Glucose transporter GLUT3: ontogeny, targeting, and role in the mouse blastocyst

The first differentiative event in mammalian development is segregation of the inner cell mass and trophectoderm (TE) lineages. The epithelial TE cells pump fluid into the spherical blastocyst to form the blastocyst cavity. This activity is fuelled by glucose supplied through facilitative glucose transporters. However, the reported kinetic characteristics of blastocyst glucose transport are inconsistent with those of the previously identified transporters and suggested the presence of a high-affinity glucose carrier. We identified and localized the primary transporter in TE cells. It is glucose transporter GLUT3, previously described in the mouse as neuron-specific. In the differentiating embryo, GLUT3 is targeted to the apical membranes of the polarized cells of the compacted morula and then to the apical membranes of TE cells where it has access to maternal glucose. In contrast, GLUT1 was restricted to basolateral membranes of the outer TE cells in both compacted morulae and blastocysts. Using antisense oligodeoxynucleotides to specifically block protein expression, we confirmed that GLUT3 and not GLUT1 is the functional transporter for maternal glucose on the apical TE. More importantly, however, GLUT3 expression is required for blastocyst formation and hence this primary differentiation in mammalian development. This requirement is independent of its function as a glucose transporter because blastocysts will form in the absence of glucose. Thus the vectorial expression of GLUT3 into the apical membrane domains of the outer cells of the morula, which in turn form the TE cells of the blastocyst, is required for blastocyst formation.     

Insulin-induced recruitment of glucose transporter 4 (GLUT4) and GLUT1 in isolated rat cardiac myocytes. Evidence of the existence of different intracellular GLUT4 vesicle populations

Using isolated rat cardiomyocytes we have examined: 1) the effect of insulin on the cellular distribution of glucose transporter 4 (GLUT4) and GLUT1, 2) the total amount of these transporters, and 3) the co-localization of GLUT4, GLUT1, and secretory carrier membrane proteins (SCAMPs) in intracellular membranes. Insulin induced 5.7- and 2.7-fold increases in GLUT4 and GLUT1 at the cell surface, respectively, as determined by the nonpermeant photoaffinity label [3H]2-N-[4(1-azi-2,2,2-trifluoroethyl)benzoyl]-1, 3-bis-(D-mannos-4-yloxy)propyl-2-amine. The total amount of GLUT1, as determined by quantitative Western blot analysis of cell homogenates, was found to represent a substantial fraction ( approximately 30%) of the total glucose transporter content. Intracellular GLUT4-containing vesicles were immunoisolated from low density microsomes by using monoclonal anti-GLUT4 (1F8) or anti-SCAMP antibodies (3F8) coupled to either agarose or acrylamide. With these different immunoisolation conditions two GLUT4 membrane pools were found in nonstimulated cells: one pool with a high proportion of GLUT4 and a low content in GLUT1 and SCAMP 39 (pool 1) and a second GLUT4 pool with a high content of GLUT1 and SCAMP 39 (pool 2). The existence of pool 1 was confirmed by immunotitration of intracellular GLUT4 membranes with 1F8-acrylamide. Acute insulin treatment caused the depletion of GLUT4 in both pools and of GLUT1 and SCAMP 39 in pool 2. In conclusion: 1) GLUT4 is the major glucose transporter to be recruited to the surface of cardiomyocytes in response to insulin; 2) these cells express a high level of GLUT1; and 3) intracellular GLUT4-containing vesicles consist of at least two populations, which is compatible with recently proposed models of GLUT4 trafficking in adipocytes.     

Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid

Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1-5 and SGLT1. The apparent Km of DHA transport via GLUT1 and GLUT3 was 1.1 +/- 0.2 and 1.7 +/- 0.3 mM, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2-4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, D-glucose, and 3-O-methylglucose among other hexoses while fructose and L-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-alpha-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2-8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA.     

Glucose transporter expression in brain: relationship to cerebral glucose utilization

Glucose is the principle energy source for mammalian brain. Delivery of glucose from the blood to the brain requires its transport across the endothelial cells of the blood-brain barrier and across the plasma membranes of neurons and glia, which is mediated by the facilitative glucose transporter proteins. The two primary glucose transporter isoforms which function in cerebral glucose metabolism are GLUT1 and GLUT3. GLUT1 is the primary transporter in the blood-brain barrier, choroid plexus, ependyma, and glia; GLUT3 is the neuronal glucose transporter. The levels of expression of both transporters are regulated in concert with metabolic demand and regional rates of cerebral glucose utilization. We present several experimental paradigms in which alterations in energetic demand and/or substrate supply affect glucose transporter expression. These include normal cerebral development in the rat, Alzheimer's disease, neuronal differentiation in vitro, and dehydration in the rat.     

Unmyelinated nerve fibers in senile nerves and in late thalidomide neuropathy: a quantitative electron microscopic study (author's transl)

Sural nerve biopsies of four patients, aged 54--76 years, with a predominantly sensory type of neuropathy following high dosages of thalidomide were examined by light and electron microscopy. The present study includes a qualitative and quantitative evaluation of unmyelinated nerve fibers. Despite severe neuropathy, increased numbers of small unmyelinated axons per endoneurial area were noted in all patients. This numerical increase appeared to be independent of aging, since it was not seen in two senile controls, studied at the age of 83 and 88 years. The increase in the endoneurial density of unmyelinated axons, especially of small sized fibers, is likely to be related to regeneration following degeneration of unmyelinated axons although endoneurial shrinkage secondary to loss of large myelinated fibers could have caused an additional increase in the number of axons per endoneurial area. Axonal sprouting, despite degeneration of large numbers of myelinated and unmyelinated fibers, appears to be consistent with some of the characteristic clinical features of thalidomide neuropathy such as paresthesias, hyperesthesia for pain and temperature, and disturbances of autonomic functions. On the other hand, a variable number of empty Schwann cells (bands of Büngner) and pockets at the surface of many Schwann cells noted in the four patients with neuropathy were also seen in both senile controls with no signs of neuropathy. Thus, it is obvious that pockets and empty Schwann cells may be related to aging or other causes of slow axonal wasting with Schwann cell proliferation and are not necessarily associated with clinically manifest neuropathy.     

Microdissection of peripheral nerve: collagen and lipid distribution with morphological correlation

Peripheral nerve is a complex tissue composed of endoneurial fascicles surrounded by perineurium and epineurium. We separated endoneurium from peri- and epineurium in human sural nerves by "endoneurial plucking", a method of microdissection. Endoneurial contents (axons, myelin sheaths, Schwann cells, vessels, and interstitial collagen) were cleanly separated in high yield from enveloping connective tissue, by both microscopic and biochemical criteria. Most of the nerve sulfatide and unesterified sterol was found in the endoneurial fraction while most of the collagen was in the peri-epineurial fraction. This microdissection method should prove useful in biochemical investigations of peripheral nerve.     

Glucose ingestion causes GLUT4 translocation in human skeletal muscle

In humans, ingestion of carbohydrates causes an increase in blood glucose concentration, pancreatic insulin release, and increased glucose disposal into skeletal muscle. The underlying molecular mechanism for the increase in glucose disposal in human skeletal muscle after carbohydrate ingestion is not known. We determined whether glucose ingestion increases glucose uptake in human skeletal muscle by increasing the number of glucose transporter proteins at the cell surface and/or by increasing the activity of the glucose transporter proteins in the plasma membrane. Under local anesthesia, approximately 1 g of vastus lateralis muscle was obtained from six healthy subjects before and 60 min after ingestion of a 75-g glucose load. Plasma membranes were isolated from the skeletal muscle and used to measure GLUT4 and GLUT1 content and glucose transport in plasma membrane vesicles. Glucose ingestion increased the plasma membrane content of GLUT4 per gram muscle (3,524 +/- 729 vs. 4,473 +/- 952 arbitrary units for basal and 60 min, respectively; P < 0.005). Transporter-mediated glucose transport into plasma membrane vesicles was also significantly increased (130 +/- 11 vs. 224 +/- 38 pmol.mg-1.s-1; P < 0.017), whereas the calculated ratio of glucose transport to GLUT4, an indication of transporter functional activity, was not significantly increased 60 min after glucose ingestion (2.3 +/- 0.4 vs. 3.0 +/- 0.5 pmol.GLUT4 arbitrary units-1.s-1; P < 0.17). These results demonstrate that oral ingestion of glucose increases the rate of glucose transport across the plasma membrane and causes GLUT4 translocation in human skeletal muscle. These findings suggest that under physiological conditions the translocation of GLUT4 is an important mechanism for the stimulation of glucose uptake in human skeletal muscle.     

Analysis of the glucose transporter compliment of metabolically important tissues from the Milan hypertensive rat

Hypertension is frequently associated with peripheral insulin resistance. An expanding body of evidence has described aberrant expression of glucose transporters in the insulin resistance associated with diabetes mellitus. Therefore, we have investigated the relative levels of expression and subcellular distribution of four members of the facilitative glucose transporter family in metabolically important tissues from the hypertensive Milan rat. Skeletal muscle is the major site of peripheral glucose disposal; skeletal muscle membranes isolated from hypertensive animals exhibited a profoundly reduced level of GLUT4 protein compared to normotensive control animals This reduction was confined to the intracellular pool which exhibited a 50% lower level of GLUT4. In contrast, adipocytes, the other major site of peripheral glucose disposal, exhibited no change in the levels of expression of either GLUT1 or GLUT4 transporter isoforms. Hepatocytes from hypertensive animals exhibit similar levels of GLUT2 protein to the normotensive controls. Patterns of expression of GLUT1, GLUT3 and GLUT4 as determined by immunoblot analysis were profoundly altered in certain brain regions in the hypertensive state. Given the importance of the GLUT4 isoform in mediating the insulin-stimulated disposal of glucose into peripheral tissues, the observation that muscle exhibits profoundly decreased levels of this transporter has important implications for the insulin-resistance associated with hypertension in these animals.     

Glucose transporter isoforms in brain: absence of GLUT3 from the blood-brain barrier

Two glucose transporter (GLUT) isoforms have been identified in brain. The GLUT1 isoform is abundant in cerebral microvessels and may be present in glia and neurons, whereas GLUT3 is probably the major neuronal glucose transporter. This study investigates whether GLUT3 is also present in microvessels from rat, human, and canine brain, by means of antisera directed against the divergent C-terminal sequences of mouse and human GLUT3. GLUT1 was detected in whole brain as two molecular mass forms: 55 kDa in microvessels and 45 kDa in cortical neuronal/glial membranes. With the aid of the appropriate antisera to the species-specific sequences, GLUT3 was detected in rat and human cortical membranes but not in isolated rat or human microvessels. These antisera failed to detect GLUT3 in either canine cortical membranes or canine microvessels, implying additional species specificity in the C-terminal sequence.     

Modulation of expression of glucose transporters GLUT3 and GLUT1 by potassium and N-methyl-D-aspartate in cultured cerebellar granule neurons

Depolarization is known to stimulate neuronal oxidative metabolism. As glucose is the primary fuel for oxidative metabolism in the brain, the entry of glucose into neural cells is a potential control point for any regulatory events in brain metabolism. Therefore, the effects of depolarizing stimuli, high K+ and N-methyl-D-aspartate (NMDA), were examined on the functional expression of glucose transporter isoforms GLUT1 and GLUT3 in primary cultured cerebellar granule neurons. Higher levels of glucose transport activity were observed in neurons cultured in 25 mM KCl (K25) compared to those in 5 and 15 mM KCl (K5 and K15). The elevated glucose transport activity correlated with increased levels of GLUT3 protein and, to a lesser extent, GLUT1. Both GLUT3 and GLUT1 were regulated at the level of mRNA expression. Addition of NMDA to K5 and K15 cultures increased both glucose uptake and GLUT3 protein levels, with smaller changes in GLUT1. NMDA effects were not additive with K25 effects. All these changes were observed only with chronic exposure of neurons to high K+ or NMDA; no acute effects on glucose uptake or transporter expression were found. Thus, chronic depolarization of primary cerebellar granule neurons acts as a stimulus for the expression of the neuronal GLUT3 glucose transporter isoform.     

Different functional domains of GLUT2 glucose transporter are required for glucose affinity and substrate specificity

GLUT2 is the major glucose transporter in pancreatic beta-cells and hepatocytes. It plays an important role in insulin secretion from beta-cells and glucose metabolism in hepatocytes. To better understand the molecular determinants for GLUT2's distinctive glucose affinity and its ability to transport fructose, we constructed a series of chimeric GLUT2/GLUT3 proteins and analyzed them in both Xenopus oocytes and mammalian cells. The results showed the following. 1) GLUT3/GLUT2 chimera containing a region from transmembrane segment 9 to part of the COOH-terminus of GLUT2 had Km values for 3-O-methylglucose similar to those of wild-type GLUT2. Further narrowing of the GLUT2 component in the chimeric GLUTs lowered the Km values to those of wild-type GLUT3. 2) GLUT3/GLUT2 chimera containing a region from transmembrane segment 7 to part of the COOH-terminus of GLUT2 retained the ability to transport fructose. Further narrowing of this region in the chimeric GLUTs resulted in a complete loss of the fructose transport ability. 3) Chimeric GLUTs with the NH2-terminal portion of GLUT2 were unable to express glucose transporter proteins in either Xenopus oocytes or mammalian RIN 1046-38 cells. These results indicate that amino acid sequences in transmembrane segments 9-12 are primarily responsible for GLUT2's distinctive glucose affinity, whereas amino acid sequences in transmembrane segments 7-8 enable GLUT2 to transport fructose. In addition, certain region(s) of the amino-terminus of GLUT2 impose strict structural requirements on the carboxy-terminus of the glucose transporter protein. Interactions between these regions and the carboxy-terminus of GLUT2 are essential for GLUT2 expression.     

A Leu-Leu sequence is essential for COOH-terminal targeting signal of GLUT4 glucose transporter in fibroblasts

In the insulin-responsive tissues, muscle and adipose, the GLUT4 glucose transporter isoform accounts for most of the increase in hexose flux in response to hormone. In these cell types, as well as in fibroblasts transfected with cDNAs encoding the transporters, GLUT1 and GLUT4 are sorted to different subcellular locations. In the latter, GLUT1 is found primarily at the cell surface whereas GLUT4 localizes to the interior of the cell in a perinuclear distribution. The construction and analysis of chimeras of these two transporter isoforms have allowed identification of the COOH-terminal 30 amino acids as a critical sorting signal for differential localization of the transporters. In this study, we show that 2 residues present in the GLUT4 COOH terminus, Leu-489 and Leu-490, are critical for the intracellular sequestration of this isoform in NIH3T3 cells.     

Glycaemic regulation of glucose transporter expression and activity in the human placenta

To determine whether the expression and activity of glucose transporters in human trophoblast are regulated by glucose, syncytiotrophoblast cells, choriocarcinoma cells, and villous fragments were incubated with a range of glucose concentrations (0-20 mM, 24 h). Expression of GLUT1 and GLUT3 glucose transporters was measured by immunoblotting, while glucose transporter activity was determined by [3H]2-deoxyglucose uptake in the cultured cells. GLUT1 expression in syncytial cells was enhanced following incubation in absence of glucose, reduced by incubation in 20 mM glucose but was not altered by incubation at 1 or 12 mM glucose. Transporter activity was inversely related to extracellular glucose over the entire range of concentrations tested (0-20 mM). Incubation of villous fragments in 20 mM glucose produced a limited suppression of GLUT1 expression, but no effects were noted following incubation at 0 or 1 mM glucose. Neither GLUT1 expression in JAr and JEG-3 choriocarcinoma cells nor transport activity in JEG-3 cells was affected by extracellular glucose concentration. Unlike syncytial cells, JAr, JEG-3 and BeWo all expressed GLUT3 protein in addition to GLUT1. These results show that while syncytiotrophoblast GLUT1 expression is altered at the extremes of extracellular glucose concentration, it is refractory to glucose alone at lower concentrations. By contrast, an inverse relationship exists between glucose transporter activity and extracellular glucose. This suggests that there are post-translational regulatory mechanisms which may respond to changes in extracellular glucose concentration.     

Persistent myocardial ischemia increases GLUT1 glucose transporter expression in both ischemic and non-ischemic heart regions

Persistently ischemic myocardium exhibits increased glucose uptake which may contribute to the preservation of myocardial function and viability. Little is known about the specific molecular events which are responsible for this increase in uptake. Therefore, we investigated whether myocardial ischemia induces the gene expression of the major cardiac facilitative glucose transporters, GLUT4 and GLUT1. We determined the expression of myocardial glucose transporter mRNAs and polypeptides after 6 h of regional ischemia in a dog model by semi-quantitative Northern blotting and immunoblotting. GLUT1 but not GLUT4 expression was significantly increased in both ischemic and non-ischemic regions from the experimental hearts when compared to surgical control and normal hearts. GLUT1 mRNA expression was increased 3.4-fold and GLUT1 polypeptide expression was increased 1.7-fold in ischemic hearts when compared to normal or surgical-control hearts. There were no significant regional differences in GLUT1 expression in either normal or ischemic hearts. However, there was a tendency for GLUT1 mRNA expression to be highest in the non-ischemic regions from the 6-h ischemia hearts. These findings suggest that myocardial ischemia induces a factor or factors which stimulate GLUT1 expression in non-ischemic as well as ischemic myocardial regions. Increased GLUT1 expression may play a role in augmenting glucose uptake during ischemia.     

Peripheral but not hepatic insulin resistance in mice with one disrupted allele of the glucose transporter type 4 (GLUT4) gene

Glucose transporter type 4 (GLUT4) is insulin responsive and is expressed in striated muscle and adipose tissue. To investigate the impact of a partial deficiency in the level of GLUT4 on in vivo insulin action, we examined glucose disposal and hepatic glucose production (HGP) during hyperinsulinemic clamp studies in 4-5-mo-old conscious mice with one disrupted GLUT4 allele [GLUT4 (+/-)], compared with wild-type control mice [WT (+/+)]. GLUT4 (+/-) mice were studied before the onset of hyperglycemia and had normal plasma glucose levels and a 50% increase in the fasting (6 h) plasma insulin concentrations. GLUT4 protein in muscle was approximately 45% less in GLUT4 (+/-) than in WT (+/+). Euglycemic hyperinsulinemic clamp studies were performed in combination with [3-3H]glucose to measure the rate of appearance of glucose and HGP, with [U-14C]-2-deoxyglucose to estimate muscle glucose transport in vivo, and with [U-14C]lactate to assess hepatic glucose fluxes. During the clamp studies, the rates of glucose infusion, glucose disappearance, glycolysis, glycogen synthesis, and muscle glucose uptake were approximately 55% decreased in GLUT4 (+/-), compared with WT (+/+) mice. The decreased rate of in vivo glycogen synthesis was due to decreased stimulation of glucose transport since insulin's activation of muscle glycogen synthase was similar in GLUT4 (+/-) and in WT (+/+) mice. By contrast, the ability of hyperinsulinemia to inhibit HGP was unaffected in GLUT4 (+/-). The normal regulation of hepatic glucose metabolism in GLUT4 (+/-) mice was further supported by the similar intrahepatic distribution of liver glucose fluxes through glucose cycling, gluconeogenesis, and glycogenolysis. We conclude that the disruption of one allele of the GLUT4 gene leads to severe peripheral but not hepatic insulin resistance. Thus, varying levels of GLUT4 protein in striated muscle and adipose tissue can markedly alter whole body glucose disposal. These differences most likely account for the interindividual variations in peripheral insulin action.     

Dissociation of GLUT4 translocation and insulin-stimulated glucose transport in transgenic mice overexpressing GLUT1 in skeletal muscle

Overexpression of the human GLUT1 glucose transporter protein in skeletal muscle of transgenic mice results in large increases in basal glucose transport and metabolism, but impaired stimulation of glucose transport by insulin, contractions, or hypoxia (Gulve, E. A., Ren, J.-M., Marshall, B. A., Gao, J., Hansen, P. A., Holloszy, J. O. , and Mueckler, M. (1994) J. Biol. Chem. 269, 18366-18370). This study examined the relationship between glucose transport and cell-surface glucose transporter content in isolated skeletal muscle from wild-type and GLUT1-overexpressing mice using 2-deoxyglucose, 3-O-methylglucose, and the 2-N-[4-(1-azi-2,2, 2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos-4-yloxy)-2-propyl amine exofacial photolabeling technique. Insulin (2 milliunits/ml) stimulated a 3-fold increase in 2-deoxyglucose uptake in extensor digitorum longus muscles of control mice (0.47 +/- 0.07 micromol/ml/20 min in basal muscle versus 1.44 micromol/ml/20 min in insulin-stimulated muscle; mean +/- S.E.). Insulin failed to increase 2-deoxyglucose uptake above basal rates in muscles overexpressing GLUT1 (4.00 +/- 0.40 micromol/ml/20 min in basal muscle versus 3.96 +/- 0.37 micromol/ml/20 min in insulin-stimulated muscle). A similar lack of insulin stimulation in muscles overexpressing GLUT1 was observed using 3-O-methylglucose. However, the magnitude of the insulin-stimulated increase in cell-surface GLUT4 photolabeling was nearly identical (approximately 3-fold) in wild-type and GLUT1-overexpressing muscles. This apparently normal insulin-stimulated translocation of GLUT4 in GLUT1-overexpressing muscle was confirmed by immunoelectron microscopy. Our findings suggest that GLUT4 activity at the plasma membrane can be dissociated from the plasma membrane content of GLUT4 molecules and thus suggest that the intrinsic activity of GLUT4 is subject to regulation.     

Distinct morphological characteristics of touch, temperature, and mechanical nociceptive neurons in the crotaline trigeminal ganglia

Intrasomal recording and horseradish peroxidase injection techniques were employed in vivo to determine the morphological characteristics of touch, temperature, and mechanical nociceptive neurons in the trigeminal ganglia of crotaline snakes. The touch neurons, with a peripheral axon conducting at the A-beta range, could be subdivided into tactile and vibrotactile neurons according to their response properties, but there were no morphological differences between them. These neurons exhibited a large and oval soma and possessed a set of large stem, peripheral, and central axons which were all myelinated and equal in diameter with a constriction at the bifurcation. The temperature neurons, which conducted peripherally at the A-delta range, were physiologically separated into thermosensitive and thermo-mechanosensitive neurons, which were also morphologically indistinguishable. The temperature neurons had a round soma of medium size and a set of medium axons with varied axonal bifurcation patterns. All axons of these neurons were myelinated, but the central axon was thinner than the stem and peripheral axons. The mechanical nociceptive neurons, which had a peripheral axon conducting at the A-delta range, were morphologically heterogeneous based on their conduction velocities. The neurons conducting at the fast A-delta range were morphologically similar to the temperature neurons in the ganglion excepting their thinner central axons, whereas those at the slow A-delta range had a thinner myelinated stem axon that gave rise to a thinner myelinated peripheral axon and an unmyelinated stem axon with a bifurcation of either a triangular expansion at the bifurcating point or a central axon arising straightforwardly from the constant stem and peripheral axons. This study revealed that distinct morphological characteristics do exist for the touch and temperature neurons and the subtypes of mechanical nociceptive neurons in the trigeminal ganglion, but not for the subfunctional types of touch neurons or temperature neurons.     

Regional variation in adipose tissue insulin action and GLUT4 glucose transporter expression in severely obese premenopausal women

Insulin action and GLUT4 expression were examined in adipose tissue of severely obese premenopausal women undergoing gastrointestinal surgery. Fat samples were taken from three different anatomical regions: the subcutaneous abdominal site, the round ligament (deep abdominal properitoneal fat), and the greater omentum (deep abdominal intraperitoneal fat). The stimulatory effect of insulin on glucose transport and the ability of the hormone to inhibit lipolysis were determined in adipocytes isolated from these three adipose depots. Insulin stimulated glucose transport 2-3 times over basal rates in all adipocytes. However, round ligament adipose cells showed a significantly greater responsiveness to insulin when compared to subcutaneous and omental adipocytes. Round ligament fat cells also displayed the greatest sensitivity and maximal antilipolytic response to insulin. We also investigated whether regional differences in fat cell insulin-stimulated glucose transport were linked to a differential expression of the GLUT4 glucose transporter. GLUT4 protein content in total membranes was 5 and 2.2 times greater in round ligament adipose tissue than in subcutaneous and omental fat depots, respectively. Moreover, GLUT4 mRNA levels were 2.1 and 3 times higher in round ligament than in subcutaneous or omental adipose tissues, respectively. Adipose tissue GLUT4 protein content was strongly and negatively associated (r = -0.79 to -0.89, p < 0.01) with the waist-to-hip ratio but not with total adiposity. In conclusion, these results demonstrate the existence of site differences in adipose tissue insulin action in morbidly obese women. The greater insulin effect on glucose transport in round ligament adipocytes was associated with a higher expression of GLUT4 when compared to subcutaneous abdominal and omental fat cells. Moreover, despite the regional variation in GLUT4 expression, an increased proportion of abdominal fat was found to be associated with lower levels of GLUT4 in all adipose regions investigated.     

Purification of the insecticidal toxin in crystals of Bacillus thuringiensis

Newly transected or denervated segments of isogeneic rat tibial nerve were implanted into the rat midbrain and sampled at weekly intervals up to 6 weeks post-operation. By 3 weeks, the peripheral nervous system (PNS) grafts were well-vascularized and contained Schwann cells, axons associated with Schwann cell processes, and macrophages. From 3 to 6 weeks, many axons within both the fresh and predegenerated grafts were myelinated by Schwann cells. The nerve fiber arrangement within the implant was similar to that of regenerating peripheral nerve in situ. The central nervous system (CNS) border of the implant was clearly demarcated by a rim of astrocytes behind which was a layer of regenerating oligodendrocytes and axons. Extending from the CNS margin were radial bridges of astroglial tissue which apprarently guided regenerating axons into the implant. Between the CNS and the PNS implant, abundant collagen deposition was present. The findings suggest that regenerating CNS axons grow via astroglial bridges into transplanted PNS tissue and are capable of stimulating the implanted Schwann cells to form myelin. Even Schwann cells deprived of axonal contact for prolonged periods were still capable of PNS myelin formation.