Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by association with a variety of tyrosine kinase receptors and intracellular tyrosine-phosphorylated substrates. We isolated a cDNA that encodes a 50-kDa regulatory subunit of PI 3-kinase with an expression cloning method using 32 P-labeled insulin receptor substrate-1 (IRS-1). This 50-kDa protein contains two SH2 domains and an inter-SH2 domain of p85␣, but the SH3 and bcr homology domains of p85␣ were replaced by a unique 6-amino acid sequence. Thus, this protein appears to be generated by alternative splicing of the p85␣ gene product. We suggest that this protein be called p50␣. Northern blotting using a specific DNA probe corresponding to p50␣ revealed 6.0-and 2.8-kb bands in hepatic, brain, and renal tissues. The expression of p50␣ protein and its associated PI 3-kinase were detected in lysates prepared from the liver, brain, and muscle using a specific antibody against p50␣. Taken together, these observations indicate that the p85␣ gene actually generates three protein products of 85, 55, and 50 kDa. The distributions of the three proteins (p85␣, p55␣, and p50␣), in various rat tissues and also in various brain compartments, were found to be different. Interestingly, p50␣ forms a heterodimer with p110 that can as well as cannot be labeled with wortmannin, whereas p85␣ and p55␣ associate only with p110 that can be wortmanninlabeled. Furthermore, p50␣ exhibits a markedly higher capacity for activation of associated PI 3-kinase via insulin stimulation and has a higher affinity for tyrosinephosphorylated IRS-1 than the other isoforms. Considering the high level of p50␣ expression in the liver and its marked responsiveness to insulin, p50␣ appears to play an important role in the activation of hepatic PI 3-kinase. Each of the three ␣ isoforms has a different function and may have specific roles in various tissues.A variety of growth factors and hormones mediate their cellular effects via interactions with cell surface receptors that possess protein kinase activity (1, 2). The interaction of most of these ligands with their receptors induces tyrosine kinase activation and autophosphorylation of the receptor, resulting in physical association of these receptors with several cytoplasmic substrates having SH2 domains. Phosphatidylinositol 3-kinase (PI 3-kinase) 1 has been identified through its ability to associate with cellular protein kinases, including numerous growth factor receptors and oncogene products (3, 4). This lipid kinase phosphorylates phosphatidylinositol at the D-3 position of the inositol ring in response to stimulation with a variety of growth factors and hormones (5). Although the role of this lipid product in cellular regulation remains unclear, recent reports suggest that the activation of PI 3-kinase leads to the activation of c-Akt, Rac, PKC-␥ isoform, and p70 S6 kinase (6 -9). As a result, PI 3-kinase has been suggested to play essential roles in the regulation of various cellular activities, including proliferation (10, 11), differen...
Phosphatidylinositol 3-kinase, which is composed of a 110-kDa catalytic subunit and a regulatory subunit, plays important roles in various cellular signaling mechanisms. We screened a rat brain cDNA expression library with 32 P-labeled human IRS-1 protein and cloned cDNAs that were very likely to be generated by alternative splicing of p85␣ gene products. These cDNAs were demonstrated to encode a 55-kDa protein (p55␣) containing two SH2 domains and an inter-SH2 domain of p85␣ but neither a bcr domain nor a SH3 homology domain. Interestingly, p55␣ contains a unique 34-amino acid sequence at its NH 2 terminus, which is not included in the p85␣ amino acid sequence. This 34-amino acid portion was revealed to be comparable with p55PIK (p55␥) in length, with a high homology between the two, suggesting that these NH 2 -terminal domains of p55␣ and p55␥ may have a specific role that p85 does not. The expression of p55␣ mRNA is most abundant in the brain, but expression is ubiquitous in most rat tissues. Furthermore, it should be noted that the expression of p85␣ mRNA in muscle is almost undetectably low by Northern blotting with a cDNA probe coding for the p85␣ SH3 domain, while the expression of p55␣ can be readily detected. These results suggest that p55␣ may play an unique regulatory role for phosphatidylinositol 3-kinase in brain and muscle.Phosphatidylinositol 3-kinase (PI 3-kinase) 1 (1, 2) has been implicated in the regulation of various cellular activities, including proliferation (3, 4), differentiation (5), membrane ruffling (6), and prevention of apoptosis (7). In addition, PI 3-kinase activation is required for insulin-stimulated glucose transport and insulin-dependent p70S6K activation (3). PI 3-kinase is a heterodimeric enzyme consisting of a regulatory subunit (1, 2, 8, 9) and a 110-kDa catalytic subunit (p110␣, ). Recently, a novel 110-kDa catalytic subunit (p110␥), which is stimulated via G␣ and G␥, was cloned (10). For the former type of PI 3-kinase, three regulatory subunit isoforms for PI 3-kinase have been identified. Among them, p55PIK is a unique protein since the SH3 domain and bcr homology domains found in p85␣ are replaced in p55PIK by a unique 34-residue NH 2 terminus (11).In this study, we isolated a novel alternatively spliced cDNA from the p85␣ gene by expression screening from a rat brain cDNA library using a 32 P-labeled human IRS-1 protein. This cDNA was demonstrated to encode a 55-kDa protein, which was designated p55␣, because it is partly identical to p85␣. In addition, we suggest changing the name of p55PIK to p55␥ to avoid confusion between p55PIK (p55␥) and p55␣. Herein, we compare the amino acid sequences of four isoforms of the regulatory subunit of rat PI 3-kinase and show their tissue distributions. These isoforms may be activated by different stimuli and/or at different intercellular locations. MATERIALS AND METHODSPreparation of Recombinant Human IRS-1-Human IRS-1 cDNA was obtained by screening the human genomic library using a 32 Plabeled DNA fragment. According to the seq...
Insulin-regulated aminopeptidase (IRAP) is an abundant cargo protein of Glut4 storage vesicles (GSVs) that traffics to and from the plasma membrane in response to insulin. We used the amino terminus cytoplasmic domain of IRAP, residues 1-109, as an affinity reagent to identify cytosolic proteins that might be involved in GSV trafficking. In this way, we identified p115, a peripheral membrane protein known to be involved in membrane trafficking. In murine adipocytes, we determined that p115 was localized to the perinuclear region by immunofluorescence and throughout the cell by fractionation. By immunofluorescence, p115 partially colocalizes with GLUT4 and IRAP in the perinuclear region of cultured fat cells. The amino terminus of p115 binds to IRAP and overexpression of a N-terminal construct results in its colocalization with GLUT4 throughout the cell. Insulin-stimulated GLUT4 translocation is completely inhibited under these conditions. Overexpression of p115 C-terminus has no significant effect on GLUT4 distribution and translocation. Finally, expression of the p115 N-terminus construct has no effect on the distribution and trafficking of GLUT1. These data suggest that p115 has an important and specific role in insulin-stimulated Glut4 translocation, probably by way of tethering insulin-sensitive Glut4 vesicles at an as yet unknown intracellular site. INTRODUCTIONInsulin normalizes blood glucose levels by mobilizing the muscle and adipocyte glucose transporter isoform, GLUT4, from intracellular storage vesicles and moving it to the plasma membrane (Simpson et al., 2001;Bryant et al., 2002). Various models of GLUT4 trafficking suggest that GLUT4 must exist in more than one intracellular compartment and the major insulin sensitive pool is localized to a compartment that is distinct from endosomal markers and is commonly referred to as glucose transporter storage vesicles (GSVs), or insulin responsive vesicle (IRVs). Despite the critical function of glucose transport in glucose homeostasis, many of the details by which adipocytes and muscle form a pathway of insulin-sensitive GLUT4 trafficking remain unknown. However, it is virtually certain that the major cargo proteins of GSVs must interact with a number of cytosolic and membrane proteins, such as adaptors and tethers, in order to be properly sorted and regulated by insulin.Insulin-responsive aminopeptidase (IRAP) was identified as an abundant cargo protein associated with GLUT4 vesicles that translocates in response to insulin in a manner seemingly identical to GLUT4 Mastick et al., 1994;Keller et al., 1995;Malide et al., 1997;Martin et al., 1997;Ross et al., 1997). In fact, it is more abundantly expressed in vesicles than the transporter (Kupriyanova et al., 2002). When the cytoplasmic N-terminus of IRAP was microinjected into 3T3-L1 adipocytes, GLUT4 was localized on the plasma membrane even in the basal state (Waters et al., 1997), suggesting IRAP can play a role in GSV movement/targeting. A chimeric protein containing the intracellular domain of IRAP and ...
OBJECTIVE-c-Cbl plays an important role in whole-body fuel homeostasis by regulating insulin action. In the present study, we examined the role of Cbl-b, another member of the Cbl family, in insulin action. RESEARCH DESIGN AND METHODS-C57BL/6(Cbl-b ϩ/ϩ ) or Cbl-b-deficient (Cbl-b Ϫ/Ϫ ) mice were subjected to insulin and glucose tolerance tests and a hyperinsulinemic-euglycemic clamp test. Infiltration of macrophages into white adipose tissue (WAT) was assessed by immunohistochemistry and flow cytometry. We examined macrophage activation using co-cultures of 3T3-L1 adipocytes and peritoneal macrophages. RESULTS-ElderlyCbl-b Ϫ/Ϫ mice developed glucose intolerance and peripheral insulin resistance; serum insulin concentrations after a glucose challenge were always higher in elderly Cbl-b Ϫ/Ϫ mice than age-matched Cbl-b ϩ/ϩ mice. Deficiency of the Cbl-b gene significantly decreased the uptake of 2-deoxyglucose into WAT and glucose infusion rate, whereas fatty liver was apparent in elderly Cbl-b Ϫ/Ϫ mice. Cbl-b deficiency was associated with infiltration of macrophages into the WAT and expression of cytokines, such as tumor necrosis factor-␣, interleukin-6, and monocyte chemoattractant protein (MCP)-1. Co-culture of Cbl-b Ϫ/Ϫ macrophages with 3T3-L1 adipocytes induced leptin expression and dephosphorylation of insulin receptor substrate 1, leading to impaired glucose uptake in adipocytes. Furthermore, Vav1, a key factor in macrophage activation, was highly phosphorylated in peritoneal Cbl-b Ϫ/Ϫ macrophages compared with Cbl-b ϩ/ϩ macrophages. Treatment with a neutralizing anti-MCP-1 antibody improved peripheral insulin resistance and macrophage infiltration into WAT in elderly Cbl-b Ϫ/Ϫ mice. O besity is a major cause of insulin resistance (1) and is considered a chronic low-grade inflammatory disease (2). Substantial evidence has accumulated in recent years that chronic infiltration and activation of macrophages in white adipose tissue (WAT) underlie the obesity-related component of these insulin-resistant states (3-5). Infiltrating macrophages secrete proinflammatory cytokines and stimulate the secretion of adipokines from adipocytes (4,5). Tumor necrosis factor (TNF)-␣ and interleukin (IL)-6 are key factors that induce insulin resistance (6,7). Leptin and adiponectin are also suggested to act as hormones that regulate insulin resistance; hyperleptinemia in obese subjects is associated with insulin resistance in tissues such as liver, WAT, and skeletal muscle (8), whereas adiponectin increases insulin sensitivity of these tissues (9). However, the molecular mechanism of macrophage activation in WAT is still unknown. CONCLUSIONS-Cbl-b is a unique ubiquitin ligase that is associated with maturation and activation of macrophages and T-cells (10,11). Expression of Cbl-b is upregulated by macrophage/monocyte differentiation of HL60 and U937 cell lines (10). Cbl-b influences CD28-dependent T-cell activation by selectively restraining T-cell receptor-mediated Vav1 activation (12)(13)(14). A recent study reported that the ...
BackgroundObesity is a major risk factor for insulin resistance, type 2 diabetes, and stroke. Flavonoids are effective antioxidants that protect against these chronic diseases. In this study, we evaluated the effects of sudachitin, a polymethoxylated flavonoid found in the skin of the Citrus sudachi fruit, on glucose, lipid, and energy metabolism in mice with high-fat diet-induced obesity and db/db diabetic mice. In our current study, we show that sudachitin improves metabolism and stimulates mitochondrial biogenesis, thereby increasing energy expenditure and reducing weight gain.MethodsC57BL/6 J mice fed a high-fat diet (40% fat) and db/db mice fed a normal diet were treated orally with 5 mg/kg sudachitin or vehicle for 12 weeks. Following treatment, oxygen expenditure was assessed using indirect calorimetry, while glucose tolerance, insulin sensitivity, and indices of dyslipidemia were assessed by serum biochemistry. Quantitative polymerase chain reaction was used to determine the effect of sudachitin on the transcription of key metabolism-regulating genes in the skeletal muscle, liver, and white and brown adipose tissues. Primary myocytes were also prepared to examine the signaling mechanisms targeted by sudachitin in vitro.ResultsSudachitin improved dyslipidemia, as evidenced by reduction in triglyceride and free fatty acid levels, and improved glucose tolerance and insulin resistance. It also enhanced energy expenditure and fatty acid β-oxidation by increasing mitochondrial biogenesis and function. The in vitro assay results suggest that sudachitin increased Sirt1 and PGC-1α expression in the skeletal muscle.ConclusionsSudachitin may improve dyslipidemia and metabolic syndrome by improving energy metabolism. Furthermore, it also induces mitochondrial biogenesis to protect against metabolic disorders.
This study, using C57BL/6J mice with streptozotocin (STZ)-induced diabetes, aimed to determine whether Bifidobacterium species (spp.) both induces the expressions of proteins in the insulin signaling pathway and enhances the expressions of certain adipocytokines. The protein expressions of IκB kinase alpha (IKKα), IκB kinase beta (IKKβ), nuclear factor-kappaB inhibitor alpha (IκBα), and the mitogen-activated protein kinase (MAPK) pathway were also investigated. Oral administration of Bifidobacterium spp. reduced blood glucose levels significantly and increased the protein expressions of insulin receptor beta, insulin receptor substrate 1, protein kinase B (Akt/PKB), IKKα, and IκBα. Extracellular-signal-regulated kinase 2 (ERK2) showed increased expression. Bifidobacterium spp. also induced the adiponectin expression and decreased both macrophage chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) expression. In addition, IKKβ, c-Jun NH 2 -terminal kinase (JNK) and p38 MAP kinase expressions showed no significant changes in both groups. In conclusion, Bifidobacterium spp. may be the promising bacteria for treating diabetes.Strains including B. bifidum, B. longum, B. infantis, and B. animalis are Bifidobacterium spp., which comprise genus of gram-positive, non-motile, often branched anaerobic bacteria. Bifidobacteria are used as probiotics for supporting digestion in many countries. To date, several studies have demonstrated the benefits of probiotics in managing metabolic disorders including diabetes. At present, there are research groups focusing on this novel concept. Dietary supplementation with multiple probiotic strains, including L. acidophilus, L. casei, L. rhamnosus, L. bulgaricus, B. breve, B. longum, and S. thermophi-
In mature adipocytes, triglyceride is stored within lipid droplets, which are coated with the protein perilipin, which functions to regulate lipolysis by controlling lipase access to the droplet in a hormone-regulatable fashion. Adipocyte differentiation-related protein (ADRP) is a widely expressed lipid droplet binding protein that is coexpressed with perilipin in differentiating fat cells but is minimally present in fully differentiated cultured adipocytes. We find that fibroblasts ectopically expressing C/EBPalpha (NIH-C/EBPalpha cells) differentiate into mature adipocytes that simultaneously express perilipin and ADRP. In response to isoproterenol, perilipin is hyperphosphorylated, lipolysis is enhanced, and subsequently, ADRP expression increases coincident with it surrounding intracellular lipid droplets. In the absence of lipolytic stimulation, inhibition of proteasomal activity with MG-132 increased ADRP levels to those of cells treated with 10 mum isoproterenol, but ADRP does not surround the lipid droplet in the absence of lipolytic stimulation. We overexpressed a perilipin A construct in NIH-C/EBPalpha cells where the six serine residues known to be phosphorylated by protein kinase A were changed to alanine (Peri A Delta1-6). These cells show no increase in ADRP expression in response to isoproterenol. We propose that ADRP can replace perilipin on existing lipid droplets or those newly formed as a result of fatty acid reesterification, under dynamic conditions of hormonally stimulated lipolysis, thus preserving lipid droplet morphology/structure.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.