Serine Phosphorylation Of Insulin Receptor Research Articles

Berberine induces lipolysis in porcine adipocytes by activating the AMP‑activated protein kinase pathway.

Lipolysis is closely associated with obesity and insulin resistance. Berberine(BBR), a natural alkaloid derived from Coptischinensis, has been shown to regulate lipolysis and improve insulin resistance. However, the underlying mechanism remains unclear. The present results suggested that BBR stimulated lipolysis in porcine adipocytes in a dose‑ and time‑dependent manner, which was independent of the cAMP/protein kinaseA pathway. Further experimental results indicated that BBR increased phosphorylation levels of AMP‑activated protein kinase (AMPK) and adipose triglyceride lipase (ATGL), along with downregulation of PerilipinA. The AMPK inhibitor compoundC significantly reversed the effect of BBR on lipolysis, PerilipinA expression and ATGL phosphorylation. Furthermore, BBR promoted expression levels of genes related to fatty acid oxidation, such as peroxisome proliferator‑activated receptor γ coactivator‑1α, mitochondrial transcription factorA, carnitine palmitoyl‑transferase‑1 and uncoupling protein2, which were abrogated by AMPKα1 knockdown. Moreover, it was found that BBR‑induced lipolysis did not elevate serine phosphorylation of insulin receptor substrate‑1 to block insulin signaling. Collectively, the present results suggested that BBR induced lipolysis in porcine adipocytes via a pathway that involves AMPK activation, but does not cause insulin resistance.

New therapeutic strategy for Alzheimer's disease using antidiabetes agents.

New therapeutic strategy for Alzheimer's disease using antidiabetes agents.

Insulin resistance in women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is associated with insulin resistance. There are also defects in pancreatic beta-cell function in affected women. These abnormalities are heritable. There are post-binding defects in insulin receptor signaling, with selective resistance to insulin's metabolic actions and constitutively activated mitogenic signaling in skeletal muscle. Intrinsic and environmental abnormalities interact to produce peripheral insulin resistance in PCOS. A susceptibility gene region for PCOS is located on chromosome 19p13.2. The susceptibility allele is also associated with a metabolic phenotype.

Pancreastatin modulates insulin signaling in rat adipocytes: mechanisms of cross-talk.

Pancreastatin (PST), a chromogranin A-derived peptide, has counterregulatory effects on insulin in the hepatocyte and the adipocyte, suggesting a possible role in insulin resistance. The mechanism of PST action on glucose and lipid metabolism is typical of a calcium-mobilizing hormone and involves a receptor Gq/11 protein-phospholipase C (PLC)-beta pathway. In the rat adipocyte, PST inhibits insulin-mediated glucose transport, glucose utilization, and lipid synthesis, and it has a lipolytic effect but stimulates basal and insulin-stimulated protein synthesis. We have also recently studied the PST receptor-effector system in adipocyte membranes. To further investigate the mechanisms of PST effect on insulin action, we studied the cross-talk of PST with insulin signaling in the rat adipocyte. We found that PST inhibits insulin-stimulated GLUT4 translocation to the membrane, which may explain the reported inhibition of glucose transport. Tyrosine phosphorylation of the activated insulin receptor, insulin receptor substrate (IRS)-1, and p60-70 was also blunted, preventing their association with p85 phosphatidylinositol 3-kinase (PI3K) and their activity. The mechanism of this inhibition involves the activation of the "classical" protein kinase C isoforms and the serine phosphorylation of insulin receptor and IRS-1. On the other hand, PST activates the mitogen-activated protein kinase (MAPK) signaling module and enhances the effect of insulin. This pathway may account for the described effect of PST on protein synthesis. In conclusion, PST seems to inhibit the insulin-stimulated PI3K pathway in the adipocyte, whereas it activates the MAPK pathway. These data provide some clues to the PST cross-talk with insulin signaling that may explain the PST effects on glucose metabolism and protein synthesis.

Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle. A potential mechanism for insulin resistance in the polycystic ovary syndrome.

We investigated the cellular mechanisms of the unique disorder of insulin action found in the polycystic ovary syndrome (PCOS). Approximately 50% of PCOS women (PCOS-Ser) had a significant increase in insulin-independent beta-subunit [32P]phosphate incorporation (3.7-fold, P < 0.05 vs other groups) in skin fibroblast insulin receptors that was present in serine residues while insulin-induced tyrosine phosphorylation was decreased (both P < 0.05 vs other groups). PCOS skeletal muscle insulin receptors had the same abnormal phosphorylation pattern. The remaining PCOS women (PCOS-n1) had basal and insulin-stimulated receptor autophosphorylation similar to control. Phosphorylation of the artificial substrate poly GLU4:TYR1 by the PCOS-Ser insulin receptors was significantly decreased (P < 0.05) compared to control and PCOS-n1 receptors. The factor responsible for excessive serine phosphorylation appeared to be extrinsic to the receptor since no insulin receptor gene mutations were identified, immunoprecipitation before autophosphorylation corrected the phosphorylation defect and control insulin receptors mixed with lectin eluates from affected PCOS fibroblasts displayed increased serine phosphorylation. Our findings suggest that increased insulin receptor serine phosphorylation decreases its protein tyrosine kinase activity and is one mechanism for the post-binding defect in insulin action characteristic of PCOS.

Studies into the identity of the sites of insulin-stimulated insulin receptor serine phosphorylation. Characterization of synthetic peptide substrates for the insulin-stimulated insulin receptor serine kinase.

The identity of the sites of insulin-stimulated serine phosphorylation in the human insulin receptor was examined by synthesizing peptides that together encompassed all the serine residues of the cytosolic portion of the beta-subunit and testing them as substrates for phosphorylation by a preparation of human insulin receptor copurified with insulin-stimulated insulin receptor serine kinase activity. Of the 14 peptides studied, only 4 (1071--1080, 1290--1298, 1253--1271, and 1313--1329) were phosphorylated on serine, with the serine phosphorylation stimulated 2--4-fold by insulin. Peptides 1071--1080 and 1290--1298 were 3--7-fold better substrates for the serine phosphorylation than the other serine-phosphorylated peptides. Peptides 1071--1080 and 1313--1329 also exhibited insulin-stimulated phosphorylation on tyrosine. Two-dimensional thin-layer tryptic mapping of the phosphorylated insulin receptor/insulin-stimulated insulin receptor serine kinase preparation or of insulin receptor phosphorylated in human Hep G2 cells yielded two major peptides, called S1 and S2, that ran as a pair of closely migrating spots, and other lesser peptides that contained phosphoserine. S1 and S2 also contained some phosphotyrosine and gave phosposerine/phosphotyrosine ratios of approximately 6 and 0.96-1.50 for the in vivo and in vitro labeled receptor, respectively. S1 and S2 were not cleaved by V8. Of the serine-phosphorylated peptides, only 1290--1298 and 1071--1080 should be V8 resistant; 1290--1298 contains serine sites 1293/4 and migrated distinctly from S1 and S2 in tryptic maps. Peptide 1071--1080 mimicked the production of S1 and S2 in tryptic maps yielding a doublet of phosphopeptides, each containing phosphoserine and phosphotyrosine, which comigrated exactly with S1 and S2. Comigration was confirmed at a different pH and by mixing experiments. Radiosequenation showed that serine 1078 was phosphorylated. Tyrosine 1075 was also phosphorylated, but it was no more than a minor site in vivo. It is concluded that serine 1078 of the insulin receptor is a major site of insulin-stimulated phosphorylation in vivo and in vitro. The peptide sequences provide a range of substrates to facilitate the study, purification, and characterization of the insulin-stimulated insulin receptor serine kinase or kinases, and the identification of a major site of insulin-stimulated serine phosphorylation will help elucidate the function of the insulin receptor serine phosphorylation.

Studies on an insulin-stimulated insulin receptor serine kinase activity: separation of the kinase activity from the insulin receptor and its reconstitution back to the insulin receptor.

In cells insulin stimulates autophosphorylation of the insulin receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Recently we have achieved co-purification of the insulin receptor with insulin-stimulated insulin receptor serine kinase activity. We now show that the co-purified serine kinase activity can be removed by NaCl washing and reconstituted by adding back the NaCl eluate. Reconstitution enabled higher serine phosphorylation than achieved with the co-purified preparation. Myelin basic protein was discovered to be a potent substrate for insulin-stimulated serine phosphorylation by the co-purified preparation, with the activity responsible having similar properties to the serine kinase activity towards the receptor. Myelin basic protein was also phosphorylated on serine by the NaCl eluate. Myelin basic protein phosphorylated by the co-purified preparation or the NaCl eluate gave the same set of phosphoserine peptides. The major myelin basic protein serine kinase activity in the NaCl eluate co-purified exactly on Mono Q with the activity that restored insulin-stimulated insulin receptor serine phosphorylation. These results provide strong evidence for the true separation of the serine kinase from the insulin receptor and the distinctiveness of the serine kinase activity from the insulin receptor tyrosine kinase and mitogen-activated protein kinases. The procedures developed for the isolation of the serine kinase and the establishment of an effective in vitro substrate should allow purification of the kinase. The protocols also provide flexible systems for identifying the functions of the insulin-stimulated serine phosphorylations and the respective kinase(s).

Protein kinase C and insulin receptor β-subunit serine phosphorylation in cultured foetal rat hepatocytes

In digitonin-permeabilized cultured foetal hepatocytes, insulin receptor β-subunit was highly phosphorylated on serine residues in the presence of [γ-32P]ATP and Ca2+, a process enhanced after short exposure to insulin with no detectable insulin receptor autophosphorylation. By contrast with this situation, experiments performed with isolated foetal insulin receptors revealed an insulin stimulation of both serine phosphorylation and tyrosine autophosphorylation. In permeabilized cells, insulin receptorβ-subunit phosphorylation was increased after a 2-min exposure to phorbol 12-myristate 13-acetate (PMA) prior to applying the permeabilization/phosphorylation step, while it was inhibited by chronic treatment with PMA leading to protein kinase C (PKC) down modulation. The PKC specific inhibitor, GF109203X, strikingly reduced basal and insulin-enhanced phosphorylation of insulin receptor β-subunit in permeabilized cells, but failed to exert any effect with isolated receptors. Labelling of glycogen from [U-14C] glucose determined l h after a 10-min transitory exposure to insulin and/or modulators of PKC activity showed that PMA prevented insulin glycogenic response, whereas GF109203X was ineffective. Thus, although not directly responsible for insulin receptor serine phosphorylation in cultured foetal hepatocytes, PKC physiologically regulates this process which may inhibit insulin receptor tyrosine kinase activity. This regulation is independent of the antagonistic effect of PMA-activated PKC on insulin glycogenic response.

Insulin-stimulated serine and threonine phosphorylation of the human insulin receptor. An assessment of the role of serines 1305/1306 and threonine 1348 by their replacement with neutral or negatively charged amino acids.

Insulin promotes insulin receptor beta-subunit phosphorylation on tyrosine, serine, and threonine residues in a variety of cells, including simian COS cells which transiently express human insulin receptors following transfection with a cDNA encoding the wild-type receptor protein. To examine the potential roles of serines 1305 and 1306 and threonine 1348 as sites of insulin-stimulated phosphorylation in these cells, these residues (i.e. either serines 1305 and 1306, or threonine 1348) were replaced with neutral (alanine) or negatively charged (aspartate) amino acids. Following transient expression of each of these mutant receptors in COS cells, two-dimensional phosphopeptide mapping reveals that threonine 1348 is the major, if not the only, insulin-stimulated threonine phosphorylation site. In contrast, while serines 1305 and/or 1306 are phosphorylated in an insulin-dependent manner, these sites comprise only a minor proportion of insulin receptor serine phosphorylation in these cells. Substitution of either serines 1305 and 1306 or threonine 1348 with neutral or negatively charged amino acids has no effect on insulin-stimulated tyrosine autophosphorylation of these mutant receptors in intact cells. Furthermore, insulin-stimulated exogenous protein-tyrosine kinase activity of the mutant receptors is unaffected, as assessed following either phosphorylation of receptors in intact cells or following immunopurification of receptors and their autophosphorylation in vitro.