Molecular and morphological alterations in uninjured skin of streptozotocin-induced diabetic mice.

Diabetes affects every tissue in the body, including the skin. The main skin problem is the increased risk of infections, which can lead to foot ulcers. Most studies evaluating the effects of diabetes on the skin are carried out in wound healing areas. There are fewer studies on uninjured skin, and some particularities of this tissue are yet to be elucidated. In general, cellular and molecular outcomes of diabetes are increased oxidative stress and lipid peroxidation. For our study, we used C57BL/6 mice that were divided into diabetic and non-diabetic groups. The diabetic group received low doses of streptozotocin on 5 consecutive days. To evaluate the effects of hyperglycemia on uninjured skin, we performed morphological analysis using hematoxylin/eosin staining, cellular analysis using Picrosirius red and Nissl staining, and immunostaining, and evaluated protein expression by polymerase chain reaction. We confirmed that mice were hyperglycemic, presenting all features related to this metabolic condition. Hyperglycemia caused a decrease in interleukin 6 (Il-6) and an increase in tumor necrosis factor alpha (Tnf-α), Il-10, F4/80, tumor growth factor beta (Tgf-ß), and insulin-like growth factor 1 (Igf-1). In addition, hyperglycemia led to a lower cellular density in the epidermis and dermis, a delay in the maturation of collagen fibers, and a decrease in the number of neurons. Furthermore, we showed a decrease in Bdnf expression and no changes in Ntrk2 expression in the skin of diabetic animals. In conclusion, chronic hyperglycemia in mice induced by streptozotocin caused disruption of homeostasis even before loss of skin continuity.

Molecular and morphological alterations in uninjured skin of streptozotocin‐induced diabetic mice

Diabetes affects every tissue in the body, including the skin. The main skin problem is the increased risk of infections, which can lead to foot ulcers. Most studies evaluating the effects of diabetes on the skin are carried out in wound healing areas. There are fewer studies on uninjured skin, and some particularities of this tissue are yet to be elucidated. In general, cellular and molecular outcomes of diabetes are increased oxidative stress and lipid peroxidation. For our study, we used C57BL/6 mice that were divided into diabetic and non-diabetic groups. The diabetic group received low doses of streptozotocin on 5 consecutive days. To evaluate the effects of hyperglycemia on uninjured skin, we performed morphological analysis using hematoxylin/eosin staining, cellular analysis using Picrosirius red and Nissl staining, and immunostaining, and evaluated protein expression by polymerase chain reaction. We confirmed that mice were hyperglycemic, presenting all features related to this metabolic condition. Hyperglycemia caused a decrease in interleukin 6 (Il-6) and an increase in tumor necrosis factor alpha (Tnf-α), Il-10, F4/80, tumor growth factor beta (Tgf-β), and insulin-like growth factor 1 (Igf-1). In addition, hyperglycemia led to a lower cellular density in the epidermis and dermis, a delay in the maturation of collagen fibers, and a decrease in the number of neurons. Furthermore, we showed a decrease in Bdnf expression and no changes in Ntrk2 expression in the skin of diabetic animals. In conclusion, chronic hyperglycemia in mice induced by streptozotocin caused disruption of homeostasis even before loss of skin continuity.

Unusual effects of nanowire-nanowire junctions on the persistent photoconductivity in SnO2 nanowire network devices.

The persistent photoconductivity (PPC) effect is a commonly observed behavior in SnO2 nanostructures. Here we described and studied this effect through a comparative study, based on measurements of electronic transport using network as well as single devices built from SnO2 nanowires under different experimental conditions. At room temperature, the PPC effect was observed to be more accentuated in single nanowire devices. It was found that nanowire-nanowire junctions play a fundamental role in the device behavior: the decay time of nanowire network (τ = 52 s) is about three orders of magnitude lower than those of single nanowire (τ = 4.57 × 104 s). Additionally, it was confirmed that the PPC effect was directly related to the amount of oxygen present in the environment and it is destroyed with increasing temperature. Furthermore, the PPC effect was interpreted based on the surface effect that depends on the capture/emission of electrons by the surface states.

Enhancement of cellular activity in hyperglycemic mice dermal wounds dressed with chitosan-alginate membranes

The use of specially designed wound dressings could be an important alternative to facilitate the healing process of wounds in the hyperglycemic state. Biocompatible dressings combining chitosan and alginate can speed up wound healing by modulating the inflammatory phase, stimulating fibroblast proliferation, and aiding in remodeling phases. However, this biomaterial has not yet been explored in chronic and acute lesions of diabetic patients. The aim of this study was to evaluate the effect of topical treatment with a chitosan-alginate membrane on acute skin wounds of hyperglycemic mice. Diabetes mellitus was induced by streptozotocin (60 mg · kg-1 · day-1 for 5 days, intraperitoneally) and the cutaneous wound was performed by removing the epidermis using a surgical punch. The results showed that after 10 days of treatment the chitosan and alginate membrane (CAM) group exhibited better organization of collagen fibers. High concentrations of interleukin (IL)-1α, IL-1β, granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor-alpha (TNF-α) were detected in the first and second days of treatment. G-CSF and TNF-α level decreased after 5 days, as well as the concentrations of TNF-α and IL-10 compared with the control group (CG). In this study, the inflammatory phase of cutaneous lesions of hyperglycemic mice was modulated by the use of CAM, mostly regarding the cytokines IL-1α, IL-1β, TNF-α, G-CSF, and IL-10, resulting in better collagen III deposition. However, further studies are needed to better understand the healing stages associated with CAM use.

Enhancement of cellular activity in hyperglycemic mice dermal wounds dressed with chitosan-alginate membranes.

The use of specially designed wound dressings could be an important alternative to facilitate the healing process of wounds in the hyperglycemic state. Biocompatible dressings combining chitosan and alginate can speed up wound healing by modulating the inflammatory phase, stimulating fibroblast proliferation, and aiding in remodeling phases. However, this biomaterial has not yet been explored in chronic and acute lesions of diabetic patients. The aim of this study was to evaluate the effect of topical treatment with a chitosan-alginate membrane on acute skin wounds of hyperglycemic mice. Diabetes mellitus was induced by streptozotocin (60 mg · kg-1 · day-1 for 5 days, intraperitoneally) and the cutaneous wound was performed by removing the epidermis using a surgical punch. The results showed that after 10 days of treatment the chitosan and alginate membrane (CAM) group exhibited better organization of collagen fibers. High concentrations of interleukin (IL)-1α, IL-1ß, granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor-alpha (TNF-α) were detected in the first and second days of treatment. G-CSF and TNF-α level decreased after 5 days, as well as the concentrations of TNF-α and IL-10 compared with the control group (CG). In this study, the inflammatory phase of cutaneous lesions of hyperglycemic mice was modulated by the use of CAM, mostly regarding the cytokines IL-1α, IL-1ß, TNF-α, G-CSF, and IL-10, resulting in better collagen III deposition. However, further studies are needed to better understand the healing stages associated with CAM use.

Defective regulation of adipose tissue autophagy in obesity.

OBJECTIVES: Autophagy is a highly regulated process that has an important role in the control of a wide range of cellular functions, such as organelle recycling, nutrient availability and tissue differentiation. A recent study has shown an increased autophagic activity in the adipose tissue of obese subjects, and a role for autophagy in obesity-associated insulin resistance was proposed. Body mass reduction is the most efficient approach to tackle insulin resistance in over-weight subjects; however, the impact of weight loss in adipose tissue autophagy is unknown. SUBJECTS: Adipose tissue autophagy was evaluated in mice and humans. RESULTS: First, a mouse model of diet-induced obesity and diabetes was maintained on a 15-day, 40% caloric restriction. At baseline, markers of autophagy were increased in obese mice as compared with lean controls. Upon caloric restriction, autophagy increased in the lean mice, whereas it decreased in the obese mice. The reintroduction of ad libitum feeding was sufficient to rapidly reduce autophagy in the lean mice and increase autophagy in the obese mice. In the second part of the study, autophagy was evaluated in the subcutaneous adipose tissue of nine obese-non-diabetic and six obese-diabetic subjects undergoing bariatric surgery for body mass reduction. Specimens were collected during the surgery and approximately 1 year later. Markers of systemic inflammation, such as tumor necrosis factor-1α, interleukin (IL)-6 and IL-1ß were evaluated. As in the mouse model, human obesity was associated with increased autophagy, and body mass reduction led to an attenuation of autophagy in the adipose tissue. CONCLUSION: Obesity and caloric overfeeding are associated with the defective regulation of autophagy in the adipose tissue. The studies in obese-diabetic subjects undergoing improved metabolic control following calorie restriction suggest that autophagy and inflammation are regulated independently.

Short-term inhibition of peroxisome proliferator-activated receptor-gamma coactivator-1alpha expression reverses diet-induced diabetes mellitus and hepatic steatosis in mice.

AIMS/HYPOTHESIS: The coactivator of nuclear receptors, peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) has been implicated in a series of events that contribute to the control of glucose metabolism. We have recently reported the use of a PGC-1alpha antisense oligonucleotide (PGC-1alphaAS) that inhibits up to 60% of PGC-1alpha expression in pancreatic islets, leading to increased insulin secretion. This oligonucleotide was used in this study to try to ameliorate diet-induced type 2 diabetes in a genetically predisposed mouse strain (Swiss mice). MATERIALS AND METHODS: Glucose and insulin tolerance tests, euglycaemic-hyperinsulinaemic clamp, immunoprecipitation assays, immunoblotting assays and immunohistochemistry were used in this investigation. RESULTS: Swiss mice became obese and overtly diabetic after 8 weeks of feeding with chow containing 24% saturated fat. One daily dose (1.0 nmol) of PGC-1alphaAS significantly reduced glucose and increased insulin blood levels without affecting food intake and body weight. These effects were accompanied by a reduced area under the glucose curve during an intraperitoneal glucose tolerance test, an increased constant of glucose decay (K(itt)) during an insulin tolerance test, and an increased glucose consumption rate during a euglycaemic-hyperinsulinaemic clamp. Moreover, mice treated with PGC-1alphaAS presented an outstanding reduction of macroscopic and microscopic features of hepatic steatosis. These effects were accompanied by reduced expression or function of a series of proteins involved in lipogenesis. CONCLUSIONS/INTERPRETATION: PGC-1alpha is an attractive target for pharmacological therapeutics in type 2 diabetes mellitus and diet-induced hepatic steatosis.

Restoration of insulin secretion in pancreatic islets of protein-deficient rats by reduced expression of insulin receptor substrate (IRS)-1 and IRS-2.

Autocrine and paracrine insulin signaling may participate in the fine control of insulin secretion. In the present study, tissue distribution and protein amounts of the insulin receptor and its major substrates, insulin receptor substrate (IRS)-1 and IRS-2, were evaluated in a model of impaired glucose-induced insulin secretion, the protein-deficient rat. Immunoblot and RT-PCR studies showed that the insulin receptor and IRS-2 expression are increased, whilst IRS-1 protein and mRNA contents are decreased in pancreatic islets of protein-deficient rats. Immunohistochemical studies revealed that the insulin receptor and IRS-1 and -2 are present in the great majority of islet cells; however, the greatest staining was localized at the periphery, suggesting a co-localization with non-insulin-secreting cells. Exogenous insulin stimulation of isolated islets promoted higher insulin receptor and IRS-1 and -2 tyrosine phosphorylation in islets from protein-deficient rats, as compared with controls. Moreover, insulin-induced IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activity are increased in islets of protein-deficient rats. The reduction of IRS-1 and IRS-2 protein expression in islets isolated from protein-deficient rats by the use of antisense IRS-1 or IRS-2 phosphorthioate-modified oligonucleotides partially restored glucose-induced insulin secretion. Thus, the impairment of insulin cell signaling through members of the IRS family of proteins in isolated rat pancreatic islets improves glucose-induced insulin secretion. The present data reinforced the role of insulin paracrine and autocrine signaling in the control of its own secretion.

Selective impairment of insulin signalling in the hypothalamus of obese Zucker rats.

AIM/HYPOTHESIS: By acting in the brain, insulin suppresses food intake. However, little is known with regard to insulin signalling in the hypothalamus in insulin-resistant states. METHODS: Western blotting, immunohistochemistry and polymerase chain reaction assays were combined to compare in vivo hypothalamic insulin signalling through the PI3-kinase and MAP kinase pathways between lean and obese Zucker rats. RESULTS: Intracerebroventricular insulin infusion reduced food intake in lean rats to a greater extent than that observed in obese rats, and pre-treatment with PI3-kinase inhibitors prevented insulin-induced anorexia. The relative abundance of IRS-2 was considerably higher than that of IRS-1 in hypothalamus of both lean and obese rats. Insulin-stimulated phosphorylation of IR, IRS-1/2, the associations of PI 3-kinase to IRS-1/2 and phosphorylation of Akt in hypothalamus were decreased in obese rats compared to lean rats. These effects seem to be mediated by increased phosphoserine content of IR, IRS-1/2 and decreased protein levels of IRS-1/2 in obese rats. In contrast, insulin stimulated the phosphorylation of MAP kinase equally in lean and obese rats. CONCLUSION/INTERPRETATION: This study provides direct measurements of insulin signalling in hypothalamus, and documents selective resistance to insulin signalling in hypothalamus of Zucker rats. These findings provide support for the hypothesis that insulin could have anti-obesity actions mediated by the PI3-kinase pathway, and that impaired insulin signalling in hypothalamus could play a role in the development of obesity in this animal model of insulin-resistance.

Peroxisome proliferator-activated receptor gamma coactivator-1-dependent uncoupling protein-2 expression in pancreatic islets of rats: a novel pathway for neural control of insulin secretion.

AIMS/HYPOTHESIS: Sympathetic inputs inhibit insulin secretion through alpha2-adrenergic receptors coupled with Gi protein. High adrenergic tonus generated by exposure of homeothermic animals to cold reduces insulin secretion. In this study we evaluate the participation of UCP-2 in cold-induced regulation of insulin secretion. METHODS: Static insulin secretion studies, western blotting and immunohistochemistry were used in this investigation. RESULTS: Exposure of rats to cold during 8 days promoted 60% ( n=15, p<0.05) reduction of basal serum insulin levels concentration accompanied by reduction of the area under insulin curve during i.p. GTT (50%, n=15, p<0.05). Isolated islets from cold-exposed rats secreted 57% ( n=6, p<0.05) less insulin following a glucose challenge. Previous sympathectomy, partially prevented the effect of cold exposure upon insulin secretion. Islets isolated from cold-exposed rats expressed 51% ( n=6, p<0.5) more UCP-2 than islets from control rats, while the inhibition of UCP-2 expression by antisense oligonucleotide treatment partially restored insulin secretion of islets obtained from cold-exposed rats. Cold exposure also induced an increase of 69% ( n=6, p<0.05) in PGC-1 protein content in pancreatic islets. Inhibition of islet PGC-1 expression by antisense oligonucleotide abrogated cold-induced UCP-2 expression and partially restored insulin secretion in islets exposed to cold. CONCLUSION/INTERPRETATION: Our data indicate that sympathetic tonus generated by exposure of rats to cold induces the expression of PGC-1, which participates in the control of UCP-2 expression in pancreatic islets. Increased UCP-2 expression under these conditions could reduce the beta-cell ATP/ADP ratio and negatively regulate insulin secretion.

Early steps of insulin action in the skin of intact rats.

Insulin is an important regulator of growth and initiates its action by binding to its receptor, which undergoes tyrosyl autophosphorylation and further enhances its tyrosine kinase activity towards other intermediate molecules, including insulin receptor substrate 1, insulin receptor substrate 2, and Shc. Insulin receptor substrate proteins can dock various src-homology-2-domain-containing signaling proteins, such as the 85 kDa subunit of phosphatidylinositol 3 kinase and growth-factor-receptor-bound protein 2. The serine-threonine kinase is activated downstream to phosphatidylinositol 3 kinase. Shc protein has been shown to directly induce the association with growth-factor-receptor-bound protein 2 and downstream the activation of the mitogen-activated protein kinase. In this study we investigated insulin signal transduction pathways in skin of intact rats by immunoprecipitation and immunoblotting with specific antibodies, and also by immunohistochemistry with anti-insulin-receptor antibody. Our results showed that skin fragments clearly demonstrated the presence of insulin receptor in cell bodies of the epidermis and hair follicles and some faint staining was also detected in fibroblasts of the dermis. It was also observed that acute stimulation with insulin can induce tyrosyl phosphorylation of insulin receptor, that the insulin receptor substrates and Shc proteins serve as signaling molecules for insulin in skin of rats, and that insulin is able to induce association of insulin receptor substrate 1/phosphatidylinositol 3 kinase and Shc/growth-factor-receptor-bound protein 2 in this tissue, as well as phosphorylation of mitogen-activated protein kinase and serine-threonine kinase, demonstrating that proteins involved in early steps of insulin action are expressed in skin of intact rats and are quickly activated after insulin stimulation.