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The effects of dietary saturated and polyunsaturated fat on the metabolism of apolipoprotein A-I (apoA-I) and apolipoprotein B (apoB) were studied in a patient with type IIb hyperlipoproteinaemia. On the saturated fat diet, the rate of synthesis of very low density lipoprotein apoprotein B (VLDL-apoB) was approximately twice normal, accounting for the increased plasma VLDL pool in this subject. However, 54% of the synthesized VLDL-apoB was catabolized by a pathway independent of low density lipoproteins (LDL). The metabolic conversion rate of VLDL-apoB to LDL-apoB was normal in this subject and his expanded plasma LDL-apoB pool resulted, not from increased input of the apoprotein from VLDL, but from a decrease in its fractional clearance rate. On the polyunsaturated diet, there was a significant fall in the plasma cholesterol and triglyceride concentrations and a change in the fatty acid composition of all plasma lipoprotein fractions. These changes were accompanied by a decrease in the plasma concentrations of apoA-I and apoB which resulted from a reduction of apoprotein synthetic rate.

Apolipoprotein-serine (apoLP-Ser or apoC-I) is one of the apoprotein constituents of human plasma very low density lipoprotein. The protein has 57 amino acid residues, including one residue of methionine and is lacking histidine, cysteine, cystine, and tyrosine. The NH2 terminus of apoLP-Ser is threonine and the COOH terminus is serine. Cleavage of apoLP-Ser with cyanogen bromide, followed by chromatography of the digest on Bio-Gel P-30 in 25% formic acid, yielded two fragments corresponding to the NH2-terminal (CNBr I) and the COOH-terminal (CNBr II) fragments and accounting for the 57 residues of the intact protein. The amino acid sequences of the tryptic peptides from CNBr I and chymotryptic peptides from CNBr II were determined by conventional methods. The amino acid sequence of apoLP-Ser is as follows: Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu-Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys-Met-Arg-Glu-Trp-Phe-Ser-Glu-Thr-Phe-Gln-Lys-Val-Lys-Glu-Lys-Leu-Lys-Ile-Asp-Ser.

Radioiodinated human apolipoprotein A-I, when incubated with plasma lipoproteins, associates exclusively with high density lipoproteins. It does not interact with very low density lipoproteins or low density lipoproteins. Binding is rapid, being complete within 10 min, and is not affected by variation of pH within the range 6.0–9.5 or of temperature over the range 0°–37°C. At equimolar concentrations of apolipoprotein A-I and high density lipoproteins, 0.58 mol of the apoprotein bind per mol of high density lipoproteins. Binding increases progressively with apolipoprotein A-I concentration up to an apolipoprotein A-I : high density lipoprotein molar ratio of 20 : 1, when each mol of lipoprotein binds 7.62 mol of apoprotein. Interaction of apolipoprotein A-I with high density lipoproteins displaces endogenous apolipoprotein A-I from the particle to a maximum of 2 mol of apolipoprotein A-I per mol of high density lipoproteins. This displacement occurs at an apoprotein : lipoprotein molar ratio of 10 : 1. The rest of the endogenous apolipoprotein A-I is not displaceable, even when the exogenous apoprotein : lipoprotein ratio is increased to 20 : 1. Incubation of apolipoprotein A-I and high density lipoproteins at molar ratios of 0.3 or less results in a mol for mol exchange of exogenous with endogenous apolipoprotein A-I on the lipoprotein. The half-life of 131I-labeled apolipoprotein A-I/high density lipoproteins in rats and rabbits is 15 h and 1.40 days respectively; the material decays exponentially from the bodies of these animals. More than 94% of the radioactivity in their plasma is associated with a component of hydrated density 1.063–1.21 kg/l. When injected into humans, the radioiodinated apoprotein incorporated into high density lipoproteins by in vitro incubation is cleared from the plasma 20% faster than endogenous apolipoprotein A-I in high density lipoproteins.

Insulin and glucagon injected into the portal vein produced rapid reciprocal changes in the activities of certain hepatic glycolytic enzymes and fructose diphosphatase in the rat. Insulin produced a rapid increase in hepatic phosphofructokinase and pyruvate kinase activities and a decrease in fructose diphosphatase activity. Glucagon produced a rapid but reciprocal response in each of these enzyme activities. Fructose diphosphate aldolase activity was unaltered by either hormone. The insulin effect on these enzymes was detected within 5 min following injection, was maximal by 10 min, and then gradually declined over the remaining 30 min of testing. The magnitude of the insulin effect was dependent on the amount of insulin injected. No effect was detected when 0.005 unit per kg (0.001 unit) was injected; phosphofructokinase and pyruvate kinase activities were slightly increased with 0.015 unit per kg (0.003 unit); phosphofructokinase, pyruvate kinase, and fructose diphosphatase activities were altered with 0.15 unit per kg (0.03 unit) and 1.5 units per kg (0.3 unit), with the higher dose giving a slightly greater effect. The insulin effect was not associated with a significant change in cyclic adenosine 3':5'-monophosphate concentrations. The glucagon effect on the enzyme activities was preceded by a significant increase in cyclic adenosine 3':5'-monophosphate concentrations, which occurred within 30 s after hormone injection. A change in enzyme activities was found at 2 to 5 min and was maximal 5 to 10 min following glucagon injection. The hormone effect on enzyme activities and cyclic adenosine 3':5'-monophosphate levels was dependent on the amount of glucagon injected, being maximal at doses of 150 to 300 µg. Pretreatment of the rat with actinomycin D or puromycin did not alter the response of the enzymes to insulin and glucagon, indicating that de novo protein synthesis was not responsible for the change in enzyme activities. Glucagon injected 5 min following insulin reversed the insulin effect; insulin given 5 min after glucagon also partially reversed the glucagon effect on cyclic adenosine 3':5'-monophosphate and the enzyme activities. Intravenous insulin produced rapid changes in the activities of renal cortical, skeletal muscle, and epididymal fat glycolytic enzymes and fructose diphosphatase in the rat. Insulin (0.15 unit per kg) produced rapid increases in pyruvate kinase and phosphofructokinase and rapid decrease in fructose diphosphatase activities in all tissues. Fructose diphosphate aldolase activity was unchanged following insulin infusion. Intravenous glucagon (0.15 mg) produced rapid changes, reciprocal to those seen with insulin, in fructose diphosphatase activity in all tissues. Glucagon significantly decreased epididymal fat phosphofructokinase activities but did not alter the activity of this enzyme in the renal cortex and skeletal muscle. Glucagon significantly decreased renal cortical pyruvate kinase but had no effect in the epididymal fat and skeletal muscle. Fructose diphosphate aldolase activity was unchanged in all tissues following glucagon infusion. Cyclic adenosine 3':5'-monophosphate infusion (0.05 mmole) produced significant changes in pyruvate kinase, phosphofructokinase, and fructose diphosphatase activities in all tissues which resembled those changes seen following glucagon infusion. These data suggest that the glucagon responses seen in the four tissues studied are mediated by cyclic adenosine 3':5'-monophosphate.

Epinephrine infusion into the portal vein produced rapid changes in the activities of certain hepatic, epididymal fat, and skeletal muscle glycolytic enzymes and fructose diphosphatase in the rat. Epinephrine produced a rapid increase in hepatic, epididymal fat, and skeletal muscle fructose diphosphatase activity. In contrast, epinephrine produced a rapid decrease in hepatic phosphofructokinase, pyruvate kinase, and glucokinase activities. Epinephrine also lowered skeletal muscle phosphofructokinase and pyruvate kinase activities. However, epinephrine had no significant effect upon epididymal fat pyruvate kinase and phosphofructokinase activities. Fructose diphosphate aldolase activities were unchanged in the liver, epididymal fat, and skeletal muscle. The epinephrine effect on the hepatic enzymes was maximal within 2 to 5 min following injection of 2 µg and then gradually returned to control levels over the remaining 35 to 38 min of testing. The magnitude of the epinephrine effect was dose-dependent. No effect was detected when 0.25 µg per min was injected. Phosphofructokinase, pyruvate kinase, and fructose diphosphatase activities were significantly altered at a dose level of 1.0 µg per min, with maximal changes occurring at the 2.0- to 4.0-µg per min dose level. The epinephrine effect was associated with a significant increase in hepatic cyclic adenosine 3′:5′-monophosphate concentrations. Actinomycin D and puromycin treatment did not alter the response of the enzymes to epinephrine. Insulin given 5 min following epinephrine partially reversed the epinephrine effect. Similarly, epinephrine given 5 min following insulin partially reversed the insulin effect. We suggest that the epinephrine effect is mediated through cyclic adenosine 3′:5′-monophosphate and may be due to phosphorylating mechanisms analogous to that responsible for the regulation of glycogen metabolism.

1. 1. Low density lipoproteins (LDL) were isolated from pig plasma by ultracentrifugal flotation in KBr between densities 1·020 and 1·060 g/ml (LDL 1 ) and between 1·060 and 1·090 g/ml (LDL 2 ). 2. 2. The chemical, immunological and physical properties of LDL 1 and LDL 2 were compared with each other and with human LDL. 3. 3. LDL 1 and LDL 2 were delipidated and fractionated by chromatography on Sephadex G-150 in 0·1 M Tris-HC1, pH 8·0, containing 2 mM sodium decyl sulfate. ApoLDL 1 and apoLDL 2 were immunochemically identical, had only minor differences of amino acid composition and had indistinguishable circular dichroic spectra. The apoproteins were very similar in amino acid composition and circular dichroic spectra to human apoLDL. 4. 4. Our findings strongly suggest that the apoproteins from pig LDL 1 and LDL 2 are identical and that this protein is similar but not identical to apoLDL from man.

High density lipoproteins (HDL) were isolated from pig plasma by ultracentrifugal flotation between densities 1.063 and 1.210 g per ml. After delipidation, the apolipoproteins were fractionated by chromatography on Sephadex G-150 and DEAE-cellulose in 5.4 m urea. One major apolipoprotein was isolated and characterized. From its chemical and physical properties, this major apolipoprotein appears very similar to apoLP-Gln-I from human HDL. Pig HDL appears to differ from human HDL in a marked decrease in the relative content of apoLP-Gln-II in the pig lipoprotein family. A distinct peak associated with apoLP-Gln-II was not identified by chromatography of pig apoHDL. A small quantity of a protein with the same mobility of human apoLPGln-II was detected in pig apoHDL after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The major apolipoprotein from pig HDL had an approximate molecular weight of 26,000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Its calculated molecular weight from amino acid analysis was 29,800. Rabbit antisera prepared against the pig apolipoprotein formed precipitin lines of partial identity between this protein and apoLP-Gln-I from human HDL. Treatment of the pig apolipoprotein with carboxypeptidase established Leu-Gln as the COOH-terminal sequence. Pig and human apoLPGln-I had similar amino acid compositions, but the pig protein contained isoleucine and had 12 more residues of alanine than did human apoLP-Gln-I. Pig and human apoLP-Gln-I had indistinguishable absorption spectra and similar circular dichroism spectra indicating a relatively high content of α-helical conformation. Both proteins activated human lecithin : cholesterol acyltransferase. These studies demonstrate that the major apolipoprotein from human and pig HDL have similar chemical, immunological, physical, and physiological properties.

Rate zonal ultracentrifugation of plasma samples from ten healthy age-matched volunteers (five males, five females) indicated that the high density lipoprotein subfraction ratio (HDL2:HDL3) in females was significantly higher than in males. The cause of this phenomenon was investigated by simultaneous examination of the metabolism of the major HDL apoproteins (apoA-I and apoA-II) in both groups. The results show that there is no significant sex-related difference in the plasma pool size, fractional catabolic rate, or synthetic rate of either apoprotein. We conclude that the increased HDL2:HDL3 ratio in females versus males does not derive from measurable differences in the metabolic handling of either apoprotein.

Subfractions of VLDL (VLDL 1 , Sf 100–400; VLDL 2 V Sf 60–100; VLDL 3 , Sf 20–60) and LDL (LDL 1 , Sf 12–20; LDL 2 , Sf 6–12; LDL 3 , Sf 3–6) were isolated from the plasma of three normal, three type in and four type IV hyperlipoproteinemic subjects. In the type IV group, all VLDL subspecies Were of normal composition but were increased in concentration in the order VLDL 1 > VLDL 2 > VLDL 3 . In the same subjects, although LDL 1 was lowered and LDL 3 increased, the total plasma LDL concentration Was normal. All VLDL subfractions were elevated in the type III group, but in this ease VLDL 3 predominated. These subfractions were enriched in cholesteryl esters and depleted in triglyceride. In the LDL density range there was a shift of mass towards the least dense fraction, LDL 1 , which was of normal composition. EPR studies of the VLDL and LDL subfractions in a type IV subject demonstrated a decrease in fluidity with increasing density. The major change occurred between VLDL 3 and LDL, and was attributed to a substantial alteration in the cholesteryl ester: triglyceride ratio in the particle. A similar argument was used to explain the reduced fluidity of type III VLDL 3 with respect to that of the same subfraction in normal or type IV subjects. Particle diameters, determined by laser light-scattering speetroscopy were-in good agreement with the values obtained by electron microscopy. This study provides a baseline for the examination of the relationship' between the physical and metabolic properties of VLDL and LDL subtractions in type in and IV hyperlipoproteinemia.

Very low density lipoproteins ere isolated from plasma of swine by ultracentrifugal flotation. After delipidation, the lipid-free proteins were separated by chromatography on Sephadex G-150 AND DEAE-cellulose. A major apoprotein was isolated and shown to activate cows' milk lipoprotein lipase. Since human very low density lipoproteins also contain an activator protein, designated, apoC-II, we have called the pig protein, pig apoC-II. Pig apoC-II had a molecular weight of approximately 10 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid composistion showed the absence of histidine, cysteine and tryptophan; there was no evidence for carbohydrate. Treatment of pig apoC-II with carboxypeptidase indicated COOH-terminal serine. Rabbit antisera prepared to the pig protein gave single precipitin lines of complete identity to very low density lipoproteins, apoC-11. Using anti-pig apoC-II, a radioimmunoassay was developed which provides a convenient and reproducible method for measuring 5-1000 ng of apoprotein.

In this study we have investigated, in four normal males the effects of dietary saturated and polyunsaturated fat on the chemical composition and thermotropic properties of human high density lipoproteins (HDL) and have measured the influence of the diets on the metabolism of that fraction of HDL apolipoprotein A-I (apoA-I) that undergoes exchange in vitro and accounts for approximately two-thirds of the lipoprotein's apoA-I complement. When compared with the saturated fat diet, the polyunsaturated diet reduced plasma cholesterol (24%, P0.01) by affecting the cholesterol content in the very low density lipoprotein ( downward arrow25%, P0.02), low density lipoprotein ( downward arrow20%, P0.01), and high density lipoprotein fractions ( downward arrow33%, P0.01). Plasma triglyceride was also lowered (by 13%, P0.01). Furthermore, polyunsaturated fat ingestion caused a significant fall in the palmitate and stearate content of HDL triglyceride (41 and 37%, respectively), cholesteryl esters (29 and 35%), and phospholipids (17 and 9%) with a concomitant increase in the linoleate content of these moieties (157, 28, and 29%, respectively). The polyunsaturated diet also produced reciprocal changes in the percentage protein ( downward arrow9%, P0.02) and phospholipid ( downward arrow11.5%, P0.01) in HDl. These compositional changes were associated with an increase in the microscopic fluidity of the polyunsaturated HDL, although both diets had little effect on the fluidity parameters of HDL at body temperature. Rate zonal ultracentrifugation indicated that the HDL(2)/HDL(3) ratio fell by 28% (P0.05) on the polyunsaturated fat diet. In addition to the above, this diet reduced plasma apoA-I by 21% (P0.01). No change was seen in the fractional catabolic rate or the distribution of the apoprotein between intravascular and extravascular compartments on the two diets. However, when compared with the saturated diet, the synthetic rate of apoA-I was reduced by 26% during polyunsaturated fat feeding. The results show that polyunsaturated fat alters the chemical composition, thermotropic properties, and subfraction distribution of HDL without changing the fractional rate of catabolism of their major protein, apoA-I.These findings deserve careful consideration in determining the applicability and efficacy of polyunsaturated fat diet therapy in the prevention of atherosclerosis in man.

Symptomatic fasting and reactive hypoglycemia in association with low iejunal and hepatic fructose-1,6-diphosphatase (FDPase) activity were found in an adult patient and her 19-month-old daughter. Plasma glucose response to glycerol ingestion was abnormal in both, showing little change in the adult and a decline in the child. FDPase activity was low in tissues from both patients. This reduced enzyme activity defines a new variant of the fructose-1,6-dipphosphatase deficiency state(s) which is mild in its manifestations and thus more resistant to the hypoglycemia-provoking effects of fructose and alanine. No difference was detected between the FDPase from these patients and unaffected individuals by polyacrylamide gel electrophoresis. The K m of the jejunal enzyme for fructose diphosphate in the adult was similar to the normal control. Glucagon rapidly increased the activity of hepatic FDPase in both patients. Folic acid, 15 mg/day, increased hepatic and jejunal FDPase activity in both patients. After folate therapy, there was improvement in the adult's symptoms and improvement in the child's ability to maintain normal plasma glucose levels. The interrelationship between the hypoglycemia, FDPase deficiency, and the response to folate therapy was discussed.

We have developed a new method for direct measurement of fluorescent probes associated with undisturbed monolayers of cultured fibroblasts. A helium-cadmium continuous wave laser produced excitation light at 325 nm to illuminate the cell monolayer on the front inner surface of a quartz sample tube. The emitted light from the cell monolayer passed through a scanning monochrometer to a low noise photomultiplier tube and was amplified with a photon-counting system. The fluorescent probe, cholesteryl 4-(3′-pyrenyl)butanoate, was incorporated into normal human low density lipoproteins (LDL). The interaction between LDL containing the fluorescent probe and the cell surface of normal human fibroblasts was examined. The uptake of the fluorescent LDL was measured as a function of temperature, concentration, time, specificity, and ability to suppress 3-hydroxy-3-methyl-glutaryl-CoA reductase. In all respects, LDL containing the fluorescent probe and native LDL were comparable. Using this technique, cell-surface interactions can be studied in situ so that changes in structure and function caused by removal of the cells from the growth surface can be avoided.

1. 1. Glucagon, insulin and epinephrine produce rapid changes (within minutes) in hepatic and jejunal formiminotransferase ( N- formimino- L -glutamate: tetrahydrofolate 5-formiminotransferase, EC 2.1.2.5) activity in the rat. Limited evidence in human subjects and patients demonstrate that similar effects also occur in man. 2. 2. Intravenous glucagon (0.0015–0.5 mg) and isnulin (0.015–1.5 units/kg) produced rapid increases and decreases, respectively, in hepatic formiminotransferase activity which were unaltered by pretreatment of the rats with either actinomycin D or puromycin. Intravenous insulin and glucagon significantly decreased and increased, respectively, rat jejunal formiminotransferase activity, as well. Intravenous epinephrine (1.0–2.0 μg/min) also produced a rapid increase in hepatic formiminotransferase activity. The stimulatory effects of glucagon and epinephrine on hepatic formiminotransferase activity were mimicked by intravenous cyclic AMP suggesting that these hormonal responses may be mediated through increased cyclic AMP production. 3. 3. In four children with fasting-induced hypoglycemia, intravenous glucagon (1 mg/min) significantly increased hepatic formiminotransferase activity within 2–3 min. In five normal human subjects, the administration of subcutaneous insulin (15 units) for three consecutive days significantly decreased jejunal formiminotransferase activity. 4. 4. We suggest that the rapid insulin, glucagon and epinephrine effects on formiminotransferase activity may be due to dephorylation— phosphorylation mechanisms analogous to that involved in the regulation of glycogen metabolism.

Glucagon and insulin given intravenously produced rapid reciprocal changes in the activity of phosphofructokinase (PFK), pyruvate kinase (PK) and fructose diphosphatase (FDPase) in rat liver. Fructose diphosphate aldolase did not change. Glucagon increased FDPase and decreased PFK and PK activity. Insulin decreased FDPase and increased PFK and PK activity. The glucagon effect was associated with a marked increase in hepatic cyclic adenosine 3′,5′-monophosphate (cyclic AMP) concentrations. Insulin did not significantly alter hepatic cyclic AMP concentrations.

A patient is reported who first sought treatment because of failing vision and hypogonadism secondary to a craniopharyngioma. This was surgically removed, and during the immediate postoperative period the patient developed hyperthermia in the absence of apparent infection, aphasia, hemiplegia and diabetes insipidus. He recovered from these and then became hypothyroid. About a year later, while taking desiccated thyroid, he developed hyperthyroidism with diffuse overactivity of the gland not suppressible with exogenous triiodothyronine. This sequence of events suggests that the hyperthyroidism resulted from an extrathyroidal disorder in this patient.