firstly treated with a controlled diet therapy; however, some of the women fail to reach the recommended glycaemia values and therefore they are passed to the second line of treatment, i.e., insulin therapy. The several protocols available in the literature regarding insulin therapy are variable and not a clear consensus is yet reached. Insulin therapy restores maternal glycaemia, but this beneficial effect is not reflected in the foetus and newborn metabolism, suggesting that other factors than d-glucose may be involved in the pathophysiology of GDM. Worryingly, insulin therapy may cause alterations in the placenta and umbilical vessels as well as the foetus and newborn additional to those seen in pregnant women with GDM treated with diet. In this review, we summarised the variable information regarding indications and protocols for administration of the insulin therapy and the possible outcomes on the function and structure of the foetoplacental unit and the neonate parameters from women with GDM.     

Modulation of the rat hepatic cytochrome P450 composition by long-term streptozotocin-induced insulin-dependent diabetes

The effects of long-term insulin-dependent diabetes on the enzymatic activities of hepatic cytochrome P450 isozymes were determined in rats rendered diabetic by the administration of streptozotocin and killed 4, 8, and 12 weeks following treatment. The O-dealkylations of ethoxy-resorufin and pentoxyresorufin were elevated in the diabetic animals throughout the study, the extent of increase being similar at all three time points. p-Nitrophenol hydroxylase activity was induced in the diabetic animals 4 weeks following treatment with streptozotocin, but the extent of increase became less pronounced with the progress of the disease. A modest increase in ethylmorphine N-demethylase activity was also observed but only in the diabetic animals killed 4 weeks after the induction of diabetes. Finally, lauric acid hydroxylase activity was elevated in the diabetic animals 4 weeks following streptozotocin administration but then declined rapidly with the duration of the disease. It is concluded that the duration of diabetes modulates the hepatic cytochrome P450 profile, with the effect being isoenzyme specific. Mechanisms that may account for these changes are discussed.     

The roles of cell-cell and organ-organ crosstalk in the type 2 diabetes mellitus associated inflammatory microenvironment

Type 2 diabetes mellitus (T2DM) is a classic metaflammatory disease, and the inflammatory states of the pancreatic islet and insulin target organs have been well confirmed. However, abundant evidence demonstrates that there are countless connections between these organs in the presence of a low degree of inflammation. In this review, we focus on cell-cell crosstalk among local cells in the islet and organ-organ crosstalk among insulin-related organs. In contrast to that in acute inflammation, macrophages are the dominant immune cells causing inflammation in the islets and insulin target organs in T2DM. In the inflammatory microenvironment (IME) of the islet, cell-cell crosstalk involving local macrophage polarization and proinflammatory cytokine production impair insulin secretion by β-cells. Furthermore, organ-organ crosstalk, including the gut-brain-pancreas axis and interactions among insulin-related organs during inflammation, reduces insulin sensitivity and induces endocrine dysfunction. Therefore, this crosstalk ultimately results in a cascade leading to β-cell dysfunction. These findings could have broad implications for therapies aimed at treating T2DM.     

Association of diabetes and microbiota: An update

Diabetes is an emerging health condition globally and is suggested to have a direct connection with the gut microbiota that determine our metabolic outcomes. Sensitivity to insulin and glucose metabolism is normal in healthy people as compared to those people who cannot maintain their glucose metabolism. One of the reasons of the differences is that healthy people have different microbiome that leads to achieve more short chain fatty acids and make up more branched amino acids, while the gut microbiota of the other group of people are more likely to produce compounds that affects glucose metabolism. Herein, this review will present the research related to the impact of gut microbes on diabetes carried out in the past decade. The review focus on the relation between gut microbiota and Type-1 Diabetes (T1D), Type-2 Diabetes (T2D), and how gut microbiota could be an alternative therapy for treatment of diabetes.     

Effects of streptozotocin-induced diabetes and insulin on calcium responsiveness of the rat vas deferens

In the present study, effects of streptozotocin-induced diabetes and insulin treatment on the reactivity of rat vas deferens to KCl and calmidazolium, a calmodulin antagonist, were evaluated and calmodulin levels in vas deferens tissue from diabetic and insulin-treated rats were determined. Diabetes was induced in rats by a single injection of streptozotocin. Five weeks after the induction of diabetes, one group of diabetic rats was injected with insulin for 3 weeks. After 8 weeks, vas deferens tissues on one side of diabetic and insulin-treated diabetic rats and their controls were mounted in organ bath to measure isometric tension, while the tissues on the other side of rats were homogenized to determine calmodulin levels by radioimmunoassay. Concentration-response curves to KCl were obtained in vas deferens tissues in the absence and presence of calmidazolium. The effects of KCl and calmidazolium on vas deferens isolated from 8-weeks diabetic rats were decreased. Calmodulin levels were also found to be decreased in vas deferens from diabetic rats. Decreased calmodulin levels in diabetic rat vas deferens were not corrected by insulin treatment. Only a partial correction following insulin treatment was observed in contractile effect of KCl on diabetic rat vas deferens, whereas insulin treatment increases the affinity of calmodulin in this muscle. Experimental diabetes causes an impairment in calcium/calmodulin-dependent contractile process of vas deferens, which is correctable partially following insulin therapy. The changes in the function of rat vas deferens due to streptozotocin diabetes seem to be related to impaired sexual functions in human diabetes.     

Plasmapheresis in the initial treatment of insulin-dependent diabetes mellitus in children.

Several factors indicate that autoimmune mechanisms may play a part in the aetiology of insulin-dependent diabetes mellitus. At the onset of the disease in 10 children (aged 11-16 years) plasmapheresis was performed four times over one to two weeks. Seventeen age-matched children with the same clinical features served as controls. The C-peptide concentrations at onset were the same in the two groups, but after one month the children treated with plasmapheresis had significantly higher values. This difference became even more pronounced after three, nine, and 18 months, both during fasting and at the maximum response to a standardised meal. The study group also had a significantly more stable metabolism, longer partial remission, and no higher insulin requirement. Of the 10 treated children islet-cell cytoplasmic antibodies were present in seven before plasmapheresis and in nine during treatment. The antibodies remained detectable in five and six out of nine patients at one and six months respectively after plasmapheresis. Although the mechanisms are obscure, plasmapheresis performed at the onset of insulin-dependent diabetes mellitus may help to preserve beta-cell function.     

Neural network modeling and control of type 1 diabetes mellitus

This paper presents a developed and validated dynamic simulation model of type 1 diabetes, that simulates the progression of the disease and the two term controller that is responsible for the insulin released to stabilize the glucose level. The modeling and simulation of type 1 diabetes mellitus is based on an artificial neural network approach. The methodology builds upon an existing rich database on the progression of type 1 diabetes for a group of diabetic patients. The model was found to perform well at estimating the next glucose level over time without control. A neural controller that mimics the pancreas secretion of insulin into the body was also developed. This controller is of the two term type: one stage is responsible for short-term and the other for mid-term insulin delivery. It was found that the controller designed predicts an adequate amount of insulin that should be delivered into the body to obtain a normalization of the elevated glucose level. This helps to achieve the main objective of insulin therapy: to obtain an accurate estimate of the amount of insulin to be delivered in order to compensate for the increase in glucose concentration.     

Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted

Mutations in the preproinsulin protein that affect processing of preproinsulin to proinsulin or lead to misfolding of proinsulin are associated with diabetes. We examined the subcellular localization and secretion of 13 neonatal diabetes-associated human proinsulin proteins (A24D, G32R, G32S, L35P, C43G, G47V, F48C, G84R, R89C, G90C, C96Y, S101C and Y108C) in rat INS-1 insulinoma cells. These mutant proinsulin proteins accumulate in the endoplasmic reticulum (ER) and are poorly secreted except for G84R and in contrast to wild-type and hyperproinsulinemia-associated mutant proteins (H34D and R89H) which were sorted to secretory granules and efficiently secreted. We also examined the effect of C96Y mutant proinsulin on the synthesis and secretion of wild-type insulin and observed a dominant-negative effect of the mutant proinsulin on the synthesis and secretion of wild-type insulin due to induction of the unfolded protein response and resulting attenuation of overall translation.     

Applications of stem cells and bioprinting for potential treatment of diabetes

Currently, there does not exist a strategy that can reduce diabetes and scientists are working towards a cure and innovative approaches by employing stem cellbased therapies. On the other hand, bioprinting technology is a novel therapeutic approach that aims to replace the diseased or lost β-cells, insulin-secreting cells in the pancreas, which can potentially regenerate damaged organs such as the pancreas. Stem cells have the ability to differentiate into various cell lines including insulinproducing cells. However, there are still barriers that hamper the successful differentiation of stem cells into β-cells. In this review, we focus on the potential applications of stem cell research and bioprinting that may be targeted towards replacing the β-cells in the pancreas and may offer approaches towards treatment of diabetes. This review emphasizes on the applicability of employing both stem cells and other cells in 3 D bioprinting to generate substitutes for diseased β-cells and recover lost pancreatic functions. The article then proceeds to discuss the overall research done in the field of stem cell-based bioprinting and provides future directions for improving the same for potential applications in diabetic research.     

Chitosan treatment for skin ulcers associated with diabetes

Infections, ulcerations, gangrene and, in severe cases, extremity amputation, are common complications among diabetic subjects. Various biomaterials have been utilized for the treatment of these lesions. Chitosan is an amino sugar with a low risk of toxicity and immune response. In this study, we evaluated chitosan topical gel and film treatments for subjects with diabetic ulcerations and wounds associated with diabetes mellitus. In a pre-experimental design, we described the result of chitosan gel and film treatment for wounds and skin ulcers among patients with long-standing diabetes mellitus. We studied 8 diabetic patients with wounds and skin ulcers (long duration and Wagner degree 1–2). Initially, most lesions had some degree of infection, tissue damage and ulceration. At the end of the treatment (topical chitosan) period, the infections were cured. All patients experienced a significant improvement in the initial injury and developed granulation tissue and a healthy skin cover. This report represents one of the few published clinical experience regarding the chitosan for the treatment of skin lesions among diabetic subjects. These results are relevant and promising for the treatment of this disease.     

Extracts of Rehmanniae radix, Ginseng radix and Scutellariae radix improve glucose-stimulated insulin secretion and β-cell proliferation through IRS2 induction

Recent studies have revealed that beta-cell dysfunction is an important factor in developing type 2 diabetes. beta-cell dysfunction is related to impairment of the insulin/IGF-1 signaling cascade through insulin receptor substrate-2 (IRS2). The induction of IRS2 in beta-cells plays an important role in potentiating beta-cell function and mass. In this study, we investigated whether herbs used for treating diabetes in Chinese medicine-Galla rhois, Rehmanniae radix, Machilus bark, Ginseng radix, Polygonatum radix, and Scutellariae radix-improved IRS2 induction in rat islets, glucose-stimulated insulin secretion and beta-cell survival. R. radix, Ginseng radix and S. radix significantly enhanced glucose-stimulated insulin secretion compared to the control, i.e., by 49, 67 and 58%, respectively. These herbs induced the expression of IRS2, pancreas duodenum homeobox-1 (PDX-1), and glucokinase. The increased level of glucokinase could explain the enhancement of glucose-stimulated insulin secretion with these extracts. Increased PDX-1 expression was associated with beta-cell proliferation, which was consistent with the cell viability assay. In conclusion, R. radix, Ginseng radix and S. radix had an insulinotropic action similar to that of exendin-4.     

Molecular docking studies of banana flower flavonoids as insulin receptor tyrosine kinase activators as a cure for diabetes mellitus

Diabetes mellitus is a metabolic disorder caused due to insulin deficiency. Banana flower is a rich source of flavonoids that exhibit anti diabetic activity. Insulin receptor is a tetramer that belongs to a family of receptor tyrosine kinases. It contains two alpha subunits that form the extracellular domain and two beta subunits that constitute the intracellular tyrosine kinase domain. Insulin binds to the extracellular region of the receptor and causes conformational changes that lead to the activation of the tyrosine kinase. This leads to autophosphorylation, a step that is crucial in insulin signaling pathway. Hence, compounds that augment insulin receptor tyrosine kinase activity would be useful in the treatment of diabetes mellitus. The 3D structure of IR tyrosine kinase was obtained from PDB database. The list of flavonoids found in banana flower was obtained from USDA database. The structures of the flavonoids were obtained from NCBI Pubchem. Docking analysis of the flavonoids was performed using Autodock 4.0 and Autodock Vina. The results indicate that few of the flavonoids may be potential activators of IR tyrosine kinase.