The Prolonged Activation of the p65 Subunit of the NF-Kappa-B Nuclear Factor Sustains the Persistent Effect of Advanced Glycation End Products on Inflammatory Sensitization in Macrophages.

Advanced glycation end products (AGEs) prime macrophages for lipopolysaccharide (LPS)-induced inflammation. We investigated the persistence of cellular AGE-sensitization to LPS, considering the nuclear content of p50 and p65 nuclear factor kappa B (NFKB) subunits and the expression of inflammatory genes. Macrophages treated with control (C) or AGE-albumin were rested for varying intervals in medium alone before being incubated with LPS. Comparisons were made using one-way ANOVA or Student t-test (n = 6). AGE-albumin primed macrophages for increased responsiveness to LPS, resulting in elevated levels of TNF, IL-6, and IL-1beta (1.5%, 9.4%, and 5.6%, respectively), compared to C-albumin. TNF, IL-6, and IL-1 beta secretion persisted for up to 24 h even after the removal of AGE-albumin (area under the curve greater by 1.6, 16, and 5.2 times, respectively). The expressions of Il6 and RelA were higher 8 h after albumin removal, and Il6 and Abca1 were higher 24 h after albumin removal. The nuclear content of p50 remained similar, but p65 showed a sustained increase (2.9 times) for up to 24 h in AGE-albumin-treated cells. The prolonged activation of the p65 subunit of NFKB contributes to the persistent effect of AGEs on macrophage inflammatory priming, which could be targeted for therapies to prevent complications based on the AGE-RAGE-NFKB axis.

Effect of resistance training on osteopenic rat bones in neonatal streptozotocin-induced diabetes: Analysis of GLUT4 content and biochemical, biomechanical, densitometric, and microstructural evaluation.

AIMS: To investigate the effect of resistance training-RT on glycemia, expression of the glucose transporter-GLUT4, bone mineral density-BMD, and microstructural and biomechanical properties of osteopenic rat bones in neonatal streptozotocin-induced diabetes. MAIN METHODS: Sixty-four 5-day-old male rats were divided into two groups: control and diabetic rats injected with vehicle or streptozotocin, respectively. After 55 days, densitometric analysis-DA of the tibia was performed. These groups were subdivided into four subgroups: non-osteopenic control-CN, osteopenic control-OC, non-osteopenic diabetic-DM, and osteopenic diabetic-OD. The OC and OD groups were suspended by their tails for 21 days to promote osteopenia in the hindlimb; subsequently, a second DA was performed. The rats were subdivided into eight subgroups: sedentary control-SC, sedentary osteopenic control-SOC, exercised control-EC, exercised osteopenic control-EOC, sedentary diabetic-SD, sedentary osteopenic diabetic-SOD, exercised diabetic-ED, and exercised osteopenic diabetic-EOD. For RT, the rats climbed a ladder with weights secured to their tails for 12 weeks. After RT, a third DA was performed, and blood samples, muscles, and tibias were assessed to measure glycemia, insulinemia, GLUT4 content, bone maximum strength, fracture energy, extrinsic stiffness, BMD, cancellous bone area, trabecular number, and trabecular width. KEY FINDINGS: After RT, glycemia, GLUT4 content, BMD, and bone microstructural and biomechanical properties were improved in diabetic rats (osteopenic and non-osteopenic). However, RT had no effect on these parameters in the EC and SC groups. SIGNIFICANCE: These results suggest that RT improves GLUT4 content, BMD, and microstructural and biomechanical properties of bone in osteopenic and non-osteopenic diabetic rats and is effective in controlling glycemia.

Resveratrol improves glycemic control in insulin-treated diabetic rats: participation of the hepatic territory.

BACKGROUND: Resveratrol is a natural polyphenol that has been proposed to improve glycemic control in diabetes, by mechanisms that involve improvement in insulin secretion and activity. In type 1 diabetes (T1D), in which insulin therapy is obligatory, resveratrol treatment has never been investigated. The present study aimed to evaluate resveratrol as an adjunctive agent to insulin therapy in a T1D-like experimental model. METHODS: Rats were rendered diabetic by streptozotocin (STZ) treatment. Twenty days later, four groups of animals were studied: non-diabetic (ND); diabetic treated with placebo (DP); diabetic treated with insulin (DI) and diabetic treated with insulin plus resveratrol (DIR). After 30 days of treatment, 24-hour urine was collected; then, blood, soleus muscle, proximal small intestine, renal cortex and liver were sampled. Specific glucose transporter proteins were analyzed (Western blotting) in each territory of interest. Solute carrier family 2 member 2 (Slc2a2), phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase catalytic subunit (G6pc) mRNAs (qPCR), glycogen storage and sirtuin 1 (SIRT1) activity were analyzed in liver. RESULTS: Diabetes induction increased blood glucose, plasma fructosamine concentrations, and glycosuria. Insulin therapy partially recovered the glycemic control; however, resveratrol as adjunctive therapy additionally improved glycemic control and restored plasma fructosamine concentration to values of non-diabetic rats. Resveratrol did not alter the expression of the glucose transporters GLUT2 and SGLT1 in the intestine, GLUT2 and SGLT2 in kidney and GLUT4 in soleus, suggesting that fluxes of glucose in these territories were unaltered. Differently, in liver, resveratrol promoted a reduction in Slc2a2, Pck1, and G6pc mRNAs, as well as in GLUT2 protein (P < 0.05, DIR vs. DI); besides, it increased (P < 0.01, DIR vs. DI) the hepatic glycogen content, and SIRT1 protein. CONCLUSIONS: Resveratrol is able to improve glycemic control in insulin-treated T1D-like rats. This effect seems not to involve changes in glucose fluxes in the small intestine, renal proximal tubule, and soleus skeletal muscle; but to be related to several changes in the liver, where downregulation of Slc2a2/GLUT2, Pck1, and G6pc expression was observed, favoring reduction of glucose production and efflux. Besides, resveratrol increased SIRT1 nuclear protein content in liver, which may be related to the observed gene expression regulations.

Maternal periodontitis decreases plasma membrane GLUT4 content in skeletal muscle of adult offspring.

AIMS: The fetal programming hypothesis suggests that intrauterine stimuli can induce metabolic changes in offspring, increasing the disease risk in adulthood. Periodontal disease may enhance serum cytokine levels. Cytokines such as tumor necrosis factor-alpha (TNF-α) have been associated with reduced glucose transporter type 4 (GLUT4) expression, decreased protein kinase B (Akt) phosphorylation, and insulin resistance. This study aimed to evaluate GLUT4 content, and Akt serine phosphorylation status in the gastrocnemius skeletal muscle (GSM), glycemia, insulinemia and change in body weight in offspring of rats with periodontal disease. MAIN METHODS: Female Wistar rats were distributed into a control group (CN) and an experimental periodontal disease group (PD), in which a ligature was placed around the mandibular first molars. Seven days after ligature placement, both groups were mated with normal male rats. The ligatures remained throughout pregnancy until weaning, after which the male offspring were distributed into groups: CN-o, control rat offspring; and PD-o, periodontal disease rat offspring. The body weight from 0 to 75days of age was measured. At 75days, the glycemia, insulinemia, TNF-α levels, Akt serine phosphorylation, and GLUT4 content in the GSM were measured in the offspring. KEY FINDINGS: The PD-o group showed a low birth weight (LBW), unchanged glycemia, increased insulinemia, insulin resistance, increased TNF-α levels, decreased Akt serine phosphorylation status, and reduced GLUT4 content in the plasma membrane and translocation index after insulin stimulation. SIGNIFICANCE: Maternal periodontal disease causes LBW, insulin resistance, and alterations in the final stage of insulin signaling in the GSM of adult offspring.

Periapical lesions decrease Akt serine phosphorylation and plasma membrane GLUT4 content in rat skeletal muscle.

OBJECTIVES: Periapical lesion (PL) promotes insulin resistance; however, the mechanisms underlying this alteration are not fully understood. Therefore, in this study, we aimed to evaluate the Akt serine phosphorylation status and GLUT4 expression levels in the gastrocnemius muscle (GM) of rats with PL. MATERIALS AND METHODS: Male Wistar rats (n = 42) were distributed equally into control (CN) and PL groups. The pulpal tissue of the PL group rats was exposed to the oral environment for 30 days. Thereafter, glucose and insulin levels were assessed, followed by homeostasis model assessment of insulin resistance (HOMA-IR). The Akt serine phosphorylation and GLUT4 levels of microsomal (M) and plasma membrane (PM) fractions were evaluated by western blotting and analyzed statistically. RESULTS: Compared to CN group rats, PL group rats had lower insulin sensitivity (as observed by HOMA-IR), lower Akt serine phosphorylation status after insulin stimulus, and lower GLUT4 levels in the PM fraction. However, the M fraction in the PL group did not differ significantly from that of the CN group. CONCLUSIONS: PL decreases insulin sensitivity, Akt phosphorylation, and PM GLUT4 content. CLINICAL RELEVANCE: The present study indicates that preventing endodontic disease can thwart insulin resistance.

Muscle IGF-1-induced skeletal muscle hypertrophy evokes higher insulin sensitivity and carbohydrate use as preferential energy substrate.

We characterized the metabolic profile of transgenic mice exhibiting enhanced muscle mass driven by increased mIGF-1 expression (MLC/mIGF-1). As expected, 6-month-old MLC/mIGF-1 mice were heavier than age-matched wild type (WT) mice (37.4 ± 0.3 versus 31.8 ± 0.6 g, resp.). MLC/mIGF-1 mice had higher respiratory quotient when compared to WT (0.9 ± 0.03 versus 0.74 ± 0.02, resp.) suggesting a preference for carbohydrate as the major fuel source. MLC/mIGF-1 mice had a higher rate of glucose disposal when compared to WT (3.25 ± 0.14 versus 2.39 ± 0.03%/min, resp.). The higher disposal rate correlated to ∼ 2-fold higher GLUT4 content in the extensor digitorum longus (EDL) muscle. Analysis of mRNA content for the glycolysis-related gene PFK-1 showed ∼ 3-fold upregulation in MLC/mIGF-1 animals. We also found a 50% downregulation of PGC1α mRNA levels in MLC/mIGF-1 mouse EDL muscle, suggesting less abundant mitochondria in this tissue. We found no difference in the expression of PPARα and PPARß/δ, suggesting no modulation of key elements in oxidative metabolism. These data together suggest a shift in metabolism towards higher carbohydrate utilization, and that could explain the increased insulin sensitivity of hypertrophied skeletal muscle in MLC/mIGF-1 mice.

Increased SGLT1 expression in salivary gland ductal cells correlates with hyposalivation in diabetic and hypertensive rats.

BACKGROUND: Oral health complications in diabetes and hypertension include decreased salivary secretion. The sodium-glucose cotransporter 1 (SGLT1) protein, which transports 1 glucose/2 Na+/264 H2O molecules, is described in salivary glands. We hypothesized that changes in SGLT1 expression in the luminal membrane of ductal cell may be related to an altered salivary flow. FINDINGS: By immunohistochemistry, we investigated SGLT1 expression in ductal cells of parotid and submandibular glands from Wistar Kyoto rats (WKY), diabetic WKY (WKY-D), spontaneously hypertensive rats (SHR) and diabetic SHR (SHR-D), as well as in parotid glands from WKY subjected to sympathetic stimulation, with or without previous propranolol blockade. Diabetes and hypertension decreased the salivary secretion and increased SGLT1 expression in the luminal membrane of ductal cells, and their association exacerbated the regulations observed. After 30 min of sympathetic stimulation, SGLT1 increased in the luminal membrane of ductal cells, and that was blocked by previous injection of propranolol. CONCLUSIONS: SGLT1 expression increases in the luminal membrane of salivary gland ductal cells and the salivary flow decreases in diabetic and hypertensive rats, which may be related to sympathetic activity. This study highlights the water transporter role of SGLT1 in salivary glands, which, by increasing ductal water reabsorption, may explain the hyposalivation of diabetic and hypertensive subjects.

Intensive insulin treatment induces insulin resistance in diabetic rats by impairing glucose metabolism-related mechanisms in muscle and liver.

Insulin replacement is the only effective therapy to manage hyperglycemia in type 1 diabetes mellitus (T1DM). Nevertheless, intensive insulin therapy has inadvertently led to insulin resistance. This study investigates mechanisms involved in the insulin resistance induced by hyperinsulinization. Wistar rats were rendered diabetic by alloxan injection, and 2 weeks later received saline or different doses of neutral protamine Hagedorn insulin (1.5, 3, 6, and 9 U/day) over 7 days. Insulinopenic-untreated rats and 6U- and 9U-treated rats developed insulin resistance, whereas 3U-treated rats revealed the highest grade of insulin sensitivity, but did not achieve good glycemic control as 6U- and 9U-treated rats did. This insulin sensitivity profile was in agreement with glucose transporter 4 expression and translocation in skeletal muscle, and insulin signaling, phosphoenolpyruvate carboxykinase/glucose-6-phosphatase expression and glycogen storage in the liver. Under the expectation that insulin resistance develops in hyperinsulinized diabetic patients, we believe insulin sensitizer approaches should be considered in treating T1DM.

Soybean and sunflower oil-induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue.

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable-oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 microL) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB- and SF-induced insulin resistance was shown to involve GLUT4. In SB- and SF-treated animals, the GLUT4 protein expression was reduced approximately 20% and 10 min after an acute in vivo stimulus with insulin, the plasma membrane GLUT4 content was approximately 60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid ( approximately 150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections.

Insulin but not phlorizin treatment induces a transient increase in GLUT2 gene expression in the kidney of diabetic rats.

BACKGROUND/AIMS: Increases in the renal glucose transporter gene expression are involved in renal tubule-glomerular diseases. Here we investigate the GLUT2 gene expression changes in the kidney of diabetic rats, by using insulin or phlorizin treatment. METHODS: Rats were rendered diabetic and studied 20 days later: 4-12 h after one single injection of insulin or phlorizin, and 1-6 days after insulin or phlorizin injection twice a day, comparing with diabetic rats injected with placebo. GLUT2 was investigated by Northern and Western analysis. RESULTS: In 20-day diabetic rats, acute treatment with insulin lowered the plasma glucose and increased the GLUT2 mRNA ( approximately 100%, p < 0.001) without changes in the protein content, while phlorizin lowered the plasma glucose, but changed neither the GLUT2 mRNA nor the protein expression. Twenty-four hours of insulin treatment increased both GLUT2 mRNA ( approximately 100%, p < 0.001) and protein ( approximately 50%, p < 0.01), but no effects of phlorizin were observed. After 6 days, insulin and phlorizin similarly reduced glycemia, with opposite effects upon plasma insulin and urinary glucose, and both treatments decreased GLUT2 mRNA and protein (p < 0.05). CONCLUSION: In kidney of diabetic rats, an initial and transient upregulation of GLUT2 was induced specifically by insulin only. The 6-day normalization of GLUT2, however, was induced by both insulin and phlorizin treatment, which seems to be related to the plasma glucose lowering.

Changes in dietary sodium consumption modulate GLUT4 gene expression and early steps of insulin signaling.

Previous studies have shown that chronic salt overload increases insulin sensitivity, while chronic salt restriction decreases it. In the present study we investigated the influence of dietary sodium on 1) GLUT4 gene expression, by No the n and Western blotting analysis; 2) in vivo GLUT4 protein translocation, by measuring the GLUT4 protein in plasma membrane and microsome, before and after insulin injection; and 3) insulin signaling, by analyzing basal and insulin-stimulated tyrosine phosphorylation of insulin receptor (IR)-beta, insulin receptor substrate (IRS)-1, and IRS-2. Wistar rats we e fed no mal-sodium (NS-0.5%), low-sodium (LS-0.06%), o high-sodium diets (HS-3.12%) fo 9 wk and were killed under pentobarbital anesthesia. Compared with NS ats, HS ats inc eased (P < 0.05) the GLUT4 protein in adipose tissue and skeletal muscle, whereas GLUT4 mRNA was increased only in adipose tissue. GLUT4 expression was unchanged in LS ats compared with NS ats. The GLUT4 translocation in HS ats was higher (P < 0.05) both in basal and insulin-stimulated conditions. On the other hand, LS ats did not increase the GLUT4 translocation after insulin stimulus. Compared with NS ats, LS ats showed reduced (P < 0.01) basal and insulin-stimulated tyrosine phosphorylation of IRS-1 in skeletal muscle and IRS-2 in live, whereas HS ats showed enhanced basal tyrosine phosphorylation of IRS-1 in skeletal muscle (P < 0.05) and of IRS-2 in live. In summary, increased insulin sensitivity in HS ats is elated to increased GLUT4 gene expression, enhanced insulin signaling, and GLUT4 translocation, whereas decreased insulin sensitivity of LS ats does not involve changes in GLUT4 gene expression but is elated to impaired insulin signaling.

O teor de sódio da dieta modula a expressão gênica dor transportadores de glicose em diferentes territórios do organismo / Dietary sodium modulates glucose transporters gene expression in different territories of organism

Verificamos o efeito do teor de sódio da dieta sobre a expressão gênica dos transportadores de glicose em tecido adiposo branco e marrom, muscular esquelético e cardíaco, intestino e rim de ratos tratados cronicamente com dietas normossódica (NS), hipossódica (LS) e hipersódica (HS).Em HS, aumento da sensibilidade insulínica se relacionou com aumentada expressão do gene do GLUT4 e translocação da sua proteína. Por outro lado, modulações na expressão gênica dos transportadores de glicose em intestino (SGLT1 e GLUT2) e rim (SGLT2, GLUT2 e GLUT1) foram observadas em HS e LS, mas não repercutiram em alterações de absorção intestinal e reabsorção renal de glicose.In the present study we verified the effect of dietary sodium content on the glucose transporters gene expression in white and brown adipose tissues, skeletal and cardiac muscles, intestine and kidney of chronically treated rats with normal- (NS), low- (LS) or hig- (HS) sodium diets. HS-treated rats showed higher insulin sensitivity, which was related to increased GLUT4 gene expression in insulin sensitive tissues, as well, to increased protein translocation. However, the modulations of HS and LS on intestinal (SGLT1 and GLUT2) and renal (SGLT2, GLUT2 and GLUT1) glucose transporters expressions did not reflect on intestinal absorption and renal reabsorption of glucose...

Esquema de restriçäo alimentar altera a resposta celular à insulina em ratos diabéticos / Scheme of food restriction change the cellular response to insulin in rats

A resistência à insulina é um elemento chave na gênese de várias alterações fisiopatológicas. O esquema de restrição alimentar meal-feeding (MF) consiste na oferta de uma única refeição diária de 2h, sem, contudo, limitar a quantidade de alimento durante esse período. Este esquema determina várias adaptações morfo-metabólicas, algumas sugerindo maior sensibilidade à insulina. No presente estudo investigou-se, em adipócitos isolados, a sensibilidade à insulina de ratos submetidos a treinamento alimentar e tornados diabéticos (MFD) comparando-os com ratos em livre curso alimentar diabéticos (FFD). A curva dose resposta da captação de 2-deoxi-D-glicose estimulada por insulina sugeriu maior sensibilidade, enquanto a captação máxima foi significativamente maior (p<0,05) nos ratos MFD. Esta maior responsividade à insulina refletiu-se nometabolismo dos adipócitos que mostraram aumento (p<0,05) na capacidade máxima de oxidar glicose a CO 2 e incorporar glicose em triacilglicerol. O aumento da resposta celular à insulina em ratos MFD refletiu-se in vivo em menor (p<0,05) insulinemia e maior (p<0,05) índice glicose/insulina (G/I). Em conjunto, os resultados indicam que o treinamento alimentar melhora a resposta à insulina em ratos diabéticos, o que pode representar uma abordagem alternativa no tratamento do diabetes mellitus.