Diabetes mellitus is associated with impaired wound healing. The topical use of insulin is a promising therapy because it may favor all phases of the wound healing process. This study aimed to investigate the therapeutic outcomes of insulin gel in wounds of hyperglycemic mice. After diabetes induction, a 1-cm2 full-thickness wound was created on each animal's dorsum. The lesions were treated daily for 14 days with insulin gel (insulin group) or vehicle gel without insulin (vehicle group). Tissue samples were extracted on days 4, 7, 10, and 14 after the creation of the lesion. The samples were analyzed with hematoxylin/eosin and Sirius red staining, immunohistochemistry, Bio-Plex immunoassays, and western blotting. Insulin gel favored re-epithelialization at day 10 and increased the organization and deposition of collagen. Additionally, it modulated the expression of cytokines (interleukin (IL)-4 and IL-10) and increased the expression of arginase I, VEGF receptor 1, and VEGF on day 10. Activation of the insulin signaling pathway occurred via IRβ, IRS1, and IKK on day 10 and activation of Akt and IRS1 on day 14. These results suggested that insulin gel improved wound healing in hyperglycemic mice by modulating the expression of inflammatory factors, growth factors, and proteins of the insulin signaling pathway.

The human gut microbiota is a complex and dynamic community of microorganisms living in our intestines and has emerged as an important factor for colorectal adenocarcinoma (CRC). The purpose of our study was to investigate the microbiota composition in Brazilian CRC patients compared with a local control population (CTL) to find out which changes could be considered universal or regional features in CRC microbiota. Fecal samples were obtained from 28 CRC and 23 CTL individuals. The 16S rRNA gene was used for metagenomic analysis. In addition to the anthropometric variables, the clinical stage (TNM 2018) was considered. Patients with CRC had a significant increase in alpha diversity and a higher percentage of genus Prevotella and a decreased proportion of Megamonas and Ruminococcus. Additionally, the proportion of Faecalibacterium prausnitzii was associated with a better prognosis in the first stages of CRC, and Fusobacterium nucleatum proved to be an important marker of colorectal carcinogenesis and tumor aggressiveness. Although regional differences influence the composition of the microbiota, in the case of CRC, the microhabitat created by the tumor seems to be a major factor. Our results contribute to a better understanding of the carcinogenic process, and even in different environments, some factors appear to be characteristic of the microbiota of patients with CRC.

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.

O estímulo mecânico sabidamente provoca deformação de proteínas da matriz extracelular e da célula óssea, as quais se tornam ativas e sinalizam reações intracelulares que determinam o aumento da massa óssea. Estudos apontam a quinase de adesão focal (FAK) como uma proteína fundamental na sinalização da deformação celular causada pelo estímulo mecânico no osso, mas há necessidade de estudos in vivo que comprovem esse papel. Objetivos - Com o ultra-som de baixa potência, investigar a influência do estímulo mecânico na expressão da FAK no osso, e a possibilidade de detectar proteínas envolvidas com a proliferação celular óssea e muscular esquelética. Material e Métodos - Tíbias e fíbulas de ratos foram estimuladas com ultra-som de baixa potência durante 20 min/uma vez ao dia, por períodos de 7, 14 e 21 dias. O grupo controle não foi estimulado. Cada grupo continha 7 ratos. Quinze horas após o último estímulo, a tíbia e a fíbula foram retiradas para realização de immunoblotting, utilizando anticorpos específicos para identificar FAK, FAK-Tyr(P)-397, extracellular signal-regulated kinase-2 (ERK-2), ERK-1/2-Tyr(P)-204, insulin receptor substrate-1 (IRS-1) e IRS-2. O músculo esquelético da região estimulada foi extraído e submetido ao mesmo procedimento. Resultados - Analisando-se os resultados de immunoblotting comparativamente com o grupo controle, não foram detectadas FAK e FAK-Tyr(P)-397 no tecido ósseo, enquanto ERK-2, ERK-1/2-Tyr(P)-204, IRS-1 e IRS-2 foram identificados. No músculo esquelético, todas as proteínas estudadas foram identificadas, exceto IRS-2. Discussão e Conclusão: Os resultados sugerem que não deve haver quantidade suficiente de FAK no osso detectável pelo método utilizado. Por outro lado, observou-se que ERK-2, IRS-1 e IRS-2 são detectáveis no osso e devem responder ao estímulo mecânico. Além disso, o encontro dessas proteínas no tecido muscular abre novas perspectivas no estudo da influência das partes moles no processo de reparação do tecido ósseo.

It’s widely known that mechanical deformation of bone promotes activation of some proteins in the extracellular matrix and bone cells. These proteins are responsible for signaling intracellular reactions that determine bone mass growth. Studies indicate focal adhesion kinase (FAK) as a crucial protein to signal mechanical deformation caused by mechanical loading on bone, but evidences in vivo are required to prove this role. Objectives - Using a low-intensity ultrasound, investigate the influence of the mechanical loading on the expression of FAK in bone, and the possibility of detecting proteins involved on cellular proliferation of bone and skeletal muscle. Material and methods - Tibiae and fibulae of 12-weeks-rats were stimulated by low-intensity ultrasound during 20 minutes/once a day for periods of 7, 14 and 21 days. There were 4 groups, each one containing 7 rats. The control group was not stimulated. Fifteen hours after the last stimulation, the tibia and fibula were extracted for immunoblotting procedures with specific antibodies in order to evaluate FAK, FAK-Tyr(P)-397, extracellular signal-regulated kinase-2 (ERK-2), ERK-1/2-Tyr(P)-204, insulin receptor substrate-1 (IRS-1) and IRS-2. The skeletal muscle of the stimulated area was subjected to the same procedures. Results - Comparing the results of the different groups, both FAK and FAK-Tyr(P)-397 were not detected in bone. However, ERK-2, ERK-1/2-Tyr(P)-204, IRS-1 and IRS-2 were identified. Moreover, all the proteins studied were detected in skeletal muscle except for IRS-2. Discussion and conclusion - The results suggest there are no significant quantities of FAK in bone detectable by the methods used. In other hand, it was noted that ERK-2, ERK-1/2-Tyr(P)-204, IRS-1 and IRS-2 are detectable in bone and may be responsive to mechanical stimulation. In addition, the detection of these proteins in skeletal muscle, including FAK, opens up new perspectives on the study of the influence of this tissue on bone repair.

Rats fed a high-fructose diet represent an animal model for insulin resistance and hypertension. We recently showed that a high-fructose diet containing vegetable oil but a normal sodium/potassium ratio induced mild insulin resistance with decreased insulin receptor substrate-1 tyrosine phosphorylation in the liver and muscle of normal rats. In the present study, we examined the mean blood pressure, serum lipid levels and insulin sensitivity by estimating in vivo insulin activity using the 15-min intravenous insulin tolerance test (ITT, 0.5 ml of 6 µg insulin, iv) followed by calculation of the rate constant for plasma glucose disappearance (Kitt) in male Wistar-Hannover rats (110-130 g) randomly divided into four diet groups: control, 1:3 sodium/potassium ratio (R Na:K) diet (C 1:3 R Na:K); control, 1:1 sodium/potassium ratio diet (CNa 1:1 R Na:K); high-fructose, 1:3 sodium/potassium ratio diet (F 1:3 R Na:K), and high-fructose, 1:1 sodium/potassium ratio diet (FNa 1:1 R Na:K) for 28 days. The change in R Na:K for the control and high-fructose diets had no effect on insulin sensitivity measured by ITT. In contrast, the 1:1 R Na:K increased blood pressure in rats receiving the control and high-fructose diets from 117 ± 3 and 118 ± 3 mmHg to 141 ± 4 and 132 ± 4 mmHg (P<0.05), respectively. Triacylglycerol levels were higher in both groups treated with a high-fructose diet when compared to controls (C 1:3 R Na:K: 1.2 ± 0.1 mmol/l vs F 1:3 R Na:K: 2.3 ± 0.4 mmol/l and CNa 1:1 R Na:K: 1.2 ± 0.2 mmol/l vs FNa 1:1 R Na:K: 2.6 ± 0.4 mmol/l, P<0.05). These data suggest that fructose alone does not induce hyperinsulinemia or hypertension in rats fed a normal R Na:K diet, whereas an elevation of sodium in the diet may contribute to the elevated blood pressure in this animal model.

Insulin stimulates the tyrosine kinase activity of its receptor resulting in the tyrosine phosphorylation of pp185, which contains insulin receptor substrates IRS-1 and IRS-2. These early steps in insulin action are essential for the metabolic effects of insulin. Feeding animals a high-fructose diet results in insulin resistance. However, the exact molecular mechanism underlying this effect is unknown. In the present study, we determined the levels and phosphorylation status of the insulin receptor and pp185 (IRS-1/2) in liver and muscle of rats submitted to a high-fructose diet evaluated by immunoblotting with specific antibodies. Feeding fructose (28 days) induced a discrete insulin resistance, as demonstrated by the insulin tolerance test. Plasma glucose and serum insulin and cholesterol levels of the two groups of rats, fructose-fed and control, were similar, whereas plasma triacylglycerol concentration was significantly increased in the rats submitted to the fructose diet (P<0.05). There were no changes in insulin receptor concentration in the liver or muscle of either group. However, insulin-stimulated receptor autophosphorylation was reduced to 72 ± 4% (P<0.05) in the liver of high-fructose rats. The IRS-1 protein levels were similar in both liver and muscle of the two groups of rats. In contrast, there was a significant decrease in insulin-induced pp185 (IRS-1/2) phosphorylation, to 83 ± 5% (P<0.05) in liver and to 77 ± 4% (P<0.05) in muscle of the high-fructose rats. These data suggest that changes in the early steps of insulin signal transduction may have an important role in the insulin resistance induced by high-fructose feeding.

Insulin induces tyrosine phosphorylation of Shc in cell cultures and in insulin-sensitive tissues of the intact rat. However, the ability of insulin receptor (IR) tyrosine kinase to phosphorylate Shc has not been previously demonstrated. In the present study, we investigated insulin-induced IR tyrosine kinase activity towards Shc. Insulin receptor was immunoprecipitated from liver extracts, before and after a very low dose of insulin into the portal vein, and incubated with immunopurified Shc from liver of untreated rats. The kinase assay was performed in vitro in the presence of exogenous ATP and the phosphorylation level was quantified by immunoblotting with antiphosphotyrosine antibody. The results demonstrate that Shc interacted with insulin receptor after infusion of insulin, and, more important, there was insulin receptor kinase activity towards immunopurified Shc. The description of this pathway in animal tissue may have an important role in insulin receptor tyrosine kinase activity toward mitogenic transduction pathways.

Insulin stimulates the tyrosine kinase activity of its receptor resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate-1 (IRS-1) which, in turn, associates with proteins containing SH2 domains. It has been shown that IRS-1 associates with the tyrosine phosphatase SHPTP2 in cell cultures. While the effect of the IRS-1/SHPTP2 association on insulin signal transduction is not completely known, this association may dephosphorylate IRS-1 and may play a critical role in the mitogenic actions of insulin. However, there is no physiological demonstration of this pathway of insulin action in animal tissues. In the present study we investigated the ability of insulin to induce association between IRS-1 and SHPTP2 in liver and muscle of intact rats, by co-immunoprecipitation with anti-IRS-1 antibody and anti-SHPTP2 antibody. In both tissues there was an increase in IRS-1 association with SHPTP2 after insulin stimulation. This association occurred when IRS-1 had the highest level of tyrosine phosphorylation and the decrease in this association was more rapid than the decrease in IRS-1 phosphorylation levels. The data provide evidence against the participation of SHPTP2 in IRS-1 dephosphorylation in rat tissues, and suggest that the insulin signal transduction pathway in rat tissues is related mainly to the mitogenic effects of the hormone.

Experiments were carried out in vitro with three viscous polysaccharides (guar gum, pectin, and carboxymethylcellulose (CMC)) of similar initial viscosity submitted to conditions that mimic events occurring in the stomach and duodenum, and their viscosity in these situations was compared to their actions on postprandial hyperglycemia in normal human subjects. Guar gum showed greater viscosity than the other gums during acidification and/or alkalinization and also showed larger effects on plasma glucose levels (35% reduction in maximum rise in plasma glucose) and on the total area under the curve of plasma glucose (control: 20,314 ± 1007 mg dl-1 180 min-1 vs guar gum: 18,277 ± 699 mg dl-1 180 min-1, P<0.01). Pectin, which showed a marked reduction in viscosity at 37oC and after events mimicking those that occur in the stomach and duodenum, did not have a significant effect on postprandial hyperglycemia. The performance of viscosity and the glycemia response to CMC were at an intermediate level between guar gum and pectin. In conclusion, these data suggest that temperature, the process of acidification, alkalinization and exposure to intestinal ions induce different viscosity changes in gums having similar initial viscosity, establishing a direct relationship between a minor decrease of gum viscosity in vitro and a reduction of postprandial hyperglycemia

Insulin stimulates the tyrosine kinase activity of its receptor, resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate 1 (IRS-1). IRS-1 is also a substrate for different peptides and growth factors, and a transgenic mouse "knockout" for this protein does not have normal growth. However, the role of IRS-1 in kidney hypertrophy and/or hyperplasia was not investigated. In the present study we investigated IRS-1 protein and tyrosine phosphorylation levels in the remnant kidney after unilateral nephrectomy (UNX) in 6-week-old male Wistar rats. After insulin stimulation the levels of insulin receptor and IRS-1 tyrosine phosphorylation were reduced to 79 ± 5% (P<0.005) and 58 ± 6% (P<0.0001), respectively, of the control (C) levels, in the remnant kidney. It is possible that a circulating factor and/or a local (paracrine) factor playing a role in kidney growth can influence the early steps of insulin action in parallel. To investigate the hypothesis of a circulating factor, we studied the early steps of insulin action in liver and muscle of unilateral nephrectomized rats. There was no change in pp185 tyrosine phosphorylation levels in liver (C 100 ± 12% vs UNX 89 ± 9%, NS) and muscle (C 100 ± 22% vs UNX 91 ± 17%, NS), and also there was no change in IRS-1 phosphorylation levels in both tissues. These data demonstrate that after unilateral nephrectomy there is a decrease in insulin-induced insulin receptor and IRS-1 tyrosine phosphorylation levels in kidney but not in liver and muscle. It will be of interest to investigate which factors, probably paracrine ones, regulate these early steps of insulin action in the contralateral kidney of unilaterally nephrectomized rats.