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.

Glucose Transport and Utilization in the Hippocampus: From Neurophysiology to Diabetes-Related Development of Dementia.

The association of diabetes with cognitive dysfunction has at least 60 years of history, which started with the observation that children with type 1 diabetes mellitus (T1D), who had recurrent episodes of hypoglycemia and consequently low glucose supply to the brain, showed a deficit of cognitive capacity. Later, the growing incidence of type 2 diabetes mellitus (T2D) and dementia in aged populations revealed their high association, in which a reduced neuronal glucose supply has also been considered as a key mechanism, despite hyperglycemia. Here, we discuss the role of glucose in neuronal functioning/preservation, and how peripheral blood glucose accesses the neuronal intracellular compartment, including the exquisite glucose flux across the blood-brain barrier (BBB) and the complex network of glucose transporters, in dementia-related areas such as the hippocampus. In addition, insulin resistance-induced abnormalities in the hippocampus of obese/T2D patients, such as inflammatory stress, oxidative stress, and mitochondrial stress, increased generation of advanced glycated end products and BBB dysfunction, as well as their association with dementia/Alzheimer's disease, are addressed. Finally, we discuss how these abnormalities are accompained by the reduction in the expression and translocation of the high capacity insulin-sensitive glucose transporter GLUT4 in hippocampal neurons, which leads to neurocytoglycopenia and eventually to cognitive dysfunction. This knowledge should further encourage investigations into the beneficial effects of promising therapeutic approaches which could improve central insulin sensitivity and GLUT4 expression, to fight diabetes-related cognitive dysfunctions.

Postmortem Brains from Subjects with Diabetes Mellitus Display Reduced GLUT4 Expression and Soma Area in Hippocampal Neurons: Potential Involvement of Inflammation.

Diabetes mellitus (DM) is an important risk factor for dementia, which is a common neurodegenerative disorder. DM is known to activate inflammation, oxidative stress, and advanced glycation end products (AGEs) generation, all capable of inducing neuronal dysfunctions, thus participating in the neurodegeneration progress. In that process, disturbed neuronal glucose supply plays a key role, which in hippocampal neurons is controlled by the insulin-sensitive glucose transporter type 4 (GLUT4). We investigated the expression of GLUT4, nuclear factor NF-kappa B subunit p65 [NFKB (p65)], carboxymethyllysine and synapsin1 (immunohistochemistry), and soma area in human postmortem hippocampal samples from control, obese, and obese+DM subjects (41 subjects). Moreover, in human SH-SY5Y neurons, tumor necrosis factor (TNF) and glycated albumin (GA) effects were investigated in GLUT4, synapsin-1 (SYN1), tyrosine hydroxylase (TH), synaptophysin (SYP) proteins, and respective genes; NFKB binding activity in the SLC2A4 promoter; effects of increased histone acetylation grade by histone deacetylase 3 (HDAC3) inhibition. Hippocampal neurons (CA4 area) of obese+DM subjects displayed reduced GLUT4 expression and neuronal soma area, associated with increased expression of NFKB (p65). Challenges with TNF and GA decreased the SLC2A4/GLUT4 expression in SH-SY5Y neurons. TNF decreased SYN1, TH, and SYP mRNAs and respective proteins, and increased NFKB binding activity in the SLC2A4 promoter. Inhibition of HDAC3 increased the SLC2A4 expression and the total neuronal content of CRE-binding proteins (CREB/ICER), and also counterbalanced the repressor effect of TNF upon these parameters. This study revealed reduced postmortem human hippocampal GLUT4 content and neuronal soma area accompanied by increased proinflammatory activity in the brains of DM subjects. In isolated human neurons, inflammatory activation by TNF reduced not only the SLC2A4/GLUT4 expression but also the expression of some genes related to neuronal function (SYN1, TH, SYP). These effects may be related to epigenetic regulations (H3Kac and H4Kac status) since they can be counterbalanced by inhibiting HDAC3. These results uncover the improvement in GLUT4 expression and/or the inhibition of HDAC3 as promising therapeutic targets to fight DM-related neurodegeneration.

Aerobic Exercise Training Reduces Atherogenesis Induced by Low-Sodium Diet in LDL Receptor Knockout Mice.

This study investigated the efficacy of aerobic exercise training (AET) in the prevention of dyslipidemia, insulin resistance (IR), and atherogenesis induced by severe low-sodium (LS) diet. LDL receptor knockout (LDLR KO) mice were fed a low-sodium (LS) (0.15% NaCl) or normal-sodium (NS; 1.27% NaCl) diet, submitted to AET in a treadmill, 5 times/week, 60 min/day, 15 m/min, for 90 days, or kept sedentary. Blood pressure (BP), plasma total cholesterol (TC) and triglyceride (TG) concentrations, lipoprotein profile, and insulin sensitivity were evaluated at the end of the AET protocol. Lipid infiltration, angiotensin II type 1 receptor (AT1), receptor for advanced glycation end products (RAGE), carboxymethyllysine (CML), and 4-hydroxynonenal (4-HNE) contents as well as gene expression were determined in the brachiocephalic trunk. BP and TC and gene expression were similar among groups. Compared to the NS diet, the LS diet increased vascular lipid infiltration, CML, RAGE, 4-HNE, plasma TG, LDL-cholesterol, and VLDL-TG. Conversely, the LS diet reduced vascular AT1 receptor, insulin sensitivity, HDL-cholesterol, and HDL-TG. AET prevented arterial lipid infiltration; increases in CML, RAGE, and 4-HNE contents; and reduced AT1 levels and improved LS-induced peripheral IR. The current study showed that AET counteracted the deleterious effects of chronic LS diet in an atherogenesis-prone model by ameliorating peripheral IR, lipid infiltration, CML, RAGE, 4-HNE, and AT1 receptor in the intima-media of the brachiocephalic trunk. These events occurred independently of the amelioration of plasma-lipid profile, which was negatively affected by the severe dietary-sodium restriction.

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.

Aerobic Exercise Training Prevents Insulin Resistance and Hepatic Lipid Accumulation in LDL Receptor Knockout Mice Chronically Fed a Low-Sodium Diet.

BACKGROUND: A low-sodium (LS) diet reduces blood pressure, contributing to the prevention of cardiovascular diseases. However, intense dietary sodium restriction impairs insulin sensitivity and worsens lipid profile. Considering the benefits of aerobic exercise training (AET), the effect of LS diet and AET in hepatic lipid content and gene expression was investigated in LDL receptor knockout (LDLr-KO) mice. METHODS: Twelve-week-old male LDLr-KO mice fed a normal sodium (NS) or LS diet were kept sedentary (S) or trained (T) for 90 days. Body mass, plasma lipids, insulin tolerance testing, hepatic triglyceride (TG) content, gene expression, and citrate synthase (CS) activity were determined. Results were compared by 2-way ANOVA and Tukey's post-test. RESULTS: Compared to NS, LS increased body mass and plasma TG, and impaired insulin sensitivity, which was prevented by AET. The LS-S group, but not the LS-T group, presented greater hepatic TG than the NS-S group. The LS diet increased the expression of genes related to insulin resistance (ApocIII, G6pc, Pck1) and reduced those involved in oxidative capacity (Prkaa1, Prkaa2, Ppara, Lipe) and lipoprotein assembly (Mttp). CONCLUSION: AET prevented the LS-diet-induced TG accumulation in the liver by improving insulin sensitivity and the expression of insulin-regulated genes and oxidative capacity.

Estrogen and Glycemic Homeostasis: The Fundamental Role of Nuclear Estrogen Receptors ESR1/ESR2 in Glucose Transporter GLUT4 Regulation.

Impaired circulating estrogen levels have been related to impaired glycemic homeostasis and diabetes mellitus (DM), both in females and males. However, for the last twenty years, the relationship between estrogen, glycemic homeostasis and the mechanisms involved has remained unclear. The characterization of estrogen receptors 1 and 2 (ESR1 and ESR2) and of insulin-sensitive glucose transporter type 4 (GLUT4) finally offered a great opportunity to shed some light on estrogen regulation of glycemic homeostasis. In this manuscript, we review the relationship between estrogen and DM, focusing on glycemic homeostasis, estrogen, ESR1/ESR2 and GLUT4. We review glycemic homeostasis and GLUT4 expression (muscle and adipose tissues) in Esr1-/- and Esr2-/- transgenic mice. We specifically address estradiol-induced and ESR1/ESR2-mediated regulation of the solute carrier family 2 member 4 (Slc2a4) gene, examining ESR1/ESR2-mediated genomic mechanisms that regulate Slc2a4 transcription, especially those occurring in cooperation with other transcription factors. In addition, we address the estradiol-induced translocation of ESR1 and GLUT4 to the plasma membrane. Studies make it clear that ESR1-mediated effects are beneficial, whereas ESR2-mediated effects are detrimental to glycemic homeostasis. Thus, imbalance of the ESR1/ESR2 ratio may have important consequences in metabolism, highlighting that ESR2 hyperactivity assumes a diabetogenic role.

AGEs-Induced and Endoplasmic Reticulum Stress/Inflammation-Mediated Regulation of GLUT4 Expression and Atherogenesis in Diabetes Mellitus.

In recent decades, complex and exquisite pathways involved in the endoplasmic reticulum (ER) and inflammatory stress responses have been demonstrated to participate in the development and progression of numerous diseases, among them diabetes mellitus (DM). In those pathways, several players participate in both, reflecting a complicated interplay between ER and inflammatory stress. In DM, ER and inflammatory stress are involved in both the pathogenesis of the loss of glycemic control and the development of degenerative complications. Furthermore, hyperglycemia increases the generation of advanced glycation end products (AGEs), which in turn refeed ER and inflammatory stress, contributing to worsening glycemic homeostasis and to accelerating the development of DM complications. In this review, we present the current knowledge regarding AGEs-induced and ER/inflammation-mediated regulation of the expression of GLUT4 (solute carrier family 2, facilitated glucose transporter member 4), as a marker of glycemic homeostasis and of cardiovascular disease (CVD) development/progression, as a leading cause of morbidity and mortality in DM.

Variants in HSD11B1 gene modulate susceptibility to diabetes kidney disease and to insulin resistance in type 1 diabetes.

BACKGROUND AND AIM: 11ß-Hydroxysteroid dehydrogenase 1 has been implicated in insulin resistance (IR) in the setting of metabolic disorders, and single nucleotide polymorphisms (SNPs) in its encoding gene (HSD11B1) have been associated with type 2 diabetes and metabolic syndrome. In type 1 diabetes (T1D), IR has been related to the development of chronic complications. We investigated the association of HSD11B1 SNPs with microvascular complications and with IR in a Brazilian cohort of T1D individuals. MATERIALS AND METHODS: Five SNPs were genotyped in 466 T1D individuals (57% women; median of 37 years old, diabetes duration of 25 years and HbA1c of 8.4%). RESULTS: The minor allele T of rs11799643 was nominally associated with diabetic retinopathy (OR = 0.52; confidence interval [CI] 95% = 0.28-0.96; P = .036). The minor allele C of rs17389016 was nominally associated with overt diabetic kidney disease (DKD) (OR = 1.90; CI 95% = 1.07-3.37; P = .028). A follow-up study revealed that 29% of the individuals lost ≥5 mL min-1 × 1.73 m2 per year of the estimated glomerular filtration rate (eGFR). In these individuals (eGFR decliners), C allele of rs17389016 was more frequent than in non-decliners (OR = 2.10; CI 95% = 1.14-3.89; P = .018). Finally, minor allele T of rs846906 associated with higher prevalence of arterial hypertension, higher body mass index and waist circumference, thus conferring risk to a lower estimated glucose disposal rate, a surrogate marker of insulin sensitivity (OR = 1.23; CI 95% = 1.06-1.42; P = .004). CONCLUSION: SNPs in the HSD11B1 gene may confer susceptibility to DKD and to IR in T1D individuals.

Alcohol Use Disorder is Associated with Upregulation of MicroRNA-34a and MicroRNA-34c in Hippocampal Postmortem Tissue.

BACKGROUND: To investigate epigenetic mechanisms potentially involved in the cognitive decline associated with chronic alcohol intake, we evaluated the expressions of three micro-RNAs (miR-34a, -34b, and -34c) highly expressed in the hippocampus and involved in neuronal physiology and pathology. MiR-34a participates in functioning and survival of mature neurons; miR-34b is associated with Alzheimer-like disorders; and miR-34c is implicated in the memory impairment of Alzheimer disease in rodents and humans. METHODS: A total of 69 cases were selected from the Biobank for Aging Studies and categorized according to the absence (n = 50) or presence (n = 19) of alcohol use disorder (AUD). Cases presenting with neuropathological diagnoses of dementias were excluded. Total RNA was extracted from hippocampal paraffinized slices, complementary DNA was synthesized from miRs, and RT-qPCR was performed with TaqMan® assays. RESULTS: Higher expressions of miR-34a and miR-34c, but not of miR-34b, were found in the group with AUD in comparison with the group without AUD after adjustment for potential confounders (age, sex, body mass index, presence of hypertension, diabetes mellitus, smoking, and physical inactivity). CONCLUSIONS: Hippocampal upregulation of miR-34a and miR-34c may be involved in the cognitive decline associated with chronic alcohol consumption.

RAGE Mediates Cholesterol Efflux Impairment in Macrophages Caused by Human Advanced Glycated Albumin.

We addressed the involvement of the receptor for advanced glycation end products (RAGE) in the impairment of the cellular cholesterol efflux elicited by glycated albumin. Albumin was isolated from type 1 (DM1) and type 2 (DM2) diabetes mellitus (HbA1c > 9%) and non-DM subjects (C). Moreover, albumin was glycated in vitro (AGE-albumin). Macrophages from Ager null and wild-type (WT) mice, or THP-1 transfected with siRNA-AGER, were treated with C, DM1, DM2, non-glycated or AGE-albumin. The cholesterol efflux was reduced in WT cells exposed to DM1 or DM2 albumin as compared to C, and the intracellular lipid content was increased. These events were not observed in Ager null cells, in which the cholesterol efflux and lipid staining were, respectively, higher and lower when compared to WT cells. In WT, Ager, Nox4 and Nfkb1, mRNA increased and Scd1 and Abcg1 diminished after treatment with DM1 and DM2 albumin. In Ager null cells treated with DM-albumin, Nox4, Scd1 and Nfkb1 were reduced and Jak2 and Abcg1 increased. In AGER-silenced THP-1, NOX4 and SCD1 mRNA were reduced and JAK2 and ABCG1 were increased even after treatment with AGE or DM-albumin. RAGE mediates the deleterious effects of AGE-albumin in macrophage cholesterol efflux.

Dual SGLT1/SGLT2 Inhibitor Phlorizin Ameliorates Non-Alcoholic Fatty Liver Disease and Hepatic Glucose Production in Type 2 Diabetic Mice.

PURPOSE: NAFLD is a hepatic component of type 2 diabetes mellitus (T2D), in which impaired hepatic glucose production plays an important role. Inhibitors of sodium glucose transporter 2 (SGLT2) reduce glycemia and exert beneficial effects on diabetic complications. Recently, dual SGLT1/2 inhibition has been proposed to be more effective in reducing glycemia. We hypothesized that improving hepatic glucose metabolism induced by SGLT1/2 inhibition could be accompanied by beneficial effects on NAFLD progression. METHODS: Glycemic homeostasis, hepatic glucose production and NAFLD features were investigated in obese T2D mice, treated with SGLT1/2 inhibitor phlorizin for 1 week. RESULTS: T2D increased glycemia; insulinemia; hepatic expression of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and glucose transporter 2 (Slc2a2 gene); hepatocyte nuclear factors 1A/4A/3B-binding activity in Slc2a2; endogenous glucose production; liver weight, plasma transaminase concentration as well as hepatic inflammation markers, and induced histological signals of non-alcoholic steatohepatitis (NASH, according to NASH-CRN Pathology Committee System). Phlorizin treatment restored all these parameters (mean NASH score reduced from 5.25 to 2.75 P<0.001); however, plasma transaminase concentration was partially reverted and some hepatic inflammation markers remained unaltered. CONCLUSION: NAFLD accompanies altered hepatic glucose metabolism in T2D mice and that greatly ameliorated through short-term treatment with the dual SGLT1/2 inhibitor. This suggests that altered hepatic glucose metabolism participates in T2D-related NAFLD and highlights the pharmacological inhibition of SGLTs as a useful approach not only for controlling glycemia but also for mitigating development and/or progression of NAFLD.

Carbenoxolone enhances peripheral insulin sensitivity and GLUT4 expression in skeletal muscle of obese rats: Potential participation of UBC9 protein.

AIM: This study investigates the insulin sensitizer effect of carbenoxolone (CBX) and potentially involved peripheral mechanisms. MAIN METHODS: Taking glucose transporter 4 (GLUT4) as a marker of glucose disposal, we investigated the CBX effects on whole-body insulin sensitivity and solute carrier 2a4 (Slc2a4)/GLUT4 expression in visceral (VAT) and subcutaneous (SAT) adipose tissues and soleus muscle of monosodium glutamate (MSG)-induced obese rats. Sterol regulatory element binding protein (SREBP1), an enhancer of Slc2a4 expression was analyzed through mRNA content and SREBP1-binding to Slc2a4 promoter. Finally, the small ubiquitin-modifier conjugating enzyme 9 (UBC9), whose low content indicates accelerated GLUT4 degradation was analyzed in soleus. KEY FINDINGS: Hypercorticosteronemia, hyperinsulinemia and low glucose decay rate in the insulin tolerance test of obese rats were restored by CBX (P < 0.05). Slc2a4/GLUT4 increased in SAT (P < 0.05) and decreased in VAT (P < 0.01) of obese rats. In soleus, obesity increased Slc2a4 but decreased GLUT4 (P < 0.01), possibly by accelerating GLUT4 degradation, as suggested by decreased UBC9 (P < 0.01). CBX restored both UBC9 and GLUT4 contents. SREBP1 did not participate in the Slc2a4 transcriptional regulation. SIGNIFICANCE: The insulin sensitizer effect of CBX involves the increase of GLUT4 expression in soleus, indicating an increased glucose disposal in skeletal muscle. This observation reinforces the skeletal muscle as the main site of insulin-induced glucose uptake and sheds new light on the metabolic effects of 11ßHSD1 inhibitors, since most of the studies so far have focused on its effects on liver and adipose tissues.

Sympathetic Regulation of Slc2a4 Gene Expression: Participation of a Putative cAMP Responsive Element (CRE) Site in the Slc2a4 Promoter.

BACKGROUND/AIMS: Studies have indicated that sympathetic activity enhances GLUT4 expression (Slc2a4 gene) by activating beta-adrenergic receptors. This could be mediated by a direct enhancer effect of cyclic AMP-responsive element binding protein (CREB) and family members upon Slc2a4 gene. However, a cAMP responsive element (CRE) in Slc2a4 promoter has never been demonstrated. METHODS: Slc2a4 CRE-site was searched by in silico analysis. In skeletal muscles from rats displaying high sympathetic activity (SHR), Slc2a4 CRE-site was investigated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay; and Slc2a4 expression was analyzed by RT-qPCR. Functional activity of the CRE-site was investigated by luciferase assay, 2 hours after 8-br-cAMP stimulation, in 3T3L1 adipocytes transientely transfected with native and mutated CRE-sites. RESULTS: In silico analysis indicated the -480/-473 segment as a putative CRE-site, with 62.5% of identity to CRE consensus sequence, and highly preserved in mouse, rat and human. CREB/CREM binding in this CRE-site was confirmed to occur in vitro (EMSA) and in vivo (ChIP assay). Enhancer activity of this segment in Slc2a4 transcription was confirmed in 3T3-L1 cells. Finally, in extensor digitorum longus muscle from SHR, 80% increase in Slc2a4 mRNA expression was observed to be accompanied by increased CREB/CREM binding into the CRE-site both in vitro and in vivo. CONCLUSION: This study demonstrates the presence of a functional CRE-site at -480/-473 sequence of the Slc2a4 gene. This CRE-site has an enhancing activity on Slc2a4 expression and participates in the Slc2a4 increased expression observed in glycolytic muscles of rats displaying high sympathetic activity.

Genetic variants in DNMT1 and the risk of cardiac autonomic neuropathy in women with type 1 diabetes.

AIMS/INTRODUCTION: Epigenetics participate in the pathogenesis of metabolic memory, a situation in which hyperglycemia exerts prolonged deleterious effects even after its normalization. We tested the hypothesis that genetic variants in an epigenetic gene could predispose to diabetes complications. MATERIAL AND METHODS: We assessed the frequency of five single-nucleotide polymorphisms in the gene encoding deoxyribonucleic acid methytransferase 1 (DNMT1; rs8112895, rs7254567, rs11085721, rs17291414 and rs10854076), and their associations with diabetic kidney disease, retinopathy, distal polyneuropathy and autonomic cardiovascular neuropathy in 359 individuals with long-term type 1 diabetes. RESULTS: None of the single-nucleotide polymorphisms studied was significantly associated with the presence of chronic complications in the overall population. However, after sex stratification, the minor allele C of rs11085721 conferred risk for cardiovascular neuropathy in women after adjustment for confounding variables (odds ratio 2.32; 95% confidence interval 1.26-4.33; P = 0.006). CONCLUSIONS: The fact that heterozygous mutations in DNMT1 are associated with hereditary sensory autonomic neuropathy provides plausibility to the present finding. If confirmed in independent samples, it suggests that genetic variants in epigenetic genes might predispose to more or fewer epigenetic changes in the face of similar metabolic derangements triggered by hyperglycemia, constituting the "genetics of epigenetics" for microvascular diabetes complications.

Palmitate-induced Slc2a4/GLUT4 downregulation in L6 muscle cells: evidence of inflammatory and endoplasmic reticulum stress involvement.

BACKGROUND: Obesity is strongly associated to insulin resistance, inflammation, and elevated plasma free fatty acids, but the mechanisms behind this association are not fully comprehended. Evidences suggest that endoplasmic reticulum (ER) stress may play a role in this complex pathophysiology. The aim of the present study was to investigate the involvement of inflammation and ER stress in the modulation of glucose transporter GLUT4, encoded by Slc2a4 gene, in L6 skeletal muscle cells. METHODS: L6 cells were acutely (2 h) and chronically (6 and 12 h) exposed to palmitate, and the expression of several proteins involved in insulin resistance, ER stress and inflammation were analyzed. RESULTS: Chronic and acute palmitate exposure significantly reduced GLUT4 protein (~ 39%, P < 0.01) and its mRNA (18%, P < 0.01) expression. Only acute palmitate treatment increased GRP78 (28%, P < 0.05), PERK (98%, P < 0.01), eIF-2A (35%, P < 0.01), IRE1a (60%, P < 0.05) and TRAF2 (23%, P < 0.05) protein content, and PERK phosphorylation (106%, P < 0.001), but did not elicit eIF-2A, IKK phosphorylation or increased XBP1 nuclear content. Additionally, acute and chronic palmitate increased NFKB p65 nuclear content (~ 30%, P < 0.05) and NFKB binding activity to Slc2a4 gene promoter (~ 45%, P < 0.05). CONCLUSION: Different pathways are activated in acute and chronic palmitate induced-repression of Slc2a4/GLUT4 expression. This regulation involves activation of initial component of ER stress, such as the formation of a IRE1a-TRAF2-IKK complex, and converges to NFKB-induced repression of Slc2a4/GLUT4. These results link ER stress, inflammation and insulin resistance in L6 cells.

AGE-albumin enhances ABCA1 degradation by ubiquitin-proteasome and lysosomal pathways in macrophages.

BACKGROUND AND AIMS: Advanced glycation end products (AGEs) induce cellular oxidative/endoplasmic reticulum stress and inflammation. We investigated its underlying mechanisms for atherogenesis focusing on regulation of ABCA1 protein decay in macrophages. METHODS: The ABCA1 decay rate was evaluated in macrophages after treatment with LXR agonist and by incubation with control (C) or AGE-albumin concomitant or not with cycloheximide, MG-132, ammonium chloride and calpain inhibitors were utilized to inhibit, respectively, proteasome, lysosome and ABCA1 proteolysis at cell surface. ABCA1 was determined by immunoblot and the protein decay rate calculated along time by the slope of the linear regression. Ubiquitination level was determined in ABCA1 immunoprecipitated from whole cell lysate or bulk cell membrane. AGE effect was also analyzed in THP-1 cells transfected with siRNA-RAGE. Carboxymethyllysine (CML) and pyrraline (PYR) were determined by LC/MS. One-way ANOVA and Student t test were utilized to compare results. RESULTS: CML and PYR-albumin were higher in AGE-albumin as compared to C. AGE-albumin reduced ABCA1 in J774 and THP-1 macrophages (20-30%) and induced a higher ABCA1 ubiquitination and a faster protein decay rate that was dependent on the presence of AGE during the kinetics of measurement in the presence of cycloheximide. Proteasomal inhibition restored and lysosomal inhibition partially recovered ABCA1 in cells treated with AGE-albumin. Calpain inhibition was not able to rescue ABCA1. RAGE knockdown prevented the reduction in ABCA1 elicited by AGE. CONCLUSIONS: AGE-albumin diminishes ABCA1 by accelerating its degradation through the proteasomal and lysosomal systems. This may increase lipid accumulation in macrophages by diminishing cholesterol efflux via RAGE signaling contributing to atherosclerosis in diabetes mellitus.

MicroRNAs-Mediated Regulation of Skeletal Muscle GLUT4 Expression and Translocation in Insulin Resistance.

The solute carrier family 2 facilitated glucose transporter member 4 (GLUT4) plays a key role in the insulin-induced glucose uptake by muscle and adipose tissues. In prediabetes and diabetes, GLUT4 expression/translocation has been detected as reduced, participating in mechanisms that impair glycemic control. Recently, a class of short endogenous noncoding RNAs named microRNAs (miRNAs) has been increasingly described as involved in the posttranscriptional epigenetic regulation of gene expression. The present review focuses on miRNAs potentially involved in the expression of GLUT4 expression, and proteins related to GLUT4 and translocation in skeletal muscle, seeking to correlate them with insulin resistance and diabetes. So far, miR-21a-5p, miR-29a-3p, miR-29c-3p, miR-93-5p, miR-106b-5p, miR-133a-3p, miR-133b-3p, miR-222-3p, and miR-223-3p have been reported to directly and/or indirectly regulate the GLUT4 expression; and their expression is altered under diabetes-related conditions. Besides, some miRNAs that have been linked to the expression of proteins involved in GLUT4 translocation machinery in muscle could also impact glucose uptake. That makes these miRNAs promising targets for preventive and/or therapeutic approaches, which could improve glycemic control, thus deserving future new investigations.

Dysregulation between TRIM63/FBXO32 expression and soleus muscle wasting in diabetic rats: potential role of miR-1-3p, -29a/b-3p, and -133a/b-3p.

Diabetes mellitus (DM) induces a variable degree of muscle sarcopenia, which may be related to protein degradation and to the expression of both E3 ubiquitin ligases and some specific microRNAs (miRNAs). The present study investigated the effect of diabetes and acute muscle contraction upon the TRIM63 and FBXO32 expression as well as the potential involvement of some miRNAs. Diabetes was induced by streptozotocin and studied after 30 days. Soleus muscles were harvested, stimulated to contract in vitro for twitch tension analysis (0.5 Hz), 30 min later for tetanic analysis (100 Hz), and 30 min later were frozen. TRIM63 and FBXO32 proteins were quantified by western blotting; Trim63 mRNA, Fbxo32 mRNA, miR-1-3p, miR-29a-3p, miR-29b-3p, miR-133a-3p, and miR-133b-3p were quantified by qPCR. Diabetes induced sarcopenia by decreasing (P < 0.05) muscle weight/tibia length index, maximum tetanic contraction and relaxation rates, and absolute twitch and tetanic forces (P < 0.05). Diabetes decreased (P < 0.05) the Trim63 and Fbxo32 mRNAs (30%) and respective proteins (60%), and increased (P < 0.01) the miR-29b-3p (2.5-fold). In muscle from diabetic rats, acute contractile stimulus increased TRIM63 protein, miR-1-3p, miR-29a-3p, and miR-133a/b-3p, but decreased miR-29b-3p (P < 0.05). Independent of the metabolic condition, after muscle contraction, both TRIM63 and FBXO32 proteins correlated significantly with miR-1-3p, miR-29a/b-3p, and miR-133a/b-3p. All diabetes-induced regulations were reversed by insulin treatment. Concluding, the results depict that muscle wasting in long-term insulinopenic condition may not be accompanied by increased proteolysis, pointing out the protein synthesis as an important modulator of muscle sarcopenia in DM.