Intracellular signalling mechanisms regulating glucose transport in insulin-sensitive tissues (review).

The rate of glucose transport into cells is of fundamental importance in whole body homeostasis and adaptation to metabolic stresses, and this review examines the signalling mechanisms controlling this process. The events that mediate the action of insulin on glucose transport, which is by far the best characterized paradigm for glucose transport regulation, are discussed. There are several excellent reviews on various aspects of this subject, which are referred to while highlighting very recent developments in the field, including the recently described CAP pathway, and emerging mechanisms for feedback regulation of insulin signalling. The manner in which hormonal signalling is modulated by stimuli such as oxidative and osmotic stress is then discussed. The second major physiological event where glucose transport regulation is critical is the contraction of skeletal muscle, due to the large metabolic demands of this activity. The mechanism of this regulation is distinct from that initiated by insulin, and recent developments will be examined that have begun to clarify how contraction stimulates glucose transport in skeletal muscle, including the roles performed by AMP-activated protein kinase and nitric oxide synthase.     

Transport and membrane binding of the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) in Xenopus laevis oocytes

Summary We have examined transport and membrane binding of 6-diazo-5-oxo-l-norleucine (DON, a photoactive diazo-analogue of glutamine) and their relationships to glutamine transport in Xenopus laevis oocytes. DON uptake was stereospecific and saturable (V _max of 0.44 pmol/oocyte · min and a K _m of 0.065 mm). DON uptake was largely Na^u+ dependent (80% at 50 m DON) and inhibited (>75%) by glutamine and arginine (substrates of the System B^0,+ transporter) at 1 mm. Glutamine and DON show mutual competitive inhibition of Na⁺-dependent transport. Preincubation of oocytes in medium containing 0.1 mm DON for 24 or 48 hr depressed the V _max for System B^0,+ transport (as measured by Na⁺-dependent glutamine uptake), this effect was highly specific (neither d-DON nor the System B^0,+ substrates glutamine and d-alanine showed any independent effect) and required Na⁺ ions. Glutamine (1 mm in preincubation medium) protected transport from inhibition by DON. The possibility that specific inactivation of System B^0,+ by DON reflects attachment of DON to the transporter was tested by examining the binding of [¹⁴C]DON to Xenopus oocyte membranes. Oocytes incubated in 100 mm NaCl in the presence of [¹⁴C]DON for up to 48 hr showed 2.4-fold higher ¹⁴C-binding to membranes than oocytes incubated in choline chloride. Na⁺-dependent DON binding (31 ± 11 fmol/g membrane protein) was suppressed by external glutamine, arginine or alanine and was largely confined to a membrane protein fraction of 48–65 kDa (as assessed by SDS-polyacrylamide gel electrophoresis). The present studies indicate that DON and glutamine uptake in oocytes are both mediated by System B^0,+ and demonstrate that DON binding to a particular membrane protein fraction is associated with inactivation of the transporter, offering the prospect of using [¹⁴C]DON as a covalent label for the transport protein in order to facilitate its isolation and subsequent biochemical characterization.This work was supported by The Wellcome Trust, Action Research for the Crippled Child, Ajinomoto GmbH, Pfrimmer GmbH, the Rank Prize Funds, the Medical Research Council and the University of Dundee. We are grateful to Dr. C.I. Pogson (Wellcome Research Laboratories) and Drs. J.C. Ellory and B. Elford (University of Oxford) for gifts of [¹⁴C]DON.     

Selective solubilization of nicotinamide nucleotide transhydrogenase from the mitochondrial inner membrane

Nicotinamide nucleotide transhydrogenase was solubilized from beef heart submitochondrial particles employing Triton X-100 or lysolecithin. Lysolecithin was considerably more efficient and selective and released over 80 % of the transhydrogenase acdtivity from the membrane together with succinate dehydrogenase. Solubilization of NADH dehydrogenase and cytochrome oxidase was more efficiently accomplished with Triton than with lysolecithin. Both detergents released ATPase to various extents. Transhydrogenase remaining bound to particles after treatment with lysolecithin still catalyzed energy-linked transhydrogenation.     

Momordica charantia fruit juice stimulates glucose and amino acid uptakes in L6 myotubes

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either 3H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (14C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1-10 microg ml(-1)) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1- 6 h. The results were expressed as pmol min(-1) (mg cell protein)(-1), n = 6-8 for each value. Basal 3H-deoxyglucose and 14C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means +/- S.E.M.) 32.14 +/- 1.34 and 13.48 +/- 1.86 pmol min(-1) (mg cell protein)(-1), respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in 3H-deoxyglucose and 14C-Me-AIB uptakes. Typically, 3H-deoxyglucose and 14C-Me-AIB uptakes in the presence of insulin were 58.57 +/- 4.49 and 29.52 +/- 3.41 pmol min(-1) (mg cell protein(-1)), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 microg ml(-1)) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in 3H-deoxy-D-glucose and 14C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 microg ml(-1). Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of 3H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin.     

Effects of corticosteroid on the transport and metabolism of glutamine in rat skeletal muscle

Intramuscular glutamine falls with injury and disease in circumstances associated with increases in blood corticosteroids. We have investigated the effects of corticosteroid administration (0.44 mg/kg dexamethasone daily for 8 days, 200 g female rats) on intramuscular glutamine and Na⁺, muscle glutamine metabolism and sarcolemmal glutamine transport in the perfused hindlimb. After dexamethasone treatment intramuscular glutamine fell by 45% and Na⁺ rose by 25% (the respective muscle/plasma distribution ratios changed from 8.6 to 4.5 and 0.12 to 0.15); glutamine synthetase and glutaminase activities were unchanged at 475 ± 75 and 60 ± 19 nmol/g muscle per min. Glutamine output by the hindlimb of anaesthetized rats was increased from 31 to 85 nmol/g per min. Sarcolemmal glutamine transport was studied by paired-tracer dilution in the perfused hindlimb: the maximal capacity (V_max) for glutamine transport into muscle (by Na⁺-glutamine symport) fell from 1058 ± 310 to 395 ± 110 nmol/g muscle per min after dexamethasone treatment, accompanied by a decrease in the K_m (from 8.1 ± 1.9 to 2.1 ± 0.4 mM glutamine). At physiological plasma glutamine concentration (0.75 mM) dexamethasone appeared to cause a proportional increase in sarcolemmal glutamine efflux over influx. Addition of dexamethasone (200 nM) to the perfusate of control rat hindlimbs caused acute changes in V_max and K_m of glutamine transport similar to those resulting from 8-day dexamethasone treatment. The reduction in muscle glutamine concentration after dexamethasone treatment may be primarily due to a reduction in the driving force for intramuscular glutamine accumulation, i.e., in the Na⁺ electrochemical gradient. The prolonged increase in muscle glutamine output after dexamethasone treatment (which occurs despite a reduction in the size of the intramuscular glutamine pool) appears to be due to a combination of (a) accelerated sarcolemmal glutamine efflux and (b) increased intramuscular synthesis of glutamine.     

The oligomycin sensitivity conferring protein (OSCP) of beef heart mitochondria: Studies of its binding to F1 and its function

The binding of oligomycin sensitivity conferring protein (OSCP) to soluble beef-heart mitochondrial ATPase (F₁) has been investigated. OSCP forms a stable complex with F₁, and the F₁ · OSCP complex is capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted submitochondrial particles. The F₁ · OSCP complex retains 50% of its ATPase activity upon cold exposure while free F₁ is inactivated by 90% or more. Both free F₁ and the F₁ · OSCP complex release upon cold exposure a part—probably 1 out of 3—of their subunits; whether subunits are also lost is uncertain. The cold-treated F₁ · OSCP complex is still capable of restoring oligomycin- and DCCD-sensitive ATPase activity to F₁- and OSCP-depleted particles. OSCP also protects F₁ against modification of its subunit by mild trypsin treatment. This finding together with the earlier demonstration that trypsin-modified F₁ cannot bind OSCP indicates that OSCP binds to the subunit of F₁ and that F₁ contains three binding sites for OSCP. The results are discussed in relation to the possible role of OSCP in the interaction of F₁ with the membrane sector of the mitochondrial ATPase system.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - OSCP oligomycin sensitivity conferring protein - SDS sodium dodecylsulfate This paper is dedicated to the memory of David E. Green—scholar, pioneer, visionary.     

CB1 receptor blockade counters age‐induced insulin resistance and metabolic dysfunction

The endocannabinoid system can modulate energy homeostasis by regulating feeding behaviour as well as peripheral energy storage and utilization. Importantly, many of its metabolic actions are mediated through the cannabinoid type 1 receptor (CB1R), whose hyperactivation is associated with obesity and impaired metabolic function. Herein, we explored the effects of administering rimonabant, a selective CB1R inverse agonist, upon key metabolic parameters in young (4 month old) and aged (17 month old) adult male C57BL/6 mice. Daily treatment with rimonabant for 14 days transiently reduced food intake in young and aged mice; however, the anorectic response was more profound in aged animals, coinciding with a substantive loss in body fat mass. Notably, reduced insulin sensitivity in aged skeletal muscle and liver concurred with increased CB1R mRNA abundance. Strikingly, rimonabant was shown to improve glucose tolerance and enhance skeletal muscle and liver insulin sensitivity in aged, but not young, adult mice. Moreover, rimonabant‐mediated insulin sensitization in aged adipose tissue coincided with amelioration of low‐grade inflammation and repressed lipogenic gene expression. Collectively, our findings indicate a key role for CB1R in aging‐related insulin resistance and metabolic dysfunction and highlight CB1R blockade as a potential strategy for combating metabolic disorders associated with aging.     

Modulating serine palmitoyl transferase (SPT) expression and activity unveils a crucial role in lipid-induced insulin resistance in rat skeletal muscle cells

Saturated fatty acids, such as palmitate, promote accumulation of ceramide, which impairs activation and signalling of PKB (protein kinase B; also known as Akt) to important end points such as glucose transport. SPT (serine palmitoyl transferase) is a key enzyme regulating ceramide synthesis from palmitate and represents a potential molecular target in curbing lipid-induced insulin resistance. In the present study we explore the effects of palmitate upon insulin action in L6 muscle cells in which SPT expression/activity has been decreased by shRNA (small-hairpin RNA) or sustained incubation with myriocin, an SPT inhibitor. Incubation of L6 myotubes with palmitate (for 16 h) increases intramyocellular ceramide and reduces insulin-stimulated PKB activation and glucose uptake. PKB inhibition was not associated with impaired IRS (insulin receptor substrate) signalling and was ameliorated by short-term treatment with myriocin. Silencing SPT expression (approximately 90%) by shRNA or chronic cell incubation with myriocin (for 7 days) markedly suppressed SPT activity and palmitate-driven ceramide synthesis; however, challenging these muscle cells with palmitate still inhibited the hormonal activation of PKB. This inhibition was associated with reduced IRS1/p85-PI3K (phosphoinositide 3-kinase) coupling that arises from diverting palmitate towards greater DAG (diacylglycerol) synthesis, which elevates IRS1 serine phosphorylation via activation of DAG-sensitive PKCs (protein kinase Cs). Treatment of SPT-shRNA cells or those treated chronically with myriocin with PKC inhibitors antagonized palmitate-induced loss in insulin signalling. The findings of the present study indicate that SPT plays a crucial role in desensitizing muscle cells to insulin in response to incubation with palmitate. While short-term inhibition of SPT ameliorates palmitate/ceramide-induced insulin resistance, sustained loss/reduction in SPT expression/activity promotes greater partitioning of palmitate towards DAG synthesis, which impacts negatively upon IRS1-directed insulin signalling.