Biogenesis of the crystalloid endoplasmic reticulum in UT-1 cells: evidence that newly formed endoplasmic reticulum emerges from the nuclear envelope.

The crystalloid endoplasmic reticulum (ER), a specialized smooth ER of the compactin-resistant UT-1 cell, is composed of multiple membrane tubules packed together in a hexagonal pattern. This membrane contains large amounts of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, an integral membrane protein that enzymatically regulates endogenous cholesterol biosynthesis. Using morphological and immunocytochemical techniques, we have traced the sequence of events in the biogenesis of this ER when compactin-withdrawn UT-1 cells, which do not have a crystalloid ER, are incubated in the presence of compactin. After 15 h of incubation in the presence of compactin, many cells had profiles of ER cisternae that were juxtaposed to the nuclear envelope and studded with ribosomes on their outer membrane. Both the outer nuclear membrane and the ER membrane contained HMG CoA reductase; however, there was little or no detectable enzyme in rough ER that was free in the cytoplasm. With longer times of incubation in the presence of compactin, these cells had lamellar stacks of smooth ER next to the nuclear envelope that contained HMG CoA reductase. Coordinate with the appearance of the smooth ER, crystalloid ER appeared in the same cell. Often regions of continuity were found between the membrane of the smooth ER and the membrane of the crystalloid ER tubules. These studies suggest that HMG CoA reductase is synthesized along the outer nuclear membrane and in response to increased enzyme synthesis, a membrane emerges from the outer nuclear membrane as smooth ER cisternae, which then transforms into crystalloid ER tubules.

Partial deletion of membrane-bound domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase eliminates sterol-enhanced degradation and prevents formation of crystalloid endoplasmic reticulum.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic NH2-terminal domain that contains seven apparent membrane-spanning regions and a single N-linked carbohydrate chain. The catalytic domain, which includes the COOH-terminal two-thirds of the protein, extends into the cytoplasm. The enzyme is normally degraded with a rapid half-life (2 h), but when cells are depleted of cholesterol, its half-life is prolonged to 11 h. Addition of sterols accelerates degradation by fivefold. To explore the requirements for regulated degradation, we prepared expressible reductase cDNAs from which we either deleted two contiguous membrane-spanning regions (numbers 4 and 5) or abolished the single site for N-linked glycosylation. When expressed in hamster cells after transfection, both enzymes retained catalytic activity. The deletion-bearing enzyme continued to be degraded with a rapid half-life in the presence of sterols, but it no longer was stabilized when sterols were depleted. The glycosylation-minus enzyme was degraded at a normal rate and was stabilized normally by sterol deprivation. When cells were induced to overexpress the deletion-bearing enzyme, they did not incorporate it into neatly arranged crystalloid ER tubules, as occurred with the normal and carbohydrate-minus enzymes. Rather, the deletion-bearing enzyme was incorporated into hypertrophied but disordered sheets of ER membrane. We conclude that the carbohydrate component of HMG CoA reductase is not required for proper subcellular localization or regulated degradation. In contrast, the native structure of the transmembrane component is required to form a normal crystalloid ER and to allow the enzyme to undergo regulated degradation by sterols.

Amylin secretion from the rat pancreas and its selective loss after streptozotocin treatment.

Amylin, a peptide copackaged with insulin in beta-cell granules, was measured in the effluent of the perfused rat pancreases by means of a newly developed specific radioimmunoassay. Its secretion parallels that of insulin in response to 20 mM glucose, 10 mM arginine, or the combination thereof. The relative molar amount of secreted amylin was estimated to be 25-37% that of insulin. Treatment with a borderline diabetogenic dose of streptozotocin reduced amylin response without significantly changing the insulin response. A severely diabetogenic dose of streptozotocin totally abolished amylin release and markedly reduced insulin release. The selective impairment of amylin secretion in streptozotocin-treated rats could represent an early manifestation of beta-cell depletion or injury.

Conservation of promoter sequence but not complex intron splicing pattern in human and hamster genes for 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Regulation of the expression of 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase is a critical step in controlling cholesterol synthesis. Previous studies in cultured Chinese hamster ovary cells have shown that HMG-CoA reductase is transcribed from a cholesterol-regulated promoter to yield a heterogeneous collection of mRNAs with 5' untranslated regions of 68 to 670 nucleotides in length. Synthesis of these molecules is initiated at multiple sites, and multiple donor sites are used to excise an intron in the 5' untranslated region. In the current paper, I report that human HMG-CoA reductase gene resembles the Chinese hamster gene in having multiple sites of transcription initiation that are subject to suppression by cholesterol. The human gene differs from the hamster gene in that a single donor splice site is used to excise the intron in the 5' untranslated region. All of the resulting RNAs have short 5' untranslated regions of 68 to 100 nucleotides. This difference in the splicing pattern of the first intron is species specific and not a peculiarity of cultured cells in that HMG-CoA reductase mRNAs from Syrian hamster livers resemble those of the cultured Chinese hamster ovary cells. Comparison of the DNA sequences of the HMG-CoA reductase promoters from three different species--humans, Syrian hamsters, and Chinese hamsters--shows a highly conserved region of 179 nucleotides that extends from 220 to 42 nucleotides upstream of the transcription initiation sites. This region is 88% identical between the human and Chinese hamster promoter. When fused to the coding region of the Escherichia coli chloramphenicol acetyltransferase gene, this highly conserved region of the reductase gene directs the cholesterol-regulated expression of chloramphenicol acetyltransferase in transfected hamster cells, further indicating the interspecies conservation of the regulatory elements.

Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA.

A recombinant plasmid containing a full-length cDNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase was introduced by calcium phosphate-mediated transfection into UT-2 cells, a mutant line of Chinese hamster ovary cells that lack 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus require low density lipoprotein-cholesterol and mevalonate for growth. We selected a line of permanently transfected cells, designated TR-36 cells, that expressed high levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus grew in the absence of low density lipoprotein and mevalonate. Constitutive synthesis of reductase mRNA in TR-36 cells was driven by the simian virus 40 early promoter, and therefore the mRNA was not suppressed by sterols, such as 25-hydroxycholesterol or cholesterol derived from low density lipoprotein, which normally suppresses transcription of reductase mRNA when the reductase gene is driven by its own promoter. Although TR-36 cells continued to synthesize large amounts of reductase mRNA and protein in the presence of sterols, reductase activity declined by 50 to 60%. This decline was caused by a twofold increase in the rate of degradation of preformed enzyme molecules. The current data demonstrate that sterols accelerate the degradation of reductase protein independently of any inhibitory effect on the synthesis of the protein.

Coordinate regulation of amylin and insulin expression in response to hypoglycemia and fasting.

Amylin is a 37-amino acid peptide synthesized in the pancreatic beta-cell and cosecreted with insulin. In situ hybridization of nondiabetic rat pancreas shows that insulin and amylin RNA are both localized within the islet of Langerhans in a similar distribution. After 12 days of insulin-induced hypoglycemia (mean blood glucose 3.0 +/- 0.4 mM [54 +/- 8 mg/dl]), both insulin and amylin RNA fell greater than 95%. However, maintenance of euglycemia by simultaneous infusion of glucose with insulin did not suppress insulin or amylin RNA. Fasting suppressed amylin and insulin secretion from the isolated, perfused pancreas 70 and 58%, respectively, and with refeeding, secretion rates recovered to fed levels. Despite these changes in the rates of secretion, the relative ratio of amylin to insulin was not significantly different in fed, fasted, or refed rats. The molar ratio of insulin to amylin was estimated to be 100:2.3-2.6. Both insulin and amylin RNA was suppressed approximately 50% in response to fasting. Thus, although the absolute amounts of insulin and amylin change substantially under the conditions tested, the relative amounts of these peptides do not change.

Human 3-hydroxy-3-methylglutaryl coenzyme A reductase. Conserved domains responsible for catalytic activity and sterol-regulated degradation.

A full length cDNA for human 3-hydroxy-3-methylglutaryl coenzyme A reductase, the membrane-bound glycoprotein that regulates cholesterol synthesis, was isolated from a human fetal adrenal cDNA library. The nucleotide sequence of this cDNA shows that the human reductase is 888 amino acids long and shares a high degree of homology with the hamster enzyme. The amino-terminal membrane-bound domain is the most conserved region between the two species (7 substitutions out of 339 amino acids). This region, which is predicted to span the endoplasmic reticulum membrane seven times, mediates accelerated degradation of reductase in the presence of sterols. The carboxyl-terminal catalytic domain is also highly conserved (22 substitutions out of 439 amino acids). However, the linker region between these two domains has diverged (32 substitutions out of 110 amino acids). Conservation of the structure of the membrane-bound domain in HMG-CoA reductase supports the hypothesis that sterol-regulated degradation is an important mechanism for suppression of reductase activity and for regulation of cholesterol metabolism in humans as well as in hamsters.

Dipeptides as inhibitors of the gelation of sickle hemoglobin.

To examine in detail a class of peptides that inhibit the polymerization of deoxyhemoglobin S, we assayed the L-amino acids and 22 dipeptides for their effect on deoxyhemoglobin S solubility. Of the amino acids, the aromatics (phenylalanine, tyrosine, and tryptophan) significantly increased deoxyhemoglobin S solubility, as did high concentration of arginine. Combinations of the hydrophobic (specifically the aromatic) amino acids with a hydrophilic amino acid, such as arginine or lysine, resulted in dipeptides which were much more soluble than the hydrophobic or aromatic amino acid alone, and also inhibited polymerization. Furthermore, samples of deoxyhemoglobin S at 26 to 27 g/dl containing some of these dipeptides such as Arg-Trp, Arg-Phe, and Lys-Trp in excess of 50 to 100 mM did not polymerize, indicating a 1.4- to 1.6-fold increase in deoxyhemoglobin S solubility. The enhancement of polymerization, i.e., decrease in deoxyhemoglobin S solubility, observed by the addition of aspartic acid, glycine, or lysine was observed or was reduced in the dipeptides containing these hydrophilic amino acids combined with hydrophobic amino acids (valine, leucine, isoleucine, or the aromatic amino acids). The effects of these dipeptides on deoxyhemoglobin S solubility were mostly linear with concentration. However, the changes in deoxyhemoglobin S solubility by addition of a dipeptide was not simply the sum of the effects observed with the individual amino acids as exemplified by the differential effect of reversing the dipeptide sequence (e.g., Arg-Phe and Phe-Arg, or Arg-Tyr and Tyr-Arg). These data provide further evidence as to the stereospecific nature of this class of noncovalent inhibitors of deoxyhemoglobin S polymerization.

Human genes involved in cholesterol metabolism: chromosomal mapping of the loci for the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase with cDNA probes.

Cellular cholesterol metabolism is regulated primarily through the coordinate expression of two proteins, the low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34). We have used cDNA probes for the human genes encoding these proteins to determine the precise chromosomal location of the two loci. By in situ hybridization we have regionally mapped the LDL receptor gene, LDLR, to the short arm of chromosome 19 in bands p13.1-p13.3. This result concurs with and extends a previous study in which LDLR was mapped to chromosome 19 by screening somatic cell hybrids with a species-specific monoclonal antibody. We have assigned the HMG-CoA reductase gene, HMGCR, to chromosome 5 by Southern blotting of DNA from a somatic cell hybrid panel and to bands 5q13.3-q14 by in situ hybridizations of the cDNA probe to human metaphase cells with normal and rearranged chromosomes.

Direct plasma radioimmunoassay for rat amylin-(1-37): concentrations with acquired and genetic obesity.

Amylin (islet-associated polypeptide) is a 37-amino acid peptide that is cosecreted with insulin from the pancreatic beta-cell. Accurate measurement of its plasma levels is important for delineating the physiological range over which amylin acts. We describe a reproducible, highly specific, and sensitive radioimmunoassay for direct measurement of plasma amylin-(1-37). We measured changes in portal and systemic plasma amylin and insulin in three groups of anesthetized rats: lean young adult and old adult Wistar rats with acquired obesity, and Wistar fatty [WDF/TaFa (fa/fa)] rats, a model of genetic obesity and insulin resistance derived from the Wistar strain. Changes in response to fasting, feeding, and intravenous stimulation with glucose plus arginine were assessed. We find that the amylin-to-insulin ratio is constant in fasted or fed young and old rats because of proportionate increases in both entities with aging. In genetically obese Wistar rats, amylin and insulin levels are three- to tenfold higher than in lean young or obese old normal controls. Islet stimulation by feeding or intravenous glucose plus arginine resulted in a decreased amylin-to-insulin molar ratio in all groups. When normalized for the degree of islet stimulation, amylin-to-insulin ratios were significantly elevated in genetically obese vs. normal rats, both in the portal and systemic circulation. These results demonstrate that aging-related weight gain in normal rats is associated with moderate and proportional increases in amylin and insulin, whereas genetic obesity is characterized by elevated amylin and an increased amylin-to-insulin ratio. Implications for the pathogenesis of insulin resistance and obesity are discussed.

Rat amylin: cloning and tissue-specific expression in pancreatic islets.

Amyloid deposits in the islets of Langerhans of the pancreas are a common finding in non-insulin-dependent diabetes mellitus. The main protein constituent of these deposits is a 37-amino acid peptide known as amylin that resembles calcitonin gene-related peptide, a neuropeptide. We have isolated cDNA clones corresponding to the rat amylin precursor from an islet cDNA library and we show that this peptide is encoded in a 0.9-kilobase mRNA that is translated to yield a 93-amino acid precursor. The amylin peptide is bordered by dibasic residues, suggesting that it is proteolyzed like calcitonin gene-related peptide. The peptide sequences flanking the amylin sequence do not resemble the calcitonin gene-related peptide flanking sequences. RNA hybridization studies show that amylin mRNA is abundant in the islets of Langerhans but is not present in the brain or seven other tissues examined. Dietary changes, such as fasting or fasting and refeeding, have little effect on amylin mRNA expression. This tissue specificity suggests that amylin is involved in specific signaling pathways related to islet function.

Loss of transcriptional repression of three sterol-regulated genes in mutant hamster cells.

Two genes that encode enzymes in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and HMG-CoA synthase, and the gene encoding the low density lipoprotein (LDL) receptor are repressed when sterols accumulate in animal cells. Their 5'-flanking regions contain a common element, designated sterol regulatory element-1 (SRE-1). In the HMG-CoA synthase and LDL receptor promoters, the SRE-1 enhances transcription in the absence of sterols and is inactivated in the presence of sterols. In the HMG-CoA reductase promoter, the region containing the SRE-1 represses transcription when sterols are present. In the current studies, we show that the SRE-1 retains enhancer function but loses sterol sensitivity in mutant Chinese hamster ovary cells that are resistant to the repressor, 25-hydroxycholesterol. In the absence of sterols, the mutant cells produced high levels of all three sterol-regulated mRNAs, and there was no repression by 25-hydroxycholesterol. When transfected with plasmids containing each of the regulated promoters fused to a bacterial reporter gene, the mutant cells showed high levels of transcription in the absence of sterols and no significant repression by sterols. When the SRE-1 in the LDL receptor and HMG-CoA synthase promoters was mutated prior to transfection into the mutant cells, transcription was markedly reduced. Thus, the 25-hydroxycholesterol-resistant cells retain a protein that enhances transcription by binding to the SRE-1 in the absence of sterols, but they have lost the function of a protein that abolishes this enhancement in the presence of sterols. Mutation of a 30-base pair segment of the HMG-CoA reductase promoter that contains the SRE-1 did not reduce transcription in the mutant cells, indicating that this promoter is driven by elements other than the SRE-1. Nevertheless, this promoter failed to be repressed by sterols in the mutant cells. These data suggest that a common factor mediates the effects of sterols on the SRE-1 in all three promoters and that this factor has been functionally lost in the 25-hydroxycholesterol-resistant cells.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and its mRNA in rat liver as studied with a monoclonal antibody and a cDNA probe.

A monoclonal antibody directed against 3-hydroxy-3-methylglutaryl Coenzyme A reductase and a cDNA to reductase mRNA were used to study the subunit structure of the enzyme and the regulation of its mRNA in rat liver. Although the monoclonal antibody and the cDNA were made with materials from cultured hamster cells, the two reagents cross-reacted with reductase protein and mRNA from rat liver. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibody, the subunit molecular weight of rat liver reductase was 90,000. When the enzyme was solubilized from microsomes by freeze-thawing, the subunit molecular weight was reduced to 52,000-58,000, owing to proteolysis. This proteolysis was inhibited by EGTA and leupeptin. The cDNA probe for reductase, radiolabeled with 32P, hybridized to restriction fragments of genomic DNA from rat liver, as visualized by Southern blot analysis. In the livers of control rats, no reductase mRNA was detected when the 32P-cDNA was blot-hybridized to poly(A+) RNA. Hepatic reductase activity was increased 45-fold when rats were fed cholestyramine and mevinolin. Under these conditions, the amount of immunodetectable reductase protein rose by 33-fold, and the reductase mRNA became visible by blot hybridization as a band of approximately 4 kilobases in length. When the mevinolin/cholestyramine-treated rats were fed cholesterol, reductase activity and immunodetectable protein declined markedly and the reductase mRNA was reduced to barely detectable levels. We conclude that treatment with cholestyramine and mevinolin increases the amount of reductase protein in rat liver by elevating the amount of its mRNA and that cholesterol feeding to such induced rats lowers the amount of hepatic reductase protein by decreasing the level of its mRNA.

Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum.

We present and evaluate a model for the secondary structure and membrane orientation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the glycoprotein of the endoplasmic reticulum that controls the rate of cholesterol biosynthesis. This model is derived from proteolysis experiments that separate the 97-kilodalton enzyme into two domains, an NH2-terminal membrane-bound domain of 339 residues and a COOH-terminal water-soluble domain of 548 residues that projects into the cytoplasm and contains the catalytic site. These domains were identified by reaction with antibodies against synthetic peptides corresponding to specific regions in the molecule. Computer modeling of the reductase structure, based on the amino acid sequence as determined by molecular cloning, predicts that the NH2-terminal domain contains 7 membrane-spanning regions. Analysis of the gene structure reveals that each proposed membrane-spanning region is encoded in a separate exon and is separated from the adjacent membrane-spanning region by an intron. The COOH-terminal domain of the reductase is predicted to contain two beta-structures flanked by a series of amphipathic helices, which together may constitute the active site. The NH2-terminal membrane-bound domain of the reductase bears some resemblance to rhodopsin, the photoreceptor protein of retinal rod disks and the only other intracellular glycoprotein whose amino acid sequence is known.

Amplification of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, in UT-1 cells.

32P-labeled cDNA probes were used to study levels of genomic DNA and regulation of mRNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase in UT-1 cells, a clone of compactin-resistant Chinese hamster ovary cells that have a 100-1000-fold increase in the amount of reductase protein. Similar measurements were made for the 53-kDa protein, a cytosolic protein of unknown function that is also expressed at high levels in UT-1 cells. The number of copies of the gene for reductase was increased by 15-fold in UT-1 cells as compared to the parental Chinese hamster ovary cells, as judged from Southern gel analysis of restriction endonuclease-digested genomic DNA. In contrast, there was no detectable increase in the number of gene copies for the 53-kDa protein. The amount of cytoplasmic mRNA for both proteins was markedly elevated in UT-1 cells, as determined by filter hybridization studies using 32P-labeled cDNA probes. The amount of mRNA for both reductase and the 53-kDa protein declined in parallel after addition of low density lipoprotein, 25-hydroxycholesterol, or mevalonate to the culture medium. The decline in reductase mRNA was associated with a marked decrease in the rate of [3H]uridine incorporation into hybridizable cytoplasmic mRNA. When UT-1 cells were grown for 3-4 months in the absence of compactin, the level of reductase mRNA and enzymatic activity decreased markedly, but the number of copies of the reductase gene did not decline. When the compactin-withdrawn cells were rechallenged with compactin, high levels of reductase mRNA and enzymatic activity promptly returned. We conclude that the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, has been stably amplified in UT-1 cells. Despite this differential gene amplification, the levels of cytoplasmic mRNA for both gene products are markedly elevated, and both are reduced in parallel by either sterols (low density lipoprotein-cholesterol or 25-hydroxycholesterol) or mevalonate, the product of the reductase-catalyzed reaction.

Domain map of the LDL receptor: sequence homology with the epidermal growth factor precursor.

The nucleotide sequence of a partial cDNA for the bovine low-density lipoprotein (LDL) receptor revealed an open reading frame of 264 amino acids that encodes the COOH-terminal 25% of the receptor protein. The sequence predicts a cytoplasmic domain of 50 amino acids at the COOH terminus, followed in order by a membrane-spanning region of 27 hydrophobic amino acids and an externally disposed stretch of 42 amino acids, that is rich in serine and threonine residues and appears to be the site of O-linked glycosylation. This orientation was confirmed by proteolysis experiments in which the relevant fragments were localized by blotting with anti-peptide antibodies and a galactose-specific lectin. The extracytoplasmic domain of the LDL receptor contains a region that is 38% identical with a 96 amino acid sequence in the precursor to mouse epidermal growth factor (EGF), a peptide hormone. This unexpected homology raises the possibility that proteins involved in growth stimulation (e.g., EGF precursor) and nutrient delivery (e.g., LDL receptor) may have a common evolutionary origin.