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L A Garza

Publications and source records attributed to L A Garza.

7 recordsLinked to original sources
PubMed2000

Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes.

The insulin-responsive aminopeptidase (IRAP/VP165/gp160) was identified originally in GLUT4-containing vesicles and shown to translocate in response to insulin, much like the glucose transporter 4 (GLUT4). This study characterizes the trafficking and kinetics of IRAP in exocytosis, endocytosis, and recycling to the membrane in 3T3-L1 adipocytes. After exposure of 3T3-L1 adipocytes to insulin, IRAP translocated to the plasma membrane as assessed by either cell fractionation, surface biotinylation, or the plasma membrane sheet assay. The rate of exocytosis closely paralleled that of GLUT4. In the continuous presence of insulin, IRAP was endocytosed with a half-time of about 3-5 min. IRAP endocytosis is inhibited by cytosol acidification, a property of clathrin-mediated endocytosis, but not by the expression of a constitutively active Akt/PKB. Arrival in an LDM fraction derived via subcellular fractionation exhibited a slower time course than disappearance from the cell surface, suggesting additional endocytic intermediates. As assayed by membrane "sheets," GLUT4 and IRAP showed similar internalization rates that are wortmannin-insensitive and occur with a half-time of roughly 5 min. IRAP remaining on the cell surface 10 min following insulin removal was both biotin- and avidin-accessible, implying the absence of thin-necked invaginations. Finally, endocytosed IRAP quickly recycled back to the plasma membrane in a wortmannin-sensitive process. These results demonstrate rapid endocytosis and recycling of IRAP in the presence of insulin and trafficking that matches GLUT4 in rate.

3T3 Cells↗
PubMed1999

Signaling pathways mediating insulin-stimulated glucose transport.

A major action of insulin is to accelerate the rate of uptake of sugar into muscle and adipose cells following a meal. The biochemical mechanism by which this is accomplished has been a subject of intense experimentation, although elucidation of the pathways has remained elusive. In recent years, numerous signaling molecules and cascades modulated by insulin have been identified, although few have been definitively established as important to the metabolic actions of the hormone. An exception to this is the lipid kinase phosphatidylinositide 3'-kinase, which, under many conditions, appears absolutely required for insulin to stimulate hexose uptake into adipocytes. Akt/PKB, a serine/threonine protein kinase activated by insulin in a phosphatidylinositide 3'-kinase-dependent manner, has been implicated as a critical mediator of insulin's actions on metabolism and cell survival. Nonetheless, Akt/PKB's role in many insulin effects, particularly accelerated glucose transport, remains controversial. Interestingly, soluble analogues of ceramide antagonize both insulin's activation of Akt/PKB as well as its stimulation of glucose transport, consistent with a causal relationship between the two.

Animals↗
PubMed1999

Identification of wortmannin-sensitive targets in 3T3-L1 adipocytes. DissociationoOf insulin-stimulated glucose uptake and glut4 translocation.

The current studies investigated the contribution of phosphatidylinositol 3-kinase (PI3-kinase) isoforms to insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation. Experiments involving the microinjection of antibodies specific for the p110 catalytic subunit of class I PI3-kinases demonstrated an absolute requirement for this form of the enzyme in GLUT4 translocation. This finding was confirmed by the demonstration that the PI3-kinase antagonist wortmannin inhibits GLUT4 and insulin-responsive aminopeptidase translocation with a dose response identical to that required to inhibit another class I PI3-kinase-dependent event, activation of pp70 S6-kinase. Interestingly, wortmannin inhibited insulin-stimulated glucose uptake at much lower doses, suggesting the existence of a second, higher affinity target of the drug. Subsequent removal of wortmannin from the media shifted this dose-response curve to one resembling that for GLUT4 translocation and pp70 S6-kinase. This is consistent with the lower affinity target being p110, which is irreversibly inhibited by wortmannin. Wortmannin did not reduce glucose uptake in cells stably expressing Myr-Akt, which constitutively induced GLUT4 translocation to the plasma membrane; this demonstrates that wortmannin does not inhibit the transporters directly. In addition to elucidating a second wortmannin-sensitive pathway in 3T3-L1 adipocytes, these studies suggest that the presence of GLUT4 on the plasma membrane is not sufficient for activation of glucose uptake.

3T3 Cells↗
PubMed1998

Regulation of insulin-stimulated glucose transporter GLUT4 translocation and Akt kinase activity by ceramide.

The sphingomyelin derivative ceramide is a signaling molecule implicated in numerous physiological events. Recently published reports indicate that ceramide levels are elevated in insulin-responsive tissues of diabetic animals and that agents which trigger ceramide production inhibit insulin signaling. In the present series of studies, the short-chain ceramide analog C2-ceramide inhibited insulin-stimulated glucose transport by approximately 50% in 3T3-L1 adipocytes, with similar reductions in hormone-stimulated translocation of the insulin-responsive glucose transporter (GLUT4) and insulin-responsive aminopeptidase. C2-ceramide also inhibited phosphorylation and activation of Akt, a molecule proposed to mediate multiple insulin-stimulated metabolic events. C2-ceramide, at concentrations which antagonized activation of both glucose uptake and Akt, had no effect on the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) or the amounts of p85 protein and phosphatidylinositol kinase activity that immunoprecipitated with anti-IRS-1 or antiphosphotyrosine antibodies. Moreover, C2-ceramide also inhibited stimulation of Akt by platelet-derived growth factor, an event that is IRS-1 independent. C2-ceramide did not inhibit insulin-stimulated phosphorylation of mitogen-activated protein kinase or pp70 S6-kinase, and it actually stimulated phosphorylation of the latter in the absence of insulin. Various pharmacological agents, including the immunosuppressant rapamycin, the protein synthesis inhibitor cycloheximide, and several protein kinase C inhibitors, were without effect on ceramide's inhibition of Akt. These studies demonstrate ceramide's capacity to inhibit activation of Akt and imply that this is a mechanism of antagonism of insulin-dependent physiological events, such as the peripheral activation of glucose transport and the suppression of apoptosis.

3T3 Cells↗
PubMed1995

The effect of monovalent cations on the self-association of cytidylyl-(3-5')-guanosine and guanylyl-(3'5')-cytidine in aqueous solution.

The hydrogen-bonding, base stacking, and formation of extended aggregates has been investigated for salts of guanylyl-3'-5')-cytidine, GpC, and cytidylyl-(3'-5')- guanosine, CpG, in which the cation was Na+, K+, or tetramethylammonium (TMA+). Variable temperature studies were done at 2-70 degrees C on aqueous solutions at pD4 and 8 using 1H NMR and FTIR. At low temperatures it has been found that at pD 8 both GpC and CpG form Watson-Crick dimers which stack upon each other to form larger species. A slight cation effect is observed below 35 degrees C which has the order: TMA+ > Na+ > K+. This order suggests that the cations are interacting with the phosphate and interactions with the bases are unlikely. The 1H NMR spectrum for TMACpG at pD 4 has been assigned and exhibits chemical shift differences from those at pD 8 which are consistent with protonation of the N3 of the cytidine residue. Based on NMR line broadening, CpG at pD 4 has a greater degree of self-association at low temperature than it or GpC have at pD 8. A different type of hydrogen bonding and self-association occur in CpG at pD 4 compared to pD 8, but the structures are uncertain. Due to hemi-protonation of the cytidine N3, parallel G-G/C-C+ base paired dimers or G-tetrads may be forming.

Cations, Monovalent↗
PubMed1994

Conformation, hydrogen bonding and aggregate formation of guanosine 5'-monophosphate and guanosine in dimethylsulfoxide.

The tetrabutylammonium salt of guanosine 5'-monophosphate (5'-GMP) dissolves in DMSO-d6 forming aggregated species which exhibit some properties of reverse micelles. 1H NOESY experiments show that the 5'-GMP adopts the syn conformation about the glycosidic bond. Molecular mechanics calculations reveal a stable structure with this conformation in which the phosphate group and the amino group of the base are in close enough proximity to hydrogen bond. In contrast inosine 5'-monophosphate in DMSO-d6, which has no NH2 group for hydrogen bond stabilization of the syn conformation, is shown by NMR to have the anti structure. Guanosine in DMSO-d6 behaves differently from 5'-GMP. Guanosine adopts the anti conformation and forms a symmetric dimer via hydrogen bonding between the N3 and NH2 of the bases.

Dimethyl Sulfoxide↗