Anchoring of protein kinase A facilitates hormone-mediated insulin secretion

Impaired insulin secretion is a characteristic of non-insulin-dependent diabetes mellitus (NIDDM). One possible therapeutic agent for NIDDM is the insulinotropic hormone glucagon-like peptide 1 (GLP-1). GLP-1 stimulates insulin secretion through several mechanisms including activation of protein kinase A (PKA). We now demonstrate that the subcellular targeting of PKA through association with A-kinase-anchoring proteins (AKAPs) facilitates GLP-1-mediated insulin secretion. Disruption of PKA anchoring by the introduction of anchoring inhibitor peptides or expression of soluble AKAP fragments blocks GLP-1 action in primary islets and cAMP-responsive insulin secretion in clonal beta cells (RINm5F). Displacement of PKA also prevented cAMP-mediated elevation of intracellular calcium suggesting that localized PKA phosphorylation events augment calcium flux.

Correlation of α-Fetoprotein Expression in Normal Hepatocytes during Development with Tyrosine Phosphorylation and Insulin Receptor Expression

The molecular mechanism of hepatic cell growth and differentiation is ill defined. In the present study, we examined the putative role of tyrosine phosphorylation in normal rat liver development and in an in vitro model, the α-fetoprotein-producing (AFP+) and AFP-nonproducing (AFP−) clones of the McA-RH 7777 rat hepatoma. We demonstrated in vivo and in vitro that the AFP+ phenotype is clearly associated with enhanced tyrosine phosphorylation, as assessed by immunoblotting and flow cytometry. Moreover, immunoprecipitation of proteins with anti-phosphotyrosine antibody showed that normal fetal hepatocytes expressed the same phosphorylation pattern as stable AFP+ clones and likewise for adult hepatocytes and AFP− clones. The tyrosine phosphorylation of several proteins, including the β-subunit of the insulin receptor, insulin receptor substrate-1, p85 regulatory subunit of phosphatidylinositol-3-kinase, and ras-guanosine triphosphatase-activating protein, was observed in AFP+ clones, whereas the same proteins were not phosphorylated in AFP− clones. We also observed that fetal hepatocytes and the AFP+ clones express 4 times more of the insulin receptor β-subunit compared with adult hepatocytes and AFP− clones and, accordingly, that these AFP+ clones were more responsive to exogenous insulin in terms of protein tyrosine phosphorylation. Finally, growth rate in cells of AFP+ clones was higher than that measured in cells of AFP− clones, and inhibition of phosphatidylinositol-3-kinase by LY294002 and Wortmannin blocked insulin- and serum-stimulated DNA synthesis only in cells of AFP+ clones. These studies provide evidences in support of the hypothesis that signaling via insulin prevents hepatocyte differentiation by promoting fetal hepatocyte growth.

The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.

Binding of Insulin-like Growth Factor (IGF)–Binding Protein-5 to Smooth-Muscle Cell Extracellular Matrix Is a Major Determinant of the Cellular Response to IGF-I

Insulin-like growth factor–binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I’s effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.

Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Phosphorylation of the α-subunit of Na+,K+-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K+-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K+-ATPase α-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the α-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat α-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive 86Rb uptake in opossum kidney cells expressing mutant rat α1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive 86Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K+-ATPase α-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Opposite Effects of Insulin on Focal Adhesion Proteins in 3T3-L1 Adipocytes and in Cells Overexpressing the Insulin Receptor

Insulin can regulate the abundance and organization of filamentous actin within cells in culture. Early studies using cell lines that overexpress the insulin receptor demonstrated that insulin caused a rapid reversible disassembly of actin filaments that coincided with the rapid tyrosine dephosphorylation of focal adhesion kinase. We have extended these studies by demonstrating that paxillin, another focal adhesion protein, and Src undergo tyrosine dephosphorylation in response to insulin in Chinese hamster ovary (CHO) and rat hepatoma (HTC) cells that overexpress the insulin receptor. This contrasted with the effect of insulin in parental CHO and HTC cells in which focal adhesion proteins were not dephosphorylated in response to the hormone. In addition, insulin caused a dispersion of focal adhesion proteins and disruption of actin filament bundles only in cells that overexpressed the insulin receptor. Moreover, in 3T3-L1 adipocytes, which are considered prototypic insulin-responsive cells, actin filament assembly was stimulated, and focal adhesion protein tyrosine phosphorylation was not altered. 3T3-L1 cells have more insulin receptors than either parental CHO or HTC cells but have fivefold less insulin receptors than the overexpressing cell lines. We hypothesize that a threshold may exist in which the overexpression of insulin receptors determines how insulin signaling pathways regulate the actin cytoskeleton.

Differential Regulation of Secretory Compartments Containing the Insulin-responsive Glucose Transporter 4 in 3T3-L1 Adipocytes

Insulin and guanosine-5′-O-(3-thiotriphosphate) (GTPγS) both stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct intracellular compartments. We show that ablation of the endosomal system using transferrin-HRP causes a modest inhibition (∼30%) of insulin-stimulated GLUT4 translocation. In contrast, the GTPγS response was significantly attenuated (∼85%) under the same conditions. Introduction of a GST fusion protein encompassing the cytosolic tail of the v-SNARE cellubrevin inhibited GTPγS-stimulated GLUT4 translocation by ∼40% but had no effect on the insulin response. Conversely, a fusion protein encompassing the cytosolic tail of vesicle-associated membrane protein-2 had no significant effect on GTPγS-stimulated GLUT4 translocation but inhibited the insulin response by ∼40%. GTPγS- and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin (∼50%) but not by GST-vesicle-associated membrane protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with GTPγS caused a marked accumulation of Rab4 and Rab5 at the cell surface, whereas other Rab proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently using distinct sets of trafficking molecules.

Interaction of Class I Human Leukocyte Antigen (HLA-I) Molecules with Insulin Receptors and Its Effect on the Insulin-Signaling Cascade

Insulin receptor (IR) and class I major histocompatibility complex molecules associate with one another in cell membranes, but the functional consequences of this association are not defined. We found that IR and human class I molecules (HLA-I) associate in liposome membranes and that the affinity of IR for insulin and its tyrosine kinase activity increase as the HLA:IR ratio increases over the range 1:1 to 20:1. The same relationship between HLA:IR and IR function was found in a series of B-LCL cell lines. The association of HLA-I and IR depends upon the presence of free HLA heavy chains. All of the effects noted were reduced or abrogated if liposomes or cells were incubated with excess HLA-I light chain, β2-microglobulin. Increasing HLA:IR also enhanced phosphorylation of insulin receptor substrate-1 and the activation of phosphoinositide 3-kinase. HLA-I molecules themselves were phosphorylated on tyrosine and associated with phosphoinositide 3-kinase when B-LCL were stimulated with insulin.

Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton

Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr774 in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Cζ, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.

Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensitivity in apolipoprotein A-II knockout mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility

Apolipoprotein (apo) A-II is the second most abundant apolipoprotein in high density lipoprotein (HDL). To study its role in lipoprotein metabolism and atherosclerosis susceptibility, apo A-II knockout mice were created. Homozygous knockout mice had 67% and 52% reductions in HDL cholesterol levels in the fasted and fed states, respectively, and HDL particle size was reduced. Metabolic turnover studies revealed the HDL decrease to be due to both decreased HDL cholesterol ester and apo A-I transport rate and increased HDL cholesterol ester and apo A-I fractional catabolic rate. The apo A-II deficiency trait was bred onto the atherosclerosis-prone apo E-deficient background, which resulted in a surprising 66% decrease in cholesterol levels due primarily to decreased atherogenic lipoprotein remnant particles. Metabolic turnover studies indicated increased remnant clearance in the absence of apo A-II. Finally, apo A-II deficiency was associated with lower free fatty acid, glucose, and insulin levels, suggesting an insulin hypersensitivity state. In summary, apo A-II plays a complex role in lipoprotein metabolism, with some antiatherogenic properties such as the maintenance of a stable HDL pool, and other proatherogenic properties such as decreasing clearance of atherogenic lipoprotein remnants and promotion of insulin resistance.

Purification of the β-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells

The β cell-specific glucose-sensitive factor (GSF), which binds the A3 motif of the rat I and human insulin promoters, is modulated by extracellular glucose. A single mutation in the GSF binding site of the human insulin promoter abolishes the stimulation by high glucose only in normal islets, supporting the suggested physiological role of GSF in the glucose-regulated expression of the insulin gene. GSF binding activity was observed in all insulin-producing cells. We have therefore purified this activity from the rat insulinoma RIN and found that a single polypeptide of 45 kDa was responsible for DNA binding. Its amino acid sequence, determined by microsequencing, provided direct evidence that GSF corresponds to insulin promoter factor 1 (IPF-1; also known as PDX-1) and that, in addition to its essential roles in development and differentiation of pancreatic islets and in β cell-specific gene expression, it functions as mediator of the glucose effect on insulin gene transcription in differentiated β cells. The human cDNA coding for GSF/IPF-1 has been cloned, its cell and tissue distribution is described. Its expression in the glucagon-producing cell line αTC1 transactivates the wild-type human insulin promoter more efficiently than the mutated construct. It is demonstrated that high levels of ectopic GSF/IPF-1 inhibit the expression of the human insulin gene in normal islets, but not in transformed βTC1 cells. These results suggest the existence of a control mechanism, such as requirement for a coactivator of GSF/IPF-1, which may be present in limiting amounts in normal as opposed to transformed β cells.

Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins

A major physiological role of insulin is the regulation of glucose uptake into skeletal and cardiac muscle and adipose tissue, mediated by an insulin-stimulated translocation of GLUT4 glucose transporters from an intracellular vesicular pool to the plasma membrane. This process is similar to the regulated docking and fusion of vesicles in neuroendocrine cells, a process that involves SNARE-complex proteins. Recently, several SNARE proteins were found in adipocytes: vesicle-associated membrane protein (VAMP-2), its related homologue cellubrevin, and syntaxin-4. In this report we show that treatment of permeabilized 3T3-L1 adipocytes with botulinum neurotoxin D, which selectively cleaves VAMP-2 and cellubrevin, inhibited the ability of insulin to stimulate translocation of GLUT4 vesicles to the plasma membrane. Furthermore, treatment of the permeabilized adipocytes with glutathione S-transferase fusion proteins encoding soluble forms of VAMP-2 or syntaxin-4 also effectively blocked insulin-regulated GLUT4 translocation. These results provide evidence of a functional role for SNARE-complex proteins in insulin-stimulated glucose uptake and suggest that adipocytes utilize a mechanism of regulating vesicle docking and fusion analogous to that found in neuroendocrine tissues.

Exercise-induced changes in expression and activity of proteins involved in insulin signal transduction in skeletal muscle: Differential effects on insulin-receptor substrates 1 and 2

Level of physical activity is linked to improved glucose homeostasis. We determined whether exercise alters the expression and/or activity of proteins involved in insulin-signal transduction in skeletal muscle. Wistar rats swam 6 h per day for 1 or 5 days. Epitrochlearis muscles were excised 16 h after the last exercise bout, and were incubated with or without insulin (120 nM). Insulin-stimulated glucose transport increased 30% and 50% after 1 and 5 days of exercise, respectively. Glycogen content increased 2- and 4-fold after 1 and 5 days of exercise, with no change in glycogen synthase expression. Protein expression of the glucose transporter GLUT4 and the insulin receptor increased 2-fold after 1 day, with no further change after 5 days of exercise. Insulin-stimulated receptor tyrosine phosphorylation increased 2-fold after 5 days of exercise. Insulin-stimulated tyrosine phosphorylation of insulin-receptor substrate (IRS) 1 and associated phosphatidylinositol (PI) 3-kinase activity increased 2.5- and 3.5-fold after 1 and 5 days of exercise, despite reduced (50%) IRS-1 protein content after 5 days of exercise. After 1 day of exercise, IRS-2 protein expression increased 2.6-fold and basal and insulin-stimulated IRS-2 associated PI 3-kinase activity increased 2.8-fold and 9-fold, respectively. In contrast to IRS-1, IRS-2 expression and associated PI 3-kinase activity normalized to sedentary levels after 5 days of exercise. Insulin-stimulated Akt phosphorylation increased 5-fold after 5 days of exercise. In conclusion, increased insulin-stimulated glucose transport after exercise is not limited to increased GLUT4 expression. Exercise leads to increased expression and function of several proteins involved in insulin-signal transduction. Furthermore, the differential response of IRS-1 and IRS-2 to exercise suggests that these molecules have specialized, rather than redundant, roles in insulin signaling in skeletal muscle.

Induction of autoantibodies to mouse CCR5 with recombinant papillomavirus particles

The vertebrate immune system has evolved to respond vigorously to microbial infection but to ignore self-antigens. Evidence has emerged that B cell responses to viruses are initiated by immune recognition of ordered arrays of antigen on the viral surface. To test whether autoantibodies against a self-antigen can be induced by placing it in a context that mimics the ordered surface of a viral particle, a peptide representing an extracellular loop of the mouse chemokine receptor CCR5 was incorporated into an immunodominant site of the bovine papillomavirus virus L1 coat protein, which self-assembles into virus-like particles. Mice inoculated with chimeric L1-CCR5 particles generated autoantibodies that bound to native mouse CCR5, inhibited binding of its ligand RANTES, and blocked HIV-1 infection of an indicator cell line expressing a human-mouse CCR5 chimera. These results suggest a general method for inducing autoantibodies against self-antigens, with diverse potential basic research and clinical applications.

Internalization of CD26 by mannose 6-phosphate/insulin-like growth factor II receptor contributes to T cell activation

CD26 is a T cell activation antigen known to bind adenosine deaminase and have dipeptidyl peptidase IV activity. Cross-linking of CD26 and CD3 with immobilized mAbs can deliver a costimulatory signal that contributes to T cell activation. Our earlier studies revealed that cross-linking of CD26 induces its internalization, the phosphorylation of a number of proteins involved in the signaling pathway, and subsequent T cell proliferation. Although these findings suggest the importance of internalization in the function of CD26, CD26 has only 6 aa residues in its cytoplasmic region with no known motif for endocytosis. In the present study, we have identified the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) as a binding protein for CD26 and that mannose 6-phosphate (M6P) residues in the carbohydrate moiety of CD26 are critical for this binding. Activation of peripheral blood T cells results in the mannose 6 phosphorylation of CD26. In addition, the cross-linking of CD26 with an anti-CD26 antibody induces not only capping and internalization of CD26 but also colocalization of CD26 with M6P/IGFIIR. Finally, both internalization of CD26 and the T cell proliferative response induced by CD26-mediated costimulation were inhibited by the addition of M6P, but not by glucose 6-phosphate or mannose 1-phosphate. These results indicate that internalization of CD26 after cross-linking is mediated in part by M6P/IGFIIR and that the interaction between mannose 6-phosphorylated CD26 and M6P/IGFIIR may play an important role in CD26-mediated T cell costimulatory signaling.