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M M Mueckler

Publications and source records attributed to M M Mueckler.

10 recordsLinked to original sources
PubMed1992

Amino acid substitutions at tryptophan 388 and tryptophan 412 of the HepG2 (Glut1) glucose transporter inhibit transport activity and targeting to the plasma membrane in Xenopus oocytes.

All 6 tryptophan residues in the human HepG2-type glucose transporter (Glut1) were individually altered by site-directed mutagenesis to investigate the role of these residues in transport function. Tryptophan residues in positions 48, 65, 186, 363, 388, and 412 of Glut1 were changed to either a glycine or leucine residue. Mutant mRNAs were synthesized and injected into Xenopus laevis oocytes. Transporter function as assessed by uptake of 2-deoxy-D-[3H]glucose or transport of 3-O-[3H]methylglucose was decreased in the 388 and 412 mutants but was unaltered in all other mutants. The amount of the mutant transporters expressed in total membrane and plasma membrane fractions was measured using Glut1-specific antibodies. Calculation of the intrinsic transport activity of each of the mutants using these data demonstrated that the reduced transport activity of the 412 mutants was caused entirely by a dramatic decrease in the intrinsic activity of the mutant proteins whereas the reduced activity of the 388 mutants was a result of a decreased level of the protein in oocytes, decreased targeting to the plasma membrane, and a modest decrease in the intrinsic activity. Protease/glycosidase mapping of in vitro translation products indicated that the effects of the 388 and 412 point mutations could not be attributed to a disruption in the ability of the mutant proteins to insert properly into the membrane. The ID50 for cytochalasin B inhibition of 2-deoxyglucose uptake was increased from 5 x 10(-7) M for the wild-type Glut1 to 4 x 10(-6) M in the 388 mutants but was unaltered in the 412 mutants. These observations suggest that 1) Trp-412 may comprise part of a hexose binding site or is involved in maintaining a local tertiary structure critical for transport function; 2) Trp-388 is involved in stabilizing the equilibrium binding of cytochalasin B to the transporter. Trp-388 may therefore lie near a substrate binding site and also appears to participate in stabilization of local tertiary structure important for full catalytic activity and efficient targeting to the Xenopus plasma membrane.

Animals↗
PubMed1992

Cellular mechanism of the insulin-like effect of growth hormone in adipocytes. Rapid translocation of the HepG2-type and adipocyte/muscle glucose transporters.

The cellular mechanism whereby growth hormone (GH) acutely stimulates adipocyte glucose uptake was studied in cultures of primary rat adipocytes differentiated in vitro. Preadipocytes were isolated by collagenase digestion of inguinal fat-pads from young rats and were differentiated in the presence of 3-isobutyl-1-methylxanthine, insulin and dexamethasone. The development of an adipocyte morphology (i.e. lipid inclusions) was observed over 6 days after initiation of differentiation. Coincident with this phenotypic change was an increase in glyceraldehyde-3-phosphate dehydrogenase (GPDH) activity and in cellular content of the HepG2-type (Glut1) and adipocyte/muscle (Glut4) glucose transporter isoforms as determined by Western immunoblotting of total cellular protein. Age-matched undifferentiated cells expressed the Glut1 transporter and low levels of GPDH, but neither accumulated lipid nor exhibited measurable expression of the Glut4 protein. On day 6 after the initiation of differentiation, GH and insulin stimulated 2-deoxy[14C]glucose uptake in a dose- and time-dependent fashion in adipocytes cultured under serum-free conditions for at least 15 h. Western-blot analysis of subcellular fractions revealed that both GH and insulin rapidly (within 20 min) stimulated translocation of the Glut1 and Glut4 proteins from a low-density microsomal fraction to the plasma membrane. Confirmatory evidence was provided in immunocytochemical experiments utilizing antisera directed against the C-terminal region of the Glut4 protein and a fluorescein isothiocyanate-labelled second antibody. Observation of the cells via confocal laser microscopic imaging was consistent with glucose transporter redistribution from an intracellular region to the plasma membrane after treatment with GH or insulin. On the basis of these data, we suggest that the insulin-like effect of GH on adipocyte glucose transport involves translocation of the Glut1 and Glut4 proteins to the plasma membrane. Furthermore, stimulation of glucose-transporter translocation by both GH and insulin may indicate a common cell signalling element between the adipocyte GH and insulin receptors or, alternatively, the existence of multiple cellular mechanisms for stimulating glucose-transporter translocation.

1-Methyl-3-isobutylxanthine↗
PubMed1992

The molecular biology of mammalian glucose transporters.

Recent advances concerning the function and regulation of the facilitative and sodium-dependent glucose transport proteins are discussed. Physiologic roles have been proposed for GLUT3 and GLUT5 in the brain and intestine, respectively. Cell biologists are beginning to elucidate the subcellular trafficking pathways of GLUT4 in insulin-responsive cells. New members of the SGLT family, including a sodium-nucleoside cotransporter, have been identified.

Amino Acid Sequence↗
PubMed1992

The mammalian glucose transporters.

We have described the properties of glucose transporters expressed in several mammalian tissues and have summarized some of the adaptations that take place involving these molecules in various normal and abnormal states. With the exception of a few cell types, such as adipocytes and skeletal muscle, glucose transport is not a rate-limiting step in cellular glucose metabolism, and other substrates may be equally important for cellular metabolism. Nevertheless, an understanding of the mechanisms behind the regulation of glucose transport in individual tissues may facilitate an understanding of in vivo glucose utilization and clearance processes as they relate to normal and disease states. Although adult studies provide an impetus toward a mechanistic approach in preventing and treating various disease states involving derangements in glucose homeostasis, there remains a need for similar studies in the fetus and newborn. These developmental studies should help unravel the fetal/neonatal responses to normal and abnormal hormonal and substrate milieu.

Animals↗
PubMed1989

Differential regulation of two distinct glucose transporter species expressed in 3T3-L1 adipocytes: effect of chronic insulin and tolbutamide treatment.

The HepG2-type glucose transporter (HepG2-GT) is expressed in 3T3-L1 fibroblasts and adipocytes. In contrast, the acutely insulin-regulatable glucose transporter (IRGT) is expressed only in the adipocytes. In the present study, the expression of the IRGT was shown to increase in parallel with the acquisition of acutely insulin-stimulated glucose uptake during differentiation of these cells, whereas the level of the HepG2-GT decreased during the course of differentiation in parallel with a decline in basal glucose uptake. We examined the effects of chronic insulin and tolbutamide treatment on glucose transporter activity in conjunction with the expression of these two glucose transporter species in 3T3-L1 adipocytes. Treatment of adipocytes with insulin, tolbutamide, or both agents in combination increased 2-deoxyglucose uptake, HepG2-GT protein, and HepG2-GT mRNA levels in parallel. The effect of combined insulin/tolbutamide administration on these three parameters was greater than the effect of either treatment alone. In contrast, these treatments either had no significant effect or decreased levels of IRGT protein and mRNA. We conclude that chronic treatment of 3T3-L1 adipocytes with insulin or tolbutamide increases glucose uptake primarily by means of a selective increase in the expression of the HepG2-GT. We suggest that part of the in vivo hypoglycemic effect of insulin and sulfonylureas may involve an increased expression of the HepG2-GT.

Adipose Tissue↗
PubMed1987

Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes.

An accelerated rate of glucose transport is among the most characteristic biochemical markers of cellular transformation. To study the molecular mechanism by which transporter activity is altered, cultured rodent fibroblasts transfected with activated myc, ras, or src oncogenes were used. In myc-transfected cells, the rate of 2-deoxy-D-glucose uptake was unchanged. However, in cells transfected with activated ras and src oncogenes, the rate of glucose uptake was markedly increased. The increased transport rate in ras- and src-transfected cells was paralleled by a marked increase in the amount of glucose transporter protein, as assessed by immunoblots, as well as by a markedly increased abundance of glucose transporter messenger RNA. Exposure of control cells to the tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) for 18 hours had a similar effect of increasing the rate of glucose transport and the abundance of transporter messenger RNA. For ras, src, and TPA, the predominant mechanism responsible for activation of the transport system is increased expression of the structural gene encoding the glucose transport protein.

Animals↗
PubMed1987

Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from rat liver as derived from the cDNA sequence.

Rat liver cDNA libraries constructed in lambda gt11 were screened for reactivity with polyclonal antibodies to native S-adenosyl-L-homocysteine (AdoHcy) hydrolase (adenosylhomocysteinase; EC 3.3.1.1). Five clones were isolated and sequenced. The amino acid sequence, deduced from the cDNA sequence, contained the sequence of eight peptides obtained by tryptic and cyanogen bromide fragmentation of rat liver AdoHcy hydrolase. Identification of the amino- and carboxyl-terminal peptides in the amino acid sequence showed that the complete sequence was obtained. A "fingerprint" sequence was found that is characteristic of dinucleotide-binding domains of many proteins. For AdoHcy hydrolase, this region from the lysine at position 213 to the aspartate at position 244, containing the sequence Gly-Xaa-Gly-Xaa-Xaa-Gly at positions 219-224, is presumably the site of binding for NAD+, which is required for the activity of the enzyme.

Adenosylhomocysteinase↗
PubMed1985

Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone.

The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase.

Amino Acid Sequence↗
PubMed1985

Levels of ornithine aminotransferase messenger RNA under conditions of cyclic AMP induction in cultured hepatocytes.

In primary cultures of adult rat hepatocytes, the relative rate of ornithine aminotransferase synthesis is increased approximately 7- to 8-fold in response to dibutyryl cyclic AMP. The corresponding increase in ornithine aminotransferase messenger RNA under these conditions was observed to be only 2- to 3-fold. The levels of ornithine aminotransferase mRNA were measured by hybridization of cellular RNA to a cloned cDNA probe. The ribosomal transit time (20-25 min) along ornithine aminotransferase mRNA was the same in both control and induced cultures. The level of ornithine aminotransferase nascent chains in cAMP-induced cultures was increased 7-fold over the level in control cultures. In the face of a 2- to 3-fold increase in the amount of ornithine aminotransferase mRNA, this suggests an approximately 3-fold increase in the rate of initiation per copy of ornithine aminotransferase messenger RNA. These results indicate that cAMP may regulate ornithine aminotransferase synthesis at both transcriptional and translational levels in cultured hepatocytes.

Animals↗