Absence of constitutively activating mutations in the GHRH receptor in GH-producing pituitary tumors.

The molecular events leading to the development of GH-producing pituitary tumors remain largely unknown. We hypothesized that activating mutations of the GHRH receptor might occur in a subset of GH-producing pituitary tumors. Genomic DNA samples from 54 GH-producing pituitary tumor tissues were screened for mutations of the GHRH receptor. Eleven homozygous or heterozygous nucleotide substitutions [169G > A (A57T), 338C > T (P113L), 363G > T (E121D), 409C > T (H137Y), 547G > A (D183N), 673G > A (V225I), 749G > A (W250X), 760G > A (V254M), 785G > A (S262N), 880G > A (G294R), 1268G > A (C423Y)] were found in 12 patients (22.2%). The 169G > A substitution (A57T) appears to be a polymorphism (4 patients, 7.4%). E121D and V225I were each found in 2 patients. In 1 patient with the V225I sequence, the substitution was not found in genomic DNA from peripheral leukocytes, suggesting a somatic mutation. A patient with a heterozygous W250X mutation was homozygous for the C423Y substitution. These variant GHRH receptors were studied in transfected TSA-201 cells to evaluate the functional consequences of the amino acid changes. None of the GHRH receptor variants was associated with basal elevation of intracellular cAMP. GHRH induced variable cAMP responses. With the W250X and G294R variants, there was no cAMP stimulation by GHRH, indicating that the mutations are inactivating. Expression of the W250X GHRH receptor on the cell membrane was severely decreased and GHRH binding to the G294R GHRH receptor was impaired. Although GHRH receptor variants are common in GH- producing pituitary adenomas, constitutively activating mutations, as a mechanism for GH-producing pituitary tumors appear to be rare.

The beta 3-adrenergic receptor gene and obesity in a population sample of African Americans.

OBJECTIVE: To examine the role of the Trp64Arg polymorphism in the beta 3-adrenergic receptor gene and the beta 3-adrenergic receptor gene locus in obesity-related traits in African Americans. SUBJECTS: A total of 687 individuals representing 193 African American families who were residents of metropolitan Chicago. MEASUREMENTS: Genotyping of the Trp64Arg polymorphism in the beta 3-adrenergic receptor gene and three microsatellite markers flanking the beta 3-adrenergic receptor gene (ADRB3) locus and measuring various obesity-related traits, including body mass index (BMI), fat-free mass, fat mass, percentage fat mass, waist circumference and serum lipid levels. RESULTS: The prevalence of obesity (defined as body mass index > or = 30 kg/m(2)) in the population was 27.3% and 51.2% in men and women, respectively. The frequency of the Arg64 allele was 10.0%. Multivariate regression analyses confirmed the existence of a significant contribution of familial variance to each of the five obesity-related traits noted above. Likelihood ratio statistics computed in a multivariate regression analysis failed to demonstrate a significant association between the Arg64 allele and any of the five obesity-related traits. Single and multipoint analyses using extended Haseman--Elston regression analyses failed to demonstrate suggestive evidence of linkage of three microsatellite markers that flank the beta 3-adrenergic receptor gene to BMI, percentage body fat, waist circumference or serum leptin levels. CONCLUSION: Given the contribution of familial variance to obesity-related traits in this population, neither the null finding for the Arg64 allele nor the lack of evidence of linkage of the ADRB3 locus to obesity-related traits could be attributed to lack of transmissibility of the traits suggesting that neither the Arg64 variant of the beta 3-adrenergic receptor gene nor another genetic variant in or near the ADRB3 locus contribute significantly to familial aggregation of obesity-related traits in African Americans. International Journal of Obesity (2001) 25, 54-60

Lack of association between the -308 polymorphism of the tumor necrosis factor-alpha gene and the insulin resistance syndrome.

BACKGROUND: Previous studies have demonstrated a role for tumor necrosis factor-alpha (TNF-alpha) in insulin resistance. A polymorphic variant of the TNF-alpha gene, the TNF2 allele, which is a guanine to adenine polymorphism at position -308 in the TNF-alpha promoter, is associated with higher basal and inducible promoter activity. The present study examined whether the TNF2 allele was associated with altered levels of different components of the insulin resistance syndrome, clustering of these components, or the 10-year change in the level of these components. METHODS: Components of the insulin resistance syndrome included insulin resistance, as determined by fasting insulin levels, body mass index, systolic blood pressure, triglycerides, uric acid, and high density lipoprotein-cholesterol. The study population was a subsample of participants from the Coronary Artery Risk Development in (Young) Adults (CARDIA) study, which included African American and white men and women aged 18-30. The sample included 243 black women, 142 black men, 392 white women, and 386 white men. Subjects were typed at the TNF-alpha locus. RESULTS: The frequency of the TNF2 allele was 12% in blacks and 16% in whites. Age-adjusted levels of the different components examined were not different at either baseline or year 10 in carriers of the TNF2 allele versus homozygotes for the wild-type allele, and the 10-year change in the level of different components was not different between the two genotype groups. There also was no evidence of increased clustering of components of the insulin resistance syndrome in carriers of the TNF2 allele. Moreover, there was no evidence of an association between the TNF2 allele and clustering across quartiles of BMI or quartiles of dietary fat intake (i.e., Key's score). CONCLUSIONS: In African Americans and whites, neither the TNF2 allele nor another polymorphism in the TNF-alpha gene or a neighboring gene with which the TNF2 allele is in linkage disequilibrium is associated with differences in the level of or increased clustering of components of the insulin resistance syndrome.

Mutational analysis of DAX1 in patients with hypogonadotropic hypogonadism or pubertal delay.

Although delayed puberty is relatively common and often familial, its molecular and pathophysiologic basis is poorly understood. In contrast, the molecular mechanisms underlying some forms of hypogonadotropic hypogonadism (HH) are clearer, following the description of mutations in the genes KAL, GNRHR, and PROP1. Mutations in another gene, DAX1 (AHC), cause X-linked adrenal hypoplasia congenita and HH. Affected boys usually present with primary adrenal failure in infancy or childhood and HH at the expected time of puberty. DAX1 mutations have also been reported to occur with a wider spectrum of clinical presentations. These cases include female carriers of DAX1 mutations with marked pubertal delay and a male with incomplete HH and mild adrenal insufficiency in adulthood. Given this emerging phenotypic spectrum of clinical presentation in men and women with DAX1 mutations, we hypothesized that DAX1 might be a candidate gene for mutation in patients with idiopathic sporadic or familial HH or constitutional delay of puberty. Direct sequencing of DAX1 was performed in 106 patients, including 85 (80 men and 5 women) with sporadic HH or constitutional delay of puberty and patients from 21 kindreds with familial forms of these disorders. No DAX1 mutations were found in these groups of patients, although silent single nucleotide polymorphisms were identified (T114C, G498A). This study suggests that mutations in DAX1 are unlikely to be a common cause of HH or pubertal delay in the absence of a concomitant history of adrenal insufficiency.

A novel mutation (R97C) in the neurophysin moiety of prepro-vasopressin-neurophysin II associated with autosomal-dominant neurohypophyseal diabetes insipidus.

Autosomal-dominant familial neurohypophyseal diabetes insipidus (adFNDI) is caused by heterozygous mutations in the gene encoding vasopressin-neurophysin II (AVP-NPII) on chromosome 20p13. We analyzed the AVP-NP II gene in a family with adFNDI by direct sequencing. A novel C to T transition (289C-->T in the cDNA, resulting in the substitution of Arg 97 by Cys (R97C) in the prepro-AVP-NPII precursor molecule) was identified in the gene region encoding neurophysin II in the index patient. This amino acid change is thought to result in the formation of an incorrectly folded hormone precursor, which may lead to chronic neurotoxicity and explain the dominant inheritance of the disease.

A mutation in the follicle-stimulating hormone receptor occurs frequently in human ovarian sex cord tumors.

A subset of ovarian tumors, referred to as sex cord-stromal tumors, produce endocrine manifestations due to the secretion of estrogens or androgens. Because gonadotropins induce the growth, differentiation, and function of the steroid-producing cells of the ovary, we hypothesized that mutations in the FSH receptor (FSH-R) might occur in this group of tumors. Ovarian sex cord tumors (n = 13), small cell carcinomas of the ovary (n = 3), and control DNA specimens (n = 116) were screened for mutations in the transmembrane domains of the FSH-R. A heterozygous T-->C mutation was found at nucleotide 1777 that converts codon 591 from phenylalanine to serine (F591S). This sixth transmembrane domain mutation was found in 9 of 13 (69%) sex cord tumors and 2 of 3 ovarian small cell carcinomas, but it was not present in control specimens, including 5 normal ovaries, 5 nonsex cord ovarian tumors, 16 thyroid tumors, or 90 specimens of peripheral blood leukocyte DNA, suggesting that this nucleotide change is not a polymorphism. The functional effects of identified mutations were assessed by expression of the wild-type or the F591S mutant FSH-R in COS-7 cells. The F591S mutation eliminated FSH-stimulated cAMP production, and a similar effect was observed when this mutation was introduced into the homologous location of the LH receptor. The high prevalence of the F591S mutation in the FSH-R suggests that it plays a role in the development of ovarian sex cord tumors.

Apoprotein-independent binding of chylomicron remnants to rat liver membranes.

Rat lymph chylomicrons and chylomicron remnants were treated with trypsin or Pronase. The ability of the resulting apoprotein-free lipoproteins to be taken up by the isolated perfused rat liver, and to bind to isolated rat liver membranes, was examined. Compared with control lipoproteins, the apoprotein-free chylomicrons and remnants retained unaltered their capacity to be differentiated by the intact liver and by the isolated membranes. Further, control remnants and apoprotein-free remnants competed for binding to the isolated membranes. We conclude that apoproteins are not required for the hepatic differentiation between chylomicrons and remnants, and suggest that the lipoprotein phospholipids may play a direct role in this process.

Phospholipids as modulators of hepatic recognition of chylomicron remnants. Observations with emulsified lipoprotein lipids.

The lipids extracted from chylomicrons, chylomicron remnants generated in vivo and hepatic-lipase-treated chylomicrons were emulsified by sonication. These emulsified particles retained the capacity of the native lipoproteins to be differentiated by the liver in vivo, i.e. only the particles derived from remnant and hepatic-lipase-treated chylomicron lipids were efficiently taken up by the liver. To investigate the role of phospholipids in this differentiation process, the phospholipids of all three lipoprotein preparations were separated from the remaining lipids by silicic acid chromatography. The phospholipid-free lipid fraction of chylomicrons was then emulsified with the phospholipids derived from each of the three lipoprotein preparations. Only the particles emulsified with phospholipids derived from remnants and hepatic-lipase-treated chylomicrons were efficiently taken up by the liver in vivo. These results support the proposition that phospholipids modulate the hepatic differentiation between chylomicrons and remnants in vivo.

Uptake of chylomicron remnants by the liver: further evidence for the modulating role of phospholipids.

Rat lymph chylomicrons were treated with rat heparin-releasable hepatic lipase (HL) or with bovine milk lipoprotein lipase (LPL). The ability of the resulting particles to be taken up by the liver in vivo was assessed following their infusion into the portal vein of partially hepatectomized animals. The following observations were made: a) the rate of phospholipid depletion, relative to the rate of triglyceride hydrolysis, induced by HL was two- to threefold higher than that observed for LPL; b) the depletion of at least 57% of phospholipids from the surface of HL-treated chylomicrons caused no major alterations in the apoprotein profile of the particles; c) for the same extent of triglyceride hydrolysis, HL-treated chylomicrons were taken up by liver at a rate significantly higher (P less than 0.005) than LPL-treated particles; d) the liver uptake of HL-treated chylomicrons was competitively inhibited by endogenously generated chylomicron remnants, indicating that these two types of lipoproteins share the same process of recognition and uptake by liver cells. It is concluded that the in vivo changes in phospholipid content, or composition, on the surface of chylomicrons during their transformation into remnants, modulate the differentiation of these two particles by the hepatic remnant receptor.

Heparin-binding apoproteins. Effects on lipoprotein lipase and hepatic uptake of remnants.

Apoprotein-free heparin-binding and non-binding chylomicrons were used as substrates to test the effects on lipoprotein lipase activity of (a) chylomicron protein I; (b) the mixture of proteins I, II and apoprotein E and (c) human beta 2-glycoprotein I. No activation of the enzyme was observed with any of those apoproteins. When rats were injected simultaneously with [3H]cholesterol-labelled heparin-binding chylomicrons (containing proteins I and II) and [14C]cholesterol-labelled non-binding chylomicrons, no differences were detected between the rates of removal from circulation of those two types of particles. Clearance of chylomicrons from circulation was accompanied by the incorporation of 3H and 14C labels into the livers at similar rates. It is concluded that proteins I, II and apoprotein E have no effect on the degradation of chylomicrons by lipoprotein lipase and that the hepatic recognition of remnants does not appear to be affected by proteins I and II.

Fractionation of chylomicrons by heparin-sepharose chromatography. Characterization of two heparin-binding proteins.

Rat lymph chylomicrons were separated into two fractions using heparin-Sepharose chromatography: a major fraction which elutes from the column with the void volume at 0.05 M NaCl, and a smaller fraction which binds to the column at 0.05 M NaCl and elutes at 0.3 M NaCl. These two fractions differ in mean particle size, and lipid and protein compositions. Both fractions share apolipoproteins B, A-IV, E, A-I, and C, but the fraction which binds to heparin-Sepharose contains two additional proteins: protein I (Mr = 6.0 X 10(4)), and protein II (Mr = 8.0 X 10(4)). Both proteins are also present in the lipoprotein-free fraction of rat serum. Proteins I and II bind to heparin-Sepharose, and are highly amphiphilic: they bind with high affinity to phospholipid surfaces and form stable monolayers at the air-water interface. The molecular weight, amino acid composition, heparin binding, and amphiphilicity of protein I resemble that of beta 2-glycoprotein I; in addition, protein I from rat lymph chylomicrons cross-reacts with rabbit antiserum to human beta 2-glycoprotein I, suggesting that these two proteins are homologous. Protein II appears to be a previously undescribed protein. The possible functions of these two proteins are discussed.

Liver uptake of chylomicron remnants with high and low apoprotein E:C ratios.

The ability of the isolated perfused rat liver to differentiate between chylomicrons and remnants with either high or low apoprotein E:C ratios was investigated. Remnants were prepared in hepatectomized rats injected with chylomicrons double-labeled with [3H]cholesterol and 14C-labeled fatty acids. By densitometric scanning of polyacrylamide gels, the apoprotein E:C ratio of the chylomicrons was 0.8 and that of the remnants was 1.5. When livers were perfused with these lipoproteins in a recirculatory system for 4 min, uptake of remnants was about 3-fold greater than that of chylomicrons. Preparation of remnants as well as chylomicrons with a low apoprotein E:C ratio was achieved by (i) removal of all apoproteins from the surface of the lipoproteins by trypsin digestion, followed by (ii) transfer of soluble apoproteins from serum lipoproteins to the apoprotein-free particles. The apoprotein E:C ratio of the reconstituted lipoproteins was decreased from 1.5 to 0.3 for remnants and from 0.8 to 0.2 for chylomicrons. In spite of these changes in apoprotein E:C ratios, the hepatic uptake of the reconstituted lipoproteins with low apoprotein E:C ratios was similar to their unmodified controls. These results indicate that the hepatic discrimination between chylomicrons and remnants is not determined by the relative amounts of apoproteins E and C on the surface of the lipoproteins.

The apoprotein B-independent hepatic uptake of chylomicron remnants.

Rat lymph chylomicrons were treated with Pronase resulting in particles completely devoid of surface apoproteins. On re-incubation with serum, the Pronase-treated chylomicrons re-acquired, by transfer from other lipoproteins, all apoproteins except apoprotein B, which is water-insoluble and non-transferable. When two groups of rats were injected with [3H]cholesterol-labelled control or Pronase-treated chylomicrons, radioactivity was incorporated into the liver of both groups at similar rates. It is concluded that the remnants of the control and Pronase-treated chylomicrons formed in the vascular space were recognized and taken up by liver cells by a process that does not require apoprotein B.

Hepatic uptake of phospholipid-depleted chylomicrons in vivo. Comparison with the uptake of chylomicron remnants.

1. Rats pretreated with Triton WR-1339 to prevent the formation of remnants were injected with [3H]cholesterol-labelled remnants, intact chylomicrons or chylomicrons depleted of most of their surface phospholipids by treatment with phospholipase A2. Within 5 min about 80% of the injected label of remnants and phospholipid-depleted chylomicrons was incorporated into the livers compared with less than 10% of the injected radioactivity of intact chylomicrons. A similar rapid hepatic uptake of radioactivity occurred when rats not pretreated with Triton were injected with [3H]cholesterol-labelled phospholipid-depleted chylomicrons. This rapid hepatic uptake of phospholipid-depleted chylomicrons occurred apparently without any alteration in the apoprotein composition of the particles. 2. The participation of hepatocytes in the uptake of remnants and phospholipid-depleted chylomicrons was examined. Both types of particles were taken up by the hepatocytes. However, small chylomicrons (Sf less than 400) were taken up more efficiently than were large chylomicrons (Sf greater than 400), but neither was taken up as efficiently as the remnants. 3. The results of this study lend support to the hypothesis that phospholipid-depleted chylomicrons and chylomicron remnants are taken up by the liver by a similar mechanism, which depends on the loss of surface phospholipids.

Uptake of phospholipid-depleted chylomicrons by the perfused rat liver.

1. Rat lymph chylomicrons were depleted of their surface phospholipids by treatment with pure phospholipase A2 from Crotalus adamanteus venom. 2. About 80% of the phospholipids could be removed from the chylomicrons without any apparent effect on their size, neutral lipid composition or qualitative profile of their tetramethylurea-soluble apoproteins. 3. Phospholipid-depleted chylomicrons were rapidly taken up whole by liver cells when perfused through isolated rat liver preparations. The rate of uptake was dependent on the extent of phospholipid depletion and reached a maximum (4-6.5-fold greater than control chylomicrons) when 80% of the phospholipids had been removed. 4. It is speculated that the hepatic uptake of phospholipid-depleted chylomicrons occurs by a mechanism to that of chylomicron-remnants uptake.

Hydrolysis of chylomicron triacylglycerol by endothelium-bound lipoprotein lipase. Effect of decreased apoprotein C-II/C-III ratio.

Chylomicrons with a decreased ratio of C-II/C-III apoproteins on their surface produced by the addition of apoproteins C-III-0 or C-III-3 to intact rat lymph chylomicrons. These chylomicrons inhibited the activity of soluble lipoprotein lipase in vitro, but had no effect on the activity of the endothelium-bound enzyme in the perfused heart.

Oscillatory changes in muscle lipoprotein lipase activity of fed and starved rats.

Lipoprotein lipase activity was measured at short time intervals in cardiac and skeletal muscles of normal and streptozotocin-treated diabetic rats fed ad libitum or deprived of food. In normal animals fed ad libitum, lipoprotein lipase activities of heart, diaphragm, soleus, and fast-twitch red fibers of the quadriceps muscle showed rhythmic oscillations that appeared to coincide with the nocturnal feeding habits of the animals. During the day (7 A.M. to 7 P.M.), when food consumption by the rats was greatly reduced, lipoprotein lipase activity in all muscles increased, followed by a decline to basal levels during the night. Similar oscillatory changes in lipoprotein lipase activity were observed in the muscles of diabetic rats fed ad libitum. In normal rats deprived of food, however, the oscillatory changes in muscle lipoprotein lipase activity were not abolished and persisted for at least 48 h. In diabetic rats starved during a 48-h period, the oscillatory changes in muscle lipoprotein lipase activity were markedly altered. In all animals, muscle lipoprotein lipase activities were not correlated to plasma glucagon levels.

The inhibition in vivo of lipoprotein lipase (clearing-factor lipase) activity by triton WR-1339.

1. Lipoprotein lipase activity was measured in heart homogenates and in heparin-releasable and non-releasable fractions of isolated perfused rat hearts, after the intravenous injection of Triton WR-1339. 2. In homogenates of hearts from starved, rats, lipoprotein lipase activity was significantly inhibited (P less than 0.001) 2h after the injection of Triton. This inhibition was restricted exclusively to the heparin-releasable fraction. Maximum inhibition occurred 30 min after the injection and corresponded to about 60% of the lipoprotein lipase activity that could be released from the heart during 30 s perfusion with heparin. 3. Hearts of Triton-treated starved rats were unable to take up and utilize 14C-labelled chylomicron triacylglycerol fatty acids, even though about 40% of heparin-releasable activity remained in the hearts. 4. It is concluded that Triton selectively inhibits the functional lipoprotein lipase, i.e. the enzyme directly involved in the hydrolysis of circulating plasma triacylglycerols. 5. Lipoprotein lipase activities measured in homogenates of soleus muscle of starved rats and adipose tissue of fed rats were decreased by 25 and 39% respectively after Triton injection. It is concluded that, by analogy with the heart, these Triton-inhibitable activities correspond to the functional lipoprotein lipase.