Stimulation Of Amino Acid Uptake Research Articles

Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.

Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status.

Oleic acid stimulation of amino acid uptake in primary human trophoblast cells is mediated by phosphatidic acid and mTOR signaling.

Normal fetal development is critically dependent on optimal nutrient supply by the placenta, and placental amino acid transport has been demonstrated to be positively associated with fetal growth. Mechanistic target of rapamycin (mTOR) is a positive regulator of placental amino acid transporters, such as System A. Oleic acid (OA) has been previously shown to have a stimulatory role on placental mTOR signaling and System A amino acid uptake in primary human trophoblast (PHT) cells. We investigated the mechanistic link between OA and System A activity in PHT. We found that inhibition of mTOR complex 1 or 2, using small interfering RNA to knock down raptor or rictor, prevented OA-stimulated System A amino acid transport indicating the interaction of OA with mTOR. Phosphatidic acid (PA) is a key intermediary for phospholipid biosynthesis and a known regulator of the mTOR pathway; however, phospholipid biosynthetic pathways have not been extensively studied in placenta. We identified placental isoforms of acyl transferase enzymes involved in de novo phospholipid synthesis. Silencing of 1-acylglycerol-3-phosphate-O-acyltransferase-4, an enzyme in this pathway, prevented OA mediated stimulation of mTOR and System A amino acid transport. These data indicate that OA stimulates mTOR and amino acid transport in PHT cells mediated through de novo synthesis of PA. We speculate that fatty acids in the maternal circulation, such as OA, regulate placental functions critical for fetal growth by interaction with mTOR and that late pregnancy hyperlipidemia may be critical for increasing nutrient transfer to the fetus.

Oleic acid stimulation of amino acid uptake in human trophoblast cells is mediated by phosphatidic acid activation of mTOR signaling

Oleic acid stimulation of amino acid uptake in human trophoblast cells is mediated by phosphatidic acid activation of mTOR signaling

Insulin action on protein synthesis and its association with eIF5A expression and hypusination.

The hormone insulin plays a central role in the metabolism of carbohydrates, lipids, and proteins. In relation to protein metabolism, insulin stimulates amino acid uptake and activates protein synthesis in responsive cells by modulation of signal transduction pathways, such as associated to Akt/PkB, mTOR, S6Ks, 4E-BP1, and several translation initiation/elongation factors. In this context, there is no information on direct cellular treatment with insulin and effects on eukaryotic translation initiation factor 5A (eIF5A) regulation. The eIF5A protein contains an exclusive amino acid residue denominated hypusine, which is essential for its activity and synthesized by posttranslational modification of a specific lysine residue using spermidine as substrate. The eIF5A protein is involved in cellular proliferation and differentiation processes, as observed for satellite cells derived from rat muscles, revealing that eIF5A has an important role in muscle regeneration. The aim of this study was to determine whether eIF5A expression and hypusination are influenced by direct treatment of insulin on L6 myoblast cells. We observed that insulin increased the content of eIF5A transcripts. This effect occurred in cells treated or depleted of fetal bovine serum, revealing a positive insulin effect independent of other serum components. In addition, it was observed that hypusination follows the maintenance of eIF5A protein content in the serum depleted cells and treated with insulin. These results demonstrate that eIF5A is modulated by insulin, contributing the protein synthesis machinery control, as observed by puromycin incorporation in nascent proteins.

Increasing Insulin Availability Does Not Augment Postprandial Muscle Protein Synthesis Rates in Healthy Young and Older Men.

Skeletal muscle protein synthesis is highly responsive to food intake. It has been suggested that the postprandial increase in circulating insulin modulates the muscle protein synthetic response to feeding. The objective of the study was to investigate whether a greater postprandial rise in circulating insulin level increases amino acid uptake in muscle and augments postprandial muscle protein synthesis rates. Forty-eight healthy young (age 22 ± 1 y; body mass index 22.0 ± 0.3 kg/m2) and older males (age 68 ± 1 y; body mass index 26.3 ± 0.4 kg/m2) ingested 20 g intrinsically L-[1-13C]-leucine- and L-[1-13C]-phenylalanine-labeled casein protein with or without local insulin infusion. Primed continuous infusions of L-[1-13C]-leucine and L-[ring-2H5]-phenylalanine were applied, with arterial and venous blood samples and muscle biopsies being collected during a 5-hour postprandial period. Insulin administration did not increase overall leg blood flow (P = .509) but increased amino acid uptake over the leg in both young and older subjects (P = .003). The greater amino acid uptake over the leg did not further increase postprandial muscle protein synthesis rates (0.050% ± 0.006% and 0.037% ± 0.004% per hour vs 0.044% ± 0.004% and 0.037% ± 0.002% per hour in the insulin-stimulated vs control condition in the young and older groups, respectively; P = .804) and did not affect postprandial deposition of dietary protein-derived amino acids in de novo muscle protein (P = .872). Greater postprandial plasma insulin availability stimulates amino acid uptake over the leg but does not further augment postprandial muscle protein synthesis rates or stimulate the postprandial deposition of protein derived amino acids into de novo muscle protein in healthy young and older men.

Regulation of the glutamate transporter EAAT3 by mammalian target of rapamycin mTOR

The serine/threonine kinase mammalian target of rapamycin (mTOR) is stimulated by insulin, growth factors and nutrients and confers survival of several cell types. The kinase has previously been shown to stimulate amino acid uptake. In neurons, the cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs) decreases excitation and thus confers protection against excitotoxicity. In epithelia, EAAT3 accomplishes transepithelial glutamate and aspartate transport. The present study explored, whether mTOR regulates EAAT3 (SLC1A1). To this end, cRNA encoding EAAT3 was injected into Xenopus oocytes with or without cRNA encoding mTOR and the glutamate induced current (Iglu), a measure of glutamate transport, determined by dual electrode voltage clamp. Moreover, EAAT3 protein abundance was determined utilizing chemiluminescence. As a result, Iglu was observed in Xenopus oocytes expressing EAAT3 but not in water injected oocytes. Coexpression of mTOR significantly increased Iglu, an effect reversed by rapamycin (100nM). mTOR coexpression increased EAAT3 protein abundance in the cell membrane. The decay of Iglu following inhibition of carrier insertion with brefeldin A in oocytes coexpressing EAAT3 with mTOR was similar in the presence and absence of rapamycin (100nM). In conclusion, mTOR is a novel powerful regulator of EAAT3 and may thus contribute to protection against neuroexcitotoxicity.

Dihydrotestosterone stimulates amino acid uptake and the expression of LAT2 in mouse skeletal muscle fibres through an ERK1/2‐dependent mechanism

Dihydrotestosterone (DHT) has acute/non-genomic actions in adult mammalian skeletal muscles whose physiological functions are still poorly understood. Therefore, the primary aim of this study was to investigate the acute/non-genomic effects of DHT on amino acid uptake as well as the cellular signal transduction events underlying these actions in mouse fast- and slow-twitch skeletal muscle fibre bundles. 14C-Labelled amino acids were used to investigate the effects of DHT and testosterone (T) on amino acid uptake and pharmacological interventions were used to determine the cellular signal transduction events mediating these actions. While T had no effect on the uptake of isoleucine (Ile) and α-methylaminoisobutyric acid (MeAIB) in both fibre types, DHT increased their uptake in the fast-twitch fibre bundles. This effect was reversed by inhibitors of protein translation, the epidermal growth factor receptor (EGFR), system A, system L, mTOR and MEK. However, it was relatively insensitive to inhibitors of transcription, androgen receptors and PI3K/Akt. Additionally, DHT treatment increased the expression of LAT2 and the phosphorylation of the EGFR in the fast-twitch fibre bundles and that of ERK1/2, RSK1/2 and ATF2 in both fibre types. Also, it decreased the phosphorylation of eEF2 and increased the incorporation of Ile into proteins in both fibre types. Most of these effects were reversed by EGFR and MEK inhibitors. From these findings we suggest that another physiological function of the acute/non-genomic actions of DHT in isolated mammalian skeletal muscle fibres is to stimulate amino acid uptake. This effect is mediated through the EGFR and involves the activation of the MAPK pathway and an increase in LAT2 expression.

Antioxidant defenses preserve membrane transport activity in Chironomus riparius larvae exposed to anoxia

Changes in enzyme activities, metabolite concentrations, and membrane transport activity underlying the Chironomus riparius larvae adaptive response to anoxia were investigated. Trehalose, malate, and aspartate degradation and alanine accumulation were recorded. During anoxia exposure, there was a boost of antioxidant defenses as shown by an increase of the specific activity of the enzymes catalase, glutathione-S-transferase, glutathione peroxidase, glutathione-synthase, malic enzyme, and NADP-dependent isocitrate dehydrogenase. The ratio, glutathione reduced over glutathione oxidized, decreased. Except for alanine and catalase, the parameters return to their basal value when larvae are transferred to normoxic conditions. To test whether antioxidant defenses had protective effects on membrane functionality, L-leucine uptake into brush border membrane vesicles and membrane lipid peroxidation was measured. No difference between membranes prepared from larvae exposed to anoxia and control larvae was found. The amino acid alanine, when present inside the vesicles, trans-stimulated leucine uptake. This effect could represent a mechanism to stimulate amino acid uptake and catabolism in vivo when free alanine concentration increases during hypoxic periods.

The uptake of amino acids by the cyanobacterium Planktothrix rubescens is stimulated by light at low irradiances

The rates of uptake of five amino acids--alanine, glutamate, glycine, leucine and serine--by axenic cultures of the cyanobacterium Planktothrix rubescens were measured over a range of irradiances using the (14)C-labelled amino acids at the nanomolar concentrations observed in Lake Zürich. The rates in the light exceeded the dark rates by as much as two- to ninefold. The light-affinity constants for stimulation were similar, indicating a similar process for each of the five amino acids. The E(k) (light saturation irradiance) for light stimulation was only 1 micromol m(-2) s(-1), less than the compensation point for photosynthesis and autotrophic growth, and much lower than the E(k) for either process. The E(k) for amino acid uptake was also less than the irradiance at which filaments obtain neutral buoyancy, which determines the depth at which they stratify and the irradiance they receive. This indicates that stimulation of amino acid uptake by light of low irradiances provides a mechanism for supplementing growth of filaments stratifying deep in the metalimnion, which, while able to grow at low irradiances, are often left with insufficient light to sustain them. Acetate uptake was also stimulated by light, but the kinetics differed.

Receptor-Activated Proteolysis of Transcription Factors

Transcription factors can be activated in various ways. One way that cells control their activity is by sequestering these proteins outside of the nucleus, denying them access to the promoters that they bind. In some cases, transcription factor activation involves cleavage to allow nuclear translocation. Andréasson et al. report that in Saccharomyces cerevisiae , receptor-activated proteolysis is the mechanism by which amino acid sensing leads to stimulation of amino acid uptake through the Ssy1-Ptr3-Ssy5 (SPS) pathway. Two cleavage events occur in the SPS pathway: autoproteolysis of Ssy5, a protein associated with the transmembrane amino acid receptor Ssy1, and stimulated cleavage of Stp1 and Stp2, the transcription factors activated in response to amino acids. Andréasson et al. show that Ssy5 interacts with Stp1 and that a fusion construct containing the region of Stp1 required for processing was cleaved when expressed in yeast exposed to leucine. Yeast deficient in Ssy5 did not cleave the Stp1 fusion construct. When an N terminally hemagglutinin-tagged form of Ssy5, which was constitutively active, was coexpressed with Stp1 in S. pombe , which lacks any orthologs of the SPS system, Stp1 was efficiently cleaved. Because tagging Ssy5 on the N terminus resulted in constitutive activation, the authors created a C terminally tagged form and found that the prodomain remained associated with the catalytic domain after cleavage. Using an internally tagged form of Ssy5, the activity of which was regulated by amino acids, the authors showed that the abundance of the prodomain diminished during a 30-minute exposure of the yeast to leucine. The authors suggest that Ssy5 autoproteolysis serves as a priming event and that upon detection of amino acids by Ssy1, an inhibitory interaction between the cleaved prodomain of Ssy5 and the catalytic domain of Ssy5 is alleviated, which allows Ssy5 to cleave Stp1, promoting nuclear translocation and transcriptional activation. They dub this mechanism of proteolytic activation "receptor-activated proteolysis" (RAP). C. Andréasson , S. Heessen, P. O. Ljungdahl, Regulation of transcription factor latency by receptor-activated proteolysis. Genes Dev. 20 , 1563-1568 (2006). [Abstract] [Full Text]

Modulation of amino acid uptake by TGF‐β in lung myofibroblasts

Hormones such as insulin, growth factors, and cell stress stimulate system A amino acid transporter. Transforming growth factor-beta (TGF-beta) stimulates amino acid uptake thereby inducing cell proliferation, cellular hypertrophy, and matrix synthesis. Insulin appears to activate amino acid in smooth muscle cells via a phosphatidylinositol 3-kinase (PI3-kinase)-dependent pathway. We examine the effect and interaction of TGF-beta, insulin, and PI3-kinase activity on amino acid uptake in human lung myofibroblasts. TGF-beta treatment induced large increases in system A activity and a small delayed increase in the phosphorylation of protein kinase B, also termed phospho-Akt. In contrast, insulin induced small increases in system A activity and large increases in phospho-Akt levels. LY294002, a PI3-kinase inhibitor, blocked the TGF-beta-induced amino acid uptake only partially, but completely blocked TGF-beta-induced Akt phosphorylation. Moreover, the level of phospho-Smad3 was found to be high even when LY294002 blocked TGF-beta-induced phospho-Akt levels. Inhibition of PI3-kinase activity resulted in increase in Km, consistent with a major change in transporter activity without change in transporter number. The PI3-kinase inhibitor also did not change the amino acid transporter 2 (ATA2) mRNA levels. Taken together, these results suggest that TGF-beta induced Smad-3 and amino acid uptake through a PI3-kinase independent pathway.

Dietary Protein to Support Anabolism with Resistance Exercise in Young Men

Resistance exercise is fundamentally anabolic and as such stimulates the process of skeletal muscle protein synthesis (MPS) in an absolute sense and relative to skeletal muscle protein breakdown (MPB). However, the net effect of resistance exercise is to shift net protein balance (NPB = MPS − MPB) to a more positive value; however, in the absence of feeding NPB remains negative. Feeding stimulates MPS to an extent where NPB becomes positive, for a transient time. When combined, resistance exercise and feeding synergistically interact to result in NPB being greater than with feeding alone. This feeding- and exercise-induced stimulation of NPB is what, albeit slowly, results in muscle hypertrophy. With this rudimentary knowledge we are now at the point where we can manipulate variables within the system to see what impact these interventions have on the processes of MPS, MPB, and NPB and ultimately and perhaps most importantly, muscle hypertrophy and strength. We used established models of skeletal muscle amino acid turnover to examine how protein source (milk versus soy) acutely affects the processes of MPS and MPB after resistance exercise. Our findings revealed that even when balanced quantities of total protein and energy are consumed that milk proteins are more effective in stimulating amino acid uptake and net protein deposition in skeletal muscle after resistance exercise than are hydrolyzed soy proteins. Importantly, the finding of increased amino acid uptake would be independent of the differences in amino acid composition of the two proteins. We propose that the improved net protein deposition with milk protein consumption is also not due to differences in amino acid composition, but is due to a different pattern of amino acid delivery associated with milk versus hydrolyzed soy proteins. If our acute findings are accurate then we hypothesized that chronically the greater net protein deposition associated with milk protein consumption post-resistance exercise would eventually lead to greater net protein accretion (i.e., muscle fiber hypertrophy), over a longer time period. In young men completing 12 weeks of resistance training (5d/wk) we observed a tendency (P = 0.11) for greater gains in whole body lean mass and whole as greater muscle fiber hypertrophy with consumption of milk. While strength gains were not different between the soy and milk-supplemented groups we would argue that the true significance of a greater increase in lean mass that we observed with milk consumption may be more important in groups of persons with lower initial lean mass and strength such as the elderly.

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.

A Possible Mechanism for the Insulin Stimulation of Alanine Uptake by Isolated Rat Hepatocytes

The mechanism underlying the insulin-induced stimulation of alanine uptake by isolated rat hepatocytes was studied by testing the involvement of the Na +-K + pump and the Na +-H + exchanger. Insulin was found to inhibit the in vitro activity of the Na +-K + ATPase in a liver homogenate. Ouabain, a specific inhibitor of the pump, enhanced alanine transport, as insulin did, and no additive effects were observed when it was added simultaneously with the hormone. When the membrane was depolarized by sodium removal from the incubation medium or by an increase in its K + concentration, a stimulation of amino acid uptake was observed. Amiloride, an inhibitor of the Na +-H + transporter enhanced also alanine uptake. The increase in transport observed when amiloride and insulin were added together was not significantly different from the one produced by amiloride alone. The stimulatory effect of the drug and the hormone disappeared, however, in presence of bicarbonate or when the membrane was hyperpolarized by potassium removal from the incubation medium. It was concluded that depolarization per se, increased alanine transport and that the effects of amiloride and insulin were mediated by intracellular acidification followed by membrane depolarization, which in turn activated the transporters.

Biophysical and Biological Properties of Naturally Occurring High Molecular Weight Insulin-like Growth Factor II Variants

A soluble form of the insulin-like growth factor II/mannose 6-phosphate receptor (sIGF-II/MPR) is present in fetal bovine serum and carries mature 7.5-kDa insulin-like growth factor II (IGF-II) and at least 12 different high molecular weight (Mr) IGF-II isoforms (Valenzano, K. J., Remmler, J., and Lobel, P. (1995) J. Biol. Chem. 270, 16441-16448). In this study, we used gel filtration and anion exchange chromatographies to resolve the isoforms into eight fractions that were characterized with respect to their biochemical, biophysical, and biological properties. Each fraction contained one to three major protein species with apparent sizes ranging from 11 to 17 kDa by SDS-polyacrylamide gel electrophoresis. The 11-kDa species contains no post-translational modifications and consists of an extended IGF-II backbone terminating at Gly-87. The remaining high Mr IGF-II isoforms are also composed of an 87-amino acid IGF-II peptide backbone but contain increasing amounts of sialated, O-linked sugars. Plasmon resonance spectroscopy experiments revealed that all the high Mr isoforms and mature 7.5-kDa IGF-II bound to immobilized recombinant soluble human IGF-I receptor, recombinant human IGF-binding protein 1, and sIGF-II/MPR with similar kinetics. In addition, radiolabeled tracer experiments demonstrated that both mature and high Mr IGF-II isoforms have similar binding profiles in fetal bovine serum and have similar affinities for IGF-II-binding proteins secreted from human fibroblasts. Finally, the biological activity of high Mr IGF-II was shown to be similar to or slightly better than mature IGF-II in stimulating amino acid uptake in fibroblasts and in inducing myoblast differentiation.

Glucocorticoid-Dependent Induction of Interleukin-6 Receptor Expression in Human Hepatocytes Facilitates Interleukin-6 Stimulation of Amino Acid Transport

The authors studied the effects of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on glutamine and alanine transport in isolated human hepatocytes. They also evaluated the role of dexamethasone in modulating this response and its effects on the expression of the plasma membrane high-affinity IL-6 receptor. Animal studies indicate that cytokines are important mediators of the increased hepatic amino acid uptake that occurs during cancer and sepsis, but studies in human tissues are lacking. The control of transport by cytokines and cytokine receptor expression in the liver may provide a mechanism by which hepatocytes can modulate amino acid availability during catabolic disease states. Human hepatocytes were isolated from wedge biopsy specimens and plated in 24-well trays. Interleukin-6 and TNF-alpha, in combination with the synthetic glucocorticoid dexamethasone, were added to hepatocytes in culture, and the transport of radiolabeled glutamine and alanine was measured. Fluorescent-activated cell sorter (FACS) analysis was used to study the effects of dexamethasone on IL-6 receptor number in the well-differentiated human hepatoma HepG2. Both IL-6 and TNF-alpha exerted a small stimulatory effect on alanine and glutamine transport. Dexamethasone alone did not alter transport rates, but pretreatment of cells augmented the effects of both cytokines on carrier-mediated amino acid uptake. Dexamethasone pretreatment and a combination of IL-6 and TNF-alpha resulted in a greater than twofold increase in transport activity. Fluorescent-activated cell sorter analysis demonstrated that dexamethasone induced a threefold increase in the expression of high-affinity IL-6 receptors. Interleukin-6 and TNF-alpha work coordinately with glucocorticoids to stimulate amino acid uptake in human hepatocytes. Dexamethasone exerts a permissive effect on cytokine-mediated increases in transport by increasing IL-6 receptor expression on the cell surface. It is likely that this upregulation of IL-6 receptors "primes" human liver cells for subsequent stimulation by cytokines. The resulting increase in hepatic amino acid transport provides the liver with substrate to support key metabolic pathways during catabolic states.

Insulin‐like growth factor‐1 stimulates amino acid uptake by the cultured human placental trophoblast

Amino acid uptake by the human placenta is known to occur via several transport mechanisms. However, regulation by extracellular factors has received relatively little attention. A recent report by this laboratory characterized the uptake of alpha-aminoisobutyric acid (AIB) stimulated by insulin in the cultured human placental trophoblast. The current study evaluated the effect of insulin-like growth factor-1 (IGF-1) on AIB uptake in cultured human placental trophoblasts. Na(+)-dependent AIB uptake was significantly stimulated by IGF-I in a time-dependent manner, as early as 30 min after hormone exposure. The maximum effect was at 2-4 hr of continuous exposure to IGF-I and the stimulation was dependent upon IGF-1 concentration approaching maximal stimulation at 50 ng.ml-1. AIB uptake was inhibited by increasing concentrations of alpha-(methylamino)isobutyric acid (MeAIB). Approximately 75% of basal (unstimulated) Na(+)-dependent AIB uptake was inhibited by MeAIB. The IGF-1-stimulated increment above basal AIB uptake was completely inhibited by MeAIB. IGF-1 increased the maximum uptake velocity but not Km. Using equimolar concentrations, stimulation was greater with IGF-1 than with IGF-2. Stimulation by IGF-1, but not insulin, was inhibited by anti-IGF-1 receptor antibody, indicating mediation via the IGF-1 receptor. H7, a nonspecific inhibitor of serine-threonine kinase, inhibited IGF-1-dependent stimulation of AIB uptake. In addition, calphostin C (a specific inhibitor of protein kinase C), but not H89 (a specific inhibitor of protein kinase A), inhibited the IGF-1 action. This study further characterizes regulated amino acid uptake by the human placental trophoblasts and demonstrates that the Na(+)-dependent component of AIB uptake is stimulated by physiologic concentrations of IGF-1.

Wortmannin inhibits the action of insulin but not that of okadaic acid in skeletal muscle: comparison with fat cells.

To look for the possible involvement of phosphatidylinositol-3-kinase (PI3-kinase) in insulin action in muscle, we have used wortmannin, described as a specific inhibitor of the enzyme, and compared its effect in muscle and in adipose cells. Both in intact mouse soleus muscle and in isolated rat adipocytes, wortmannin blocked insulin effect on glucose uptake, without markedly altering basal glucose uptake. In adipocyte, this effect results from a blockade of the translocation process because wortmannin inhibited the stimulatory action of insulin on both the Glut 4 movement from the internal compartment to the plasma membranes and the Rab4 departure from the microsomes. In a similar fashion, two other insulin effects, the activation of glycogen synthase and the stimulation of amino acid uptake, were blocked by wortmannin in skeletal muscle. Lipogenesis from acetate was also inhibited by wortmannin in adipocytes. By contrast, wortmannin did not affect muscle deoxglucose uptake when it was stimulated either by okadaic acid or by the protein kinase C activator tumor promoting agent. These results suggest that, in muscle and adipocyte, PI3-kinase inhibition causes a blockade of all insulin effects studied. By contrast, wortmannin did not affect the same responses elicited in muscle by okadaic acid or tumor promoting agent.

Biological activity of immunoreactive insulin-like activity extracted from rat submandibular gland

Earlier studies indicate the presence of an insulin-like immunoreactivity (ILI) in rat submandibular salivary glands (SSG). Previous observations also showed that streptozotocin (STZ)-induced diabetes was accompanied by an increase in SSG ILI concentrations. In the present work we studied the effect of SSG ILI from normal and STZ diabetic rats (ILI-N and ILI-D, respectively) on insulin receptor binding and function in LMH cell line. ILI-N and ILI-D inhibited 125I-insulin binding to intact cells and wheat germ agglutinin (WGA)-purified insulin receptors with a high affinity. Furthermore, ILI-N and ILI-D activated, although weakly, the beta-subunit autophosphorylation of solubilized and WGA-purified insulin receptors. An ATP hydrolytic activity was present in ILI-N and, to a greater extent, in ILI-D extracts, which can at least in part explain their low potency for activating autophosphorylation and kinase activity of insulin receptors in vitro. However, after ILI treatment of intact cells and immunoprecipitation of insulin receptors, ILI induced a dose-dependent tyrosine phosphorylation of the insulin receptor beta-subunit. Finally, ILI-N and ILI-D stimulated amino acid uptake and lipogenesis in LMH cells. These findings suggest that SSG ILI is biologically active and can participate in metabolic regulations.

Cell polarity of the insulin-like growth factor system in human intestinal epithelial cells. Unique apical sorting of insulin-like growth factor binding protein-6 in differentiated human colon cancer cells.

In this study, we have used enterocyte-like differentiated HT29-D4 human colonic carcinoma cells cultured in a glucose-free medium (HT29-D4-GAL cells) on semi-permeable supports in order to investigate the polarity of the insulin-like growth factor (IGF) system. We report that these cells secrete endogenous IGF-II predominantly (66%) from the basolateral cell surface where type I IGF receptors are almost all (> 96%) localized. HT29-D4-GAL cells also secrete IGF-binding protein (IGFBP) -2, -4, and -6 as evidenced by Western ligand and immunoblot analyses of conditioned medium. IGFBP-2 and IGFBP-4 are secreted primarily into the basolateral side (71 and 87%, respectively), whereas IGFBP-6 is targeted to the apical surface (76%) as a possible consequence of an active sorting. Finally, HT29-D4-GAL cells are found to display responses to IGF-II added to the basolateral but not the apical membrane side in terms of intracellular tyrosine phosphorylation and long-term stimulation of amino acid uptake. This study indicates (a) that IGF-II is potentially capable of autocrine regulation on the basolateral side of HT29-D4-GAL cell, and (b) that IGFBP-6 has a unique pattern of secretory polarity. It supports the concept that a differential sorting of the various forms of IGFBPs might play a modulatory role in the maintenance of a functional polarity in the differentiated HT29-D4-GAL cells.