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ABSTRACT

Lipoatrophic diabetes has been produced in rabbits by injection of a fraction prepared from the urine from patients with congenital generalized lipodystrophy. Both these conditions are considered to be hypothalamic syndromes. The animals, and a patient with congenital generalized lipodystrophy and latent diabetes were treated with the dopamine receptor blocker, pimozide, for 4 and 17 months, respectively. The results were discouraging even though the patient got a daily dose of 16 mg pimozide. Fenfluramine has a lowering effect on brain serotonin, and peripheral effects on glucose and triglyceride metabolism. This drug improved the general condition of the rabbits with lipoatrophic diabetes, as well as that of the patient with congenital generalized lipodystrophy. The rabbits became normoglycaemic and insulin sensitive. In the patient a normalization of the urinary excretion of the serotonin metabolite 5-OH-indole acetic acid was observed.

His voracious hunger and profuse perspiration were reduced, the hyperkeratotic layer of the skin peeled off, and the pigmentations of the skin decreased. There was observed an improvement of ALAT and ASAT, normalization of the fasting blood glucose, and increased sensitivity to exogenous insulin. After 11 months of 200 mg fenfluramine daily addtitional administration of 2 g clofibrate per day produced normalization of the serum triglyceride concentration and a marked reduction of the resistance to insulin. Three more patients with congenital generalized lipodystrophy, two of whom have manifest diabetes, have now started treatment with fenfluramine and are improving. The rabbits got relapse of their lipoatrophic diabetes when the fenfluramine treatment was stopped. It is suggested that a disturbance in the serotonin metabolism of the central nervous system may be of pathogenetic importance in congenital generalized lipodystrophy.

ABSTRACT

Urine from 5 patients with congenital generalized lipodystrophy has been fractionated by protein precipitation and Sephadex gel filtration. A fraction with a molecular weight in the range of 1000 was observed to be metabolically active in mice, rats, and rabbits. Hypophysectomized rats got hypoglycaemia following an injection, and the lipolytic-hyperglycaemic effect of ACTH was reduced after injection into intact mice. This effect was probably due to insulin release, because no insulin-like activity was observed on isolated fat cells in vitro. Persistant changes were observed in the animals after 3 weeks of daily injections of the urinary fraction. Adult mice and rabbits developed lipoatrophy with decrease of body weight in spite of a doubling of the food consumption. The metabolic rate and the body temperature were raised. Infantile animals developed a lipodystrophic state with increased growth velocity, and 50 per cent increase of the body weight, although no fat depots were observed. The treated animals got hyperglycaemia, hypertriglyceridaemia, hyperinsulinaemia, and insulin resistance. The rabbits developed manifest diabetes. The corresponding fraction prepared from the urine from the lipoatrophic rabbits produced lipoatrophy after injection into the mice. It is suggested that the lipodystrophic urinary fraction is of hypothalamic origin, and that it acts through the pituitary gland. The fraction is still heterogenous, and was observed to contain thyrotrophin releasing activity.

ABSTRACT

A lipid mobilizing factor (LMF) with an adipotrophic effect in man and animal adipose tissue has been prepared from human pituitary glands. In vitro studies have demonstrated that the lipolytic effect was dependent on the albumin used in the incubation medium. Two of the six albumins studied depressed the release of non-esterified fatty acids from human fat pads. Polyacrylamide gel disc-electrophoresis demonstrated the presence of contaminations in purchased serum albumins. They were eliminated by DEAE-cellulose chromatography, which gave one fraction that inhibited the adipokinetic effect of LMF, and a more homogeneous albumin fraction. In vitro lipolysis in rabbit as well as in human fat pads occurred more readily in the media containing purified serum albumin than in media with purchased serum albumin. It was concluded that in vitro lipolysis gives comparable results when studied in media containing the same batch of serum albumin only. Furthermore an agent should not be claimed to be non-lipolytic until it has been assayed in several, preferably homogeneous serum albumin media.

ABSTRACT

A lipid-mobilizing factor (LMF) with an adipotrophic effect in human and animal fat tissue has been prepared from human pituitary glands. The addition of normal human serum to LMF reduced its lipolytic effect, and it was completely abolished by serum from a group of obese patients, whereas the lipolysis was not influenced by serum from patients with generalized lipodystrophy.

By DEAE-cellulose chromatography of human serum the inhibitory effect on LMF was found to be present in a protein fraction less acidic than the main serum albumin fraction. The inhibitory fraction was deprived of some contaminants by Sephadex gel filtration. Disc electrophoresis demonstrated the presence of three components in the inhibitory protein (IP), and they were identified as albumin, transferin, and haemopexin by immuno-electrophoresis. Precipitation of these proteins by their rabbit antisera demonstrated that the inhibitory effect was present in the albumin fraction. Insulin like activity was not observed in IP. A protein binding of LMF by IP could not be demonstrated. Incubation at 37^°C for one hour of a mixture of LMF and IP eliminated the electrophoretic picture of LMF. It is concluded that the inhibitory effect of human serum may be due to proteolysis of LMF.

ABSTRACT

A comparison has been made of some physicochemical and metabolic characteristics of a human growth hormone preparation with somatotrophic and adipokinetic-diabetogenic activity (r.HGH), of a purified r.HGH with potent somatotrophic effect but negligible adipokinetic-diabetogenic effect in the fed animal (t.STH), and a human pituitary-hyperglycaemic and lipid-mobilizing factor (LMF) which was prepared from the crude pituitary extract prior to the preparation of t.STH and was without somatotrophic activity.

The t.STH was separated into two bands on polyacrylamide gel electrophoresis, and an additional band was observed for r.HGH in the area of the single band obtained for LMF. The r.HGH, t.STH, and LMF preparations had maximum optical absorption at 278 mμ up to pH 10. At a more alkaline pH the absorption spectrum of t.STH was unchanged, whereas an additional absorption peak at 290 mμ appeared for r.HGH and LMF. Tentative molecular weights determined by Sephadex gel filtration were in the range of 25 000 for r.HGH, 21 000 for t.STH, and 5500 for LMF.

The t.STH had a significantly higher growth promoting activity in the tibia test than r.HGH. LMF had no such activity in doses of 1 mg. The adipokinetic effect in fed rabbits was strong for LMF and r.HGH, but negligible for t.STH, which, however, caused an increase of nonesterified fatty acids in animals deprived of food. The in vitro lipolytic effect on human fat, obtained from fasting females, for LMF, r.HGH, and t.STH was on a per weight basis in the ratio of 100:10:1, respectively. In rabbits daily injections of r.HGH and LMF, but not t.STH, induced a hyperglycaemia which continued for 9 and 23 days respectively. Rabbits given daily injections of r.HGH and LMF got a diabetic response of blood glucose after injection of adrenalin and prednisone, although an increased sensitivity to insulin. In contrast to this, a single injection of LMF gave a decreased sensitivity to insulin in the rabbit. Rabbits treated with t.STH behaved as untreated controls.

Preparative ultracentrifugation of r.HGH dissolved in physiological saline gave a precipitate with the characteristics of t.STH, and a supernatant with potent hyperglycaemic-adipokinetic effect in rabbits. It was furthermore possible to subdivide r.HGH into t.STH and an adipotrophic component by acetone precipitation and Sephadex gel filtration.

It is discussed whether the r.HGH is an aggregation product of t.STH and a hyperglycaemic-adipokinetic agent, or whether the r.HGH is original and made up of these two components.

ABSTRACT

A lipid-mobilizing factor (LMF) has previously been prepared from human pituitary glands. The LMF was strongly adipokinetic in rabbits. Subcutaneous injection of 0.1 mg gave an increase of serum nonesterified fatty acids (NEFA) in the range of 5 meq./l. Addition of 3 ml normal human serum to LMF reduced its adipokinetic effect. The inhibition of LMF by human serum was specific, since serum from mouse or pig did not reduce the increase of serum NEFA induced by LMF, and human serum had no influence on the adipokinesis given by whale, pig, or synthetic ACTH preparations in rabbits.

The human serum inhibition of LMF was located to the gamma G globulin fraction by DEAE-cellulose chromatography.

Serum from six children with generalized lipodystrophy had a negligible influence on the adipokinesis in rabbits induced by LMF. Sera from one group of obese patients reduced the adipokinetic effect of LMF as normal control sera, while sera from another group of obese patients made the increase of serum NEFA negligible. It is suggested that the first group of obese patients may be related to Mayer's simple regulatory or 'hyperphagic' type of obesity and the latter to his metabolic type of obesity. Furthermore possible mechanisms for the human serum inhibition of LMF are discussed.

ABSTRACT

The methods described for the preparation of a purified HGH*, a crude HPG, and an adipotrophic fraction are simple, and suitable for the preparation of relatively small batches of pituitary glands.

The crude fractions were obtained by alterations in the hydrogen ion concentration of a saline extract of frozen pituitary glands, and a subsequent precipitation with acetone and ammonium sulphate. The crude HGH fraction was purified by Sephadex filtration. The yield of purified HGH was approximately 7 mg per g of fresh pituitary glands. Acrylamide gel electrophoresis demonstrated two bands in the HGH preparation. The bands could be separated by DEAE-cellulose chromatography. The homogeneous HGH preparations, however, had a significantly reduced growth-promoting activity.

The crude HPG was also obtained in a yield of approximately 7 mg per g of pituitary glands, and was used clinically without further purification.

The HGH and HPG preparations have been used therapeutically with success for seven years. The hormones are easily soluble, and well tolerated. No lack of response to HGH because of antibody production has been observed in more than 30 treated hyposomatotrophic children. The purified HGH is suitable for radioimmunoassay. Furthermore an adipotrophic-diabetogenic fractions can be obtained.

Modifications of the preparation procedure are discussed.

ABSTRACTS

A "family" of peptides which produced metabolic or behavioural effects following injections into mice or rabbits had been isolated from the urine from patients with the hypothalamic syndrome congenital, generalized lipodystrophy. Anorexia nervosa is associated with hypothalamic disturbances. Precipitates from urine specimens from 25 patients diagnosed as anorexia nervosa were chromatographed on Sephadex G-25 gel columns, and could be divided into 4 different patterns: One was similar to that for normal controls (4), one similar to that observed for patients with schizophrenia (6), 5 patients with a hysteriform type of neurosis had a third form of pattern, and 10 girls considered to have a primary "hypothalamic" type of anorexia nervosa also had typical chromatograms. Fractions influencing appetite in mice were found in the latter group, only. Two peptides influencing appetite were purified through several steps of chromatography. The sequence of the anorexigenic peptide was verified by the synthesis of the tripeptide, pyroGlu-His-GlyOH. A total dose of 12 nmole of this peptide injected daily over 20 days induced food refusal in mice with reduction of food consumption from 5.7 to about 3 g per day, and the mean body weight decreased from 35 to a minimum of 24.1 g. Food consumption did not normalize until 6 months later, at the same time body weights increased to the same level as in the controls. An appetite stimulating peptide increased the daily consumption of food to more than 10 g, and the mean body weight increased to 57.2 g. We provide evidence for the existence of a hypothalamic form of anorexia nervosa, and that the appetite may be regulated by humoral factors. Screening of peptides present in the urine from patients with anorexia nervosa may give diagnostic and therapeutic information.