The MEF2A isoform is required for striated muscle-specific expression of the insulin-responsive Glut4 glucose transporter

Previously, we have demonstrated that an MEF2 consensus sequence located between −473/−464 in the human GLUT4 gene was essential for both tissue-specific and hormonal/metabolic regulation of GLUT4 expression (Thai, M. V., Guruswamy, S., Cao, K. T., Pessin, J. E., and Olson, A. L. (1998)J. Biol. Chem. 273, 14285-14292). To identify the specific MEF2 isoform(s) responsible for GLUT4 expression, we studied the pattern of expression of the MEF2 isoforms in insulin-sensitive tissues. Both heart and skeletal muscle were found to express the MEF2A, MEF2C, and MEF2D isoforms but not MEF2B. However, only the MEF2A protein was selectively down-regulated in insulin-deficient diabetes. Co-immunoprecipitation with isoform-specific antibodies revealed that, in the basal state, essentially all of the MEF2A protein was presented as a MEF2A-MEF2D heterodimer without any detectable MEF2A-MEF2A homodimers or MEF2A-MEF2C and MEF2C-MEF2D heterodimers. Electrophoretic mobility shift assays revealed that nuclear extracts from diabetic animals had reduced binding to the MEF2 binding site compared with extracts from control or insulin-treated animals. Furthermore, immunodepletion of the MEF2A-MEF2D complex from control extracts abolished binding to the MEF2 element. However, addition of MEF2A to diabetic nuclear extracts fully restored binding activity to the MEF2 element. These data strongly suggest that the MEF2A-MEF2D heterodimer is selectively decreased in insulin-deficient diabetes and is responsible for hormonally regulated expression of the GLUT4 gene

Cbl downreguation increases RBP4 expression in adipocytes of female mice

Obesity leads to adipose tissue dysfunction, insulin resistance and diabetes. Adipose tissue produces adipokines that contribute to regulate insulin sensitivity. In turn, insulin stimulates the production and release of some adipokines. Casitas-b-lymphoma proteins (c-Cbl, Cbl-b and Cbl3) are intracellular adaptor signalling proteins that are rapidly phosphorylated by activation of tyrosine kinase receptors. c-Cbl is rapidly phosphorylated by insulin in adipocytes. Here, we tested the hypothesis that Cbl signalling regulates adipokine expression in adipose tissue. We determined the adipokine profile of WAT of Cbl−/− and Cbl+/+ mice in the C57BL6 background. Female Cbl−/− mice exhibited altered expression of adiponectin, leptin and RBP4 in visceral adipose tissue, while no significant changes were seen in male mice. TNFα and IL6 levels were unaffected by Cbl depletion. RBP4 expression was unchanged in liver. Adipose tissue of Cbl−/− animals showed increased basal activation of extracellular regulated kinases (ERK1/2) compared to Cbl+/+. c-Cbl knockdown in 3T3L1 adipocytes also increased basal ERK phosphorylation and RBP4 expression. Inhibition of ERK1/2 phosphorylation in Cbl-depleted 3T3L1 adipocytes or in adipose tissue explants of Cbl−/− mice reduced RBP4 mRNA. 17β-Estradiol increased RBP4 mRNA in adipocytes. Cbl depletion did not change ER expression but increased phosphorylation of ERα at S118, a target site for ERK1/2. ERK1/2 inhibition reduced phosphoER and RBP4 levels. These findings suggest that Cbl contributes to regulate RBP4 expression in adipose of female mice through ERK1/2-mediated activation of ERα. Since Cbl signalling is compromised in diabetes, these data highlight a novel mechanism that upregulates RBP4 locally

Evaluation of macrophage plasticity in brown and white adipose tissue

There are still questions about whether macrophage differentiation is predetermined or is induced in response to tissue microenvironments. C2D macrophage cells reside early in the macrophage lineage in vitro, but differentiate to a more mature phenotype after adoptive transfer to the peritoneal cavity (PEC-C2D). Since C2D macrophage cells also traffic to adipose tissue after adoptive transfer, we explored the impact of white adipose tissue (WAT), brown adipose tissue (BAT) and in vitro cultured adipocytes on C2D macrophage cells. When PEC-C2D macrophage cells were cultured with preadipocytes the cells stretched out and CD11b and Mac-2 expression was lower compared to PEC-C2D macrophage cells placed in vitro alone. In contrast, PEC-C2D cells co-cultured with adipocytes maintained smaller, round morphology and more cells expressed Mac-2 compared to PEC-C2D co-cultured with preadipocytes. After intraperitoneal injection, C2D macrophage cells migrated into both WAT and BAT. A higher percentage of C2D macrophage cells isolated from WAT (WAT-C2D) expressed Ly-6C (33%), CD11b (11%), Mac-2 (11%) and F4/80 (29%) compared to C2D macrophage cells isolated from BAT (BAT-C2D). Overall, BAT-C2D macrophage cells had reduced expression of many cytokine, chemokine and receptor gene transcripts when compared to in vitro grown C2D macrophages, while WAT-C2D macrophage cells and PEC-C2D up-regulated many of these gene transcripts. These data suggest that the C2D macrophage phenotype can change rapidly and distinct phenotypes are induced by different microenvironments

The MEF2A and MEF2D isoforms are differentially regulated in muscle and adipose tissue during states of insulin deficiency

Previously we have demonstrated that striated muscle GLUT4 gene expression decreased following streptozotocin-induced diabetes due to a loss of MEF2A transcription factor expression without any significant effect on the MEF2D isoform (Mora, S. and J. E. Pessin (2000) J Biol Chem, 275:16323-16328). In contrast to both cardiac and skeletal muscle, adipose tissue displays a selective decrease in MEF2D expression in diabetes without any significant alteration in MEF2A protein content. Adipose tissue also expresses very low levels of the MEF2 transcription factors and nuclear extracts from white adipose tissue exhibit poor in vitro binding to the MEF2 element. However, addition of in vitro synthesized MEF2A to adipose nuclear extracts results in the formation of the expected MEF2/DNA complex. More importantly, binding to the MEF2 element was also compromised in the diabetic condition. Furthermore, in vivo overexpression of MEF2A selectively in adipose tissue did not affect GLUT4 or MEF2D expression and was not sufficient to prevent GLUT4 down-regulation that occurred in insulin-deficient states

The Insulin receptor catalyzes the tyrosine phosphorylation o Caveolin 1

Our previous studies revealed that insulin stimulates the tyrosine phosphorylation of caveolin in 3T3L1 adipocytes. To explore the mechanisms involved in this event, we evaluated the association of the insulin receptor with caveolin. The receptor was detected in a Triton-insoluble low density fraction, co-sedimenting with caveolin and flotillin on sucrose density gradients. We also detected the receptor in caveolin-enriched rosette structures by immunohistochemical analysis of plasma membrane sheets from 3T3L1 adipocytes. Insulin stimulated the phosphorylation of caveolin-1 on Tyr14. This effect of the hormone was not blocked by overexpression of mutant forms of the Cbl-associated protein that block the translocation of phospho-Cbl to the caveolin-enriched, lipid raft microdomains. Moreover, this phosphorylation event was also unaffected by inhibitors of the MAPK and phosphatidylinositol 3-kinase pathways. Although previous studies demonstrated that the Src family kinase Fyn was highly enriched in caveolae, an inhibitor of this kinase had no effect on insulin-stimulated caveolin phosphorylation. Interestingly, overexpression of a mutant form of caveolin that failed to interact with the insulin receptor did not undergo phosphorylation. Taken together, these data indicate that the insulin receptor directly catalyzes the tyrosine phosphorylation of caveolin. Previous article in issu

Atypical Protein Kinase C (PKCλ/ζ) is a convergent downstream target of the insulin stimulated phosphatidylinositol 3-kinase and TC10 signalling pathways

Insulin stimulation of adipocytes resulted in the recruitment of atypical PKC (PKCζ/λ) to plasma membrane lipid raft microdomains. This redistribution of PKCζ/λ was prevented by Clostridium difficile toxin B and by cholesterol depletion, but was unaffected by inhibition of phosphatidylinositol (PI) 3-kinase activity. Expression of the constitutively active GTP-bound form of TC10 (TC10Q/75L), but not the inactive GDP-bound mutant (TC10/T31N), targeted PKCζ/λ to the plasma membrane through an indirect association with the Par6-Par3 protein complex. In parallel, insulin stimulation as well as TC10/Q75L resulted in the activation loop phosphorylation of PKCζ. Although PI 3-kinase activation also resulted in PKCζ/λ phosphorylation, it was not recruited to the plasma membrane. Furthermore, insulin-induced GSK-3β phosphorylation was mediated by both PI 3-kinase-PKB and the TC10-Par6-atypical PKC signaling pathways. Together, these data demonstrate that PKCζ/λ can serve as a convergent downstream target for both the PI 3-kinase and TC10 signaling pathways, but only the TC10 pathway induces a spatially restricted targeting to the plasma membrane

Intracellular trafficking and secretion of adiponectin is dependent on GGA coated vesicles

Adiponectin (Acrp30) is an insulin-sensitizing hormone produced and secreted exclusively by adipose tissue. Confocal fluorescent microscopy demonstrated the colocalization of adiponectin with the Golgi membrane markers p115, β-COP, and the trans-Golgi network marker, syntaxin 6. Treatment of cells with brefeldin A redistributed adiponectin to the endoplasmic reticulum where it colocalized with the chaperone protein BIP and inhibited secretion of adiponectin demonstrating a requirement for a functional Golgi apparatus for adiponectin release. Confocal fluorescent microscopy also demonstrated a colocalization of endogenous adiponectin with that of expressed GGA1myc (Golgi-localizing γ-adaptin ear homology ARF-binding protein) but with no significant overlap between adiponectin and the GGA2myc or GGA3myc isoforms. Consistent with confocal fluorescent microscopy, transmission electron microscopy demonstrated the colocalization of GGA1 with adiponectin. Although GGA1 did not directly interact with the adiponectin protein, the adiponectin enriched membrane compartments of adipocyte were precipitated by a GST-GGA1 cargo binding domain (VHS) fusion protein but not with a GST-GGA2 VHS or GST-GGA3 VHS fusion proteins. Moreover, co-expression of adiponectin with a GGA1 dominant-interfering mutant (GGA1-VHS GAT domain) resulted in a marked inhibition of adiponectin secretion in both 3T3L1 adipocytes and HEK293 cells, whereas no inhibition was detected with the truncated mutants GGA2-VHSGAT or GGA3-VHSGAT. Moreover, co-expression of wild type GGA1 with adiponectin enhanced secretion of adiponectin. Interestingly, leptin secretion was unaffected by neither the wild type form or GGA1 mutant. Taken together these data demonstrate that the trafficking of adiponectin through its secretory pathway is dependent on GGA-coated vesicles

Hypoxia‑induced HIF1α activation regulates small extracellular vesicle release in human embryonic kidney cells.

Extracellular vesicles (EVs) are membrane enclosures released by eukaryotic cells that carry bioactive molecules and serve to modulate biological responses in recipient cells. Both increased EV release and altered EV composition are associated with the development and progression of many pathologies including cancer. Hypoxia, a feature of rapidly growing solid tumours, increases the release of EVs. However, the molecular mechanisms remain unknown. The hypoxia inducible factors (HIFs) are transcription factors that act as major regulators of the cellular adaptations to hypoxia. Here, we investigated the requirement of HIF pathway activation for EV release in Human Embryonic Kidney Cells (HEK293). Time course experiments showed that EV release increased concomitantly with sustained HIF1α and HIF2α activation following the onset of hypoxia. shRNA mediated knock‑down of HIF1α but not HIF2α abrogated the effect of hypoxia on EV release, suggesting HIF1α is involved in this process. However, stabilization of HIF proteins in normoxic conditions through: (i) heterologous expression of oxygen insensitive HIF1α or HIF2α mutants in normoxic cells or (ii) chemical inhibition of the prolyl hydroxylase 2 (PHD2) repressor protein, did not increase EV release, suggesting HIF activation alone is not sufficient for this process. Our findings suggest HIF1α plays an important role in the regulation of EV release during hypoxia in HEK293 cells, however other hypoxia triggered mechanisms likely contribute as stabilization of HIF1α alone in normoxia is not sufficient for EV release

NCS-1 Inhibits insulin stimulated GLUT4 translocation in 3T3L1 adipocytes through a phosphatidylinositol 4-kinase dependent pathway

Expression of NCS-1 (neuronal calcium sensor-1, also termed frequenin) in 3T3L1 adipocytes strongly inhibited insulin-stimulated translocation of GLUT4 and insulin-responsive aminopeptidase. The effect of NCS-1 was specific for GLUT4 and the insulin-responsive aminopeptidase translocation as there was no effect on the trafficking of the cation-independent mannose 6-phosphate receptor or the GLUT1 glucose transporter isoform. Moreover, NCS-1 showed partial colocalization with GLUT4-EGFP in the perinuclear region. The inhibitory action of NCS-1 was independent of calcium sequestration since neither treatment with ionomycin nor endothelin-1, both of which elevated the intracellular calcium concentration, restored insulin-stimulated GLUT4 translocation. Furthermore, NCS-1 did not alter the insulin-stimulated protein kinase B (PKB/Akt) phosphorylation or the recruitment of Cbl to the plasma membrane. In contrast, expression of the NCS-1 effector phosphatidylinositol 4-kinase (PI 4-kinase) inhibited insulin-stimulated GLUT4 translocation, whereas co-transfection with an inactive PI 4-kinase mutant prevented the NCS-1-induced inhibition. These data demonstrate that PI 4-kinase functions to negatively regulate GLUT4 translocation through its interaction with NCS-1

Extracellular vesicles from hypoxic adipocytes and obese subjects reduce insulin-stimulated glucose uptake

Scope We investigate the effects of extracellular vesicles (EVs) obtained from in vitro adipocyte cell models and from obese subjects on glucose transport and insulin responsiveness. Methods and results EVs are isolated from the culture supernatant of adipocytes cultured under normoxia, hypoxia (1% oxygen), or exposed to macrophage conditioned media (15% v/v). EVs are isolated from the plasma of lean individuals and subjects with obesity. Cultured adipocytes are incubated with EVs and activation of insulin signalling cascades and insulin‐stimulated glucose transport are measured. EVs released from hypoxic adipocytes impair insulin‐stimulated 2‐deoxyglucose uptake and reduce insulin mediated phosphorylation of AKT. Insulin‐mediated phosphorylation of extracellular regulated kinases (ERK1/2) is not affected. EVs from individuals with obesity decrease insulin stimulated 2‐deoxyglucose uptake in adipocytes (p = 0.0159). Conclusion EVs released by stressed adipocytes impair insulin action in neighboring adipocytes