Macrophages and Mitochondria: A Critical Interplay Between Metabolism, Signaling, and the Functional Activity.

Macrophages are phagocytic cells that participate in a broad range of cellular functions and they are key regulators of innate immune responses and inflammation. Mitochondria are highly dynamic endosymbiotic organelles that play key roles in cellular metabolism and apoptosis. Mounting evidence suggests that mitochondria are involved in the interplay between metabolism and innate immune responses. The ability of these organelles to alter the metabolic profile of a cell, thereby allowing an appropriate response to each situation, is crucial for the correct establishment of immune responses. Furthermore, mitochondria act as scaffolds for many proteins involved in immune signaling pathways and as such they are able to modulate the function of these proteins. Finally, mitochondria release molecules, such as reactive oxygen species, which directly regulate the immune response. In summary, mitochondria can be considered as core components in the regulation of innate immune signaling. Here we discuss the intricate relationship between mitochondria, metabolism, intracellular signaling, and innate immune responses in macrophages.

The DEXLIFE study methods: identifying novel candidate biomarkers that predict progression to type 2 diabetes in high risk individuals.

The incidence of type 2 diabetes (T2D) is rapidly increasing worldwide and T2D is likely to affect 592 million people in 2035 if the current rate of progression is continued. Today, patients are diagnosed with T2D based on elevated blood glucose, either directly or indirectly (HbA1c). However, the information on disease progression is limited. Therefore, there is a need to identify novel early markers of glucose intolerance that reflect the underlying biology and the overall physiological, metabolic and clinical characteristics of progression towards diabetes. In the DEXLIFE study, several clinical cohorts provide the basis for a series of clinical, physiological and mechanistic investigations in combination with a range of--omic technologies to construct a detailed metabolic profile of high-risk individuals across multiple cohorts. In addition, an exercise and dietary intervention study is conducted, that will assess the impact on both plasma biomarkers and specific functional tissue-based markers. The DEXLIFE study will provide novel diagnostic and predictive biomarkers which may not only effectively detect the progression towards diabetes in high risk individuals but also predict responsiveness to lifestyle interventions known to be effective in the prevention of diabetes.

Central venous catheter colonization with Staphylococcus aureus is not always an indication for antimicrobial therapy.

Whether patients whose catheter tip grows Staphylococcus aureus but who have no concomitant bacteraemia should receive antimicrobials remains an unresolved issue. However, a proportion of patients with catheter tips colonized by S. aureus have no blood cultures taken because of low suspicion of sepsis and the meaning of this microbiological finding is unknown. We have analysed all catheter tips growing S. aureus during a 6-year period and have selected patients without blood cultures taken 7 days before or after central vascular catheter removal. Patient's evolution was classified into good and poor outcome. Poor outcome was defined as S. aureus infection within 3 months after catheter withdrawal or death in the same period with no obvious cause. Patients with good and poor outcomes were compared to assess whether antimicrobial therapy influenced evolution. Sixty-seven patients fulfilled our inclusion criteria and five (7.4%) had a poor outcome. The administration of early anti-staphylococcal therapy had no impact on the outcome of this population (p 0.99). The only factor independently associated with a poor outcome was the presence of clinical signs of sepsis when the catheter was removed (OR 20.8; 95% CI 2.0-206.1; p 0.009). Our data suggest that patients with central vascular catheter tips colonized with S. aureus should be closely monitored for signs and symptoms of ongoing infection, but if these are not present then antimicrobial therapy does not seem justified.

Identification of novel GH-regulated genes in C2C12 cells.

Growth hormone (GH) is the main regulator of longitudinal growth before puberty, and treatment with human recombinant (rh) GH can increase muscle strength. Nevertheless, molecular mechanisms responsible remain mostly unknown. Many physiological effects of GH require hormone-mediated changes in gene expression. In an attempt to gain insight into the mechanism of GH action in muscle cells we evaluated the effects of rhGH on gene expression profile in a murine skeletal muscle cell line C2C12. The objective of the work was to identify changes in gene expression in the murine skeletal muscle cell line C2C12 after rGH treatment using microarray assays. C2C12 murine skeletal muscle cell cultures were differentiated during 4 days. After 16 h growing in serum-free medium, C2C12 myotubes were stimulated during 6 h with 500 ng/ml rhGH. Four independent sets of experiments were performed to identify GH-regulated genes. Total RNA was isolated and subjected to analysis. To validate changes candidate genes were analyzed by real-time quantitative polymerase chain reaction. One hundred and fifty-four differentially expressed genes were identified; 90 upregulated and 64 downregulated. Many had not been previously identified as GH-responsive. Real-time PCR in biological replicates confirmed the effect of rGH on 15 genes: Cish, Serpina3g, Socs2, Bmp4, Tnfrsf11b, Rgs2, Tgfbr3, Ugdh, Npy1r, Gbp6, Tgfbi, Tgtp, Btc, Clec3b, and Bcl6. This study shows modifications in the gene expression profile of the C2C12 cell line after rhGH exposure. In vitro and gene function analysis revealed genes involved in skeletal and muscle system as well as cardiovascular system development and function.

Genes involved in mitochondrial biogenesis/function are induced in response to bilio-pancreatic diversion in morbidly obese individuals with normal glucose tolerance but not in type 2 diabetic patients.

AIMS/HYPOTHESIS: The mechanisms allowing normalisation of insulin sensitivity and reversal of type 2 diabetes after bilio-pancreatic diversion (BPD) have not been elucidated. We studied whether the expression of genes relevant to mitochondrial biogenesis/function is induced in response to BPD and whether the response differs between morbidly obese patients with normal glucose tolerance (NGT) and patients with type 2 diabetes. METHODS: The effect of stable weight reduction after BPD on metabolic variables and expression of nuclear genes encoding for mitochondrial proteins or regulators of mitochondrial function was investigated in skeletal muscle. Insulin sensitivity was assessed by euglycaemic-hyperinsulinaemic clamp and substrate oxidation by indirect calorimetry. RESULTS: Both NGT and type 2 diabetic patients showed a net improvement of insulin sensitivity, with the latter also showing blood glucose normalisation. NGT patients had a large increase in glucose oxidation and substantial reduction in lipid oxidation. In contrast, type 2 diabetic patients had a blunted response to BPD in terms of glucose oxidation. NGT patients showed increased expression of genes encoding mitofusin 2, porin or citrate synthase; no significant changes were detected in diabetic patients. The expression of genes regulating mitochondrial activity (PGC-1beta [also known as PPARGC1B], PGC-1alpha [also known as PPARGC1A], PPARdelta [also known as PPARD], SIRT1) was induced only in NGT patients. CONCLUSIONS/INTERPRETATION: These findings indicate that weight loss after BPD exerts a beneficial effect on insulin sensitivity via mechanisms that are independent of the expression of genes involved in mitochondrial biogenesis/activity. Furthermore, the observation that gene expression is not altered with weight loss in type 2 diabetic patients while it is induced in NGT patients suggests a heritable component.

Protection from inflammatory disease in insulin resistance: the role of mannan-binding lectin.

AIMS/HYPOTHESIS: Decreased sensing of the innate immune system may lead to chronic activation of the inflammatory cascade. We hypothesised that mannan-binding lectin (MBL) deficiency may confer risk of obesity and insulin resistance. MATERIALS AND METHODS: We performed a cross-sectional study of MBL protein concentration (n=434) and MBL2 gene mutations (exon 1) (n=759) in association with obesity, markers of inflammation and insulin action (euglycaemic clamp, n=113), and a longitudinal study of MBL protein before and after weight loss in obese patients (n=10). We also studied the effects of MBL in vitro in muscle cells and circulating MBL-A (mouse equivalent of human MBL) in a mouse model. RESULTS: Among 434 consecutive non-diabetic men, the age-adjusted serum MBL concentration was lower in obese subjects than in lean subjects (median: 959 microg/ml [interquartile range: 116.8-2,044 microg/ml] vs 1,365 [467-2,513] microg/ml; p=0.01) and was accompanied by increased serum inflammatory markers. Insulin action correlated significantly with serum MBL (r=0.49, p<0.0001). Serum MBL concentration increased by a median of 110.2% after weight loss. The change in serum concentration of MBL was positively associated with the increase in insulin sensitivity (r=0.713, p=0.021). At least one MBL2 gene mutation was present in 48.2% of obese vs 39.3% of non-obese subjects (p=0.037). The plasma concentration of MBL-A was lower in insulin-resistant obese ob/ob mice, as was the glucose/insulin ratio. Incubation of rat soleus muscle with human MBL markedly increased fatty acid oxidation. CONCLUSIONS/INTERPRETATION: These findings suggest that MBL, previously thought only to be involved in inflammation and immune system function, affects metabolic pathways.

Prolonged treatment with the beta3-adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 2) modulation of glucose uptake and monoamine oxidase activity.

Beta3-adrenergic agonists are well-recognited to promote lipid mobilisation and adipose tissue remodeling in rodents, leading to multilocular fat cells enriched in mitochondria. However, effects of beta3-adrenergic agonists on glucose transport are still controversial. In this work, we studied in white adipose tissue (WAT) the influence of sustained beta3-adrenergic stimulation on the glucose transport and on the mitochondrial monoamine oxidase (MAO) activity. As one-week administration of CL 316243 (CL, 1 mg/kg/d) induces beta-adrenergic desensitization in rat but not in guinea pig adipocytes, attention was paid to compare these models. When expressing glucose uptake as nmoles of 2-deoxyglucose/100 mg cell lipids, maximally stimulated uptake was increased in adipocytes of WAT from treated rats but not from treated guinea pigs. However, basal hexose uptake was also increased in CL-treated rats and, as a consequence, the dose-dependent curves for insulin stimulation were similar in control and CL-treated rats when expressed as fold increase over basal. Insulin-induced lipogenesis was unchanged in rat or guinea pig adipocytes after CL-treatment. The glucose carriers GLUT4 and corresponding mRNA were increased in subcutaneous WAT or in brown adipose tissue (BAT) but not in visceral WAT or muscles of CL-treated rats. There was an increase of MAO activity in WAT and BAT, but not in liver, of CL-treated rats while no change was detected in guinea pigs. These findings show that only rat adipocytes, which are beta3-adrenergic-responsive, respond to chronic beta3-AR agonist by an increase of GLUT4 content and MAO activity, despite a desensitization of all beta-adrenoceptor subtypes.

The release of soluble VAP-1/SSAO by 3T3-L1 adipocytes is stimulated by isoproterenol and low concentrations of TNFalpha.

Plasma level of the protein VAP-1/SSAO (Vascular Adhesion Protein-1/Semicarbazide-Sensitive Amine Oxidase) is increased in diabetes and/or obesity and may be related to vascular complications associated to these pathologies. The aim of this work was to complete a preceding study where we described the role played by some hormones or metabolites, implicated in diabetes and/or obesity, in the regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes. Here we focused on the previously observed effect produced by TNFalpha in the release of VAP-1/SSAO and studied the effect of a beta-adrenergic compound, isoproterenol. Both compounds stimulated the release of VAP-1/SSAO to the culture medium but had a different effect on the VAP-1/SSAO membrane form. While TNFalpha produced a decrease on VAP-1/SSAO membrane form content, isoproterenol did not modify it. We thus observed two different ways of regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes by metabolites implicated in diabetes and adipose tissue physiopathology. Our work permits a better understanding of this increased plasma VAP-1/SSAO levels observed in diabetes.

Could the low level of expression of the gene encoding skeletal muscle mitofusin-2 account for the metabolic inflexibility of obesity?

AIMS/HYPOTHESIS: In obesity the cellular capacity to switch from using lipid to carbohydrate and vice versa as the energy substrate, known as 'metabolic flexibility', is impaired. Mitofusin 2 (MFN2), a mitochondrial membrane protein, seems to contribute to the maintenance and operation of the mitochondrial network, and its expression is reduced in obesity. The aim of this study was to verify whether MFN2 might be implicated in the metabolic inflexibility of obesity. MATERIALS AND METHODS: Insulin sensitivity was measured in six morbidly obese women before and 2 years after malabsorptive bariatric surgery (BMI 53.3+/-10.5 vs 30.3+/-4.0 kg/m2). Skeletal muscle MFN2, SLC2A4 (formerly known as GLUT4), COX3 (encoding cytochrome c oxidase subunit III) and CS (encoding citrate synthase) mRNA levels were measured by real-time PCR. RESULTS: Following bilio-pancreatic surgery, significant increases in MFN2 mRNA (from 0.4+/-0.2 to 1.7+/-1.1 arbitrary units [AU], p=0.019) and SLC2A4 mRNA (0.38+/-0.12 to 0.76+/-0.24 AU, p=0.04) were observed, while increases in COX3 mRNA (from 14.2+/-6.4 to 20.2+/-12.5 AU) and CS mRNA (from 0.4+/-0.1 to 0.7+/-0.3 AU) failed to reach statistical significance. Insulin-mediated whole-body glucose uptake significantly (p<0.0001) increased from 21.2+/-4.1 to 52.8+/-5.9 micromol kg fat-free mass(-1) min(-1) and glucose oxidation rose from 11.1+/-2.1 to 37.7+/-4.7 micromol kg fat-free mass(-1) min(-1) (p<0.0001). Levels of MFN2 mRNA were strongly correlated with the absolute values for the glucose oxidation rate, both during fasting (glucose oxidation =3.55 MFN2 mRNA + 3.93; R2=0.92, p<0.0001) and during the clamp (glucose oxidation=18.8 MFN2 mRNA+34.7; R2=0.80, p<0.0001). The percentage changes in MFN2 mRNA were positively correlated with the percentage change in glucose oxidation during the clamp (glucose oxidation percent (%) change=0.3 MFN2 mRNA percent (%) change+153.2; R2=0.61, p<0.001). CONCLUSIONS/INTERPRETATION: We propose that the significant increase in MFN2 mRNA levels may explain the increase in glucose oxidation observed in morbid obesity following bariatric surgery.

The release of soluble VAP-1/SSAO by 3T3-L1 adipocytes is stimulated by isoproterenol and low concentrations of TNFá

Plasma level of the protein SSAO/VAP-1 (samicarbazide-sensitive amine oxidase / vascular-adhesion protein-1) is increased in diabetes and/or obesity and may be related to vascular complications associated to these pathologies. The aim of this work was to complete a preceding study where we described the role played by some hormones or metabolites, implicated in diabetes and/or obesity, in the regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes. Here we focused on the previously observed effect produced by TNFa in the release of VAP-1/SSAO and studied the effect of a beta-adrenergic compound, isoproterenol. Both compounds stimulated the release of VAP-1/SSAO to the culture medium but had a different effect on the SSAO/VAP-1 membrane form. While TNFa produced a decrease on SSAO/VAP-1 membrane form content, isoproterenol did not modify it. We thus observed two different ways of regulation of the release of SSAO/VAP-1 by 3T3-L1 adipocytes by metabolites implicated in diabetes and adipose tissue physiopathology. Our work permits a better understanding of this increased plasma SSAO/VAP-1 levels observed in diabetes

Los niveles plasmáticos de la proteina SSAO/VAP-1 están aumentados en la diabetes y la obesidad, lo que podría estar relacionado con las complicaciones vasculares asociadas a estas patologías. En continuidad con trabajos anteriores acerca del papel de algunas hormonas o metabolitos, implicados en la diabetes y obesidad. Se estudia en este trabajo el efecto producido por el TNFa y del agonista beta-adrenérgico, isoproterenol en la regulación de la liberación de VAP-1/SSAO por adipocitos 3T3-L1. Ambos compuestos estimularon la liberación de VAP-1/SSAO al medio de cultivo, pero tuvieron un efecto diferente sobre la isoforma ligada a la membrana de SSAO/VAP-1. Así, mientras que el TNFa produjo una disminución significativa en la actividad SSAO/VAP-1 ligada a la membrana, no se modificó por el isoproterenol. Además, observamos dos maneras diferentes de regulación de la liberación de SSAO/VAP-1 por adipocitos 3T3-L1 a través de metabolitos implicados en diabetes y fisiopatología del tejido adiposo. Nuestro trabajo permite un mejor entendimiento de estos niveles plasmáticos aumentados de SSAO/VAP-1 observados en diabetes

Mechanisms regulating GLUT4 glucose transporter expression and glucose transport in skeletal muscle.

Skeletal muscle is a major glucose-utilizing tissue in the absorptive state and the major glucose transporter expressed in muscle in adulthood is GLUT4. GLUT4 expression is exquisitely regulated in muscle and this seems important in the regulation of insulin-stimulated glucose uptake by this tissues. Thus, muscle GLUT4 overexpression in transgenic animals ameliorates insulin resistance associated with obesity or diabetes. Recent information indicates that glut4 gene transcription is regulated by a number of factors in skeletal muscle that include MEF2, MyoD myogenic proteins, thyroid hormone receptors, Kruppel-like factor KLF15, NF1, Olf-1/Early B cell factor and GEF/HDBP1. In addition, studies in vivo indicate that under normal conditions the activity of the muscle-specific GLUT4 enhancer is low in adult skeletal muscle compared with the maximal potential activity that it can attain at high levels of the MRF transcription factors, MEF2, and TRalpha1. This finding indicates that glut4 transcription may be greatly up-regulated via activation of this enhancer through an increase in the levels of expression or activity of these transcription factors. Understanding the molecular basis of the expression of glut4 will be useful for the appropriate therapeutic design of treatments for insulin-resistant states. The nature of the intracellular signals that mediate the stimulation of glucose transport in response to insulin or exercise is also reviewed.

[Role of novel mitochondrial proteins in energy balance]. / Papel de nuevas proteínas mitocondriales en el balance energético.

Mitochondrial activity plays a key role in the control of thermogenesis and energy expenditure in homeothermic organisms. Recently, much attention has been paid to the novel protein members of UCP family as putative regulators of uncoupled respiration. Nevertheless, it is likely that other aspects, as yet unrecognized, of mitochondrial biology are also crucial to comprehend mitochondrial function. Thus, mitochondria are organized in filaments or in networks in many cell types as a result of the operation of fusion and fission events. The demonstration that mitofusin-2, a protein involved in mitochondrial fusion, stimulates the mitochondrial oxidation of substrates, cell respiration and mitochondrial membrane potential suggests that this protein may play an important role in mitochondrial metabolism, and as a consequence, in energy balance. Furthermore, the observation that mitofusin-2 expression is repressed in obese skeletal muscle suggests a posible role in the pathophysiology and/or etiopathogenesis of obesity.

A novel muscle DNA-binding activity in the GLUT1 promoter.

GLUT1 glucose transporters are highly expressed in proliferating and transformed cells and serum and cAMP or the transcription factor Sp1 induce GLUT1 gene transcription. Here we identified a cis element situated at -46/-37 (MG1E - muscle-specific GLUT1 element) to which muscle-specific nuclear factors bind, and the DNA-protein complexes showed electrophoretic mobility of 41 and 32 kDa. MyoD over-expression induced the generation of MG1E-protein complexes characteristic of myoblast cells. MG1E does not bind any known factors defined in databases. Mutation of the MG1E sequence impaired transcriptional activity of the GLUT1 promoter specifically in skeletal or cardiac muscle cells. The transcriptional activity of the GLUT1 promoter induced by either Sp1, cAMP or serum was markedly reduced when MG1E was inactivated. We propose that the MG1E sequence permits the binding of muscle-specific nuclear factors and a maximal transcriptional activity in muscle cells in response to Sp1, cAMP or serum.

Adipocytes release a soluble form of VAP-1/SSAO by a metalloprotease-dependent process and in a regulated manner.

AIMS/HYPOTHESIS: Vascular adhesion protein-1 (VAP-1), which is identical to semicarbazide-sensitive amine oxidase (SSAO), is a dual-function membrane protein with adhesion properties and amine oxidase activity. A soluble form of VAP-1 is found in serum, where concentrations are enhanced in diabetes and obesity. In vitro, soluble VAP-1 enhances lymphocyte adhesion to endothelial cells, thus possibly participating in the enhanced lymphocyte adhesion capacity that is implicated in the cardiovascular complications associated with diabetes or obesity. In both, the tissue origin of the soluble VAP-1/SSAO is unknown. We examined whether adipose tissue, which has abundant expression of VAP-1/SSAO, is a source of soluble VAP-1. METHODS: We detected VAP-1/SSAO in plasma of diabetic animals, with or without VAP-1 immunoprecipitation, and in culture medium from 3T3-L1 adipocytes and human adipose tissue explants. VAP-1 protein glycosylation was measured. RESULTS: Diabetic and obese animals have increased plasma SSAO activity associated with VAP-1 protein. We also found that 3T3-L1 adipocytes and human adipose tissue explants release a soluble form of VAP-1/SSAO, which derives from the membrane. The release of soluble VAP-1 was enhanced by exposure of murine and human adipocytes to TNF-alpha and blocked by batimastat, a metalloprotease inhibitor. Partial ablation of adipose tissue reduced plasma SSAO activity in normal and diabetic rats. CONCLUSIONS/INTERPRETATION: Adipose cells are a source of soluble VAP-1/SSAO released by shedding of the membrane form. The release of SSAO is regulated by TNF-alpha and insulin. By releasing VAP-1/SSAO, adipose cells could contribute to the atherogenesis and vascular dysfunction associated with diabetes and obesity.

Terapia génica en endocrinología:una aproximación realista / Gene therapy in endocrinology: a realistic approach

El número de ensayos clínicos que pretenden demostrar la eficacia de nuevos métodos de tratamiento, curación o prevención de enfermedades mediante la utilización de la terapia génica aumentan año tras año; así, en el año 2002 se registraron en el mundo un total de 636 protocolos clínicos de terapia génica en marcha. En la actualidad, los principales tipos de enfermedades objeto de protocolos clínicos de terapia génica son el cáncer, las enfermedades vasculares, el sida, las enfermedades genéticas monogénicas como la deficiencia en adenosina desaminasa, la hipercolesterolemia familiar o la fibrosis quística. La terapia génica aplicada a la curación de enfermedades endocrinas representa un potencial nuevo tipo de intervención que podría aplicarse a enfermedades endocrina de origen monogénico, como la deficiencia de hormona del crecimiento, la diabetes insípida hipotalámica o las enfermedades multifactoriales, como la diabetes, la obesidad o los tumores endocrinos. En esta minirrevisión se comentan aspectos generales relativos a la terapia génica y su posible aplicación en el tratamiento, curación o prevención de enfermedades endocrinas en general y de enfermedades multigénicas, como es el caso de la diabetes, en particular (AU)

Stimulation of glucose transport by semicarbazide-sensitive amine oxidase activity in adipocytes from diabetic rats.

Semicarbazide-sensitive amine oxidase (SSAO) is highly expressed in adipose cells, and substrates of SSAO such as benzylamine in combination with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 recruitment in mouse 3T3-L1 adipocytes and in isolated rat adipocytes. Here we examined whether this combination of molecules also stimulates glucose transport in adipocytes from streptozotocin-induced diabetic rats and from Goto-Kakizaki diabetic rats. As previously reported, adipocytes obtained from streptozotocin-induced diabetic rats, showed a reduced stimulation of glucose transport in response to insulin. Under these conditions, the combination of benzylamine and vanadate caused a marked stimulation of glucose transport that was similar to the stimulation detected in control adipocytes. Adipocytes isolated from Goto-Kakizaki diabetic rats also showed a defective response to insulin; however, acute incubation in the presence of benzylamine and vanadate stimulated glucose transport in these cells to the same extent than in adipocytes from non-diabetic rats. These data indicate that adipocytes obtained from two different models of animal diabetes do not show resistance to the activation of glucose transport by SSAO activity, which is in contrast to the well reported resistance to insulin action. It seems to suggest that SSAO activity in combination with vanadate triggers a glucose transport-activating intracellular pathway that remains intact in the diabetic state. Further, our data support the view that the combination of benzylamine and vanadate could be an effective therapy in diabetes.

The endosomal compartment is an insulin-sensitive recruitment site for GLUT4 and GLUT1 glucose transporters in cardiac myocytes.

In nonstimulated cardiomyocytes, the glucose transporter GLUT4 is confined to intracellular vesicles forming at least two populations: a storage pool enriched in GLUT4 (pool 1) and an endosomal pool containing both GLUT4 and GLUT1 (pool 2). We have now studied the dynamics of these pools in response to insulin or the mitochondrial inhibitor rotenone in rat cardiomyocytes. Rotenone recruited GLUT4 and GLUT1 to the cell surface from endosomal pool 2 without affecting pool 1. Kinetic experiments were consistent with rotenone acting on an intracellular compartment that is in close connection with the plasma membrane. In contrast, insulin caused rapid, complete depletion of GLUT4 from pool 1 and reduced the GLUT1 content of pool 2 by approximately 50%, whereas, surprisingly, no net decrease in GLUT4 occurred in this pool. Subsequent insulin withdrawal resulted in slow replenishment of pool 2 with GLUT1 and of pool 1 with GLUT4. When pool 1 was still largely depleted of GLUT4, a second insulin challenge did reduce GLUT4 in pool 2 and stimulated glucose transport to the same extent as the first insulin treatment. In conclusion, the storage pool is the primary source of GLUT4 in response to insulin, but not to rotenone. In addition, the endosomal compartment is an important recruitment site of both GLUT1 and GLUT4 when the storage pool is either unaffected (rotenone) or depleted (by a previous insulin challenge). GLUT4 mobilized by insulin from the storage pool may pass through an intermediary (possibly endosomal) compartment on its way to the cell surface.

A novel functional co-operation between MyoD, MEF2 and TRalpha1 is sufficient for the induction of GLUT4 gene transcription.

We report tripartite co-operation between MyoD, myocyte enhancer factor-2 (MEF2) and the thyroid hormone receptor (TRalpha1) that takes place in the context of an 82-bp muscle-specific enhancer in the rat insulin-responsive glucose transporter (GLUT4) gene that is active in both cardiac and skeletal muscle. In the L6E9 skeletal muscle cell line and in 10T1/2 fibroblasts, a powerful synergistic activation of the GLUT4 enhancer relied on the over-expression of MyoD, MEF2 and TRalpha1 and the integrity of their respective binding sites, and occurred when linked to either a heterologous promoter or in the context of the native GLUT4 promoter. In cardiac myocytes, enhancer activity was dependent on the binding sites for MEF2 and TRalpha1. Furthermore, we show that in 10T1/2 fibroblasts, the forced expression of MyoD, MEF2 and TRalpha1 induced the expression of the endogenous, otherwise silent, GLUT4 gene. In all, our results indicate a novel functional co-operation between these three factors which is required for full activation of GLUT4 transcription.

Heteromeric amino acid transporters: biochemistry, genetics, and physiology.

The heteromeric amino acid transporters (HATs) are composed of two polypeptides: a heavy subunit (HSHAT) and a light subunit (LSHAT) linked by a disulfide bridge. HSHATs are N-glycosylated type II membrane glycoproteins, whereas LSHATs are nonglycosylated polytopic membrane proteins. The HSHATs have been known since 1992, and the LSHATs have been described in the last three years. HATs represent several of the classic mammalian amino acid transport systems (e.g., L isoforms, y(+)L isoforms, asc, x(c)(-), and b(0,+)). Members of the HAT family are the molecular bases of inherited primary aminoacidurias cystinuria and lysinuric protein intolerance. In addition to the role in amino acid transport, one HSHAT [the heavy subunit of the cell-surface antigen 4F2 (also named CD98)] is involved in other cell functions that might be related to integrin activation. This review covers the biochemistry, human genetics, and cell physiology of HATs, including the multifunctional character of CD98.

Lipid rafts are required for GLUT4 internalization in adipose cells.

It has been recently reported that insulin recruits a novel signaling machinery to lipid rafts required for insulin-stimulated GLUT4 translocation [Baumann, A., Ribon, V., Kanzaki, M., Thurmond, D. C., Mora, S., Shigematsu, S., Bickel, P. E., Pessin, J. E. & Saltiel, A. R. (2001) Nature 407, 202-207, 2000; Chiang, S. H., Baumann, C. A., Kanzaki, M., Thurmond, D. C., Watson, R. T., Neudauer, C. L., Macara, I. G., Pessin, J. E. & Saltiel, A. R. (2001) Nature 410, 944-948]. We have assessed the role of lipid rafts on GLUT4 traffic in adipose cells. High GLUT4 levels were detected in caveolae from adipocytes by two approaches, the mechanical isolation of purified caveolae from plasma membrane lawns and the immunogold analysis of plasma membrane lawns followed by freeze-drying. The role of lipid rafts in GLUT4 trafficking was studied by adding nystatin or filipin at concentrations that specifically disrupt caveolae morphology and inhibit caveolae function without altering clathrin-mediated endocytosis. These caveolae inhibitors did not affect the insulin-stimulated glucose transport. However, they blocked both the GLUT4 internalization and the down-regulation of glucose transport triggered by insulin removal in 3T3-L1 adipocytes. Our data indicate that lipid rafts are crucial for GLUT4 internalization after insulin removal. Given that high levels of GLUT4 were detected in caveolae from insulin-treated adipose cells, this transporter may be internalized from caveolae or caveolae may operate as an obligatory transition station before internalization.