Studies on lipolysis in human adipose cells.

Epinephrine stimulated lipolysis and the uptake of oxygen by subcutaneous adipose cells of man. When glucose-(14)C was present in the medium, its utilization was not increased by epinephrine, although lipolysis was accelerated. Insulin did not reduce the production of fatty acids that had been stimulated by epinephrine. The combination of human growth hormone and cortisol stimulated the production of fatty acids by isolated human adipose cells to a lesser extent than epinephrine. When human growth hormone or cortisol was used singly, or when bovine growth hormone was added in combination with cortisol, no effect on fatty acid production was observed. Furthermore, an acetone-dried preparation of human pituitary glands, which was shown to stimulate lipolysis in rat adipose cells, had no effect on fatty acid formation in human adipose cells. This suggested that the human pituitary gland contained no more potent lipolytic agents than growth hormone and was supported by the lack of response of human adipose cells to purified corticotropin.

An abnormal triglyceride-rich lipoprotein containing excess sialylated apolipoprotein C-III.

An abnormal triglyceride-rich lipoprotein has been isolated from some patients with chronic renal failure or severe hypertriglyceridemia. The abnormal lipoprotein was characterized by an increased content of apolipoprotein (apo) C-III-2 (57.5% of total apo C-III peptides compared with 35.5% for controls, P less than 0.001) as characterized by isoelectric focusing and scanning densitometry. As determined by a substrate competition assay, the abnormal lipoprotein was a less efficient substrate for purified bovine milk lipoprotein lipase than control lipoproteins. Neuraminidase digestion of abnormal or control lipoprotein resulted in a reduction of the apo C-III-2 band with a corresponding increase in the region of apo C-III-0, which suggests that the increased content of apo C-III-2 in the abnormal is due to excessive sialylation of the C-III peptide. Limited incubation of the abnormal lipoproteins with neuraminidase caused a partial loss of sialic acid and resulted in a triglyceride-rich lipoprotein with a normal C-III-2:C-III-1 ratio. This preparation displayed normal substrate interaction with lipoprotein lipase. Three severely hypertriglyceridemic patients with the abnormal lipoprotein showed a marked reduction in serum triglyceride concentration, which is associated with a reversion to a normal C-peptide profile after dietary therapy. The results suggest that the extent of sialylation of the apo C-III peptide carried on triglyceride-rich lipoproteins may be critical for their interaction with lipoprotein lipase.

Deoxyribonucleic acid polymorphism in the apolipoprotein A-1-C-III gene cluster. Association with hypertriglyceridemia.

A DNA sequence polymorphism, revealed by digestion of human DNA with the restriction endonuclease Sst-1 and hybridization with an apolipoprotein A-I complementary DNA clone, has been shown to be located in or close to the 3' noncoding region of the apolipoprotein C-III gene. This polymorphism is found in significantly increased prevalence (P less than 0.001) in Caucasian hypertriglyceridemic subjects compared with race-matched controls, and its distribution in normal individuals of differing racial origins is reported. Furthermore, no alteration of high density lipoprotein or apolipoprotein A-I and apolipoprotein C-III phenotypes was observed in individuals with or without the polymorphism.

Regulation of sterol synthesis in human lymphocytes: evidence for post-transcriptional control by low density lipoprotein.

The effect of cordycepin (3'-deoxyadenosine), an inhibitor of messenger RNA synthesis, on the induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase mediated by lipid-depleted serum was studied in isolated human lymphocytes. 50 micrograms/ml cordycepin, although inhibiting messenger RNA synthesis by more than 50%, had no inhibitory effect on the two and four-fold induction of hydroxymethylglutaryl-CoA reductase when cells were incubated in a medium containing lipid-depleted serum for 8 and 16 h, respectively. This result suggests that newly synthesises messenger RNA is not required for the effect of lipid-depleted serum on the induction of hydroxymethylglutaryl-CoA reductase in human lymphocytes.

Genetic polymorphisms at the human liver/islet glucose transporter (GLUT2) gene locus in Caucasian and West Indian subjects with type 2 (non-insulin-dependent) diabetes mellitus.

The liver/islet glucose transporter (GLUT2) is mainly expressed in the hepatocytes of the liver and the beta-cells of the pancreatic islets and a defect in this transporter could lead to diabetic phenotypes, such as relative hypoinsulinaemia and reduced uptake and metabolism of glucose in the liver. DNA from unrelated individuals was digested with the restriction endonucleases Bgl-I and Taq-I followed by blotting and hybridisation with a 32P-labelled GLUT2 cDNA which revealed three restriction fragment length polymorphisms (RFLPs) (B1, T1 and T2) in both Caucasian and West Indian populations. Linkage analysis between these variant sites demonstrated that the alleles of these polymorphisms were in strong linkage disequilibrium. Disease association of genetic variants at the GLUT2 locus with type 2 diabetes was examined with these RFLPs in both Caucasian (n = 54) and West Indian (n = 46) populations with type 2 diabetes. There were no significant differences in the frequency of alleles, genotypes or haplotypes between diabetic patients and non-diabetic controls. However, there were significant differences in the allele frequencies of all these three polymorphisms between Caucasian and West Indian populations.

Sib-pair analysis of adenosine deaminase locus in NIDDM.

Recently, linkage between the ADA gene locus and MODY, a subtype of NIDDM, has been reported. The possibility that the region of chromosome 20q containing the ADA locus also may play a role in susceptibility to NIDDM needs to be investigated. Therefore, we examined the linkage between the ADA locus and NIDDM in affected siblings of 50 European white diabetic pedigrees--21 Italian and 29 British. Departure from independent segregation of the disease and an Alu VpA polymorphism within the 5' flanking region of the ADA locus was tested in the affected sib-pairs with the APM statistical method. After DNA amplification by the PCR and PAGE, five alleles were identified in the ALU VpA tract at the ADA locus in the two populations. Allele frequencies did not differ significantly between the two populations (chi 2 = 2.426, P > 0.05 [NS]). Analysis of the 50 diabetic sib sets, and independently of the Italian and British groups of affected sib pairs, revealed no segregation distortion between the marker locus and NIDDM. We conclude that mutations within or around the ADA locus are unlikely to play a major role in the etiology of NIDDM.

Lipoprotein lipase activity in human adipose tissue during induced hypertriglyceridaemia.

Needle biopsies of adipose tissue and blood samples were obtained before and at short intervals after a "bolus" injection of 10% Intralipid. Lipoprotein lipase activities were measured in acetone--ether extracts of the tissue samples. Levels of serum triglyceride began to fall less than 5 min after the injection of the Intralipid with a half-life of 20 min. During this time interval, no significant changes were observed in the activities of lipoprotein lipase in adipose tissue. A patient with a severe hypertriglyceridaemia (Type V) underwent plasma exchange with a reduction in serum triglyceride levels from 11 to 4 mm/l. There was a parallel fall in adipose tissue lipoprotein lipase activity. We conclude that lipoprotein lipase in adipose tissue is unaltered during experimental hypertriglyceridaemia and that the activity of the enzyme in adipose tissue is probably not reduced as a secondary feature of an elevated plasma triglyceride level.

An abnormal triglyceride-rich lipoprotein carrying excess apolipoprotein C-II.

The polypeptide composition of a variant lipoprotein (d less than 1.006) carrying a relative excess of apolipoprotein C-II has been characterised by polyacrylamide gel electrophoresis and isoelectric focussing. The apo-C peptides of the variant lipoprotein contained 45.2 +/- 1.3 (n = 9) % of apo C-II compared with 21.5 +/- 5.4 (n = 30) % for hypertriglyceridaemic controls. The variant lipoprotein activated purified bovine milk lipoprotein lipase normally, but was an inefficient substrate for this enzyme as assessed by direct release of fatty acids from the lipoprotein or by a substrate competition assay. Electron microscopy revealed the variant lipoprotein as non-spherical flattened particles compared with the more spherical appearance of control triglyceride-rich lipoproteins. We suggest that the relative proportion of apo C peptides associated with the lipoprotein particle may be critical for optimal enzyme-substrate interaction.

DNA polymorphisms at the lipoprotein lipase gene: associations in normal and hypertriglyceridaemic subjects.

Lipoprotein lipase is a rate determining enzyme for the removal of triglyceride-rich lipoproteins from the blood stream. We examined whether genetic variation at the lipoprotein lipase gene locus was related to the fasting plasma level of triglycerides in both a normal and hypertriglyceridaemic population. Two restriction fragment length polymorphisms revealed by the enzymes PvuII and HindIII generated alleles designated H1, 17.5 kb;H2, 8.7 kb;P1, 7.0 kb;P2, 4.4 and 2.5 kb, respectively. These were studied in 46 Caucasian hypertriglyceridaemic subjects in comparison with 86 normolipidaemic controls. The respective allelic frequencies were H1 0.211, H2 0.789 and H1 0.414, H2 0.586 (p less than 0.01). Similar differences in allelic frequencies were found in a smaller group of Japanese hypertriglyceridaemic subjects (n = 29) compared to Japanese controls (n = 41, p less than 0.01). Ninety-three healthy Caucasians were genotyped for both polymorphic sites to relate to levels of plasma triglyceride. We found that individuals with genotype P1P1 had fasting triglyceride levels of 0.96 +/- 0.31 mmol/l (n = 20) compared to genotype P2P2 with levels of 1.31 +/- 0.66 mmol/l (n = 30, p less than 0.02); heterozygous subjects (P1P2) had intermediate levels of plasma triglyceride (1.15 +/- 0.46 mmol/l, n = 43). The HindIII alleles were not significantly associated with variation in levels of plasma triglyceride, cholesterol, or HDL-cholesterol. We conclude that DNA variations at, or around, the lipoprotein lipase gene may constitute genetic determinants for both the population variation in plasma triglyceride levels as well as for the common metabolic disorder of primary hypertriglyceridaemia.

DNA polymorphisms in the apolipoprotein C-III and insulin genes and atherosclerosis.

A total of 167 patients undergoing investigation for suspected coronary artery disease (CAD) were genotyped for restriction fragment length polymorphisms (RFLP) at the apo A-1/C-III locus and the insulin gene locus using cloned human apo A-1 and insulin gene probes. The study group was subdivided into patients with absent or minimal CAD, intermediate CAD and severe obstructive CAD. An Sst-1 polymorphism located in the 3' non-coding region of the apo C-III gene identifies two alleles. One of the alleles (S2) showed a significantly increased frequency in the subjects with severe obstructive CAD (18%) compared with patients with minimal or absent CAD (6%) (P less than 0.025) and normolipidaemic control subjects. This A-1/C-III polymorphism may be a marker for an abnormality in the A-1/C-III genes predisposing to atherosclerosis. In contrast to a previous report, we found no increase in the frequency of the Class 3 insulin alleles in subjects with severe CAD.

Lipoprotein and hepatic lipase gene variants in coronary atherosclerosis.

Lipoprotein lipase is the rate determining enzyme for the removal of triglyceride rich lipoproteins from the blood stream. We examined whether genetic variation at the lipoprotein lipase gene locus is related to the occurrence of premature coronary artery disease. Two restriction fragment length polymorphisms, revealed by the enzymes HindIII and PvuII, demonstrated alleles designated H1 (17.5 kb), H2 (8.7 kb), P1 (7.0 kb), P2 (4.4 kb and 2.5 kb) respectively. These were studied in 70 Caucasian subjects with severe coronary atherosclerosis in comparison with 122 Caucasian healthy controls. The allelic frequencies for cases and controls were respectively: H2 0.770, 0.579 (P less than 0.001); P2 0.575, 0.554 (P NS). The allelic frequencies of the HindIII and BglII polymorphic sites at the hepatic lipase gene locus were also studied in the same groups of subjects. These showed no differences between cases and controls. We conclude that DNA variation at or adjacent to the lipoprotein lipase gene may contain genetic determinants for the occurrence of premature coronary artery disease.

The use of polymorphic DNA and protein markers for the third complement component for determining linkage of familial hypercholesterolaemia.

We have used DNA and protein polymorphisms for the third complement component (C3) to assess the potential of DNA markers in the diagnosis and study of familial hypercholesterolaemia (FH), and to confirm the reported linkage between FH and C3. The inheritance of FH and the C3 gene has been studied in 10 families by combining information from both the protein and DNA polymorphisms. Our results confirm that the C3 gene is loosely linked to the gene causing FH (lod score maximum of 2.0) at a recombination distance of 0.15. When these results are combined with previously published data the overall lod score maximum is 4.75 at a recombination distance of 0.2, meaning that the two genes will be inherited together in only about 80% of children. These results confirm that the gene that causes familial hypercholesterolaemia is linked to C3 and is therefore on chromosome 19, but C3 is not close enough to be used as a diagnostic marker.

Enhanced in vivo platelet release reaction and malondialdehyde formation in patients with hyperlipidemia.

Plasma level of beta-thromboglobulin (beta TG), a useful marker of in vivo platelet "release reaction,"was determined by radioimmunoassay in 69 patients, with three types of primary hyperlipidemia (IIa, IIb, IV) and compared with the findings in age- and sex-matched healthy controls and 57 patients with established atherosclerosis and peripheral vascular disease. Malondialdehyde (MDA) formation, used for assessment of prostaglandin synthesis, was determined in 51 and plasma platelet factor 4 (PF4), measured by radioimmunoassay, in 48 of the patients with hyperlipidemia. Results were correlated to five serum lipids and lipoprotein levels in the patients with hyperlipidemia. beta TG was significantly increased in the patients with hyperlipidemia and peripheral vascular disease, compared to those in the controls (p < 0.001); it was significantly higher in the patients with hyperlipidemia than in those with peripheral vascular disease. PF4 and MDA formation were also increased in the patients with hyperlipidemia, and significantly higher levels of MDA were obtained in patients with type IIb and type IV hyperlipidemia than in those with type IIa hyperlipidemia (p < 0.02). beta TG and MDA correlated weakly with total serum cholesterol triglycerides and very low density lipoprotein-triglyceride. There was also a significant correlation between beta TG and PF4, and MDA production. These results indicate that in vivo platelet "release reaction" and MDA formation are increased in hyperlipidemic patients. The release reaction is more enhanced in those with hyperlipidemia than in the patients with peripheral vascular disease. They suggest that the abnormal platelet function is related to the elevated levels of serum lipids and lipoproteins in the hyperlipidemic patients and not only to the atherosclerotic changes associated with hyperlipidemia.

Triglyceride storage disease: a defect in activation of lipolysis in adipose tissue.

A child, aged 6 years 3 months, with a triglyceride storage disorder in peripheral adipose tissue, microcephaly, and gross emaciation has been studied at autopsy. The mean triglyceride content of adipocytes in hand and foot was 0.17 +/- 0.02 mug/cell and 0.18 +/- 0.02 mug/cell. Adipocytes from abdominal tissue were small, irregular, and difficult to measure accurately, reflecting the degree of cachexia. Basal tissue contents of cyclic AMP and release of glycerol and fatty acid from peripheral tissue of the child were in the same range as adult tissues. None of these measurements, however, were increased by incubation with isoprenaline (10(-5 M), compared to a three- to seven-fold increment in adult subcutaneous tissues and to a four- to ten-fold increment of glycerol and cyclic AMP in peripheral adipose tissue of a control child aged 10 years. We postulate that the proband may have had a defect of adenyl cyclase or catecholamine receptor, which has a role in the abnormal storage of triglyceride in peripheral adipose tissue.

Alpha-adrenergic receptor activity, cyclic AMP and lipolysis in adipose tissue of hypothyroid man and rat.

Lipolysis and intracellular levels of cyclic AMP of adipose tissue from man and rat in both hypothyroid and euthyroid states were studied in response to stimulation by catecholamines in vitro. Hypothyroid patients were studied before and after treatment, and were also compared with euthyroid obese controls. The experimental group of rats was rendered hypothyroid by the addition of 2.9 mM-propylthiouracil to their drinking water, and their status confirmed by plasma thyroid function tests. Evidence for alpha-adrenergic receptor activity was found in rat adipose tissue, but was less marked than the pronounced alpha-adrenergic activity in human adipose tissue. Glycerol release from adipose tissue in response to noradrenaline stimulation was less marked in hypothyroidism in both species, and was related to an increased alpha-adrenergic activity. No evidence was found for increased alpha-adrenergic effects on cyclic AMP level in hypothyroid subjects, and little evidence was found in adipose tissue from hypothyroid rats. This discrepancy may be due to the presence of the phosphodiesterase inhibitor, theophylline, in the incubation system. The possible modulatory role of thyroid hormones on receptor and phosphodiesterase activity, and on lipolysis, is discussed.

Coronary artery disease and dyslipidemia within Europe: genetic variants in lipid transport gene loci in German subjects with premature coronary artery disease.

Fifteen polymorphisms in six lipid transport genes were studied in a German population for relationships with dyslipidemia and coronary artery disease (CAD), to investigate a possible genetic basis for the marked differences in mortality rates from coronary heart disease within Europe. In other populations these polymorphisms have all been associated with CAD or with phenotypes known to predispose to CAD. The apoAI PstI polymorphism (P<0.005) and the lipoprotein lipase Ser(447)-Ter mutation (P<0.005) were associated with plasma triglyceride concentrations. Additionally, the apoAI PstI polymorphism (P<0.05), the apoB XbaI polymorphism (P<0.05) and apoE phenotypes (P<0.05) were associated with plasma cholesterol concentrations. However, none of the allele frequencies of the polymorphisms studied were related to the presence, or absence, of coronary artery disease. Associations between five polymorphisms representing four lipid transport gene loci and dyslipidemia were demonstrated in this German population. It is possible that predisposition to dyslipidemia in Germany involves a particular selection of polymorphic loci, which are different from those identified in other European countries.

Genetic markers to predict polygenic disease: a new problem for social genetics.

Many genetic markers that relate to common multifactorial disease in adults have been identified during the past 15 years. Their use as adjuncts for the diagnosis, prognosis, prediction of disease or targeting therapy for these disorders has begun, good examples being the Factor V Leiden mutation for venous-thromboembolism, lipoprotein lipase mutations for hypertriglyceridaemia and the apolipoprotein E4 variant for Alzheimer's dementia. However, extensive gene-gene and gene-environment interactions make their use more complex than markers for the simpler monogenic disorders (such as cystic fibrosis, or Duchenne's muscular dystrophy). Possible misapplication of the genetic markers for multifactorial disease in the fields of risk prediction, direct sales to the public, life assurance, employment rights, and legislation for regulation of their use are discussed.

Genetic determinants of atherosclerosis-related dyslipidemias and their clinical implications.

New approaches to the study of the genetics of premature coronary heart disease (CHD) have been made possible by the introduction of DNA probes for the apolipoproteins, the receptors and enzymes involved in lipid transport. Initially these were used to identify restriction fragment length polymorphisms for use as genetic markers to locate genes involved in the pathogenesis of atherosclerosis. More recent developments have allowed direct analysis of etiological mutations by such methods as single stranded conformational polymorphism (SSCP) analysis and denaturing gradient gel electrophoresis (DGGE). Loci that have been incriminated by such techniques include the apolipoprotein A-I--C-III--A-IV gene cluster, the apo B gene and the lipoprotein lipase locus. It is to be anticipated that these and other techniques will eventually identify all the major determinants involved in disorders of lipid transport and the development of premature atherosclerosis.

Sorbitol and other polyols in lens, adipose tissue and urine in diabetes mellitus.

Sugars and polyols (glucose, fructose, sorbitol, inositol and pentitols) have been measured in urine, adipose tissue and lens in groups of diabetic and non-diabetic patients. The daily excretions of glucose, sorbitol and inositol were raised in the diabetics. There was a linear relation between 24 hour urinary excretion of glucose and hexitols (r = +0.87, p less than 0.001). In lens of diabetics there was an increase in glucose concentration, but not of fructose, sorbitol or inositol. Compounds readily detected in adipose tissue were inositol and glucose; in addition an unidentified carbohydrate was detected in adipose tissue that related to the concentration of tissue glucose. These results are discussed in relation to the possibility that tissue accumulation of polyols could be responsible for the secondary complications of diabetes such as cataracts.