Sir Philip Randle, who died aged 80 on 26 September 2006 after a brief illness, was one of the world's foremost researchers into mammalian metabolism. In a career spanning some five decades, he provided a series of brilliant insights into the fundamental mechanisms that determine the selection of metabolic fuels by muscle and other tissues. Many of his findings were concerned with the role of insulin, including the control of its secretion from the β-cells in the pancreatic islets of Langerhans, and with the regulation of glucose oxidation through changes in the activity of pyruvate dehydrogenase. The ideas generated by his investigations laid the foundations for countless subsequent studies and have had a direct bearing on the understanding of diabetes. His lifelong enthusiasm for good research has had a great influence on all who worked with him.

The 2-oxoglutarate dehydrogenase (OGDH) complex is an important control point in vertebrate mitochondrial oxidative metabolism, including in the citrate cycle and catabolism of alternative fuels including glutamine. It is subject to allosteric regulation by NADH and the ATP/ADP ratio, and by Ca2+ through binding to the E1 subunit. The latter involves a unique Ca2+ -binding site which includes D(114)ADLD (site 1). Here, we describe three splice variants of E1 in which either the exon expressing this site is replaced with another exon (loss of site 1, LS1) or an additional exon is expressed leading to the insertion of 15 amino acids just downstream of site 1 (Insert), or both changes occur together (LS1/Insert). We show that all three variants are essentially Ca2+ -insensitive. Comparison of massive parallel sequence (RNA-Seq) databases demonstrates predominant expression of the Ca2+ -sensitive archetype form in heart and skeletal muscle, but substantial expression of the Ca2+ -insensitive variants in brain, pancreatic islets and other tissues. Detailed proteomic and activity studies comparing OGDH complexes from rat heart and brain confirmed the substantial difference in expression between these tissues. The evolution of OGDH variants was explored using bioinformatics, and this indicated that Ca2+ -sensitivity arose with the emergence of chordates. In all species examined, this was associated with the co-emergence of Ca2+ -insensitive variants suggesting a retained requirement for the latter in some settings. Tissue-specific expression of OGDH splice variants may thus provide a mechanism that tunes the control of the enzyme to the specialized metabolic and signalling needs of individual cell types.

The regulation of the 2-oxoglutarate dehydrogenase complex is central to intramitochondrial energy metabolism. In the present study, the active full-length E1 subunit of the human complex has been expressed and shown to be regulated by Ca2+, adenine nucleotides and NADH, with NADH exerting a major influence on the K0.5 value for Ca2+. We investigated two potential Ca2+-binding sites on E1, which we term site 1 (D114ADLD) and site 2 (E139SDLD). Comparison of sequences from vertebrates with those from Ca2+-insensitive non-vertebrate complexes suggest that site 1 may be the more important. Consistent with this view, a mutated form of E1, D114A, shows a 6-fold decrease in sensitivity for Ca2+, whereas variant ∆site1 (in which the sequence of site 1 is replaced by A114AALA) exhibits an almost complete loss of Ca2+ activation. Variant ∆site2 (in which the sequence is replaced with A139SALA) shows no measurable change in Ca2+ sensitivity. We conclude that site 1, but not site 2, forms part of a regulatory Ca2+-binding site, which is distinct from other previously described Ca2+-binding sites.

The discovery of the mitogen-activated protein (MAP) kinase family of protein kinases has sparked off an intensive effort to elucidate their role in the regulation of many cellular processes. These protein kinases were originally identified based on their rapid activation by insulin. In this review we concentrate on examining the evidence for and against a role for the MAP kinases Erk-1 and Erk-2 in mediating the effects of insulin. While then is good evidence in favour of a direct role for MAP kinase in the growth-promoting effects of insulin and the regulation of Glut-1 and c-Sos expression, and AP-1 transcriptional complex activity, this is by no means conclusive. MAP kinase may also play a role in the control of mRNA translation by insulin. On the other hand, the evidence suggests that MAP kinase is not sufficient for the acute regulation of glucose transport (Glut-4 translocation), glycogen synthesis, acetyl-CoA carboxylase or pyruvate dehydrogenase activity. The findings suggest that insulin may utilise at least three distinct signalling pathways which do not involve MAP kinase.

1. Isolated fat-cells and intact epididymal fat-pads were incubated in medium containing 45Ca2+ and the incorporation of 4"Ca into mitochondrial and extramitochondrial fractions was studied. Redistribution of 45Ca between these fractions wag essentially prevented by the addition of EGTA [ethanedioxybis(ethylamine)tetra-acetate] and Ruthenium Red to the sucrose-based extraction medium. 2. Incorporation of 45Ca into mitochondrial fractions of both fat-ells and fat-pads was found to be complete within 2-5 min, suggesting that mitochondria contain a pool ofcalcium in rapid isotopic exchange with extracellular Ca2+. This pool was about 20 times larger in mitochondria within fat-cells than within fat-pads. In fat-cells, 4Ca incorporation into the mitochondrial fraction accounted for about 34% of the total 5Ca incorporation into cells after 20min and about 50% of the total mitochondrial calcium content measured by atomic absorption; values in fat-pads were about 7 and 20% respectively. 3. Total 45Ca incorporation into both fat-cells and fat-pads continued to increase after the first few minutes, when there was no further increase into mitochondrial fractions, suggesting the presence of an extramitochondrial pool of calcium into which 4"Ca is only slowly incorporated. 4. Parallel changes in 45Ca incorporation into mitochondrial and extramitochondrial fractions were not observed in general. With fat-cells, Ruthenium Red, NiC12, MnCI2, ionophore A23187, high Pi concentration and Li+ replacement all markedly increased 45Ca incorporation into the extramitochondrial fraction but caused modest or no increase into the mitochondrial fraction. Uncoupler (dinitrophenol) decreased and respiratory substrates increased incorporation into the mitochondrial fraction, with little change into the extramitochondrial fraction. 5. No changes in 4"Ca incorporation into the mitochondrial fraction of fat-pads were found with insulin under conditions where pyruvate dehydrogenase activity was increased. However, addition of adrenaline to fatpads incubated with insulin lowered 4"Ca incorporation into the mitochondrial fraction and also decreased pyruvate dehydrogenase activity. Incubation of fat-pads in Ca2+free medium containing EGTA after preincubation with medium containing "Ca2+ caused an equally marked lowering of "Ca incorporation into the mitochondrial fraction without altering the pyruvate dehydrogenase activity. In view of these findings, it is concluded that a rise in the mitochondrial concentration of Ca2+ is unlikely to be important in the increase in pyruvate dehydrogenase activity seen with insulin, but the possibility remains that the effects of adrenaline may involve a decrease in mitochondrial Ca2+ concentration.

In isolated rat adipocytes, the insulin stimulation of pyruvate dehydrogenase can be partially inhibited by inhibitors of PI3K (phosphoinositide 3-kinase) and MEK1/2 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase). In combination, U0126 and wortmannin completely block the insulin stimulation of pyruvate dehydrogenase. It is concluded that the effect of insulin on pyruvate dehydrogenase in rat adipocytes involves two distinct signalling pathways: one is sensitive to wortmannin and the other to U0126. The synthetic phosphoinositolglycan PIG41 can activate pyruvate dehydrogenase but the activation is only approx. 30% of the maximal effect of insulin. This modest activation can be completely blocked by wortmannin alone, suggesting that PIG41 acts through only one of the pathways leading to the activation of pyruvate dehydrogenase.

Signalling by the insulin receptor substrate (IRS) proteins is critically dependent on the tyrosine phosphorylation of specific binding sites that recruit Src homology 2 (SH2)-domain-containing proteins, such as the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase), the tyrosine phosphatase SHP-2 and the adapter protein Grb2. Here we show that stimulation by insulin of freshly isolated primary adipocytes resulted in the expected rapid tyrosine phosphorylation of the insulin receptor, IRS-1 and IRS-3. Inhibition of PI 3-kinase enhanced the insulin-stimulated phosphorylation of IRS-1 on (i) Tyr(612) and Tyr(941) (p85 binding sites), concomitant with an increased association of the p85 subunit of PI 3-kinase; (ii) Tyr(896) (a Grb2 binding site); and (iii) Tyr(1229) (an SHP-2 binding site), although little or no binding of SHP-2 to IRS-1 was detectable under any conditions. In contrast, inhibition of PI 3-kinase led to a decrease in insulin-stimulated p85 binding to IRS-3, but had no effect on SHP-2 binding. Furthermore, insulin-induced insulin receptor tyrosine phosphorylation, phosphorylation of Tyr(1158) and insulin receptor tyrosine kinase activity were all reduced by inhibition of PI 3-kinase at later time points (>or=20 min). The results demonstrate that, in primary adipocytes, PI 3-kinase feedback control of signalling by the insulin receptor and IRS proteins is multifaceted and reciprocal, illustrating the complexity of predicting the net flux of the insulin signal(s) through the IRS proteins.

Conference Abstract| June 01 2001 Different roles and regulation of insulin receptor substrate-1 and insulin receptor substrate-3 in insulin signalling in primary adipocytes I. Hers; I. Hers 1Department of Biochemistry, University Walk, Bristol, BS8 1TD, UK Search for other works by this author on: This Site PubMed Google Scholar C. J. Bell; C. J. Bell 1Department of Biochemistry, University Walk, Bristol, BS8 1TD, UK Search for other works by this author on: This Site PubMed Google Scholar J. M. Tavare; J. M. Tavare 1Department of Biochemistry, University Walk, Bristol, BS8 1TD, UK Search for other works by this author on: This Site PubMed Google Scholar R. M. Denton R. M. Denton 1Department of Biochemistry, University Walk, Bristol, BS8 1TD, UK Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (2001) 29 (3): A67. https://doi.org/10.1042/bst029a067c Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation I. Hers, C. J. Bell, J. M. Tavare, R. M. Denton; Different roles and regulation of insulin receptor substrate-1 and insulin receptor substrate-3 in insulin signalling in primary adipocytes. Biochem Soc Trans 1 June 2001; 29 (3): A67. doi: https://doi.org/10.1042/bst029a067c Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 2001 Biochemical Society2001 Article PDF first page preview Close Modal You do not currently have access to this content.

A fundamental control point in the regulation of the initiation of protein synthesis is the formation of the eukaryotic initiation factor 4F (eIF-4F) complex. The formation of this complex depends upon the availability of the mRNA cap binding protein, eIF-4E, which is sequestered away from the translational machinery by the tight association of eIF-4E binding proteins (4E-BPs). Phosphorylation of 4E-BP1 is critical in causing its dissociation from eIF-4E, leaving 4E available to form translationally active eIF-4F complexes, switching on mRNA translation. In this report, we provide the first evidence that the phosphorylation of 4E-BP1 increases during mitosis and identify Ser-65 and Thr-70 as phosphorylated sites. Phosphorylation of Thr-70 has been implicated in the regulation of 4E-BP1 function, but the kinase phosphorylating this site was unknown. We show that the cyclin-dependent kinase, cdc2, phosphorylates 4E-BP1 at Thr-70 and that phosphorylation of this site is permissive for Ser-65 phosphorylation. Crucially, the increased phosphorylation of 4E-BP1 during mitosis results in its complete dissociation from eIF-4E.

mTOR immunoprecipitates contain two 4E-BP1 protein kinase activities. One appears to be due to mTOR itself and results in the phosphorylation of 4E-BP1 on residues T36 and T45, as shown previously by others. The other is a kinase which can be separated from mTOR and which phosphorylates 4E-BP1 within a peptide(s) containing residues S64 and T69. This phosphorylation, which occurs predominantly on S64, results in the dissociation of 4E-BP1 from eIF-4E.

Here we report that the widely used protein kinase C inhibitors, bisindolylmaleimide I and IX, are potent inhibitors of glycogen synthase kinase‐3 (GSK‐3). Bisindolylmaleimide I and IX inhibited GSK‐3 in vitro, when assayed either in cell lysates (IC 50 360 nM and 6.8 nM, respectively) or in GSK‐3β immunoprecipitates (IC 50 170 nM and 2.8 nM, respectively) derived from rat epididymal adipocytes. Pretreatment of adipocytes with bisindolylmaleimide I (5 μM) and IX (2 μM) reduced GSK‐3 activity in total cell lysates, to 25.1±4.3% and 12.9±3.0% of control, respectively. By contrast, bisindolylmaleimide V (5 μM), which lacks the functional groups present on bisindolylmaleimide I and IX, had little apparent effect. We propose that bisindolylmaleimide I and IX can directly inhibit GSK‐3, and that this may explain some of the previously reported insulin‐like effects on glycogen synthase activity.

Here we report that the β‐adrenergic agonist isoproterenol increases the activity of the stress‐activated kinase p38 MAPK over 10‐fold in freshly isolated rat epididymal fat cells. Stimulation of the kinase was rapid, sustained for at least 60 min and sensitive to the specific p38 MAPK inhibitor, SB 203580. Half‐maximal stimulation of p38 MAPK by isoproterenol occurred at 13 nM isoproterenol. The cell permeable cyclic AMP analogue, chlorophenylthio‐cyclic AMP increased p38 MAPK activity to a similar extent to isoproterenol, suggesting that the effect of the β‐adrenergic agonist is mediated via increases in the activity of cyclic‐AMP dependent protein kinase. Although it had little or no effect on the activity of c‐Jun N‐terminal kinase, isoproterenol and a number of other treatments which activated p38 MAPK were found to stimulate AMP‐activated protein kinase in fat cells. Activation of AMPK and p38 MAPK were not, however, found to be directly linked.

Mitochondria are strategically localized at sites of Ca2+ release, such that increases in cytosolic free Ca2+ ([Ca2+]c) from either internal Ca2+ stores or Ca2+ influx across the plasma membrane can be rapidly transported into the mitochondrial matrix. The consequent elevation in mitochondrial Ca2+ ([Ca2+]m) stimulates the Ca2+-sensitive intramitochondrial dehydrogenases, resulting in elevation of NAD(P)H. The preferential coupling between increases in [Ca2+]c and [Ca2+]m is one proposed mechanism to coordinate mitochondrial ATP production with cellular energy demand. In liver cells, hormones that act through the second messenger inositol 1,4,5-trisphosphate (IP3) generate oscillatory [Ca2+]c signals, which result from a periodic Ca2+- and IP3-mediated activation/deactivation of intracellular Ca2+ release channels. The [Ca2+]c spiking frequency increases with agonist dose, whereas the amplitude of each [Ca2+]c spike is constant. This frequency modulation of [Ca2+]c spiking encodes the signal from the extracellular agonist, which is then decoded by the internal Ca2+-sensitive proteins such as the Ca2+-sensitive intramitochondrial dehydrogenases. Our studies have investigated the relationship between IP3-dependent [Ca2+]c signals and [Ca2+]m in primary cultured hepatocytes. In addition, the changes in cellular [Ca2+] levels have been correlated with the regulation of intramitochondrial NAD(P)H levels, pyruvate dehydrogenase activity and the magnitude of the mitochondrial proton motive force.