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J L Goldstein

Publications and source records attributed to J L Goldstein.

At least 73 records · Page 4Linked to original sources
PubMed1999

Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy.

Congenital generalized lipodystrophy (CGL) is a rare autosomal recessive disorder characterized by a paucity of adipose (fat) tissue which is evident at birth and is accompanied by a severe resistance to insulin, leading to hyperinsulinaemia, hyperglycaemia and enlarged fatty liver. We have developed a mouse model that mimics these features of CGL: the syndrome occurs in transgenic mice expressing a truncated version of a nuclear protein known as nSREBP-1c (for sterol-regulatory-element-binding protein-1c) under the control of the adipose-specific aP2 enhancer. Adipose tissue from these mice was markedly deficient in messenger RNAs encoding several fat-specific proteins, including leptin, a fat-derived hormone that regulates food intake and energy metabolism. Here we show that insulin resistance in our lipodystrophic mice can be overcome by a continuous systemic infusion of low doses of recombinant leptin, an effect that is not mimicked by chronic food restriction. Our results support the idea that leptin modulates insulin sensitivity and glucose disposal independently of its effect on food intake, and that leptin deficiency accounts for the insulin resistance found in CGL.

Adipocytes↗
PubMed1999

Autocatalytic processing of site-1 protease removes propeptide and permits cleavage of sterol regulatory element-binding proteins.

Site-1 protease (S1P) is a subtilisin-related protease that cleaves sterol regulatory element-binding proteins (SREBPs) in the endoplasmic reticulum lumen, thereby initiating a process by which the transcriptionally active NH(2)-terminal fragments of SREBPs are released from membranes. In the current experiments, we transfected cDNAs encoding epitope-tagged hamster S1P into HEK-293 cells or mutant hamster cells that lack S1P. Protease protection assays showed that the bulk of S1P is in the endoplasmic reticulum lumen, anchored by a COOH-terminal membrane-spanning segment. Cleavage of the NH(2)-terminal signal sequence of S1P generates S1P-A (amino acids 23-1052), which is inactive. The protein is self-activated by an intramolecular cleavage at Site-B, generating S1P-B (amino acids 138-1052) and liberating a 115-amino acid propeptide that is secreted intact into the medium. The sequence at Site-B is RSLK, which differs from the RSVL sequence at the cleavage site in SREBP-2. S1P-B is further cleaved at an internal RRLL sequence to yield S1P-C (amino acids 187-1052). Mutational analysis suggests that S1P-B and S1P-C are both active in cleaving SREBP-2 in a fashion that requires SREBP cleavage-activating protein. The activity of S1P-C may be short-lived because it appears to be transported to the Golgi, a site at which SREBP-2 cleavage may not normally occur. These data provide the initial description of the processing of a subtilisin-related protease that controls the level of cholesterol in blood and cells. In an accompanying paper (Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. (1999), J. Biol. Chem., 274, 22805-22812), we develop an in vitro assay to characterize the activity of purified recombinant S1P.

Amino Acid Sequence↗
PubMed1999

Secreted site-1 protease cleaves peptides corresponding to luminal loop of sterol regulatory element-binding proteins.

We describe a permanent line of Chinese hamster ovary cells transfected with a cDNA encoding a truncated form of Site-1 protease (S1P) that is secreted into the culture medium in an enzymatically active form. S1P, a subtilisin-like protease, normally cleaves the luminal loop of sterol regulatory element-binding proteins (SREBPs). This cleavage initiates the two-step proteolytic process by which the NH(2)-terminal domains of SREBPs are released from cell membranes for translocation to the nucleus, where they activate transcription of genes involved in the biosynthesis and uptake of cholesterol and fatty acids. Truncated S1P (amino acids 1-983), produced by the transfected Chinese hamster ovary cells, lacks the COOH-terminal membrane anchor. Like native S1P, this truncated protein undergoes normal autocatalytic processing after residue 137 to release an NH(2)-terminal propeptide, thereby generating an active form, designated S1P-B. Prior to secretion, truncated S1P-B, like native S1P-B, is cleaved further after residue 186 to generate S1P-C, which is the only form that appears in the culture medium. The secreted enzyme, designated S1P(983)-C, cleaves a synthetic peptide that terminates in a 7-amino-4-methyl-coumarin fluorochrome. This peptide, RSLK-MCA, corresponds to the internal propeptide cleavage site that generates S1P-B as described in the accompanying paper (Espenshade, P. J., Cheng, D., Goldstein, J. L., and Brown, M. S. (1999), J. Biol. Chem. 274, 22795-22804). The secreted enzyme does not cleave RSVL-MCA, a peptide corresponding to the physiologic cleavage site in SREBP-2. However, S1P(983)-C does cleave after this leucine when the RSVL sequence is contained within a 16-residue peptide corresponding to the central portion of the SREBP-2 luminal loop. The catalytic activity of S1P(983)-C differs from that of furin/prohormone convertases, two related proteases, in its more alkaline pH optimum (pH 7-8), its relative resistance to calcium chelating agents, and its ability to cleave after lysine or leucine rather than arginine. These data provide direct biochemical evidence that S1P is the protease that cleaves SREBPs and thereby functions to control lipid biosynthesis and uptake in animal cells.

Amino Acid Sequence↗
PubMed1999

Membrane topology of S2P, a protein required for intramembranous cleavage of sterol regulatory element-binding proteins.

In sterol-depleted mammalian cells, a two-step proteolytic process releases the NH(2)-terminal domains of sterol regulatory element-binding proteins (SREBPs) from membranes of the endoplasmic reticulum (ER). These domains translocate into the nucleus, where they activate genes of cholesterol and fatty acid biosynthesis. The SREBPs are oriented in the membrane in a hairpin fashion, with the NH(2)- and COOH-terminal domains facing the cytosol and a single hydrophilic loop projecting into the lumen. The first cleavage occurs at Site-1 within the ER lumen to generate an intermediate that is subsequently released from the membrane by cleavage at Site-2, which lies within the first transmembrane domain. A membrane protein, designated S2P, a putative zinc metalloprotease, is required for this cleavage. Here, we use protease protection and glycosylation site mapping to define the topology of S2P in ER membranes. Both the NH(2) and COOH termini of S2P face the cytosol. Most of S2P is hydrophobic and appears to be buried in the membrane. All three of the long hydrophilic sequences of S2P can be glycosylated, indicating that they all project into the lumen. The HEIGH sequence of S2P, which contains two potential zinc-coordinating residues, is contained within a long hydrophobic segment. Aspartic acid 467, located approximately 300 residues away from the HEIGH sequence, appears to provide the third coordinating residue for the active site zinc. This residue, too, is located in a hydrophobic sequence. The hydrophobicity of these sequences suggests that the active site of S2P is located within the membrane in an ideal position to cleave its target, a Leu-Cys bond in the first transmembrane helix of SREBPs.

Amino Acid Sequence↗
PubMed1999

Disruption of LDL receptor gene in transgenic SREBP-1a mice unmasks hyperlipidemia resulting from production of lipid-rich VLDL.

Transgenic mice that overexpress the nuclear form of sterol regulatory element binding protein-1a (SREBP-1a) in liver (TgBP-1a mice) were shown previously to overproduce cholesterol and fatty acids and to accumulate massive amounts of cholesterol and triglycerides in hepatocytes. Despite the hepatic overproduction of lipids, the plasma levels of cholesterol ( approximately 45 mg/dl) and triglycerides ( approximately 55 mg/dl) were not elevated, perhaps owing to degradation of lipid-enriched particles by low-density lipoprotein (LDL) receptors. To test this hypothesis, in the current studies we bred TgBP-1a mice with LDL receptor knockout mice. As reported previously, LDLR-/- mice manifested a moderate elevation in plasma cholesterol ( approximately 215 mg/dl) and triglycerides ( approximately 155 mg/dl). In contrast, the doubly mutant TgBP-1a;LDLR-/- mice exhibited marked increases in plasma cholesterol ( approximately 1,050 mg/dl) and triglycerides ( approximately 900 mg/dl). These lipids were contained predominantly within large very-low-density lipoprotein (VLDL) particles that were relatively enriched in cholesterol and apolipoprotein E. Freshly isolated hepatocytes from TgBP-1a and TgBP-1a;LDLR-/- mice overproduced cholesterol and fatty acids and secreted increased amounts of these lipids into the medium. Electron micrographs of livers from TgBP-1a mice showed large amounts of enlarged lipoproteins within the secretory pathway. We conclude that the TgBP-1a mice produce large lipid-rich lipoproteins, but these particles do not accumulate in plasma because they are degraded through the action of LDL receptors.

Animals↗
PubMed1999

Quality assurance in the endoscopy unit: An emphasis on outcomes.

This article identifies and reviews key elements of quality improvement programs for an endoscopy unit. The new paradigm of quality improvement seeks to improve all performance, and does not simply focus on the low outliers as in the traditional "quality assurance" program. The organization and key elements are emphasized, including selection of key indicators and methodologies for the development of standards of practice and benchmarks. Examples of quality improvement initiatives are discussed, demonstrating their value in improved patient care and the process or function of an endoscopy unit.

Endoscopy, Gastrointestinal↗
PubMed1998

Blunted feedback suppression of SREBP processing by dietary cholesterol in transgenic mice expressing sterol-resistant SCAP(D443N).

Feedback regulation of cholesterol biosynthesis is mediated by membrane-bound transcription factors designated sterol regulatory element-binding proteins (SREBP)-1 and -2. In sterol-deprived cultured cells, SREBPs are released from membranes by a proteolytic process that is stimulated by SREBP cleavage-activating protein (SCAP), a membrane protein containing a sterol-sensing domain. Sterols suppress SREBP cleavage by blocking the action of SCAP, thereby decreasing cholesterol synthesis. A point mutation in SCAP(D443N) causes resistance to sterol suppression. In this article, we produced transgenic mice that express mutant SCAP(D443N) in liver. In these livers the nuclear content of SREBP-1 and -2 was increased, mRNAs encoding proteins involved in uptake and synthesis of cholesterol and fatty acids were elevated, and the livers were engorged with cholesteryl esters and triglycerides enriched in monounsaturated fatty acids. When the mice were challenged with a high cholesterol diet, cleavage of SREBP-1 and -2 was reduced in wild-type livers and less so in transgenic livers. We conclude that SCAP(D443N) stimulates proteolytic processing of native SREBPs in liver and decreases the normal sterol-mediated feedback regulation of SREBP cleavage, suggesting a central role for SCAP as a sterol sensor in liver.

Animals↗
PubMed1998

Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N.

SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.

Amino Acid Sequence↗
PubMed1998

Isolation of cholesterol-requiring mutant Chinese hamster ovary cells with defects in cleavage of sterol regulatory element-binding proteins at site 1.

The synthesis and uptake of cholesterol requires transcription factors designated sterol regulatory element-binding proteins (SREBPs). SREBPs are bound to membranes in a hairpin orientation with their transcriptionally active NH2-terminal segments facing the cytosol. The NH2-terminal segments are released from membranes by two-step proteolysis initiated by site 1 protease (S1P), which cleaves in the luminal loop between two membrane-spanning segments. Next, site 2 protease (S2P) releases the NH2-terminal fragment of SREBP. The S2P gene was recently isolated by complementation cloning using Chinese hamster ovary cells that require cholesterol for growth, due to a mutation in the S2P gene. A similar approach cannot be used for S1P because all previous cholesterol auxotrophs manifest defects in S2P, which is encoded by a single copy gene. To circumvent this problem, in the current studies we transfected Chinese hamster ovary cells with the S2P cDNA, assuring multiple copies. We mutagenized the cells, selected for cholesterol auxotrophy, and identified two mutant cell lines (SRD-12A and -12B) that fail to cleave SREBPs at site 1. Complementation analysis demonstrated that the defects in both cell lines are recessive and noncomplementing, indicating a mutation in the same gene. These cells should now be useful for expression cloning of the sterol-regulated S1P gene.

Acetates↗
PubMed1998

Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy.

Overexpression of the nuclear form of sterol regulatory element-binding protein-1c (nSREBP-1c/ADD1) in cultured 3T3-L1 preadipocytes was shown previously to promote adipocyte differentiation. Here, we produced transgenic mice that overexpress nSREBP-1c in adipose tissue under the control of the adipocyte-specific aP2 enhancer/promoter. A syndrome with the following features was observed: (1) Disordered differentiation of adipose tissue. White fat failed to differentiate fully, and the size of white fat depots was markedly decreased. Brown fat was hypertrophic and contained fat-laden cells resembling immature white fat. Levels of mRNA encoding adipocyte differentiation markers (C/EBPalpha, PPARgamma, adipsin, leptin, UCP1) were reduced, but levels of Pref-1 and TNFalpha were increased. (2) Marked insulin resistance with 60-fold elevation in plasma insulin. (3) Diabetes mellitus with elevated blood glucose (>300 mg/dl) that failed to decline when insulin was injected. (4) Fatty liver from birth and elevated plasma triglyceride levels later in life. These mice exhibit many of the features of congenital generalized lipodystrophy (CGL), an autosomal recessive disorder in humans.

Adipose Tissue↗
PubMed1998

Differential stimulation of cholesterol and unsaturated fatty acid biosynthesis in cells expressing individual nuclear sterol regulatory element-binding proteins.

Three sterol regulatory element-binding proteins (SREBP-1a, -1c, and -2) stimulate transcription of genes involved in synthesis and receptor-mediated uptake of cholesterol and fatty acids. Here, we explore the individual roles of each SREBP by preparing lines of Chinese hamster ovary (CHO) cells that express graded amounts of nuclear forms of each SREBP (designated nSREBPs) under control of a muristerone-inducible nuclear receptor system. The parental hamster cell line (M19 cells) lacks its own nSREBPs, owing to a deletion in the gene encoding the Site-2 protease, which releases nSREBPs from cell membranes. By varying the concentration of muristerone, we obtained graded expression of individual nSREBPs in the range that restored lipid synthesis to near physiologic levels. The results show that nSREBP-2 produces a higher ratio of synthesis of cholesterol over fatty acids than does nSREBP-1a. This is due in part to a selective ability of low levels of nSREBP-2, but not nSREBP-1a, to activate the promoter for squalene synthase. nSREBP-1a and -2 both activate transcription of the genes encoding stearoyl-CoA desaturase-1 and -2, thereby markedly enhancing the production of monounsaturated fatty acids. nSREBP-1c was inactive in stimulating any transcription at the concentrations achieved in these studies. The current data support the emerging view that the nSREBPs act in complementary ways to modulate the lipid composition of cell membranes.

Animals↗
PubMed1998

Second-site cleavage in sterol regulatory element-binding protein occurs at transmembrane junction as determined by cysteine panning.

In response to sterol deprivation, two sequential proteolytic cleavages release the NH2-terminal fragments of sterol regulatory element-binding proteins (SREBPs) from cell membranes. The fragments translocate to the nucleus where they activate genes involved in cholesterol and fatty acid metabolism. The SREBPs are bound to membranes in a hairpin fashion. The NH2-terminal and COOH-terminal domains face the cytoplasm, separated by two membrane spanning segments and a short lumenal loop. The first cleavage occurs at Site-1 in the lumenal loop. The NH2-terminal fragment is then released by cleavage at Site-2, which is believed to lie within the first transmembrane segment. Here, we use a novel cysteine panning method to identify the second cleavage site (Site-2) in human SREBP-2 as the Leu484-Cys485 bond that lies at the junction between the cytoplasmic NH2-terminal fragment and the first transmembrane segment. We transfected cells with cDNAs encoding fusion proteins with single cysteine residues at positions to the NH2-terminal and COOH-terminal sides of cysteine 485. The NH2-terminal fragments were tested for susceptibility to modification with Nalpha-(3-maleimidylpropionyl)biocytin, which attaches a biotin group to cysteine sulfhydryls. Cysteines to the NH2-terminal side of cysteine 485 were retained on the NH2-terminal fragment, but cysteines to the COOH-terminal side of leucine 484 were lost. Leucine 484 is three residues to the COOH-terminal side of the tetrapeptide Asp-Arg-Ser-Arg, which immediately precedes the first transmembrane segment and is required for Site-2 cleavage.

Amino Acid Sequence↗
PubMed1998

Topology of SREBP cleavage-activating protein, a polytopic membrane protein with a sterol-sensing domain.

The NH2-terminal fragments of sterol regulatory element-binding proteins (SREBPs) are released from endoplasmic reticulum membranes by proteases whose activities depend upon SREBP cleavage-activating protein (SCAP), a polytopic endoplasmic reticulum membrane protein. The activity of SCAP is inhibited by sterols, which appear to interact with the polytopic membrane domain of SCAP. Here, we use protease protection and N-linked glycosylation site-mapping techniques to define the topology of the eight membrane-spanning domains of SCAP. The data indicate that the NH2 terminus and COOH terminus of SCAP face the cytosol. The long intralumenal loops after membrane-spanning segments 1 and 7 are glycosylated, confirming their lumenal location. The region comprising membrane-spanning segments 2-6 shows sequence resemblance to putative sterol-sensing domains in three other proteins: 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), the Niemann-Pick C1 protein, and the morphogen receptor Patched. The orientation of the eight membrane-spanning segments in SCAP is consistent with the model proposed for HMG-CoA reductase (Olender, E. H., and Simoni, R. D. (1992) J. Biol. Chem. 267, 4223-4235). The membrane-spanning domains of SCAP and HMG-CoA reductase confer sterol sensitivity upon the functional activities of the two molecules. The common membrane topology of the two proteins is consistent with the notion that sterols regulate both proteins by a common mechanism.

Animals↗
PubMed1998

Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2.

We produced transgenic mice that express a dominant-positive truncated form of sterol regulatory element-binding protein-2 (SREBP-2) in liver and adipose tissue. The encoded protein lacks the membrane-binding and COOH-terminal regulatory domains, and it is therefore not susceptible to negative regulation by cholesterol. Livers from the transgenic mice showed increases in mRNAs encoding multiple enzymes of cholesterol biosynthesis, the LDL receptor, and fatty acid biosynthesis. The elevations in mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase were especially marked (13-fold and 75-fold, respectively). As a result, the transgenic livers showed a 28-fold increase in the rate of cholesterol synthesis and a lesser fourfold increase in fatty acid synthesis, as measured by intraperitoneal injection of [3H]water. These results contrast with previously reported effects of dominant-positive SREBP-1a, which activated fatty acid synthesis more than cholesterol synthesis. In adipose tissue of the SREBP-2 transgenics, the mRNAs for cholesterol biosynthetic enzymes were elevated, but the mRNAs for fatty acid biosynthetic enzymes were not. We conclude that SREBP-2 is a relatively selective activator of cholesterol synthesis, as opposed to fatty acid synthesis, in liver and adipose tissue of mice.

Adipose Tissue↗
PubMed1998

Cleavage of sterol regulatory element-binding proteins (SREBPs) at site-1 requires interaction with SREBP cleavage-activating protein. Evidence from in vivo competition studies.

Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that promote lipid synthesis in animal cells. They are embedded in the membranes of the endoplasmic reticulum (ER) in a helical hairpin orientation and are released from the ER by a two-step proteolytic process. Proteolysis begins when the SREBPs are cleaved at Site-1, which is located at a leucine residue in the middle of the hydrophobic loop in the lumen of the ER. Sterols suppress Site-1 cleavage, apparently by interacting with a polytopic membrane protein designated SREBP cleavage-activating protein (SCAP). SREBPs and SCAP are joined together in ER membranes through interaction of their cytoplasmic COOH-terminal domains. Here we use an in vivo competition assay in transfected cells to show that the SREBP.SCAP complex is essential for Site-1 cleavage. Overexpression of the truncated COOH-terminal domains of either SREBP-2 or SCAP disrupted the complex between full-length SREBP-2 and SCAP as measured by co-immunoprecipitation. This resulted in a complete inhibition of Site-1 cleavage that was restored by concomitant overexpression of full-length SCAP. The transfected COOH-terminal domains also inhibited the transcription of a reporter gene driven by an SRE-containing promoter, and this, too, was restored by overexpression of full-length SCAP. We interpret these data to indicate that the SREBP.SCAP complex directs the Site-1 protease to its target in the lumenal domain of SREBP and that disruption of this complex inactivates the Site-1 cleavage reaction.

Amino Acid Sequence↗
PubMed1998

Acute acid exposure increases rabbit esophageal cell proliferation.

In the present study we examined whether an acute infusion of HCl into the esophagus of rabbits would cause an increase in esophageal cellular proliferation independent of morphologic evidence of cell injury. To examine this question, the distal two thirds of the rabbit esophagus was infused for 1 hour with either 40 mmol/L HCl or NSS (control), and cellular proliferation was studied 24 and 48 hours later by using bromodeoxyuridine (BrDu) to label the nuclei of dividing cells and ornithine decarboxylase (ODC) enzyme activity as a biochemical index of cell division. Although there was no gross or microscopic evidence of cell necrosis or mucosal inflammation 24 hours after H+ infusion, BrDu labeling of basal cell nuclei was significantly greater 24 hours after H+ infusion (31%+/-6%) as compared with that in control animals infused with NSS (15%+/-4%). This increase in labeling index was paralleled by a threefold greater ODC enzyme activity at 24 hours with H+ infusion. Rete pegs were infrequent in control tissues (4+/-4 rete pegs per 100 microm of esophageal length) or in animals examined 24 hours after acid exposure (4+/-2 rete pegs per 100 microm). However, rete pegs were very prominent 48 hours after acid infusion (22+/-6 rete pegs per 100 microm). A short exposure to acid can cause a significant increase in mucosal proliferation independent of injury, suggesting that esophageal cell acidification either directly or indirectly acts as a tissue mitogen.

Animals↗