Factors governing the assignment of visual consequence to the corresponding action.

To achieve fast feedback control of voluntary movements, the visual consequences of our motor commands need to be quickly identified and analyzed by the motor control processes in the brain. These processes work remarkably well even in complex visual environments and in the face of discrepancies between physical actuator and visually perceived effect, e.g. when moving a computer mouse on a visually crowded screen. Here, we use an ambiguous situation in which a single cursor could be controlled by either the left or the right hand to determine the visual and cognitive factors that determine the assignment of a visual stimulus to the corresponding motor command. Our results demonstrate that the visuomotor system is exquisitely sensitive to the spatio-temporal correlation between cursor and hands, learning the appropriate mapping implicitly within several minutes. In contrast, spatial proximity between end effector and visual consequence has an immediate but only transient effect on the assignment process. Finally, an explicit instruction about which hand controls the cursor only has a minor influence when the instruction is presented first. These findings provide insight into the relative importance of the factors that determine the binding of visual information to the corresponding motor structures to enable fast feedback control.NEW & NOTEWORTHY For efficient visuomotor online control, the brain needs to solve the correspondence problem between an ongoing movement and its visual consequences. Here, we challenge the visuomotor system with an ambiguous reaching task, in which the visual feedback was controlled by either hand or by a combination of both. Our findings characterize the properties of a flexible assignment process that quickly takes into account the spatio-temporal properties of movements and the visual scene.

Unmet expectations delay sensory processes.

Expectations strongly affect and shape our perceptual decision-making processes. Specifically, valid expectations speed up perceptual decisions, and determine what we see in a noisy stimulus. Despite the well-established effects of expectations on decision-making, whether and how they affect low-level sensory processes remain elusive. To address this problem, we investigated the effect of expectation on temporal thresholds in an individuation task (detection of the position of an intact image, a house or face). We found that compared to a neutral baseline, thresholds increase when the intact images are of the unexpected category, but remain unchanged when they are of the expected category. Using a recursive Bayesian model with dynamic priors we show that delay in sensory processes is the result of further processing, consequently longer time, required in case of violated expectations. Expectations, however, do not alter internal parameters of the system. These results reveal that sensory processes are delayed when expectations are not met, and a simple parsimonious computational model can successfully explain this effect.

Homozygous LAMC3 mutation links to structural and functional changes in visual attention networks.

The occipital lobe contains a substantial part of the neural machinery involved in visual perception. Mutations in the LAMC3 gene have recently been shown to cause complex bilateral occipital cortical gyration abnormalities. However, to what extent these structural changes impact visual behavior is not known. We recorded responses for two screening test batteries targeting visual function (Leuven - Perceptual Organization Screening Test, Cortical Vision Screening Test) and measured eye fixation performance in a visual attention experiment from a patient with homozygous LAMC3 gene mutation. Using voxel-based morphometry (VBM) we quantitatively assessed the extent of structural changes brought on by the genetic mutation by comparing mean cortical curvature, cortical thickness, and gray matter volume in 34 cortical areas between patient and an age-, sex-, and education-matched control group. Anatomical connectivity between these cortical areas was investigated by a structural covariance analysis. Visual screening-, and behavioral results revealed that the patient's impairments were predominantly in visuo-spatial attention. Consistent with this, VBM and structural connectivity results revealed significant structural changes in cortical regions subserving attentional functions. We conclude that the LAMC3 gene mutation affects cortical areas beyond the occipital lobe and primarily those visual functions that involve heavily distributed networks - such as visuo-spatial attention.

Meconial peritonitis in a rare association of partial ileal apple-peel atresia with small abdominal wall defect.

Intestinal atresia type III B (apple peel) and gastroschisis are both congenital malformations who require early surgical correction in neonatal age. Their association is very rare. We present the case of a full term infant with partial apple peel ileal atresia and a small defect of the anterior abdominal wall, complicated by in utero intestinal perforation and subsequent meconial peritonitis. We observed a partial atresia of small intestine, with involvement of terminal ileus savings of jejunum and a large part of the proximal ileum, small anterior abdominal wall defect with herniation of few bowel loops, intestinal malrotation. Paralytic ileus and infections are the main causes of morbidity and mortality at neonatal age. In our case, in spite of the mild phenotype, prognosis has been complicated by the onset of functional bowel obstruction, caused by chemical peritonitis resulting from contact with either amniotic fluid and meconium.

A novel variant of glutamine: fructose-6-phosphate amidotransferase-1 (GFAT1) mRNA is selectively expressed in striated muscle.

Glutamine:fructose-6-phosphate amidotransferase(GFAT) is the rate-limiting enzyme of the hexosamine synthesis pathway. Products of this pathway have been implicated in insulin resistance and glucose toxicity. GFAT1 is ubiquitous, whereas GFAT2 is expressed mainly in the central nervous system. In the course of developing a competitive reverse transcriptase-polymerase chain reaction assay, we noted that GFAT1 cDNA from muscle but not from other tissues migrated as a doublet. Subsequent cloning and sequencing revealed two GFAT1 mRNAs in both mouse and human skeletal muscles. The novel GFAT1 mRNA (GFAT1Alt [muscle selective variant of GFAT1]) is likely a splice variant. It is identical to GFAT1 except for a 48 or 54 bp insert in the mouse and human, respectively, at nucleotide position 686 of the coding sequence, resulting in a 16 or 18 amino acid insert at position 229 of the protein. GFAT1Alt is the predominant GFAT1 mRNA in mouse hindlimb muscle, is weakly expressed in the heart, and is undetectable in the brain, liver, kidney, lung, intestine, spleen, and 3T3-L1 adipocytes. In humans, it is strongly expressed in skeletal muscle but not in the brain. GFAT1 and GFAT1Alt expressed by recombinant adenovirus infection in COS-7 cells displayed robust enzyme activity and kinetic differences. The apparent K(m) of GFAT1Alt for fructose-6-phosphate was approximately twofold higher than that of GFAT1, whereas K(i) for UDP-N-acetylglucosamine was approximately fivefold lower. Muscle insulin resistance is a hallmark and predictor of type 2 diabetes. Variations in the expression of GFAT isoforms in muscle may contribute to predisposition to insulin resistance.

alpha-Lipoic acid prevents the development of glucose-induced insulin resistance in 3T3-L1 adipocytes and accelerates the decline in immunoreactive insulin during cell incubation.

Oxidative stress has been implicated in glucose toxicity. We tested the hypothesis that certain antioxidants may prevent insulin-resistant glucose transport that develops in adipocytes after sustained exposure to high glucose, provided insulin is present. The antioxidant alpha-lipoic acid has been proposed as an insulin sensitizer. 3T3-L1 adipocytes were preincubated 18 hours in media containing insulin (0.6 nmol/L) with low (5 mmol/L) or high (25 mmol/L) glucose with or without alpha-lipoate, dihydrolipoate (each 0.1 to 0.5 mmol/L), or N-acetylcysteine (1 to 5 mmol/L). After extensive re-equilibration in insulin and antioxidant-free media, basal and maximally insulin-stimulated (100 nmol/L) glucose transport was measured. Insulin was quantified by radioimmunoassay. Preincubation with alpha-lipoate and dihydrolipoate but not N-acetylcysteine increased subsequent basal glucose transport; the effect was much smaller than that of acute maximal insulin stimulation. Preincubation in high glucose without antioxidants inhibited acutely insulin-stimulated glucose transport by 40% to 50% compared with low glucose. This down- regulation was partially or completely prevented by each antioxidant. In cell-free media, the 2 reductants, dihydrolipoate and N-acetylcysteine, rapidly decreased immunoreactive insulin, but alpha-lipoate was ineffective. However, during incubation with adipocytes, alpha-lipoate, and dihydrolipoate promoted the decline in immunoreactive insulin nearly equally. Because insulin and high glucose are synergistic in inducing insulin resistance in this model, the reduction in immunoreactive insulin probably contributed to the protective effect of the antioxidants. 3T3-L1 adipocytes efficiently metabolize alpha-lipoate to dihydrolipoate, which may be released into the medium. The stimulation of glucose transport by alpha-lipoic acid may represent redox effects in subcellular compartments that are accessible to dihydrolipoate.

Excessive hexosamines block the neuroprotective effect of insulin and induce apoptosis in retinal neurons.

In addition to microvascular abnormalities, neuronal apoptosis occurs early in diabetic retinopathy, but the mechanism is unknown. Insulin may act as a neurotrophic factor in the retina via the phosphoinositide 3-kinase/Akt pathway. Excessive glucose flux through the hexosamine biosynthetic pathway (HBP) is implicated in the development of insulin resistance in peripheral tissues and diabetic complications such as nephropathy. We tested whether increased glucose flux through the HBP perturbs insulin action and induces apoptosis in retinal neuronal cells. Exposure of R28 cells, a model of retinal neurons, to 20 mm glucose for 24 h attenuated the ability of 10 nm insulin to rescue them from serum deprivation-induced apoptosis and to phosphorylate Akt compared with 5 mm glucose. Glucosamine not only impaired the neuroprotective effect of insulin but also induced apoptosis in R28 cells in a dose-dependent fashion. UDP-N-acetylhexosamines (UDP-HexNAc), end products of the HBP, were increased approximately 2- and 15-fold after a 24-h incubation in 20 mm glucose and 1.5 mm glucosamine, respectively. Azaserine, a glutamine:fructose-6-phosphate amidotransferase inhibitor, reversed the effect of 20 mm glucose, but not that of 1.5 mm glucosamine, on attenuation of the ability of insulin to promote cell survival and phosphorylate Akt as well as accumulation of UDP-HexNAc. Glucosamine also impaired insulin receptor processing in a dose-dependent manner but did not decrease ATP content. By contrast, in L6 muscle cells, glucosamine impaired insulin receptor processing but did not induce apoptosis. These results suggest that the excessive glucose flux through the HBP may direct retinal neurons to undergo apoptosis in a bimodal fashion; i.e. via perturbation of the neuroprotective effect of insulin mediated by Akt and via induction of apoptosis possibly by altered glycosylation of proteins. The HBP may be involved in retinal neurodegeneration in diabetes.

High glucose and glucosamine induce insulin resistance via different mechanisms in 3T3-L1 adipocytes.

Sustained hyperglycemia induces insulin resistance, but the mechanism is still incompletely understood. Glucosamine (GlcN) has been extensively used to model the role of the hexosamine synthesis pathway (HSP) in glucose-induced insulin resistance. 3T3-L1 adipocytes were preincubated for 18 h in media +/- 0.6 nmol/l insulin containing either low glucose (5 mmol/l), low glucose plus GlcN (0.1-2.5 mmol/l), or high glucose (25 mmol/l). Basal and acute insulin-stimulated (100 nmol/l) glucose transport was measured after re-equilibration in serum and insulin-free media. Preincubation with high glucose or GlcN (1-2.5 mmol/l) inhibited basal and acute insulin-stimulated glucose transport only if insulin was present during preincubation. However, only preincubation with GlcN plus insulin inhibited insulin-stimulated GLUT4 translocation. GLUT4 and GLUT1 protein expression were not affected. GlcN (2.5 mmol/l) increased cellular UDP-N-acetylhexosamines (UDP-HexNAc) by 400 and 900% without or with insulin, respectively. High glucose plus insulin increased UDP-HexNAc by 30%. GlcN depleted UDP-hexoses, whereas high glucose plus insulin increased them. Preincubation with 0.5 mmol/l GlcN plus insulin maximally increased UDP-HexNAc without affecting insulin-stimulated or basal glucose transport. GlcN plus insulin (but not high glucose plus insulin) caused marked GlcN dose-dependent accumulation of GlcN-6-phosphate, which correlated with insulin resistance of glucose transport (r = 0.935). GlcN plus insulin (but not high glucose plus insulin) decreased ATP (10-30%) and UTP (>50%). GTP was not measured, but GDP increased. Neither high glucose plus insulin nor GlcN plus insulin prevented acute insulin stimulation (approximately 20-fold) of insulin receptor substrate 1-associated phosphatidylinositol (PI)-3 kinase. We have come to the following conclusions. 1) Chronic exposure to high glucose or GlcN in the presence of low insulin caused insulin resistance of glucose transport by different mechanisms. 2) GlcN inhibited GLUT4 translocation, whereas high glucose impaired GLUT4 "intrinsic activity" or membrane intercalation. 3) Both agents may act distally to PI-3 kinase. 4) GlcN has metabolic effects not shared by high glucose. GlcN may not model HSP appropriately, at least in 3T3-L1 adipocytes.

Lower calorie intake enhances muscle insulin action and reduces hexosamine levels.

Previous studies have demonstrated enhanced insulin sensitivity in calorie-restricted [CR, fed 60% ad libitum (AL) one time daily] compared with AL-fed rats. To evaluate the effects of reduced food intake, independent of temporal differences in consumption, we studied AL (unlimited food access)-fed and CR (fed one time daily) rats along with groups temporally matched for feeding [fed 3 meals (M) daily]: MAL and MCR, eating 100 and 60% of AL intake, respectively. Insulin-stimulated glucose transport by isolated muscle was increased in MCR and CR vs. AL and MAL; there was no significant difference for MCR vs. CR or MAL vs. AL. Intramuscular triglyceride concentration, which is inversely related to insulin sensitivity in some conditions, did not differ among groups. Muscle concentration of UDP-N-acetylhexosamines [end products of the hexosamine biosynthetic pathway (HBP)] was lower in MCR vs. MAL despite unaltered glutamine-fructose-6-phosphate aminotransferase activity (rate-limiting enzyme for HBP). These results indicate that the CR-induced increase in insulin-stimulated glucose transport in muscle is attributable to an altered amount, not timing, of food intake and is independent of lower triglyceride concentration. They further suggest that enhanced insulin action might involve changes in HBP.

Decreased hexosamine biosynthesis in GH-deficient dwarf rat muscle. reversal with GH, but not IGF-I, therapy.

Enhanced glucose flux via the hexosamine biosynthesis pathway (HNSP) has been implicated in insulin resistance. We measured L-glutamine:D-fructose-6-phosphate amidotransferase activity (GFAT, a rate-limiting enzyme) and concentrations of UDP-N-acetyl hexosamines (UDP-HexNAc, major products of HNSP) in muscle and liver of growth hormone (GH)-deficient male dwarf (dw) rats. All parameters measured, except body weight, were similar in 5-wk-old control and dw rats. Muscle GFAT activity declined progressively with age in controls and dw rats but was consistently 30-60% lower in 8- to 14-wk-old dw rats vs. age-matched controls; UDP-HexNAc concentrations in muscle were concomitantly 30% lower in dw rats vs. controls (P < 0.01). Concentrations of UDP-hexoses, GDP-mannose, and UDP in muscle were similar in control and dw rats. Muscle HNSP activity was similarly diminished in fed and fasted dw rats. In liver, only a small difference in GFAT activity was evident between controls and dw rats, and no differences in UDP-HexNAc concentrations were observed. Treatment with recombinant human GH (rhGH) for 5 days restored UDP-HexNAc to control levels in dw muscles (P < 0.01) and partially restored GFAT activity. Insulin-like growth factor I treatment was ineffective. We conclude that GH participates in HNSP regulation in muscle.

Hyperglycemia inhibits insulin activation of Akt/protein kinase B but not phosphatidylinositol 3-kinase in rat skeletal muscle.

Sustained hyperglycemia impairs insulin-stimulated glucose utilization in the skeletal muscle of both humans and experimental animals--a phenomenon referred to clinically as glucose toxicity. To study how this occurs, a model was developed in which hyperglycemia produces insulin resistance in vitro. Rat extensor digitorum longus muscles were preincubated for 4 h in Krebs-Henseleit solution containing glucose or glucose + insulin at various concentrations, after which insulin action was studied. Preincubation with 25 mmol/l glucose + insulin (10 mU/ml) led to a 70% decrease in the ability of insulin (10 mU/ml) to stimulate glucose incorporation into glycogen and a 30% decrease in 2-deoxyglucose (2-DG) uptake, compared with muscles incubated with 0 mmol/l glucose. Glucose incorporation into lipid and its oxidation to CO2 were marginally diminished, if at all. The alterations of glycogen synthesis and 2-DG uptake were first evident after 1 h and were maximal after 2 h of preincubation; they were not observed in muscles preincubated with 25 mmol/l glucose + insulin for 5 min. Preincubation for 4 h with 25 mmol/l glucose in the absence of insulin produced a similar although somewhat smaller decrease in insulin-stimulated glycogen synthesis; however, it did not alter 2-DG uptake, glucose oxidation to CO2, or incorporation into lipids. Studies of insulin signaling in the latter muscles revealed that activation of Akt/protein kinase B (PKB) was diminished by 60%, compared with that of muscles preincubated in a glucose-free medium; whereas activation of phosphatidylinositol (PI) 3-kinase, an upstream regulator of Akt/PKB in the insulin-signaling cascade, and of mitogen-activated protein (MAP) kinase, a parallel signal, was unaffected. Immunoblots demonstrated that this was not due to a change in Akt/PKB abundance. The results indicate that hyperglycemia-induced insulin resistance can be studied in rat skeletal muscle in vitro. They suggest that impairment of insulin action in these muscles is related to inhibition of Akt/PKB by events that do not affect PI 3-kinase.

Congenital thyroid disease revisited: migrational anomalies and dyshormonogenesis.

Nuclear medicine scintigraphy, in conjunction with other diagnostic tools, plays a vital role in assessing patients with congenital migrational anomalies. We present 5 patients, 4 of whom were hypothyroid and 1 patient who was euthyroid. Scintigraphic and ultrasonographic images were examined and correlated with laboratory results. These patients demonstrate the spectrum of migrational anomalies of the thyroid. Nuclear medicine scintigraphy was a valuable aid in establishing the location of the ectopic thyroid as well as its function.

Diabetes research in South Carolina: past, present, and future.

Medical investigators in South Carolina have been on the "cutting edge" of diabetes research for a number of decades. Despite this fact, our state ranks second in the nation in diabetes prevalence, and diabetes complications are more severe here than anywhere else. It is from the efforts of these investigators that our hope for a brighter future comes. Through a concerted effort toward prevention, improvements in care, and investigation of the pathophysiology of diabetes and its complications, researchers may reduce the substantial burden of diabetes in our state and throughout the world.

Human recombinant alpha 2-HS glycoprotein is produced in insect cells as a full length inhibitor of the insulin receptor tyrosine kinase.

Human Alpha 2-HS glycoprotein (AHSG), a glycoprotein synthesized by hepatocytes, was expressed in insect cells using the recombinant baculovirus system. The protein was purified from the cell supernatant, and appeared as a single band at about 52 kDa. Western blot using a specific antibody to the B-chain of AHSG indicated that the connecting peptide was present in the protein. When incubated with solubilized insulin receptors, recombinant AHSG inhibited the tyrosine kinase activity of the receptors in a dose-dependent fashion at concentrations in the range of those of the circulating protein. AHSG did not interfere with the binding of insulin to its receptor. These results indicate that human AHSG represents a natural inhibitor of the insulin receptor tyrosine kinase, is active as a single-chain protein and possesses a biological role similar to that of its homologue in rats, pp63, described by Auberger et al. (1).

Increased activity of the hexosamine synthesis pathway in muscles of insulin-resistant ob/ob mice.

Enhanced glucose flux via the hexosamine biosynthetic pathway has been implicated in insulin resistance. We measured products of this pathway, UDP-N-acetyl hexosamines (UDP-HexNAc), and activity of the rate-limiting enzyme L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) in tissues of ob/ob mice and lean controls. Ob/ob mice were obese, hyperglycemic, and hyperinsulinemic. Resistance to the effect of insulin on glucose transport was demonstrated in isolated soleus muscles, although total GLUT-4 concentration was mildly increased in muscles from ob/ob mice. UDP-HexNAc concentrations in hindlimb muscles decreased between 8 and 17 wk but were always higher in ob/ob vs. controls (P < 0.001, mean increase 67%). Concentrations of UDP-hexoses and GDP-mannose were similar in ob/ob and control muscles. Muscle GFAT activity declined with age but was increased in ob/ob vs. controls at each age examined (P < 0.001, mean increase 108%). UDP-HexNAc concentrations and GFAT activity were similar in livers of ob/ob and controls. These data suggest that glucose flux via the hexosamine pathway is selectively increased in muscle but not liver of ob/ob mice and may contribute to muscle insulin resistance in this model of non-insulin-dependent diabetes mellitus.

Effects of exercise and feeding on the hexosamine biosynthetic pathway in rat skeletal muscle.

Products of the hexosamine biosynthesis pathway (HSNP) have been implicated in glucose-induced insulin resistance. We measured the major products of HSNP, UDP-N-acetyl hexosamines (UDP-HexNAc), and the activity of L-glutamine: D-fructose-6-phosphate amidotransferase (GFAT, rate-limiting enzyme) in rat hindlimb muscles immediately after exercise and 1, 3, and 16 h postexercise (swimming) in fed and fasted rats and sedentary controls. Muscle glycogen decreased by 50-75% postexercise. In sedentary rats, muscle GFAT activity decreased by approximately 30% (P < 0.002) after an 18-h fast. GFAT activity was not affected by exercise under any condition. Muscle UDP-HexNAc increased approximately 30% postexercise (P < 0.01) in ad libitum-fed but not in fasted rats. UDP-HexNAc remained elevated (approximately 30%, P < 0.002) for 16 h if animals were fed postexercise. Concentrations of UDP-hexoses, GDP-mannose, and UDP were unchanged postexercise. Conclusions are that muscle GFAT activity is regulated by the nutritional state but not by acute exercise. Glucose flux via HNSP may be increased postexercise in muscles of ad libitum-fed rats. Increased HSNP products may serve as negative feedback regulators to limit excessive muscle glycogen deposition postexercise.

Effects of overexpression of glutamine:fructose-6-phosphate amidotransferase (GFAT) and glucosamine treatment on translocation of GLUT4 in rat adipose cells.

Insulin resistance is associated with diabetes. Hyperglycemia per se causes insulin resistance as well as increased flux of glucose through the hexosamine biosynthetic pathway. The rate-limiting enzyme for entry of glucose into this pathway is glutamine:fructose-6-phosphate amidotransferase (GFAT). To directly evaluate the role of GFAT in modulating insulin-stimulated glucose transport, we co-transfected primary cultures of rat adipose cells with expression vectors for human GFAT as well as an epitope-tagged GLUT4 and examined the effect of overexpressed GFAT on insulin-stimulated translocation of GLUT4. When we measured cell surface tagged GLUT4 in response to insulin, cells overexpressing GFAT and tagged GLUT4 had an insulin-dose response curve that was similar to that of control cells expressing only tagged GLUT4. As an alternative means of increasing flux through the hexosamine biosynthetic pathway, we incubated adipose cells with glucosamine (a substrate of the pathway downstream from GFAT) and insulin. Interestingly, for short incubation times (4 h) we observed a decrease in both basal and insulin-stimulated glucose transport without a detectable effect on insulin-stimulated translocation of GLUT4. However, for longer incubation times (16 h), we observed a significant decrease in the amount of GLUT4 in the plasma membrane. Our data suggest that products of the hexosamine biosynthetic pathway may cause insulin resistance, in part, by acutely decreasing intrinsic activity of GLUT4 as well as chronically altering the amount of GLUT4 at the cell surface.

Differential effects of GLUT1 or GLUT4 overexpression on hexosamine biosynthesis by muscles of transgenic mice.

Transgenic mice that overexpress GLUT1 or GLUT4 in skeletal muscle were studied; the former but not the latter develop insulin resistance. Because increased glucose flux via the hexosamine biosynthesis pathway has been implicated in glucose-induced insulin resistance, we measured the activity of glutamine:fructose-6-phosphate amidotransferase (GFAT; rate-limiting enzyme) and the concentrations of UDP-N-acetyl hexosamines (major products of the pathway) as well as UDP-hexoses and GDP-mannose in hind limb muscles and liver in both transgenic models and controls. GFAT activity was increased 60-70% in muscles of GLUT1 but not in GLUT4 transgenics. GFAT mRNA abundance was unchanged. The concentrations of all nucleotide-linked sugars were increased 2-3-fold in GLUT1 and were unchanged in GLUT4-overexpressing muscles. Similar results were obtained in fed and fasted mice. GFAT and nucleotide sugars were unchanged in liver, where the transgene is not expressed. We concluded that 1) glucose transport appears to be rate limiting for synthesis of nucleotide sugars; 2) chronically increased glucose flux increases muscle GFAT activity posttranscriptionally; 3) increased UDP-glucose likely accounts for the marked glycogen accumulation in muscles of GLUT1-overexpressing mice; and 4) glucose flux via the hexosamine biosynthetic pathway is increased in muscles of GLUT1-overexpressing but not GLUT4-overexpressing mice; products of the pathway may contribute to insulin resistance in GLUT1 transgenics.

Characterization of a mutant GLUT4 lacking the N-glycosylation site: studies in transfected rat adipose cells.

GLUT4, the insulin-responsive glucose transporter expressed primarily in muscle and adipose tissue, contains a single N-glycosylation site. We characterized a mutant GLUT4 lacking the N-glycosylation site (Asn57-->Gln) in primary cultures of rat adipose cells. We transiently transfected cells with expression vectors for epitope-tagged GLUT4 containing either wild-type (GLUT4-HA) or mutant (GLN57-HA) cDNA sequences. Expression of GLN57-HA in adipose cells was approximately 10-fold lower than for GLUT4-HA even though mRNA levels for both recombinant transporters were comparable. Biosynthetic labeling studies showed markedly decreased incorporation of [35S]-methionine/cysteine into GLN57-HA relative to GLUT4-HA consistent with either a decreased synthetic rate or accelerated degradation of GLN57-HA. Interestingly, transient transfection of GLUT4-HA and GLN57-HA in COS-7 cells (which do not express endogenous GLUT4) resulted in comparable levels of protein expression for both transporters. Thus, in the physiologically relevant adipose cell, glycosylation of GLUT4 appears to play an important functional role.

Synergistic effects of insulin and phorbol ester on mitogen-activated protein kinase in Rat-1 HIR cells.

Regulation of the activity of the extracellular signal regulated kinase (ERK) mitogen-activated protein kinases was examined in Rat-1 HIR, a fibroblast cell line overexpressing the human insulin receptor. Insulin or phorbol ester induced partial activations of ERKs, while a combination of insulin and phorbol ester resulted in a synergistic activation. Preincubation with phorbol ester increased the subsequent response to insulin. Phorbol ester did not enhance tyrosine phosphorylation of the insulin receptor. Insulin did not enhance activation of phospholipase D in response to phorbol ester. Lysophosphatidic acid also acted synergistically with insulin to induce ERK activation. Lysophosphatidic acid alone had little effect on ERK, and did not activate phospholipase D. The combination of phorbol ester and insulin maintained tyrosine phosphorylation of focal adhesion kinase, while insulin alone decreased its tyrosine phosphorylation. Phorbol ester induced phosphorylation of She on serine/threonine, while insulin induced tyrosine phosphorylation of She and She-Grb2 binding. These results suggest that full activation of ERKs in fibroblasts can require the cooperation of at least two signaling pathways, one of which may result from a protein kinase C-dependent phosphorylation of effectors regulating ERK activation. In this manner, phorbol esters may enhance mitogenic signals initiated by growth factor receptors.