Sylvia Y. Low

Modulation of glycogen synthesis in rat skeletal muscle by changes in cell volume.

1. The hypothesis that cellular hydration state modulates muscle glycogen synthesis was tested by measuring the incorporation of [14C]glucose into glycogen (glycogen synthesis) in primary rat myotubes after experimentally induced volume changes. 2. Glycogen synthesis in myotubes increased (by 75%, P < 0.01) after swelling induced by 60 min exposure to hyposmotic media (170 mosmol kg‐1) relative to isosmotic control (300 mosmol kg‐1) values, it decreased (by 31%, P < 0.05) after shrinkage induced by 60 min exposure to hyperosmotic (430 mosmol kg‐1) media. Myotube 2‐deoxy‐D‐glucose (0.05 mM) uptake was unaffected by changes in external osmolality. 3. Wortmannin (100 nM; 60 min), a phosphatidylinositol 3‐kinase inhibitor, decreased basal glycogen synthesis by 28% whereas rapamycin (100 nM; 60 min), which blocks the activation of p70 S6 kinase, had no effect. Both wortmannin (100 nM; 60 min) and rapamycin (100 nM; 60 min) blocked the changes in glycogen synthesis resulting from hypo‐ and hyperosmotic exposure. 4. Myotube glycogen synthesis is modulated by volume changes independently of changes in glucose uptake. The phenomenon may be physiologically important in promoting glycogen storage during circumstances of myofibrillar swelling, e.g. after feeding or exercise.

Signaling elements involved in amino acid transport responses to altered muscle cell volume.

Skeletal muscle glutamine uptake via the transport system Nm is subject to rapid (t1/2= ≍1 min) regulation after changes in cell volume by mechanisms that remain to be elucidated. Wortmannin (phosphatidylinositol 3‐kinase inhibitor) but not rapamycin (inhibitor of p70S6 kinase activation) prevents both hypo‐osmotic swelling‐induced stimulation and hyperosmotic shrinkage‐induced inhibition of Na+‐dependent glutamine uptake in primary culture of rat skeletal muscle. G‐protein inhibitors (cholera, pertussis toxins) also abolished responses of glutamine transport to cell volume changes whereas these responses were sustained in the presence of G‐protein activators (MAS 7, lysophosphatidic acid). Swelling‐induced activation of glutamine transport does not seem to involve release of autocrine factors because “conditioned” medium from swollen cells has no effect on previously unstimulated cells. System A amino acid transport exhibits responses to cell volume change that are opposite to those of system Nm, but these are also blocked by wortmannin. Active phosphatidylinositol 3‐kinase appears to be required to enable muscle cells to exhibit rapid, volume‐induced changes in amino acid transport when suitably stimulated.—Low, S. Y., Rennie, M. J., Taylor, P. M. Signaling elements involved in amino acid transport responses to altered muscle cell volume. FASEB J. 11, 1111–1117 (1997)

Responses of glutamine transport in cultured rat skeletal muscle to osmotically induced changes in cell volume.

1. In order to investigate the relationship between cellular hydration state and the rate of glutamine transport, tracer glutamine uptake into primary rat myotubes was studied at external osmolalities of 170, 320 or 430 mosmol kg‐1. 2. Incubation of myotubes with glutamine (2 mM; 30 min) at 320 mosmol kg‐1 increased cell volume and glutamine transport (by 35 and 36%, respectively); insulin (66 nM; 30 min) also increased cell volume and glutamine transport (by 22 and 40%, respectively) and the effects of insulin and glutamine combined were additive. The increase in glutamine uptake following glutamine pre‐incubation represented an increase in Vmax of Na(+)‐dependent glutamine transport. 3. There was an inverse relationship between myotube glutamine transport and external osmolality after 30 min exposure. 4. During hyposmotic (170 mosmol kg‐1) exposure there were large, rapid increases of cell volume and glutamine transport; the latter increased transiently (during the cell swelling phase) by a maximum of approximately 80% at 2 min, (due to an increased Vmax for Na(+)‐dependent glutamine transport) then decayed to a new elevated steady state after 30 min exposure. 5. During hyperosmotic (430 mosmol kg‐1) exposure there were rapid decreases in glutamine transport and myotube cell volume (both by approximately 30%) to values which were maintained for at least 15 min. 6. The volume‐sensitive glutamine transport process features characteristics of the insulin‐sensitive system Nm transporter. 7. Modulation of Na(+)‐dependent glutamine transport by insulin and cell volume changes may contribute towards regulation of muscle metabolism.

Mechanisms of glutamine transport in rat adipocytes and acute regulation by cell swelling.

Adipose tissue is a major site for whole-body glutamine synthesis and we are investigating mechanisms and regulation of glutamine transport across the adipocyte membrane. Glutamine transport in adipocytes includes both high- and low-affinity Na+-dependent components (consistent with observed expression of ASCT2 and ATA2/SAT2 transporter mRNAs respectively) and a Na+-independent transport component (consistent with observed expression of LAT1/2 transporter mRNAs). Hypo-osmotic (235 mosmol/kg) swelling of adipocytes transiently stimulated glutamine uptake (180% increase at 0.05 mM glutamine) within 5 mins. Stimulation was blocked by the tyrosine kinase inhibitor genistein and the MAP kinase pathway inhibitors PD98059 and SB203580, but not by wortmannin (PI 3-kinase inhibitor) or rapamycin (mTOR pathway inhibitor). Cell-swelling also stimulated uptake of glucose but not MeAIB (indicating that ASCT2 rather than ATA2 was stimulated by swelling). Insulin (66 nM) treatment for up to 1 h stimulated Na+-dependent glutamine transport and increased adipocyte water space. Activation of the ERK1-2 MAP kinase pathway by cell swelling or insulin may be important for rapid activation of the ASCT2 glutamine transporter in adipocytes. Insulin may also exert a minor additional stimulatory effect on adipocyte glutamine transport indirectly via cell swelling. The mechanisms regulating glutamine transport in adipose tissue are distinct from those in other major sites of glutamine turnover in the body (notably liver and skeletal muscle).

Sodium-dependent glutamate transport in cultured rat myotubes increases after glutamine deprivation.

Glutamine produced and stored in skeletal muscle is an important source of nitrogen and energy for the whole body in health and disease and, unsurprisingly, glutamine turnover in muscle is subject to substantial metabolic control. l‐Glutamate, a necessary substrate for glutamine synthetase, is transported into muscle cells by Na+‐dependent and ‐independent transport systems. In primary cultures of rat skeletal muscle myotubes (a useful model system for studies of muscle metabolism and membrane transport), Na+‐dependent glutamate transport (Km ≈ 0.7 mM glutamate) shows adaptive up‐regulation (65% increase in transport Vmax from 2.7 to 4.4 nmol · min–1 · mg protein–1) in cells within 24 h of glutamine depletion (t1/2 for increase of ≈ 4 h), whereas Na+‐ndependent glutamate uptake remains unaltered. Up‐regulation of transport is suppressed by inhibitors of gene transcription (actinomycin‐D) and translation (cycloheximide) and is reversed by glutamine supplementation. Increased glutamate transport capacity should provide extra substrate for glutamine synthesis in muscle cells. Thus, in concert with previously discovered increases in cell glutamine transport capacity and glutamine synthetase activity, it may represent part of a coordinated response to decreased glutamine availability (e.g., under circumstances of increased glutamine utilization by other tissues in vivo).— Low, S. Y., Rennie, M. J., Taylor, P. M. Sodium‐dependent glutamate transport in cultured rat myotubes increases after glutamine deprivation. FASEB J. 8: 127‐131; 1994.

Integrin and cytoskeletal involvement in signalling cell volume changes to glutamine transport in rat skeletal muscle

1 Muscle glutamine transport is modulated in response to changes in cell volume by a mechanism dependent on active phosphatidylinositol 3‐kinase. We investigated the possibility that this mechanism requires interactions between the extracellular matrix (ECM), integrins and the cytoskeleton as components of a mechanochemical transduction system. 2 Using skeletal muscle cells, we studied effects of (a) inactivating integrin‐substratum interactions by using integrin‐binding peptide GRGDTP with inactive peptide GRGESP as control, and (b) disrupting the cytoskeleton using colchicine or cytochalasin D, on glutamine transport after brief exposure to hypo‐osmotic, isosmotic or hyperosmotic medium (170, 300 and 430 mosmol kg−1, respectively). 3 Neither GRGDTP nor GRGESP significantly affected basal glutamine uptake (0.05 mm; 338 ± 58 pmol min−1 (mg protein)−1) but GRGDTP specifically prevented the increase (71 %) and decrease (39 %) in glutamine uptake in response to hypo‐ and hyperosmotic exposure, respectively. 4 Colchicine and cytochalasin D prevented the increase and decrease in glutamine uptake in response to changes in external osmolality. They also increased basal glutamine uptake by 59 ± 19 and 85 ± 16 %, respectively, in a wortmannin‐sensitive manner. 5 These results indicate involvement of ECM‐integrin‐mediated cell adhesion and the cytoskeleton in mechanochemical transduction of cell volume changes to chemical signals modulating glutamine transport in skeletal muscle. Phosphatidylinositol 3‐kinase may function to maintain the mechanotransducer in an active state.

Amino Acid Transport during Muscle Contraction and Its Relevance to Exercise

The functional significance of amino acid transport in skeletal muscle has been explored by the use of a variety of techniques including work in isolated perfused organs, isolated incubated organs and tissue culture of muscle cells. The results suggest that although there is a wide variety of amino acid transport systems of different characteristics and with different responses to ionic, hormonal and nervous modulation, the amino acid glutamine (transported by system Nm) demonstrates some unusual properties not observed with amino acids transported by other systems. Glutamine is transported at very high rates in skeletal muscle and heart and both the glutamate and glutamine transporter appear to be adaptively regulated by the availability of glutamine. Glutamine appears to be involved in the regulation of a number of important metabolic processes in heart and skeletal muscle (e.g., regulation of the glutathione reduced/oxidised ratio and regulation of protein and glycogen synthesis). Furthermore, glutamine transport appears to interact with systems for regulation of volume control and many of the metabolic features attributable to changes in glutamine concentration appear to be modulated via alteration in cytoskeletal status.

Biomembrane Transport and Interorgan Nutrient Flows: The Amino Acids

This chapter provides an introduction to current concepts in interorgan nutrition as they relate to protein and amino acid metabolism in the whole body. Methodological issues and their limitations are discussed together with assessments of the scope and importance of amino acid flows between tissues and organs. The chapter describes specific interorgan amino acid flows together with the influence of a variety of physiological and pathophysiological conditions upon them. It also highlights the vital role of biomembrane transport of amino acids in the facilitation and control of flows between compartments. It later illustrates the use of metabolic control theory to determine the quantitative importance of transport processes to regulation of amino acid metabolism. The past 5 years have seen very rapid advances in our knowledge of the molecular structure of amino acid transporters. Furthermore, cDNA-encoding transport proteins in important amino acid transport systems, such as A and N, remain to be cloned. Nevertheless, there are already significant challenges and opportunities for the integration of the new molecular knowledge into a holistic view of interorgan amino acid nutrition. Many of the phenomenological descriptions of interorgan nutrition contained in this chapter are capable of being explained on a mechanistic basis by combining the knowledge gained from classical flux measurements with the new molecular information.

Thermodynamics and Transport

The total free energy change of a transport process or a series of processes may have numerous obvious components. Knowledge of these components frequently has practical implications for the interpretation of transport experiments. In addition, knowledge of the free energy changes associated with each component of transport is required in order to fully understand how transport contributes to the work performed by cells. It is proposed in this chapter that the free energy changes associated with the components of primary active transport processes may include much larger changes in the magnitude of enthalpy and entropy than are associated with ATP synthesis or hydrolysis. In addition to the free energy available from solute transport, the cell depends on transport occurring rapidly enough to do work that is useful to it. Transport proteins are the catalysts that permit solutes and the solvent to migrate across biomembranes much more rapidly than they can by permeating phospholipid bilayers. Although ordinary diffusion of solutes and the solvent may be relatively rapid over short distances in intracellular and extracellular aqueous solutions, such diffusion is virtually halted across thin biomembranes.

A role for membrane transport in modulation of intramuscular free glutamine turnover in streptozotocin diabetic rats.

We wished to examine the effects of diabetes on muscle glutamine kinetics. Accordingly, female Wistar rats (200 g) were made diabetic by a single injection of streptozotocin (85 mg/kg) and studied 4 days later; control rats received saline. In diabetic rats, glutamine concentration of gastrocnemius muscle was 33% less than in control rats: 2.60 +/- 0.06 mumol/g vs. 3.84 +/- 0.13 mumol/g (P < 0.001). In gastrocnemius muscle, glutamine synthetase activity (Vmax) was unaltered by diabetes (approx. 235 nmol/min per g) but glutaminase Vmax increased from 146 +/- 29 to 401 +/- 94 nmol/min per g; substrate Km values of neither enzyme were affected by diabetes. Net glutamine efflux (A-V concentration difference x blood flow) from hindlimbs of diabetic rats in vivo was greater than control values (-30.0 +/- 3.2 vs. -1.9 +/- 2.6 nmol/min per g (P < 0.001)) and hindlimb NH3 uptake was concomitantly greater (about 27 nmol/min per g). The glutamine transport capacity (Vmax) of the Na-dependent System Nm in perfused hindlimb muscle was 29% lower in diabetic rats than in controls (820 +/- 50 vs. 1160 +/- 80 nmol/min per g (P < 0.01)), but transporter Km was the same in both groups (9.2 +/- 0.5 mM). The difference between inward and net glutamine fluxes indicated that glutamine efflux in perfused hindlimbs was stimulated in diabetes at physiological perfusate glutamine (0.5 mM); ammonia (1 mM in perfusate) had little effect on net glutamine flux in control and diabetic muscles. Intramuscular Na+ was 26% greater in diabetic (13.2 mumol/g) than control muscle, but muscle K+ (100 mumol/g) was similar. The accelerated rate of glutamine release from skeletal muscle and the lower muscle free glutamine concentration observed in diabetes may result from a combination of: (i), a diminished Na+ electrochemical gradient (i.e., the net driving force for glutamine accrual in muscle falls); (ii), a faster turnover of glutamine in muscle and (iii), an increased Vmax/Km for sarcolemmal glutamine efflux.