Biophysical assessment of human aquaporin-7 as a water and glycerol channel in 3T3-L1 adipocytes

The plasma membrane aquaporin-7 (AQP7) has been shown to be expressed in adipose tissue and its role in glycerol release/uptake in adipocytes has been postulated and correlated with obesity onset. However, some studies have contradicted this view. Based on this situation, we have re-assessed the precise localization of AQP7 in adipose tissue and analyzed its function as a water and/or glycerol channel in adipose cells. Fractionation of mice adipose tissue revealed that AQP7 is located in both adipose and stromal vascular fractions. Moreover, AQP7 was the only aquaglyceroporin expressed in adipose tissue and in 3T3-L1 adipocytes. By overexpressing the human AQP7 in 3T3-L1 adipocytes it was possible to ascertain its role as a water and glycerol channel in a gain-of-function scenario. AQP7 expression had no effect in equilibrium cell volume but AQP7 loss of function correlated with higher triglyceride content. Furthermore it is also reported for the first time a negative correlation between water permeability and the cell non-osmotic volume supporting the observation that AQP7 depleted cells are more prone to lipid accumulation. Additionally, the strong positive correlation between the rates of water and glycerol transport highlights the role of AQP7 as both a water and a glycerol channel and reflects its expression levels in cells. In all, our results clearly document a direct involvement of AQP7 in water and glycerol transport, as well as in triglyceride content in adipocytes

Neuregulin, an effector on mitochondria metabolism that preserves insulin sensitivity

Various external factors modulate the metabolic efficiency of mitochondria. This review focuses on the impact of the growth factor neuregulin and its ErbB receptors on mitochondria and their relationship with several physiopathological alterations. Neuregulin is involved in the differentiation of heart, skeletal muscle and the neuronal system, among others, and its deficiency is deleterious for the health. Information gathered over the last two decades suggests that neuregulin plays a key role regulating mitochondrial oxidative machinery, which sustain cell survival and insulin sensitivity

Insulin-induced redistribution of GLUT4 glucose carriers in the muscle fiber

Insulin rapidly stimulates glucose transport in muscle fiber. This process controls the utilization of glucose in skeletal muscle, and it is deficient in various insulin-resistant states, such as non-insulin-dependent diabetes mellitus. The effect of insulin on muscle glucose transport is mainly due to the recruitment of GLUT4 glucose carriers to the cell surface of the muscle fiber. There is increasing evidence that the recruitment of GLUT4 carriers triggered by insulin affects selective domains of sarcolemma and transverse tubules. In contrast, GLUT1 is located mainly in sarcolemma and is absent in transverse tubules, and insulin does not alter its cellular distribution in muscle fiber. The differential distribution of GLUT1 and GLUT4 in the cell surface raises new questions regarding the precise endocytic and exocytic pathways that are functional in the muscle fiber. The current view of insulin-induced GLUT4 translocation is based mainly on studies performed in adipocytes. These studies have proposed the existence of intracellular compartments of GLUT4 that respond to insulin in a highly homogeneous manner. However, studies performed in skeletal muscle have identified insulin-sensitive as well as insulin-insensitive intracellular GLUT4-containing membranes. These data open a new perspective on the dynamics of intracellular GLUT4 compartments in insulin-sensitive cells

Identification of a membrane protein, LAT-2, that co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids

We have identified a new human cDNA, L-amino acid transporter-2 (LAT-2), that induces a system L transport activity with 4F2hc (the heavy chain of the surface antigen 4F2, also named CD98) in oocytes. Human LAT-2 is the fourth member of the family of amino acid transporters that are subunits of 4F2hc. The amino acid transport activity induced by the co-expression of 4F2hc and LAT-2 was sodium-independent and showed broad specificity for small and large zwitterionic amino acids, as well as bulky analogs (e.g. BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)). This transport activity was highly trans-stimulated, suggesting an exchanger mechanism of transport. Expression of tagged N-myc-LAT-2 alone in oocytes did not induce amino acid transport, and the protein had an intracellular location. Co-expression of N-myc-LAT-2 and 4F2hc gave amino acid transport induction and expression of N-myc-LAT-2 at the plasma membrane of the oocytes. These data suggest that LAT-2 is an additional member of the family of 4F2 light chain subunits, which associates with 4F2hc to express a system L transport activity with broad specificity for zwitterionic amino acids. Human LAT-2 mRNA is expressed in kidney >>> placenta >> brain, liver > spleen, skeletal muscle, heart, small intestine, and lung. Human LAT-2 gene localizes at chromosome 14q11.2-13 (13 cR or approximately 286 kb from marker D14S1349). The high expression of LAT-2 mRNA in epithelial cells of proximal tubules, the basolateral location of 4F2hc in these cells, and the amino acid transport activity of LAT-2 suggest that this transporter contributes to the renal reabsorption of neutral amino acids in the basolateral domain of epithelial proximal tubule cells

Aquaglyceroporins are differenctially expressed in beige and white adipocytes

Browning of white adipocytes has been proposed as a powerful strategy to overcome metabolic complications, since brown adipocytes are more catabolic, expending energy as a heat form. However, the biological pathways involved in the browning process are still unclear. Aquaglyceroporins are a sub-class of aquaporin water channels that also permeate glycerol and are involved in body energy homeostasis. In the adipose tissue, aquaporin-7 (AQP7) is the most representative isoform, being crucial for white adipocyte fully differentiation and glycerol metabolism. The altered expression of AQP7 is involved in the onset of obesity and metabolic disorders. Herein, we investigated if aquaglyceroporins are implicated in beige adipocyte differentiation, similar to white cells. Thus, we optimized a protocol of murine 3T3-L1 preadipocytes browning that displayed increased beige and decreased white adipose tissue features at both gene and protein levels and evaluated aquaporin expression patterns along the differentiation process together with cellular lipid content. Our results revealed that AQP7 and aquaporin-9 (AQP9) expression was downregulated throughout beige adipocyte differentiation compared to white differentiation, which may be related to the beige physiological role of heat production from oxidative metabolism, contrasting with the anabolic/catabolic lipid metabolism requiring glycerol gateways occurring in white adipose cells

Developmental regulation of GLUT-1 (Erytroid/HepG2) and GLUT-4 (Muscle/Fat) glucose transporter expression in rat heart, skeletal muscle, and brown adipose tissue

The expression of GLUT-1 (erythroid/Hep G2) and GLUT-4 (muscle/fat) glucose transporters was assessed during development in rat heart, skeletal muscle, and brown adipose tissue. GLUT-4 protein expression was detectable in fetal heart by day 21 of pregnancy; it increased progressively after birth. attaining levels close to those of adults at day 15 post natal.'In contrast, GLUT-4 messenger RNA (mRNA)was already present in hearts from 17 day-old fetuses. GLUT-4 mRNA stayed low during early postnatal life in heart and brown adipose tissue and only increased after day 10 post natal. The expression pattern for GLUT-4 protein in skeletal muscle during development was comparable to that observed in heart. In contrast to heart and skeletal muscle, GLUT-4 protein in brown adipose tissue was detected in high levels (30% of adult) during late fetal life. During fetal life, GLUT-l presented a very high expression level in brown adipose tissue, heart, and skeletal muscle. Soon after birth, GLUT-1 protein diminished progressively, attaining adult levels at day 10 in heart and skeletal muscle. GLUT-1 mRNA levels in heart followed a similar pattern to the GLUT- 1 protein, being very high during fetal life and decreasing early in post natal life. GLUT-1 protein showed a complex pattern in brown adipose tissue: fetal levels were high, decreased after birth, and increased subsequently in post natal life, reaching a peak by day 9. Progesterone-induced postmaturity protected against the decrease in GLUT-1 protein associated with post natal life in skeletal muscle and brown adipose tissue. However, GLUT-4 induction was not blocked by postmaturity in any of the tissues subjected to study. These results indicate that: 1) during fetal and early post natal life, GLUT-1 is a predominant glucose transporter isotype expressed in heart, skeletal muscle, and brown adipose tissue; 2) during early post natal life there is a generalized GLUT-1 repression; 3) during development, there is a close correlation between protein and mRNA levels for GLUT-l, and therefore regulation at a pretranslational level plays a major regulatory role; 4) the onset of GLUT-4 protein induction occurs between days 20-21 of fetal life; based on data obtained in rat heart and brown adipose tissue, there is a dissociation during development between mRNA and protein levels for GLUT-4, suggesting modifications at translational or posttranslational steps; and 5) postmaturity blocks the decrease in GLUT-l expression but not the induction of GLUT-4. observed soon after birth. All these findings suggest that GLUT-1 repression and GLUT-4 induction are mediated by different mechanisms

Vanadate stimulates system A amino acid transport activity in skeletal muscle. Evidence for the involvement of intracellular pH as a mediator of vanadate action

Sodium orthovanadate caused a 2-fold stimulation of system A transport activity in soleus muscle, as assessed by the uptake of the nonmetabolizable analog 2-(methylamino)isobutyric acid (MeAIB). The effect of vanadate on system A was rapid, concentration-dependent and was characterized by an increased Vmax without modification of Km for MeAIB. Under these conditions, vanadate also activated 3-O-methylglucose uptake and lactate production. The effects of vanadate on muscle metabolism showed a complex interaction with the effects of insulin. Thus, the stimulatory effects of vanadate and insulin on MeAIB and 3-O-methylglucose uptake were not additive; however, the effects of insulin and vanadate on lactate production were additive. In spite of the lack of additivity, insulin- and vanadate-induced stimulation of system A differed in their sensitivity to gramicidin D, being the vanadate effect more susceptible to inhibition by gramicidin D than the insulin effect. System A transport activity shows a dependence on pH, and recent results suggest the presence of critical histidine residues on the A carrier that may be responsible for its pH dependence (Bertran, J., Roca, A., Pola, E., Testar, X., Zorzano, A. & Palacín, M. (1991) J. Biol. Chem. 266, 798-802). In this regard, a rise in extracellular pH led to a substantial activation of system A. Furthermore, lowering of muscle intracellular pH induced by ethylisopropylamiloride (EIPA), a specific inhibitor of sodium/proton exchange activity, led to inhibition of system A. This suggests that critical histidine residues are present in an intracellular localization on the A carrier. Furthermore, the rate of muscle glycolysis was also altered in response to a rise in extracellular pH or to EIPA treatment. Regarding the mechanisms involved in vanadate action, vanadate treatment in the incubated soleus muscle did not cause any significant stimulation of tyrosine kinase activity after partial purification of muscle insulin receptors. On the other hand, vanadate but not insulin caused a substantial increase in muscle intracellular pH as assessed by 5,5'-dimethyloxazolidine-2,4-dione equilibrium. This effect of vanadate on intracellular pH was not due to activation of the sodium/proton exchanger, since it was not blocked by EIPA. Based on these findings, we suggest that alkalinization of muscle intracellular pH might mediate the effects of vanadate on system A and on glycolysis

The relevance of EGFR, ErbB receptors and neuregulins in human adipocytes and adipose tissue in obesity

Objective: To investigate the potential role of EGFR, ErbBs receptors and neuregulins in human adipose tissue physiology in obesity. Methods: Gene expression analysis in human subcutaneous (SAT) and visceral (VAT) adipose tissue in three independent cohorts [two cross-sectional (N = 150, N = 87) and one longitudinal (n = 25)], and in vitro gene knockdown and overexpression experiments were performed. Results: While both SAT and VAT ERBB2 and ERBB4 mRNA increased in obesity, SAT EGFR mRNA was negatively correlated with insulin resistance, but did not change in obesity. Of note, both SAT and VAT EGFR mRNA were significantly associated with adipogenesis and increased during human adipocyte differentiation. In vitro experiments revealed that EGFR, but not ERBB2 and ERBB4, gene knockdown in preadipocytes and in fully differentiated human adipocytes resulted in decreased expression of adipogenic-related genes. ERBB2 gene knockdown also reduced gene expression of fatty acid synthase in fully differentiated adipocytes. In addition, neuregulin 2 (NRG2) mRNA was associated with expression of adipogenic genes in human adipose tissue and adipocytes, and its overexpression increased expression of EGFR and relevant adipogenic genes. Conclusions: This study demonstrates the association between adipose tissue ERBB2 and obesity, confirms the relevance of EGFR on human adipogenesis, and suggests a possible adipogenic role of NRG2

Caveolin interaction governs Kv1.3 lipid raft targeting

The spatial localization of ion channels at the cell surface is crucial for their functional role. Many channels localize in lipid raft microdomains, which are enriched in cholesterol and sphingolipids. Caveolae, specific lipid rafts which concentrate caveolins, harbor signaling molecules and their targets becoming signaling platforms crucial in cell physiology. However, the molecular mechanisms involved in such spatial localization are under debate. Kv1.3 localizes in lipid rafts and participates in the immunological response. We sought to elucidate the mechanisms of Kv1.3 surface targeting, which govern leukocyte physiology. Kv1 channels share a putative caveolin-binding domain located at the intracellular N-terminal of the channel. This motif, lying close to the S1 transmembrane segment, is situated near the T1 tetramerization domain and the determinants involved in the Kvβ subunit association. The highly hydrophobic domain (FQRQVWLLF) interacts with caveolin 1 targeting Kv1.3 to caveolar rafts. However, subtle variations of this cluster, putative ancillary associations and different structural conformations can impair the caveolin recognition, thereby altering channel's spatial localization. Our results identify a caveolin-binding domain in Kv1 channels and highlight the mechanisms that govern the regulation of channel surface localization during cellular processes