Summary Here, we demonstrate that the fractalkine(FKN)/CX3CR1 system represents a previously undescribed regulatory mechanism for pancreatic islet beta cell function and insulin secretion. CX3CR1 KO mice exhibited a marked defect in glucose and GLP1-stimulated insulin secretion, and this defect was also observed in vitro in isolated islets from CX3CR1 KO mice. In vivo administration of FKN improved glucose tolerance with an increase in insulin secretion. In vitro treatment of islets with FKN increased intracellular Ca2+ and potentiated insulin secretion in both mouse and human islets. The KO islets exhibited reduced expression of a set of genes necessary for the fully functional, differentiated beta cell state, whereas, treatment of WT islets with FKN led to increased expression of these genes. Lastly, expression of FKN in islets was decreased by aging and HFD/obesity, suggesting that decreased fractalkine/CX3CR1 signaling could be a mechanism underlying beta cell dysfunction in type 2 diabetes.

The SDF-1α/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1α and CXCR4 expression in fetal pancreas. We have found that SDF-1α and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) γ–nonobese diabetic mouse. We show that SDF-1α stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1α on ductal cell migration. Importantly, blocking the SDF-1α/CXCR4 axis in IFNγ-nonobese diabetic mice resulted in diminished proliferation and increased apoptosis in the pancreatic ductal cells. Together, these data indicate that the SDF-1α–CXCR4 ligand receptor axis is an obligatory component in the maintenance of duct cell survival, proliferation, and migration during pancreatic regeneration.

Previous studies have reported that prolonged administration of pharmacological doses of glucocorticoids in young rats results in a rise in urinary 3-methyl-L-histidine (3-MH) excretion followed by a fall to initial levels by 8 days. To determine whether this response reflects events in skeletal muscle, protein breakdown in this tissue was evaluated using the perfused hindquarter preparation with rats treated with corticosterone (10 mg X 100 g-1 X day-1) for 2, 4, or 8 days. Myofibrillar and total cell proteolysis were evaluated by measuring the release of 3-MH and tyrosine, respectively, after inhibition of protein synthesis with cycloheximide. Corticosterone treatment resulted in an early increase (1-4 days) followed by a fall (4-8 days) in 3-MH excretion. 3-MH release by the perfused hindquarter of treated rats responded in a similar manner, in that its release increased at days 2 and 4 and decreased to control levels by day 8. On the other hand, corticosterone treatment did not affect the release of tyrosine by the perfused hindquarter. Corticosterone treatment diminished protein synthesis in muscle by 30-50% (P less than 0.01), which unlike 3-MH release by perfused muscle persisted throughout the treatment period. The data indicate that glucocorticoids specifically augment the breakdown of myofibrillar proteins in skeletal muscle. This response is attenuated with prolonged treatment and is unrelated to a loss of metabolic effectiveness of the steroid. Also our findings suggest that the breakdown of myofibrillar and nonmyofibrillar proteins might be regulated independently.

Phospholipase C-␥ (PLC␥) is the isozyme of PLC phosphorylated by multiple tyrosine kinases including epidermal growth factor, platelet-derived growth factor, nerve growth factor receptors, and nonreceptor tyrosine kinases. In this paper, we present evidence for the association of the insulin receptor (IR) with PLC␥. Precipitation of the IR with glutathione S-transferase fusion proteins derived from PLC␥ and coimmunoprecipitation of the IR and PLC␥ were observed in 3T3-L1 adipocytes. To determine the functional significance of the interaction of PLC␥ and the IR, we used a specific inhibitor of PLC, U73122, or microinjection of SH2 domain glutathione S-transferase fusion proteins derived from PLC␥ to block insulin-stimulated GLUT4 translocation. We demonstrate inhibition of 2-deoxyglucose uptake in isolated primary rat adipocytes and 3T3-L1 adipocytes pretreated with U73122. Antilipolytic effect of insulin in 3T3-L1 adipocytes is unaffected by U73122. U73122 selectively inhibits mitogen-activated protein kinase, leaving the Akt and p70 S6 kinase pathways unperturbed. We conclude that PLC␥ is an active participant in metabolic and perhaps mitogenic signaling by the insulin receptor in 3T3-L1 adipocytes. The insulin receptor (IR)1 is a hetero-tetramer consisting of two ␣-subunits that are entirely extracellular and two ␤-subunits that span the plasma membrane and contain intrinsic tyrosine kinase activity (1, 2). One of the major metabolic effects of insulin in fat and skeletal muscle is the stimulation of glucose uptake (3). This occurs through the translocation of glucose transporters (GLUT4) from intracellular vesicles to the plasma membrane (4). Neither the molecular mechanism by which GLUT4 vesicles fuse with the plasma membrane nor the signaling proteins downstream of the IR leading to the stimulation of glucose transport have been clearly elucidated. An involvement of IRS-1 is indicated by both in vitro studies where primary rat adipocytes were transfected with an antisense ribozyme directed against rat IRS-1 (5) and in vivo studies where insulin-mediated glucose transport was attenuated in mice with targeted disruption of the IRS-1 gene (6). The ability of IRS-1 knock-out mice to transport glucose in response to insulin implies alternative mechanisms of glucose transport activation by insulin. PI 3-kinase has been demonstrated to be required for the insulin effect on glucose transport (7-10).Protein kinase C has been studied extensively as a mediator of insulin-stimulated glucose transport (11). The insulinomimetic effect of phorbol esters on glucose uptake implicates DAG as a potentiator of glucose uptake. Phorbol ester down-regulation reportedly inhibits insulin-stimulated glucose uptake in mouse soleus (12), rat heart (13), and rat adipocytes (14 -16). In 3T3-L1 adipocytes, however, insulin-stimulated glucose uptake has been reported to be refractory to down-regulation by phorbol esters (17,18). There are a number of ways that DAG can be generated in the cell in response to cell-surface receptors. An imme...

Osmotic shock and insulin stimulate GLUT4 translocation and glucose transport via mechanisms that are for the most part distinct yet convergent. In this article, we investigated the effect of osmotic shock and insulin on the activation of the mitogen-activated protein kinase (MAPK) cascades in differentiated 3T3-L1 adipocytes. The MAPKs are activated by phosphorylation on conserved tyrosine and threonine residues. Both sorbitol and insulin strongly stimulated extracellular regulated kinase (ERK) 1 and 2 phosphorylation (8-and 18-fold, respectively). In contrast, c-jun-NH 2 -terminal kinase (JNK)/stress-activated protein kinase (SAPK) phosphorylation was stimulated only by sorbitol (sevenfold) and not by insulin. Phosphorylation of p38 MAPK was stimulated strongly by sorbitol (22-fold) but weakly by insulin (2.7-fold). Measurement of intrinsic JNK and p38 MAPK activity confirmed the phosphorylation studies. JNK and p38 MAPK were activated only significantly by sorbitol. (1-3). The ERKs are strongly activated by polypeptide growth factors and phorbol esters but are weakly activated by environmental stresses, such as osmotic or heat shock, UV light, and inhibitors of protein synthesis. In contrast, JNK and p38 MAPKs are strongly activated by cytokines and adverse stimuli, but are poorly activated by growth factors.All MAPKs are activated by phosphorylation on both threonine and tyrosine residues within the motif Thr-Xaa-Tyr. The Xaa represents Glu in the ERK subfamily, Pro in the JNK subfamily, and Gly in the p38 MAPK subfamily. Both the threonine and tyrosine residues are phosphorylated by a dualspecificity kinase or MAPK (mitogen-activated ERK-activating kinase [MEK] or MAPK kinase [MKK]). The central residue in the Thr-Xaa-Tyr motif allows for selective activation by different MKKs, such that MEK1 and MEK2 selectively phosphorylate and activate the ERKs; MKK4/SAPK kinase (SEK) 1 and MKK7 phosphorylate and activate JNK; MKK3 and MKK6 phosphorylate and activate p38 MAPK (4-9). Despite the prevailing view of the selectivity of these kinases, a growing body of evidence indicates that these pathways overlap. In a study examining interleukin-1␤-induced cyclooxygenase-2 expression in rat renal mesangial cells, Guan et al. (10) have found that overexpressing the dominant negative form of MKK4 inhibited both JNK and p38 MAPK phosphorylation in their system. This finding indicates that MKK4 can act upstream of p38 in certain systems (10). In another study, the investigators coexpressed p38␦ and MKK3, MKK4, and MKK6 in 293 cells and found that, although MKK3 and MKK6 were the dominant regulators, MKK4 could also phosphorylate p38␦ (11).