Glucose Control in Acute Myocardial Infarction: A Pilot Randomized Study Controlled by Continuous Glucose Monitoring System Comparing the Use of Insulin Glargine with Standard of Care

Background: This pilot study aimed to verify if glycemic control can be achieved in type 2 diabetes patients after acute myocardial infarction (AMI), using insulin glargine (iGlar) associated with regular insulin (iReg), compared with the standard intensive care unit protocol, which uses continuous insulin intravenous delivery followed by NPH insulin and iReg (St. Care). Patients and Methods: Patients (n = 20) within 24 h of AMI were randomized to iGlar or St. Care. Therapy was guided exclusively by capillary blood glucose (CBG), but glucometric parameters were also analyzed by blinded continuous glucose monitoring system (CGMS). Results: Mean glycemia was 141 +/- 39 mg/dL for St. Care and 132 +/- 42 mg/dL for iGlar by CBG or 138 +/- 35 mg/dL for St. Care and 129 +/- 34 mg/dL for iGlar by CGMS. Percentage of time in range (80-180 mg/dL) by CGMS was 73 +/- 18% for iGlar and 77 +/- 11% for St. Care. No severe hypoglycemia (<= 40 mg/dL) was detected by CBG, but CGMS indicated 11 (St. Care) and seven (iGlar) excursions in four subjects from each group, mostly in sulfonylurea users (six of eight patients). Conclusions: This pilot study suggests that equivalent glycemic control without increase in severe hyperglycemia may be achieved using iGlar with background iReg. Data outputs were controlled by both CBG and CGMS measurements in a real-life setting to ensure reliability. Based on CGMS measurements, there were significant numbers of glycemic excursions outside of the target range. However, this was not detected by CBG. In addition, the data indicate that previous use of sulfonylurea may be a potential major risk factor for severe hypoglycemia irrespective of the type of insulin treatment.

Kinin B1 Receptor in Adipocytes Regulates Glucose Tolerance and Predisposition to Obesity

Background: Kinins participate in the pathophysiology of obesity and type 2 diabetes by mechanisms which are not fully understood. Kinin B-1 receptor knockout mice (B-1(-/-)) are leaner and exhibit improved insulin sensitivity. Methodology/Principal Findings: Here we show that kinin B-1 receptors in adipocytes play a role in controlling whole body insulin action and glucose homeostasis. Adipocytes isolated from mouse white adipose tissue (WAT) constitutively express kinin B-1 receptors. In these cells, treatment with the B-1 receptor agonist des-Arg(9)-bradykinin improved insulin signaling, GLUT4 translocation, and glucose uptake. Adipocytes from B-1(-/-) mice showed reduced GLUT4 expression and impaired glucose uptake at both basal and insulin-stimulated states. To investigate the consequences of these phenomena to whole body metabolism, we generated mice where the expression of the kinin B-1 receptor was limited to cells of the adipose tissue (aP2-B-1/B-1(-/-)). Similarly to B-1(-/-) mice, aP2-B-1/B-1(-/-) mice were leaner than wild type controls. However, exclusive expression of the kinin B1 receptor in adipose tissue completely rescued the improved systemic insulin sensitivity phenotype of B-1(-/-) mice. Adipose tissue gene expression analysis also revealed that genes involved in insulin signaling were significantly affected by the presence of the kinin B-1 receptor in adipose tissue. In agreement, GLUT4 expression and glucose uptake were increased in fat tissue of aP2-B-1/B-1(-/-) when compared to B-1(-/-) mice. When subjected to high fat diet, aP2-B-1/B-1(-/-) mice gained more weight than B-1(-/-) littermates, becoming as obese as the wild types. Conclusions/Significance: Thus, kinin B-1 receptor participates in the modulation of insulin action in adipocytes, contributing to systemic insulin sensitivity and predisposition to obesity.

Omega-3 improves glucose tolerance but increases lipid peroxidation and DNA damage in hepatocytes of fructose-fed rats

The high consumption of fructose is linked to the increase in various characteristics of the metabolic syndrome. Fish oil is beneficial for the treatment of these comorbidities, such as insulin resistance, dyslipidemia, and hepatic steatosis. The objective of this study was to evaluate the consequences of the administration of fish oil concomitant to fructose ingestion during the experiment (45 days) and during the final 15 days in high-fructose-fed rats. Male Wistar rats were divided into 5 groups: control; those receiving 10% fish oil (FO); those receiving 60% fructose (Fr); those receiving 60% fructose and 10% fish oil for 45 days (FrFO); and those receiving fructose plus soybean oil for 30 days and fish oil for the final 15 days of the study (FrFO15). There was an increase in triacylglycerol, serum total cholesterol, and hepatic volume in the Fr group. The FO and FrFO groups experienced an increase in lipid peroxidation and a decrease in serum reduced glutathione. The FrFO group suffered greater hepatic injury, with increased alanine aminotransferase levels and DNA damage. Marked n-3 incorporation occurred in the groups receiving fish oil, favoring a better response to the oral glucose tolerance test. Fructose induced comorbidities of the metabolic syndrome, and the use of fish oil promoted a better glucose tolerance, although it was accompanied by more hepatocyte damage.

T-3 Rapidly Increases SLC2A4 Gene Expression and GLUT4 Trafficking to the Plasma Membrane in Skeletal Muscle of Rat and Improves Glucose Homeostasis

Background: Glucose transporter 4 (GLUT4) is highly expressed in muscle and fat tissue, where triiodothyronine (T-3) induces solute carrier family 2 facilitated glucose transporter member 4 (SLC2A4) gene transcription. T-3 was also shown to rapidly increase glucose uptake in myocytes exposed to cycloheximide, indicating that it might act nongenomically to regulate GLUT4 availability. We tested this hypothesis by evaluating, in thyroidectomized rats (Tx rats), the acute and/or chronic T-3 effects on GLUT4 mRNA expression and polyadenylation, protein content, and trafficking to the plasma membrane (PM) in skeletal muscle, as well as on blood glucose disappearance rate (kITT) after insulin administration. Methods: Rats were surgically thyroidectomized and treated with T-3 (0.3 to 100 mu g/100 g body weight) from 10 minutes to 5 days, and killed thereafter. Sham-operated (SO) rats were used as controls. Total RNA was extracted from the skeletal muscles (soleus [SOL] and extensorum digitalis longus [EDL]) and subjected to Northern blotting analysis using rat GLUT4 cDNA probe. Total protein was extracted and subjected to specific centrifugations for subcellular fractionation, and PM as well as microsomal (M) fractions were subjected to Western blotting analysis, using anti-GLUT4 antiserum as a probe. GLUT4 mRNA polyadenylation was examined by a rapid amplification of cDNA ends-poly(A) test (RACE-PAT). Results: Thyroidectomy reduced skeletal muscle GLUT4 mRNA, mRNA poly(A) tail length, protein content, and trafficking to the PM, as well as the kITT. The acute T-3 treatment rapidly (30 minutes) increased all these parameters compared with Tx rats. The 5-day T-3 treatment increased GLUT4 mRNA and protein expression, and restored GLUT4 trafficking to the PM and kITT to SO values. Conclusions: The results presented here show for the first time that, in parallel to its transcriptional action on the SLC2A4 gene, T-3 exerts a rapid post-transcriptional effect on GLUT4 mRNA polyadenylation, which might increase transcript stability and translation efficiency, leading to the increased GLUT4 content and availability to skeletal muscle, as well as on GLUT4 translocation to the PM, improving the insulin sensitivity, as shown by the kITT.

Plasma kinetics of an LDL-like nanoemulsion and lipid transfer to HDL in subjects with glucose intolerance

OBJECTIVE: Glucose intolerance is frequently associated with an altered plasma lipid profile and increased cardiovascular disease risk. Nonetheless, lipid metabolism is scarcely studied in normolipidemic glucose-intolerant patients. The aim of this study was to investigate whether important lipid metabolic parameters, such as the kinetics of LDL free and esterified cholesterol and the transfer of lipids to HDL, are altered in glucose-intolerant patients with normal plasma lipids. METHODS: Fourteen glucose-intolerant patients and 15 control patients were studied; none of the patients had cardiovascular disease manifestations, and they were paired for age, sex, race and co-morbidities. A nanoemulsion resembling a LDL lipid composition (LDE) labeled with C-14-cholesteryl ester and H-3-free cholesterol was intravenously injected, and blood samples were collected over a 24-h period to determine the fractional clearance rate of the labels by compartmental analysis. The transfer of free and esterified cholesterol, triglycerides and phospholipids from the LDE to HDL was measured by the incubation of the LDE with plasma and radioactivity counting of the supernatant after chemical precipitation of non-HDL fractions. RESULTS: The levels of LDL, non-HDL and HDL cholesterol, triglycerides, apo A1 and apo B were equal in both groups. The 14 C-esterified cholesterol fractional clearance rate was not different between glucose-intolerant and control patients, but the H-3-free- cholesterol fractional clearance rate was greater in glucose-intolerant patients than in control patients. The lipid transfer to HDL was equal in both groups. CONCLUSION: In these glucose-intolerant patients with normal plasma lipids, a faster removal of LDE free cholesterol was the only lipid metabolic alteration detected in our study. This finding suggests that the dissociation of free cholesterol from lipoprotein particles occurs in normolipidemic glucose intolerance and may participate in atherogenic signaling.

Triiodothyronine Acutely Stimulates Glucose Transport into L6 Muscle Cells Without Increasing Surface GLUT4, GLUT1, or GLUT3

Background: Thyroid hormones (THs) act genomically to stimulate glucose transport by elevating glucose transporter (Slc2a) expression and glucose utilization by cells. However, nongenomic effects of THs are now emerging. Here, we assess how triiodothyronine (T-3) acutely affects glucose transport and the content of GLUT4, GLUT1, and GLUT3 at the surface of muscle cells, and possible interactions between T-3 and insulin action. Methods: Differentiated L6 myotubes transfected with myc-tagged Slc2a4 (L6-GLUT4myc) or Slc2a1 (L6-GLUT1myc) and wild-type L6 myotubes were studied in the following conditions: control, hypothyroid (Tx), Tx plus T3, Tx plus insulin, and Tx plus insulin and T-3. Results: Glucose uptake and GLUT4 content at the cell surface decreased in the Tx group relative to controls. T-3 treatment for 30 minutes increased glucose transport into L6-GLUT4myc cells without altering surface GLUT4 content, which increased only thereafter. The total amount of GLUT4 protein remained unchanged among the groups studied. The surface GLUT1 content of L6-GLUT1myc cells also remained unaltered after T-3 treatment; however, in these cells glucose transport was not stimulated by T-3. In wild-type L6 cells, although T-3 treatment increased the total amount of GLUT3, it did not change the surface GLUT3 content. Moreover, within 30 minutes, T-3 stimulation of glucose uptake was additive to that of insulin in L6-GLUT4myc cells. As expected, insulin elevated surface GLUT4 content and glucose uptake. However, interestingly, surface GLUT4 content remained unchanged or even dropped with T-3 plus insulin. Conclusions: These data reveal that T-3 rapidly increases glucose uptake in L6-GLUT4myc cells, which, at least for 30 minutes, did not depend on an increment in GLUT4 at the cell surface yet potentiates insulin action. We propose that this rapid T-3 effect involves activation of GLUT4 transporters at the cell surface, but cannot discount the involvement of an unknown GLUT.

Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens

Among trypanosomatids, the genus Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of P. serpens, that displays high amino acid sequence similarity with phytobacteria and Leishmania enzymes. No orthologous PDC/IPDC genes were found in Trypanosoma cruzi or T. brucei. Conservation of the PDC/IPDC gene sequence was verified in 14 Phytomonas isolates. A phylogenetic analysis shows that Phytomonas protein is robustly monophyletic with Leishmania spp. and C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of gamma-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between Phytomonas. Leishmania and Crithidia. We have measured the PDC activity in P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4 mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation. (C) 2012 Elsevier B.V. All rights reserved.

Maternal Melatonin Programs the Daily Pattern of Energy Metabolism in Adult Offspring

Background: Shift work was recently described as a factor that increases the risk of Type 2 diabetes mellitus. In addition, rats born to mothers subjected to a phase shift throughout pregnancy are glucose intolerant. However, the mechanism by which a phase shift transmits metabolic information to the offspring has not been determined. Among several endocrine secretions, phase shifts in the light/dark cycle were described as altering the circadian profile of melatonin production by the pineal gland. The present study addresses the importance of maternal melatonin for the metabolic programming of the offspring. Methodology/Principal Findings: Female Wistar rats were submitted to SHAM surgery or pinealectomy (PINX). The PINX rats were divided into two groups and received either melatonin (PM) or vehicle. The SHAM, the PINX vehicle and the PM females were housed with male Wistar rats. Rats were allowed to mate and after weaning, the male and female offspring were subjected to a glucose tolerance test (GTT), a pyruvate tolerance test (PTT) and an insulin tolerance test (ITT). Pancreatic islets were isolated for insulin secretion, and insulin signaling was assessed in the liver and in the skeletal muscle by western blots. We found that male and female rats born to PINX mothers display glucose intolerance at the end of the light phase of the light/dark cycle, but not at the beginning. We further demonstrate that impaired glucose-stimulated insulin secretion and hepatic insulin resistance are mechanisms that may contribute to glucose intolerance in the offspring of PINX mothers. The metabolic programming described here occurs due to an absence of maternal melatonin because the offspring born to PINX mothers treated with melatonin were not glucose intolerant. Conclusions/Significance: The present results support the novel concept that maternal melatonin is responsible for the programming of the daily pattern of energy metabolism in their offspring.

Altered Glucose Homeostasis and Hepatic Function in Obese Mice Deficient for Both Kinin Receptor Genes

The Kallikrein-Kinin System (KKS) has been implicated in several aspects of metabolism, including the regulation of glucose homeostasis and adiposity. Kinins and des-Arg-kinins are the major effectors of this system and promote their effects by binding to two different receptors, the kinin B2 and B1 receptors, respectively. To understand the influence of the KKS on the pathophysiology of obesity and type 2 diabetes (T2DM), we generated an animal model deficient for both kinin receptor genes and leptin (obB1B2KO). Six-month-old obB1B2KO mice showed increased blood glucose levels. Isolated islets of the transgenic animals were more responsive to glucose stimulation releasing greater amounts of insulin, mainly in 3-month-old mice, which was corroborated by elevated serum C-peptide concentrations. Furthermore, they presented hepatomegaly, pronounced steatosis, and increased levels of circulating transaminases. This mouse also demonstrated exacerbated gluconeogenesis during the pyruvate challenge test. The hepatic abnormalities were accompanied by changes in the gene expression of factors linked to glucose and lipid metabolisms in the liver. Thus, we conclude that kinin receptors are important for modulation of insulin secretion and for the preservation of normal glucose levels and hepatic functions in obese mice, suggesting a protective role of the KKS regarding complications associated with obesity and T2DM.

CTAB, Triton X-100 and freezing-thawing treatments of Candida guilliermondii: Effects on permeability and accessibility of the glucose-6-phosphate dehydrogenase, xylose reductase and xylitol dehydrogenase enzymes

Cells of Candida guilliermondii (ATCC 201935) were permeabilised with surfactant treatment (CTAB or Triton X-100) or a freezing-thawing procedure. Treatments were monitored by in situ activities of the key enzymes involved in xylose metabolism, that is, glucose-6-phosphate dehydrogenase (G6PD), xylose reductase (XR) and xylitol dehydrogenase (XD). The permeabilising ability of the surfactants was dependent on its concentration and incubation time. The optimum operation conditions for the permeabilisation of C. guilliermondii with surfactants were 0.41 mM (CTAB) or 2.78 mM (Triton X-100), 30 degrees C, and pH 7 at 200 rpm for 50 min. The maximum permeabilisation measured in terms of the in situ G6PD activity observed was, in order, as follows: CTAB (122.4 +/- 15.7 U/g(cells)) > freezing-thawing, , (54.3 +/- 1.9 U/g(cells)) > Triton X-100 (23.5 +/- 0.0 U/g(cells)). These results suggest that CTAB surfactant is more effective in the permeabilisation of C. guilliermondii cells in comparison to the freezing-thawing and Triton X-100 treatments. Nevertheless, freezing-thawing was the only treatment that allowed measurable in situ XR activity. Therefore, freezing-thawing permeabilised yeast cells could be used as a source of xylose reductase for analytical purposes or for use in biotransformation process such as xylitol preparation from xylose. The level of in situ xylose reductase was found to be 13.2 +/- 0.1 U/g(cells).

Lipoprotein metabolism in patients with type 1 diabetes under intensive insulin treatment

Background Type 1 diabetes (T1DM) is frequently accompanied by dyslipidemia related with insulin-dependent steps of the intravascular lipoprotein metabolism. T1DM dyslipidemia may predispose to precocious cardiovascular disease and the lipid status in T1DM under intensive insulin treatment has not been sufficiently explored. The aim was to investigate the plasma lipids and the metabolism of LDL and HDL in insulin-treated T1DM patients with high glycemic levels. Methods Sixteen male patients with T1DM (26 ± 7 yrs) with glycated hemoglobin >7%, and 15 control subjects (28 ± 6 yrs) were injected with a lipid nanoemulsion (LDE) resembling LDL and labeled with 14C-cholesteryl ester and 3H-free-cholesterol for determination of fractional clearance rates (FCR, in h-1) and cholesterol esterification kinetics. Transfer of labeled lipids from LDE to HDL was assayed in vitro. Results LDL-cholesterol (83 ± 15 vs 100 ± 29 mg/dl, p=0.08) tended to be lower in T1DM than in controls; HDL-cholesterol and triglycerides were equal. LDE marker 14C-cholesteryl ester was removed faster from plasma in T1DM patients than in controls (FCR=0.059 ± 0.022 vs 0.039 ± 0.022h-1, p=0.019), which may account for their lower LDL-cholesterol levels. Cholesterol esterification kinetics and transfer of non-esterified and esterified cholesterol, phospholipids and triglycerides from LDE to HDL were also equal. Conclusion T1DM patients under intensive insulin treatment but with poor glycemic control had lower LDL-cholesterol with higher LDE plasma clearance, indicating that LDL plasma removal was even more efficient than in controls. Furthermore, HDL-cholesterol and triglycerides, cholesterol esterification and transfer of lipids to HDL, an important step in reverse cholesterol transport, were all normal. Coexistence of high glycemia levels with normal intravascular lipid metabolism may be related to differences in exogenous insulin bioavailabity and different insulin mechanisms of action on glucose and lipids. Those findings may have important implications for prevention of macrovascular disease by intensive insulin treatment.

Plasma kinetics of an LDL-like nanoemulsion and lipid transfer to HDL in subjects with glucose intolerance

OBJECTIVE: Glucose intolerance is frequently associated with an altered plasma lipid profile and increased cardiovascular disease risk. Nonetheless, lipid metabolism is scarcely studied in normolipidemic glucose-intolerant patients. The aim of this study was to investigate whether important lipid metabolic parameters, such as the kinetics of LDL free and esterified cholesterol and the transfer of lipids to HDL, are altered in glucose-intolerant patients with normal plasma lipids. METHODS: Fourteen glucose-intolerant patients and 15 control patients were studied; none of the patients had cardiovascular disease manifestations, and they were paired for age, sex, race and co-morbidities. A nanoemulsion resembling a LDL lipid composition (LDE) labeled with 14C-cholesteryl ester and ³H-free cholesterol was intravenously injected, and blood samples were collected over a 24-h period to determine the fractional clearance rate of the labels by compartmental analysis. The transfer of free and esterified cholesterol, triglycerides and phospholipids from the LDE to HDL was measured by the incubation of the LDE with plasma and radioactivity counting of the supernatant after chemical precipitation of non-HDL fractions. RESULTS: The levels of LDL, non-HDL and HDL cholesterol, triglycerides, apo A1 and apo B were equal in both groups. The 14C-esterified cholesterol fractional clearance rate was not different between glucose-intolerant and control patients, but the ³H-free-cholesterol fractional clearance rate was greater in glucose-intolerant patients than in control patients. The lipid transfer to HDL was equal in both groups. CONCLUSION: In these glucose-intolerant patients with normal plasma lipids, a faster removal of LDE free cholesterol was the only lipid metabolic alteration detected in our study. This finding suggests that the dissociation of free cholesterol from lipoprotein particles occurs in normolipidemic glucose intolerance and may participate in atherogenic signaling.

Impaired glucose tolerance in low-carbohydrate diet: maybe only a physiological state

Conselho Nacional de Desenvolvimento da Pesquisa (National Council of Research Development) - 476148/2010-3