Autologous stem cell transplantation for early type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) is the result of the autoimmune response against pancreatic insulin producing cells. This autoimmune response begins months or even years before the first presentation of signs and symptoms of hyperglycemia and at the time of clinical diagnosis near 30% of -cell mass still remains. In daily clinical practice, the main therapeutic option for T1DM is multiple subcutaneous insulin injections that are shown to promote tight glucose control and reduce much of diabetic chronic complications, especially microvascular complications. Another important aspect related to long-term complications of diabetes is that patients with initially larger -cell mass suffer less microvascular complications and less hypoglycemic events than those patients with small -cell mass. In face of this, -cell preservation is another important target in the management of type 1 diabetes and its related complications. For many years, various immunomodulatory regimens were tested aiming at blocking autoimmunity against -cell mass and at promoting -cell preservation, mainly in secondary prevention trials. In this review, we summarize some of the most important studies involving -cell preservation by immunomodulation and discuss our preliminary data on autologous nonmyeloablative hematopoietic stem cell transplantation in newly-diagnosed T1DM.

High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets

Rafacho A, Cestari TM, Taboga SR, Boschero AC, Bosqueiro JR. High doses of dexamethasone induce increased beta-cell proliferation in pancreatic rat islets. Am J Physiol Endocrinol Metab 296: E681-E689, 2009. First published January 21, 2009; doi:10.1152/ajpendo.90931.2008.-Activation of insulin signaling and cell cycle intermediates is required for adult beta-cell proliferation. Here, we report a model to study beta-cell proliferation in living rats by administering three different doses of dexamethasone (0.1, 0.5, and 1.0 mg/kg ip, DEX 0.1, DEX 0.5, and DEX 1.0, respectively) for 5 days. Insulin sensitivity, insulin secretion, and histomorphometric data were investigated. Western blotting was used to analyze the levels of proteins related to the control of beta-cell growth. DEX 1.0 rats, which present moderate hyperglycemia and marked hyperinsulinemia, exhibited a 5.1-fold increase in beta-cell proliferation and an increase (17%) in beta-cell size, with significant increase in beta-cell mass, compared with control rats. The hyperinsulinemic but euglycemic DEX 0.5 rats also showed a significant 3.6-fold increase in beta-cell proliferation. However, DEX 0.1 rats, which exhibited the lowest degree of insulin resistance, compensate for insulin demand by improving only islet function. Activation of the insulin receptor substrate 2/phosphatidylinositol 3-kinase/serine-threoninekinase/ribosomalprotein S6 kinase pathway, as well as protein retinoblastoma in islets from DEX 1.0 and DEX 0.5, but not in DEX 0.1, rats was also observed. Therefore, increasing doses of dexamethasone induce three different degrees of insulin requirement in living rats, serving as a model to investigate compensatory beta-cell alterations. Augmented beta-cell mass involves beta-cell hyperplasia and, to a lower extent, beta-cell hypertrophy. We suggest that alterations in circulating insulin and, to a lesser extent, glucose levels could be the major stimuli for beta-cell proliferation in the dexamethasone-induced insulin resistance.

A randomized controlled trial comparing a computer-assisted insulin infusion protocol with a strict and a conventional protocol for glucose control in critically ill patients

Purpose: The objective of this study is to evaluate blood glucose (BG) control efficacy and safety of 3 insulin protocols in medical intensive care unit (MICU) patients. Methods: This was a multicenter randomized controlled trial involving 167 MICU patients with at least one BG measurement +/- 150 mg/dL and one or more of the following: mechanical ventilation, systemic inflammatory response syndrome, trauma, or burns. The interventions were computer-assisted insulin protocol (CAIP), with insulin infusion maintaining BG between 100 and 130 mg/dL; Leuven protocol, with insulin maintaining BG between 80 and 110 mg/dL; or conventional treatment-subcutaneous insulin if glucose > 150 mg/dL. The main efficacy outcome was the mean of patients` median BG, and the safety outcome was the incidence of hypoglycemia (<= 40 mg/dL). Results: The mean of patients` median BG was 125.0, 127.1, and 158.5 mg/dL for CAIP, Leuven, and conventional treatment, respectively (P = .34, CAIP vs Leuven; P < .001, CAIP vs conventional). In CAIP, 12 patients (21.4%) had at least one episode of hypoglycemia vs 24 (41.4%) in Leuven and 2 (3.8%) in conventional treatment (P = .02, CAIP vs Leuven; P = .006, CAIP vs conventional). Conclusions: The CAIP is safer than and as effective as the standard strict protocol for controlling glucose in MICU patients. Hypoglycemia was rare under conventional treatment. However, BG levels were higher than with IV insulin protocols. (C) 2009 Elsevier Inc. All rights reserved.

Effects of Lidocaine Infusion during Experimental Endotoxemia in Horses

Background The clinical efficacy of IV infusion of lidocaine for treatment of equine endotoxemia has not been studied. Hypothesis Lidocaine infusion after exposure to lipopolysaccharide (LPS) will inhibit the inflammatory response and have inhibitory effects on the hemodynamic and cytokine responses to endotoxemia. Animals Twelve horses. Methods Two equal groups (n = 6): saline (GI) and lidocaine (GII). In all animals, endotoxin (500 ng/kg body weight [BW]) was injected intraperitoneally over 5 minutes. Twenty minutes later, animals received a bolus of GI or GII (1.3 mg/kg BW) over 5 minutes, followed by a 6-hour continuous rate infusion of GI or GII (0.05 mg/kg BW/min). Treatment efficacy was judged from change in arterial blood pressure, peripheral blood and peritoneal fluid (PF) variables (total and differential cell counts, enzyme activities, and cytokine concentrations), and clinical scores (CS) for behavioral evidence of abdominal pain or discomfort during the study. Results Compared with the control group, horses treated with lidocaine had significantly lower CS and serum and PF tumor necrosis factor-alpha (TNF-alpha) activity. At several time points in both groups, total and differential cell counts, glucose, total protein and fibrinogen concentrations, and alkaline phosphatase, creatine kinase, and TNF-alpha activities were significantly different from baseline values both in peripheral blood and in PF. Conclusions and Clinical Importance Lidocaine significantly decreased severity of CS and inhibited TNF-alpha activity in PF.

The Na+/glucose cotransporters: from genes to therapy

Glucose enters eukaryotic cells via two types of membrane-associated carrier proteins, the Na+/glucose cotransporters (SGLT) and the facilitative glucose transporters (GLUT). The SGLT family consists of six members. Among them, the SGLT1 and SGLT2 proteins, encoded by the solute carrier genes SLC5A1 and SLC5A2, respectively, are believed to be the most important ones and have been extensively explored in studies focusing on glucose fluxes under both physiological and pathological conditions. This review considers the regulation of the expression of the SGLT promoted by protein kinases and transcription factors, as well as the alterations determined by diets of different compositions and by pathologies such as diabetes. It also considers congenital defects of sugar metabolism caused by aberrant expression of the SGLT1 in glucose-galactose malabsorption and the SGLT2 in familial renal glycosuria. Finally, it covers some pharmacological compounds that are being currently studied focusing on the interest of controlling glycemia by antagonizing SGLT in renal and intestinal tissues.

Steviol effect, a glycoside of Stevia rebaudiana, on glucose clearances in rats

Stevia rebaudiana, a South American plant normally used as a natural herbal sweetener, has been suggested as exerting beneficial effects on human health, including as an antihypertensive and antihyperglycemic. The present experiment was undertaken to evaluate the renal excretion of steviol, the aglycone of several natural products extracted from the leaves of S. rebaudiana, and to clarify the actual participation of this compound on the renal excretion of glucose in rats, which has been previously suggested as the preferential action of steviol on the Na+-glucose renal tubular transport system. Steviol was obtained by enzymatic hydrolysis of stevioside with pectinase. Thirty normal male Wistar rats weighing 345 g were used. After a control period, steviol was infused iv at three doses (0.5, 1.0 and 3.0 mg.kg-1/h), according to classical clearance techniques. During all the experiments no significant changes in inulin clearance (Cin) and p-aminohipuric acid clearance (C PAH) were observed. Administration of steviol resulted in a statistically significant increase in the fractional sodium excretion (FeNa+), fractional potassium excretion (FeK+), urinary flow as percent of glomerular filtration rate (V/GFR) and glucose clearance (C G) when compared to controls, but these effects were absent with the dose of 0.5 mg.kg-1/h. The steviol clearance (C S) was higher than the Cin and lower than the C PAH at all the doses employed in this study. The data suggest that steviol is secreted by renal tubular epithelium, causing diuresis, natriuresis, kaliuresis and a fall in renal tubular reabsorption of glucose.

Influência do local de nascimento e do transporte sobre a morbimortalidade de recém-nascidos prematuros

OBJETIVO: Verificar a influência do local de nascimento e do transporte sobre a morbimortalidade de recém-nascidos prematuros na Região Sul do Brasil. MÉTODOS: Estudo de coorte com recém-nascidos prematuros transferidos para a unidade de tratamento intensivo de referência (grupo transporte = 61), tendo sido acompanhados até a alta. Os dados sobre o atendimento no hospital de origem e transporte foram obtidos no momento da internação. Esse grupo foi comparado com neonatos da maternidade de referência, pareados por idade gestacional (grupo controle = 123), tendo como desfecho primário o óbito e desfechos secundários as alterações da glicemia, temperatura e saturação de oxigênio no momento da internação e a incidência de enterocolite necrosante, displasia broncopulmonar e sepses. Na associação entre as variáveis e o desfecho, foi utilizado o risco relativo. Foi adotado um nível de significância de α = 5% e β = 90%. RESULTADOS: A distância média percorrida foi de 91 km. A idade gestacional média foi de 34 semanas. Entre os recém-nascidos transferidos, 23% (n = 14) não tiveram atendimento pediátrico na sala de parto. No transporte, 33% dos recém-nascidos foram acompanhados por pediatra, e os equipamentos utilizados foram: incubadora (57%), bomba de infusão (13%), oxímetro (49%) e aparelho para aferição da glicemia (21%). O grupo transporte apresentou maior incidência de hiperglicemia, risco relativo (RR) = 3,2 (2,3-4,4), hipoglicemia, RR = 2,4 (1,4-4,0), hipertermia, RR = 2,5 (1,6-3,9), e hipoxemia, RR = 2,2 (1,6-3,0). Foram observados 18% de óbitos no grupo dos transferidos e 8,9% no grupo controle, RR = 2,0 (1,0-2,6). CONCLUSÕES: A pesquisa expõe deficiências no atendimento e transporte dos recém-nascidos, sendo necessária uma melhor organização do atendimento perinatal e do transporte na região nordeste do Rio Grande do Sul.

Changes in Glucose and Glutamine Lymphocyte Metabolisms Induced by Type I Interferon alpha

In lymphocytes (LY), the well-documented antiproliferative effects of IFN-alpha are associated with inhibition of protein synthesis, decreased amino acid incorporation, and cell cycle arrest. However, the effects of this cytokine on the metabolism of glucose and glutamine in these cells have not been well investigated. Thus, mesenteric and spleen LY of male Wistar rats were cultured in the presence or absence of IFN-alpha, and the changes on glucose and glutamine metabolisms were investigated. The reduced proliferation of mesenteric LY was accompanied by a reduction in glucose total consumption (35%), aerobic glucose metabolism (55%), maximal activity of glucose-6-phosphate dehydrogenase (49%), citrate synthase activity (34%), total glutamine consumption (30%), aerobic glutamine consumption (20.3%) and glutaminase activity (56%). In LY isolated from spleen, IFN alpha also reduced the proliferation and impaired metabolism. These data demonstrate that in LY, the antiproliferative effects of IFN alpha are associated with a reduction in glucose and glutamine metabolisms.

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

Background: In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties. Results: The highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e. g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity. Conclusions: Taken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.

Differential regulation of PGC-1 alpha expression in rat liver and skeletal muscle in response to voluntary running

Background: The beneficial actions of exercise training on lipid, glucose and energy metabolism and insulin sensitivity appear to be in part mediated by PGC-1 alpha. Previous studies have shown that spontaneously exercised rats show at rest enhanced responsiveness to exogenous insulin, lower plasma insulin levels and increased skeletal muscle insulin sensitivity. This study was initiated to examine the functional interaction between exercise-induced modulation of skeletal muscle and liver PGC-1 alpha protein expression, whole body insulin sensitivity, and circulating FFA levels as a measure of whole body fatty acid (lipid) metabolism. Methods: Two groups of male Wistar rats (2 Mo of age, 188.82 +/- 2.77 g BW) were used in this study. One group consisted of control rats placed in standard laboratory cages. Exercising rats were housed individually in cages equipped with running wheels and allowed to run at their own pace for 5 weeks. At the end of exercise training, insulin sensitivity was evaluated by comparing steady-state plasma glucose (SSPG) concentrations at constant plasma insulin levels attained during the continuous infusion of glucose and insulin to each experimental group. Subsequently, soleus and plantaris muscle and liver samples were collected and quantified for PGC-1 alpha protein expression by Western blotting. Collected blood samples were analyzed for glucose, insulin and FFA concentrations. Results: Rats housed in the exercise wheel cages demonstrated almost linear increases in running activity with advancing time reaching to maximum value around 4 weeks. On an average, the rats ran a mean (Mean +/- SE) of 4.102 +/- 0.747 km/day and consumed significantly more food as compared to sedentary controls (P < 0.001) in order to meet their increased caloric requirement. Mean plasma insulin (P < 0.001) and FFA (P < 0.006) concentrations were lower in the exercise-trained rats as compared to sedentary controls. Mean steady state plasma insulin (SSPI) and glucose (SSPG) concentrations were not significantly different in sedentary control rats as compared to exercise-trained animals. Plantaris PGC-1 alpha protein expression increased significantly from a 1.11 +/- 0.12 in the sedentary rats to 1.74 +/- 0.09 in exercising rats (P < 0.001). However, exercise had no effect on PGC-1 alpha protein content in either soleus muscle or liver tissue. These results indicate that exercise training selectively up regulates the PGC-1 alpha protein expression in high-oxidative fast skeletal muscle type such as plantaris muscle. Conclusion: These data suggest that PGC-1 alpha most likely plays a restricted role in exercise-mediated improvements in insulin resistance (sensitivity) and lowering of circulating FFA levels.

Prevalence of diabetes mellitus and impaired glucose tolerance in a rural community of Angola

Background: To determine the prevalence of diabetes mellitus (DM) and impaired glucose tolerance (IGT) in a rural community (Bengo) of Angola. Methods: A random sample of 421 subjects aged 30 to 69 years (30% men and 70% women) was selected from three villages of Bengo province. This cross-sectional home survey was conducted using a sampling design of stage conglomerates. First, clinical and anthropometric data were obtained and fasting capillary glucose level was determined. Subjects who screened positive (fasting capillary glucose >= 100 mg/dl and < 200 mg/dl) and each sixth consecutive subject who screened negative (fasting capillary glucose < 100 mg/dl) were submitted to the second phase of survey, consisting of the 75 g oral glucose tolerance test. Data was analyzed by the use of SAS statistical software. Results: The prevalence rates of diabetes mellitus and IGT were 2.8% and 8.1%, respectively. The age group with the highest prevalence of diabetes was 60 to 69 years (42%). Impaired glucose tolerance prevalence was 38% in the 40 to 49 year age group and it increased with age, considering that the 50 to 59 and 60 to 69 year age groups as a whole represent 50% of all subjects with impaired glucose tolerance. The prevalence of diabetes mellitus did not differ significantly between men (3.2%) and women (2.7%) (p = 0.47). On the other hand, the prevalence of impaired glucose tolerance among women showed almost twice that found in men (9.1% vs. 5.6%, respectively). Overweight was present in 66.7% of the individuals with diabetes mellitus and 26.5% of individuals with impaired glucose tolerance showed overweight or obesity. Conclusions: Although the prevalence of diabetes mellitus was low, the prevalence of impaired glucose tolerance is considered to be within an intermediary range, suggesting a future increase in the frequency of diabetes in this population.

Predictive factors of non-deterioration of glucose tolerance following a 2-year behavioral intervention

Aim: To identify predictive factors associated with non-deterioration of glucose metabolism following a 2-year behavioral intervention in Japanese-Brazilians. Methods: 295 adults (59.7% women) without diabetes completed 2-year intervention program. Characteristics of those who maintained/improved glucose tolerance status (non-progressors) were compared with those who worsened (progressors) after the intervention. In logistic regression analysis, the condition of non-progressor was used as dependent variable. Results: Baseline characteristics of non-progressors (71.7%) and progressors were similar, except for the former being younger and having higher frequency of disturbed glucose tolerance and lower C-reactive protein (CRP). In logistic regression, non-deterioration of glucose metabolism was associated with disturbed glucose tolerance impaired fasting glucose or impaired glucose tolerance - (p < 0.001) and CRP levels <= 0.04 mg/dL (p = 0.01), adjusted for age and anthropometric variables. Changes in anthropometry and physical activity and achievement of weight and dietary goals after intervention were similar in subsets that worsened or not the glucose tolerance status. Conclusion: The whole sample presented a homogeneous behavior during the intervention. Lower CRP levels and diagnosis of glucose intolerance at baseline were predictors of non-deterioration of the glucose metabolism after a relatively simple intervention, independent of body adiposity.

Leucine supplementation improves adiponectin and total cholesterol concentrations despite the lack of changes in adiposity or glucose homeostasis in rats previously exposed to a high-fat diet

Background: Studies suggest that leucine supplementation (LS) has a therapeutic potential to prevent obesity and to promote glucose homeostasis. Furthermore, regular physical exercise is a widely accepted strategy for body weight maintenance and also for the prevention of obesity. The aim of this study was to determine the effect of chronic LS alone or combined with endurance training (ET) as potential approaches for reversing the insulin resistance and obesity induced by a high-fat diet (HFD) in rats. Methods: Forty-seven rats were randomly divided into two groups. Animals were fed a control diet-low fat (n = 10) or HFD (n = 37). After 15 weeks on HFD, all rats received the control diet-low fat and were randomly divided according to treatment: reference (REF), LS, ET, and LS+ET (n = 7-8 rats per group). After 6 weeks of treatment, the animals were sacrificed and body composition, fat cell volume, and serum concentrations of total cholesterol, HDL-cholesterol, triacylglycerol, glucose, adiponectin, leptin and tumor necrosis factor-alpha (TNF-alpha) were analyzed. Results: At the end of the sixth week of treatment, there was no significant difference in body weight between the REF, LS, ET and LS+ET groups. However, ET increased lean body mass in rats (P = 0.019). In addition, ET was more effective than LS in reducing adiposity (P = 0.019), serum insulin (P = 0.022) and TNF-alpha (P = 0.044). Conversely, LS increased serum adiponectin (P = 0.021) levels and reduced serum total cholesterol concentration (P = 0.042). Conclusions: The results showed that LS had no beneficial effects on insulin sensitivity or adiposity in previously obese rats. On the other hand, LS was effective in increasing adiponectin levels and in reducing total cholesterol concentration.

Control of the Intracellular Redox State by Glucose Participates in the Insulin Secretion Mechanism

Background: Production of reactive oxygen species (ROS) due to chronic exposure to glucose has been associated with impaired beta cell function and diabetes. However, physiologically, beta cells are well equipped to deal with episodic glucose loads, to which they respond with a fine tuned glucose-stimulated insulin secretion (GSIS). In the present study, a systematic investigation in rat pancreatic islets about the changes in the redox environment induced by acute exposure to glucose was carried out. Methodology/Principal Findings: Short term incubations were performed in isolated rat pancreatic islets. Glucose dose- and time-dependently reduced the intracellular ROS content in pancreatic islets as assayed by fluorescence in a confocal microscope. This decrease was due to activation of pentose-phosphate pathway (PPP). Inhibition of PPP blunted the redox control as well as GSIS in a dose-dependent manner. The addition of low doses of ROS scavengers at high glucose concentration acutely improved beta cell function. The ROS scavenger N-acetyl-L-cysteine increased the intracellular calcium response to glucose that was associated with a small decrease in ROS content. Additionally, the presence of the hydrogen peroxide-specific scavenger catalase, in its membrane-permeable form, nearly doubled glucose metabolism. Interestingly, though an increase in GSIS was also observed, this did not match the effect on glucose metabolism. Conclusions: The control of ROS content via PPP activation by glucose importantly contributes to the mechanisms that couple the glucose stimulus to insulin secretion. Moreover, we identified intracellular hydrogen peroxide as an inhibitor of glucose metabolism intrinsic to rat pancreatic islets. These findings suggest that the intracellular adjustment of the redox environment by glucose plays an important role in the mechanism of GSIS.

High Glucose-Mediated Oxidative Stress Impairs Cell Migration

Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we evaluate the hypothesis that high glucose concentrations inhibit cell migration. Using CHO.K1 cells, NIH-3T3 fibroblasts, mouse embryonic fibroblasts and primary skin fibroblasts from control and diabetic rats cultured in 5 mM D-glucose (low glucose, LG), 25 mM D-glucose (high glucose, HG) or 25 mM L-glucose medium (osmotic control - OC), we analyzed the migration speed, protrusion stability, cell polarity, adhesion maturation and the activity of the small Rho GTPase Rac1. We also analyzed the effects of reactive oxygen species by incubating cells with the antioxidant N-Acetyl-Cysteine (NAC). We observed that HG conditions inhibited cell migration when compared to LG or OC. This inhibition resulted from impaired cell polarity, protrusion destabilization and inhibition of adhesion maturation. Conversely, Rac1 activity, which promotes protrusion and blocks adhesion maturation, was increased in HG conditions, thus providing a mechanistic basis for the HG phenotype. Most of the HG effects were partially or completely rescued by treatment with NAC. These findings demonstrate that HG impairs cell migration due to an increase in oxidative stress that causes polarity loss, deficient adhesion and protrusion. These alterations arise, in large part, from increased Rac1 activity and may contribute to the poor wound healing observed in diabetic patients.