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1.
Biochim Biophys Acta ; 1499(1-2): 74-84, 2000 Dec 11.
Article in English | MEDLINE | ID: mdl-11118640

ABSTRACT

Glucose deprivation leads to the synthesis of an aberrantly glycosylated ('alternative') and inefficiently processed form of the insulin proreceptor in 3T3-L1 adipocytes. To further explore the effect of aberrant (rather than absent) N-linked glycosylation of the insulin receptor, we examined the relationship of processing to function. Our studies show that the alternative form of the proreceptor does not oligomerize nor does it acquire the ability to undergo insulin-sensitive autophosphorylation. This along with an interaction with the glucose-regulated stress protein GRP78/BiP implies inappropriate folding/dimerization and retention in the ER. Glucose refeeding causes the post-translational modification of the alternative form of the proreceptor to a novel 'intermediate' form which is independent of new protein synthesis. As little as 100 microM glucose (or mannose) can induce this modification. In vitro digestion of the alternative and intermediate proreceptors with SPC1/furin shows that both the alpha- and beta-subunit domains are glycosylated, albeit aberrantly. This implies that the aberrantly glycosylated proreceptor could serve as a substrate for SPC1 in a physiological setting if the receptor was able to interact with the enzyme in the appropriate compartment (i.e., the trans-Golgi network). Based on inhibitor studies, however, both the alternative and intermediate forms of the proreceptor appear to be primarily targeted to the proteasome for degradation.


Subject(s)
Heat-Shock Proteins , Receptor, Insulin/metabolism , Schizosaccharomyces pombe Proteins , 3T3 Cells , Animals , Carrier Proteins/metabolism , Cell Compartmentation , Cysteine Endopeptidases/chemistry , Dose-Response Relationship, Drug , Endoplasmic Reticulum Chaperone BiP , Glucose/deficiency , Glucose/pharmacology , Glycosylation , Mice , Mitogen-Activated Protein Kinases/chemistry , Molecular Chaperones/metabolism , Multienzyme Complexes/chemistry , Phosphorylation , Proteasome Endopeptidase Complex , Protein Folding , Protein Precursors/biosynthesis , Protein Precursors/metabolism , Receptor, Insulin/biosynthesis , Receptor, Insulin/chemistry , Signal Transduction
2.
Biochem Biophys Res Commun ; 273(3): 859-64, 2000 Jul 14.
Article in English | MEDLINE | ID: mdl-10891337

ABSTRACT

We have previously demonstrated that glucose deprivation alters the glycosylation of the GLUT1 glucose transporter in 3T3-L1 adipocytes. Many aberrantly glycosylated proteins are retained in the endoplasmic reticulum by interaction with chaperones. Herein, we use three independent procedures to show that GLUT1 is targeted to the plasma membrane, despite alterations in glycosylation. While earlier experiments revealed that plasma membrane targeting of aglyco GLUT 1 transporter was significantly reduced, our data show for the first time that altered glycosylation provides sufficient information to drive appropriate trafficking.


Subject(s)
Adipocytes/drug effects , Glucose/pharmacology , Monosaccharide Transport Proteins/metabolism , 3T3 Cells , Adipocytes/metabolism , Animals , Biotin/metabolism , Cell Fractionation , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Glucose Transporter Type 1 , Glycosylation , Mice
3.
J Biol Chem ; 274(32): 22813-20, 1999 Aug 06.
Article in English | MEDLINE | ID: mdl-10428866

ABSTRACT

The mature insulin receptor is a cell surface heterotetrameric glycoprotein composed of two alpha- and two beta-subunits. In 3T3-L1 adipocytes as in other cell types, the receptor is synthesized as a single polypeptide consisting of uncleaved alpha- and beta-subunits, migrating as a 190-kDa glycoprotein. To examine the importance of N-linked glycosylation on insulin receptor processing, we have used glucose deprivation as a tool to alter protein glycosylation. Western blot analysis shows that glucose deprivation led to a time-dependent accumulation of an alternative proreceptor of 170 kDa in a subcellular fraction consistent with endoplasmic reticulum localization. Co-precipitation assays provide evidence that the alternative proreceptor bound GRP78, an endoplasmic reticulum molecular chaperone. N-Glycosidase F treatment shows that the alternative proreceptor contained N-linked oligosaccharides. Yet, endoglycosidase H insensitivity indicates an aberrant oligosaccharide structure. Using pulse-chase methodology, we show that the synthetic rate was similar between the normal and alternative proreceptor. However, the normal proreceptor was processed into alpha- and beta-subunits (t((1)/(2)) = 1.3 +/- 0.6 h), while the alternative proreceptor was degraded (t((1)/(2)) = 5.1 +/- 0.6 h). Upon refeeding cells that were initially deprived of glucose, the alternative proreceptor was processed to a higher molecular weight form and gained sensitivity to endoglycosidase H. This "intermediate" form of the proreceptor was also degraded, although a small fraction escaped degradation, resulting in cleavage to the alpha- and beta-subunits. These data provide evidence for the first time that glucose deprivation leads to the accumulation of an alternative proreceptor, which can be post-translationally glycosylated with the readdition of glucose inducing both accelerated degradation and maturation.


Subject(s)
Endoplasmic Reticulum/metabolism , Glycoproteins/metabolism , Heat-Shock Proteins , Protein Precursors/metabolism , Protein Processing, Post-Translational , Receptor, Insulin/metabolism , 3T3 Cells , Adenosine Triphosphate/pharmacology , Adipocytes , Animals , Carrier Proteins/metabolism , Cell Compartmentation , Cell Fractionation , Electrophoresis, Gel, Two-Dimensional , Endoplasmic Reticulum Chaperone BiP , Glucose/deficiency , Glycoproteins/chemistry , Glycosylation/drug effects , Hexosaminidases/pharmacology , Mice , Models, Biological , Molecular Chaperones/metabolism , Oligosaccharides/chemistry , Precipitin Tests , Protein Binding/drug effects , Protein Precursors/chemistry , Receptor, Insulin/chemistry , Tunicamycin/pharmacology
4.
Clin J Sport Med ; 9(1): 40-5, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10336051

ABSTRACT

BACKGROUND: Clinicians often use the talar tilt (TT) and anterior drawer (AD) stress x-rays to diagnose acute or chronic mechanical ankle instability. However, the wide range of TT and AD values in normal and injured ankles makes interpretation of the test results difficult. OBJECTIVE: To critically review the literature and determine the accuracy of stress radiography in the diagnosis of mechanical ankle instability. DATA SOURCES: MEDLINE was searched for relevant articles published since 1966 using MEDLINE subject headings (MeSH) and textwords for English articles related to ankle injuries and radiography. Additional references were reviewed from the bibliographies of the retrieved articles. The total number of articles reviewed was 67. Of these, 8 studies met criteria for inclusion and were analyzed. STUDY SELECTION: Only clinical studies that used surgical exploration as the gold standard for diagnosing lateral ligament rupture were evaluated for this study. Cadaver or laboratory studies were excluded. DATA EXTRACTION AND SYNTHESIS: In reviewing the literature, pertinent strengths of the different study designs were emphasized. From these data, particular attention was paid to the diagnostic accuracy of each study in comparing TT and AD stress x-rays to surgical confirmation of lateral ligament rupture. MAIN RESULTS: A total of eight prospective clinical series satisfied the inclusion criteria. Seven of the eight assessed acute ankle instability as the outcome and one assessed chronic ankle instability. Of the seven studies that focused on acute ankle injuries, only one concluded significant benefit in using stress views to diagnose lateral ligament rupture. Three of the seven reported a positive relationship between stress radiography and surgical findings, although all six studies concluded that TT and AD stress x-rays are not reliable enough to make the diagnosis. The authors who assessed chronic ankle instability stated that TT and AD stress views combined were not useful in defining ankle instability. CONCLUSION: The published data regarding TT and AD stress x-rays are too variable to determine accepted normal values compared with injured values. There are insufficient data for comparison of the use of mechanical versus manual techniques, or use of local anesthetic to facilitate the stress test. Because the treatment evolution of all acute ankle sprains is toward functional nonoperative treatment and because treatment does not depend on the degree of ankle instability on stress views, the TT and AD stress x-rays have no clinical relevance in the acute situation. In cases of chronic instability, the large variability in TT and AD values in both injured and noninjured ankles precludes their routine use.


Subject(s)
Ankle Joint , Joint Instability/diagnostic imaging , Acute Disease , Ankle Joint/diagnostic imaging , Chronic Disease , Humans , Joint Instability/diagnosis , Magnetic Resonance Imaging , Radiography
6.
J Biol Chem ; 272(12): 7759-64, 1997 Mar 21.
Article in English | MEDLINE | ID: mdl-9065437

ABSTRACT

Insulin resistance is a manifestation of both diabetes mellitus and obesity. However, the mechanism is still not clearly identified. Herein, we describe a procedure that allows us to evaluate the development of insulin resistance in 3T3-L1 adipocytes. Under these conditions, we show that the concentration of insulin required for 50% desensitization of glucose transport activity is 100 pM; maximal desensitization could be achieved with 1 nM. This demonstrates for the first time that 3T3-L1 adipocytes develop insulin resistance in response to physiologically relevant concentrations of insulin. Glucose (or glucosamine), in addition to insulin, was required to establish desensitization. The expression of GLUT4 protein decreased by 50% with exposure to 10 nM insulin. The dose-dependent loss of GLUT4 was similar to the dose dependence for insulin-resistant transport activity. Translocation in the presence of acute insulin was apparent, but the extent of recruitment directly reflected the decrease in GLUT4 protein. GLUT4 mRNA also declined, but the ED50 was approximately 5 nM. Together, these data suggest that the loss of GLUT4 protein likely underlies the cause of desensitization. However, the loss of GLUT4 protein did not correlate with the loss in GLUT4 mRNA suggesting post-translational control of GLUT4 expression.


Subject(s)
Adipocytes/metabolism , Insulin Resistance , Muscle Proteins , 3T3 Cells , Animals , Biological Transport , Gene Expression Regulation/drug effects , Glucosamine/pharmacology , Glucose/metabolism , Glucose/pharmacology , Glucose Transporter Type 4 , Insulin/pharmacology , Mice , Monosaccharide Transport Proteins/genetics , Protein Processing, Post-Translational , RNA, Messenger/genetics , RNA, Messenger/metabolism
7.
Nutr Rev ; 54(10): 329-31, 1996 Oct.
Article in English | MEDLINE | ID: mdl-9063024

ABSTRACT

Although muscle is thought to be a primary assimilator of glucose, adipose may provide a substantial amount of substrate for gluconeogenesis, even in the fed state.


Subject(s)
Adipose Tissue/physiology , Glucose/metabolism , Insulin/physiology , Animals , Humans
8.
Mol Cell Biochem ; 162(1): 51-8, 1996 Sep 06.
Article in English | MEDLINE | ID: mdl-8905625

ABSTRACT

We tested the hypothesis that the constitutive glucose transporter (GLUT1) in 3T3-L1 adipocytes belongs to the family of glucose-regulated proteins which are transcriptionally regulated by glucose deprivation. Using cDNA probes for both GRP78 (BiP) and GLUT1, we show that the level of GRP78 mRNA increased by 15-fold within 24 h of glucose deprivation with little change in GLUT1 mRNA. The elevated GRP78 mRNA in turn led to a time-dependent increase in GRP78 protein. While glucose deprivation did not alter the expression of the normal glycoform of GLUT1, a lower molecular weight glycoform accumulated with extended deprivation. Mannose and fructose, but not galactose, prevented the induction of GRP78 and accumulation of the abnormal GLUT1. Because GRP78 acts as a chaperone in other cell systems, we also sought evidence to support this activity in 3T3-L1 adipocytes. Using the technique of co-immunoprecipitation, we demonstrate that GRP78 bound several proteins unique to the glucose-deprived state. No deprivation-specific proteins could be detected in association with GLUT1. These data lead us to conclude that GLUT1 does not display characteristics of the glucose-regulated proteins, at least in 3T3-L1 adipocytes, a widely used model for differentiation, hormone action, and nutrient control. However, the mechanisms for activating traditional members of this family appear intact.


Subject(s)
Adipocytes/metabolism , Carrier Proteins/biosynthesis , Heat-Shock Proteins , Molecular Chaperones/biosynthesis , Monosaccharide Transport Proteins/biosynthesis , 3T3 Cells , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , DNA, Complementary , Endoplasmic Reticulum Chaperone BiP , Gene Expression Regulation , Glucose/metabolism , Glucose Transporter Type 1 , Glycosylation , Mice , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tunicamycin/pharmacology
9.
J Biol Chem ; 271(20): 11806-9, 1996 May 17.
Article in English | MEDLINE | ID: mdl-8662621

ABSTRACT

Glucose deprivation increases the rate of glucose transport in 3T3-L1 adipocytes in a protein synthesis-dependent fashion. To determine if translocation of either GLUT1 or GLUT4 is responsible for this phenomenon, we adapted existing fractionation procedures toward isolating 3T3-L1 adipocyte membranes. By Western blot analysis of equal protein, GLUT1 was distributed between plasma membranes, high density "microsomal" membranes, and low density "microsomal" membranes isolated from control cells. GLUT4 comigrated with high density and low density membranes. Glucose deprivation for 12 h did not alter the distribution of either GLUT1 or GLUT4, despite an 8-10-fold increase in glucose transport activity in intact cells. Importantly, increased transport activity was retained in plasma membrane vesicles isolated from glucose-deprived cells. These data show for the first time that the increase in transport activity associated with glucose deprivation does not result from the translocation of either of the glucose transporters known to exist in 3T3-L1 adipocytes. As GLUT4 is excluded from the plasma membrane, these data provide evidence for activation of GLUT1.


Subject(s)
Adipocytes/metabolism , Glucose/metabolism , Monosaccharide Transport Proteins/metabolism , Muscle Proteins , 3T3 Cells , Animals , Biological Transport , Glucose Transporter Type 1 , Glucose Transporter Type 4 , Mice
10.
Am J Physiol ; 270(4 Pt 1): E640-5, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8928771

ABSTRACT

In 3T3-L1 adipocytes, the glycosylation of the GLUT-1 transporter is altered beyond 12 h of glucose deprivation. To determine whether glycogen degradation provides substrate for normal protein glycosylation during this delay, we measured the glycogen content of 3T3-L1 adipocytes. From an initial value of 0.537 +/- 0.097 mumol glucose/10(6) cells, glycogen was depleted in a time-dependent manner in response to glucose deprivation, exhibiting a half-time of 6 h. Surprisingly, fructose did not prevent glycogen depletion. However, in such glycogen-depleted adipocytes, the alteration of GLUT-1 glycosylation in response to glucose deprivation was more rapid than in normal adipocytes. Chinese hamster ovary (CHO) cells, which synthesize abbreviated dolichol-linked oligosaccharides within minutes of glucose deprivation (J. I. Rearick, A. Chapman, and S. Kornfeld. J. Biol. Chem. 256: 6255-6261, 1981), contained only 1% of the level of glycogen found in 3T3-L1 adipocytes. Glycosylation of GLUT-1 was altered in CHO cells within 3 h of glucose deprivation. These data demonstrate that, during glucose stress, glycogen may serve as a buffer for oligosaccharide biosynthesis and provide a potential explanation for varying sensitivities of different cell types to glucose deprivation.


Subject(s)
Adipocytes/metabolism , Carbohydrate Metabolism , Glucose/deficiency , Glycogen/metabolism , Monosaccharide Transport Proteins/metabolism , 3T3 Cells , Animals , CHO Cells , Cricetinae , Fructose/pharmacology , Glucose Transporter Type 1 , Glycogen/deficiency , Glycosylation/drug effects , Mice
11.
J Biol Chem ; 270(20): 12094-9, 1995 May 19.
Article in English | MEDLINE | ID: mdl-7744857

ABSTRACT

Metabolic labeling and immunoprecipitation were used to analyze the glucose-dependent regulation of GLUT1 synthesis, processing, and turnover in a murine adipocyte cell line. Metabolically labeled GLUT1 from control cells migrated as a 46-kDa protein, while GLUT1 from cells deprived of glucose for more than 12 h migrated as a 37-kDa protein. On the basis of tunicamycin sensitivity, both GLUT1 species arose from a common protein migrating at 36 kDa. In addition, the rate of synthesis of GLUT1 in control and glucose-deprived cells was similar. In short pulse-chase experiments, we distinguished two species arising from the core GLUT1 protein in control cells; an intermediate and the mature 46-kDa species. In contrast, only one glycoform, the 37-kDa species, arose from the core protein in glucose-deprived cells, which was not further processed in either the presence or absence of glucose. Although 12-18 h of glucose deprivation were required to affect GLUT1 glycosylation, glucose-deprived cells quickly recovered the ability to correctly glycosylate GLUT1 upon the readdition of glucose (t1/2 < 1 h). GLUT1 in control adipocytes exhibited a half-life of approximately 14 h, while that in glucose-deprived adipocytes was greater than 50 h. This effect was readily reversed upon the readdition of glucose. In total, these data show that glucose deprivation alters both the processing (glycosylation) and turnover (degradation) of GLUT1. These results are discussed in light of transport function.


Subject(s)
3T3 Cells/metabolism , Adipose Tissue/metabolism , Monosaccharide Transport Proteins/metabolism , Protein Processing, Post-Translational , 3T3 Cells/drug effects , Amino Acid Sequence , Animals , Cell Differentiation/drug effects , Energy Metabolism , Glucose/metabolism , Glucose/pharmacology , Glucose Transporter Type 1 , Glycosylation , Mice , Molecular Sequence Data , Molecular Weight , Monosaccharide Transport Proteins/immunology , Peptide Fragments/immunology , Precipitin Tests
13.
J Biol Chem ; 268(2): 1320-5, 1993 Jan 15.
Article in English | MEDLINE | ID: mdl-7678253

ABSTRACT

Elevated glucose transport rates during glucose deprivation are phenomena that have been observed in several different types of cells in culture. We show here that glucose transport rates in 3T3-L1 adipocytes increased by 10-fold within 18 h in response to glucose deprivation, confirming earlier work by Van Putten and Krans (Van Putten, J. P. M., and Krans, H. M. J. (1985) J. Biol. Chem. 260, 7996-8001). Mannose and 3-O-methylglucose (a nonmetabolizable glucose analog), but not fructose or galactose, blocked the increase in transport activity. Although the increase in transport was dependent on new protein synthesis, only a small and transient increase in GLUT 1 mRNA (less than 2-fold) was observed. In addition, the level of the normal isoform of GLUT 1 (46 kDa) did not increase. A lower molecular mass isoform (37 kDa) was observed but not until 15 h after glucose removal, the appearance of which was clearly not correlated with the increase in activity. Further, the extracellular glucose concentration required to elicit accumulation of this form (p37) was 2 orders of magnitude less than that required for transport stimulation (5 microM versus 500 microM glucose; p37 accumulation and transport activation, respectively). Interestingly, p37 was seen in the presence of galactose, but not fructose, despite elevated transport activity with either sugar. The p37 isoform was slightly larger than N-glycosidase F-treated GLUT 1 (36 kDa), implying that this form is still glycosylated, albeit incompletely. It is not known if p37 is functional, but the time- and sugar-dependent appearance of the lower isoform suggests that p37 is not responsible for starvation-induced transport but potentially represents an underglycosylated precursor of the normal, 46-kDa isoform of GLUT 1.


Subject(s)
Adipose Tissue/metabolism , Glucose/metabolism , Glucose/pharmacology , Monosaccharide Transport Proteins/biosynthesis , 3T3 Cells , Adipose Tissue/drug effects , Animals , Biological Transport/drug effects , Blotting, Northern , Blotting, Western , Cycloheximide/pharmacology , Kinetics , Mice , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/isolation & purification , Monosaccharides/pharmacology , Oligonucleotide Probes , RNA/genetics , RNA/isolation & purification , RNA, Messenger/genetics , RNA, Messenger/metabolism , Time Factors
14.
Am J Physiol ; 262(1 Pt 1): C91-7, 1992 Jan.
Article in English | MEDLINE | ID: mdl-1370745

ABSTRACT

The differentiation of 3T3-L1 fibroblasts to adipocytes can be accelerated by the addition of 1-methyl-3-isobutylxanthine (MIX), insulin, and dexamethasone to the culture medium. During differentiation, we have demonstrated that the level of both annexin I mRNA and protein decreases. The half-times for this reduction were 2 h and 10 h for annexin I mRNA and protein, respectively. Of the added agents in the differentiation medium, only MIX caused a decline in annexin I expression in 3T3-L1 fibroblasts. The MIX effect in fibroblasts was reversible and required de novo transcription but not protein synthesis. Although MIX could be replaced by high levels of theophylline, neither agonists of the beta-adrenergic receptor nor intracellular second messengers, cAMP and cGMP, were able to reduce annexin I. The potential role of annexin I in cellular differentiation is discussed.


Subject(s)
1-Methyl-3-isobutylxanthine/pharmacology , Adipose Tissue/physiology , Calcium-Binding Proteins/metabolism , Cyclic AMP/pharmacology , 3T3 Cells , Adipose Tissue/cytology , Animals , Annexins , Calcium-Binding Proteins/genetics , Cell Differentiation , Dexamethasone/pharmacology , Fibroblasts/cytology , Fibroblasts/metabolism , Fibroblasts/physiology , Insulin/pharmacology , Mice , Phenotype , RNA, Messenger/metabolism , Time Factors
15.
Biochem J ; 275 ( Pt 2): 313-9, 1991 Apr 15.
Article in English | MEDLINE | ID: mdl-1827255

ABSTRACT

We demonstrate that annexin I/lipocortin I (lipo I) gene expression is regulated by dexamethasone (DEX) in mouse 3T3-L1 fibroblasts and LA-4 lung epithelial cells. We have characterized this induction further in 3T3-L1 fibroblasts. At 24 h after addition of DEX, the levels of lipo I mRNA and protein increased 5-fold and 1.5-fold respectively. Time-course experiments revealed that the induction was delayed by 2-4 h after DEX addition. Half-maximal induction of both lipo I mRNA and protein was achieved with 10 nM-DEX. Both actinomycin D and cycloheximide blocked the DEX effect on lipo I mRNA expression. These results indicate that the induction of lipo I by DEX has a transcriptional component and requires protein synthesis de novo.


Subject(s)
Calcium-Binding Proteins/biosynthesis , Dexamethasone/pharmacology , Animals , Annexins , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/isolation & purification , Cell Line , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Gene Expression Regulation/drug effects , Kinetics , Membrane Proteins/biosynthesis , Mice , Mice, Inbred BALB C , Molecular Weight , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Time Factors
16.
Endocrinology ; 128(3): 1574-9, 1991 Mar.
Article in English | MEDLINE | ID: mdl-1825630

ABSTRACT

Expression of annexin-I was investigated in the rat mammary gland during pregnancy, lactation, and involution. Both the mRNA and protein were very abundant in the mature virgin gland, but declined significantly by midpregnancy. In the lactating gland, little or no annexin-I was detected. After weaning, mRNA and protein levels increased dramatically, corresponding to glandular involution. We also show that premature removal of the suckling stimulus caused a rapid increase in mRNA expression, but that translation of message was delayed, possibly until the gland was irreversibly committed to involution. Since high levels of annexin-I are associated with the quiescent epithelial cell, annexin-I may play an important role in blocking differentiation of the mammary gland.


Subject(s)
Calcium-Binding Proteins/physiology , Lactation/physiology , Mammary Glands, Animal/metabolism , Animals , Annexins , Calcium-Binding Proteins/genetics , Cell Differentiation , Female , Histones/genetics , Mammary Glands, Animal/cytology , Pregnancy , RNA, Messenger/metabolism , Rats , Rats, Inbred Strains , Superoxide Dismutase/genetics , Weaning
18.
Biochem J ; 269(3): 589-95, 1990 Aug 01.
Article in English | MEDLINE | ID: mdl-2202293

ABSTRACT

Phenylarsine oxide (PAO), a trivalent arsenical, has been shown to inhibit insulin-stimulated glucose transport in 3T3-L1 adipocytes, implicating vicinal dithiols in signal transmission [Frost & Lane (1985) J. Biol. Chem. 260, 2646-2652]. To assist in the direct identification of a PAO-binding protein which might be involved in this process, we have synthesized [3H]acetylaminophenylarsine oxide [( 3H]APAO) from the amino derivative of phenylarsine oxide (NPAO). To assess the inhibitory effect of the product, a dual-labelling experiment was performed which showed that [3H]APAO inhibited insulin-stimulated 2-deoxy[1-14C]glucose transport in 3T3-L1 adipocytes with a Ki of 21 microM, identical with that of the parent compound, NPAO. Further characterization revealed that over a wide concentration range, uptake of the labelled arsine oxide was linear. Although the dithiol reagent 2,3-dimercaptopropanol (DMP) reversed PAO-induced inhibition of transport, it had no effect on the uptake of [3H]APAO. In a simple fractionation experiment approx. 50% of the radioactivity was associated with the cytosolic fraction and 50% with the total membrane fraction. Identification of radiolabelled proteins by non-reducing SDS/PAGE revealed fraction-specific binding, although many proteins were observed. Covalent modification was time-dependent and could be reversed by addition of DMP. These data further support a role for vicinal dithiols in insulin-stimulated glucose transport. Additionally, the probe described may offer a new means with which to identify the inhibitory protein or, more globally, to investigate mechanisms of action of vicinal dithiol-containing proteins.


Subject(s)
Adipose Tissue/metabolism , Arsenicals/pharmacokinetics , Adipose Tissue/cytology , Animals , Arsenicals/metabolism , Biological Transport , Cell Fractionation , Deoxyglucose/pharmacokinetics , Insulin/pharmacology , Intracellular Fluid/metabolism , Kinetics , Stimulation, Chemical , Time Factors , Tritium
19.
Mol Cell Endocrinol ; 72(1): 55-61, 1990 Jul 30.
Article in English | MEDLINE | ID: mdl-2272403

ABSTRACT

For the first time, we demonstrate here the ability of human relaxin to block cell division. During the induction of differentiation of 3T3-L1 fibroblasts to adipocytes, the cells typically undergo two rounds of cell division followed by accumulation of lipid droplets and expression of insulin-stimulated glucose transport as the cells attain the adipocyte phenotype. Human relaxin added during induction had no effect on the development of the adipocyte phenotype or insulin-stimulated glucose transport. However, it blocked cell division at a half-maximal concentration of 1.25 nM, well within physiological range. This could be reversed by the addition of antibodies specific for human relaxin. Thus relaxin joins a select number of hormones with growth inhibitory properties such as transforming growth factor-beta (TGF beta) and mammastatin. Potentially, this is an important but until now unidentified function of relaxin. Unlike other inhibitory polypeptides, like TGF beta, relaxin does not prevent differentiation but rather uncouples it from cell division.


Subject(s)
Fibroblasts/cytology , Relaxin/pharmacology , Adipose Tissue/cytology , Adipose Tissue/metabolism , Animals , Biological Transport , Cell Differentiation/drug effects , Cell Division/drug effects , Cell Line , DNA/biosynthesis , Fibroblasts/drug effects , Fibroblasts/metabolism , Glucose/metabolism , Humans , Insulin/pharmacology , Swine
20.
J Cell Physiol ; 141(3): 467-74, 1989 Dec.
Article in English | MEDLINE | ID: mdl-2687296

ABSTRACT

We have shown previously that insulin stimulates fluid phase endocytosis in 3T3-L1 adipocytes (Gibbs et al., 1986). Using [14C]sucrose as an endocytotic marker, we show here that phenylarsine oxide, a trivalent arsenical which binds neighboring dithiols, blocked not only insulin-stimulated fluid phase endocytosis, but basal endocytosis as well. The Ki for this process was 6 microM in the presence or absence of insulin and the time required for inhibition was less than 2.5 min, the limit of detection in our assay system. These results can be compared with the inhibitory effect of phenylarsine oxide on insulin-stimulated glucose transport. Although the Ki for insulin-stimulated transport (7 microM) was similar to that for inhibition of endocytosis, basal glucose transport was not affected by the inhibitor. Further, when cells were prestimulated with insulin causing maximal stimulation of the glucose transport rate, phenylarsine oxide induced a time-dependent reduction to the basal rate (t 1/2 of 10 min), despite the fact that endocytosis was blocked immediately. This observation suggests that if the transporter is recycled by an exocytotic/endocytotic mechanism, it is distinct from fluid-phase endocytosis/exocytosis, which is a vesicle-mediated process, and provides further evidence that the transporter may undergo intrinsic activation/inactivation which does not require vesicle movement.


Subject(s)
Arsenicals/pharmacology , Endocytosis/drug effects , Adipose Tissue/cytology , Adipose Tissue/metabolism , Adipose Tissue/ultrastructure , Animals , Biological Transport/drug effects , Biological Transport/physiology , Cell Line , Cell Membrane/drug effects , Cell Membrane/physiology , Endocytosis/physiology , Exocytosis/drug effects , Exocytosis/physiology , Glucose/pharmacokinetics , Insulin/pharmacology , Membrane Fluidity/drug effects , Membrane Fluidity/physiology , Microscopy, Electron , Monosaccharide Transport Proteins/physiology , Rats , Time Factors
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