The physiology and pharmacology of adipose tissue lipolysis: Its inhibition and implications for the treatment of diabetes

Summary The regulation of adipose tissue lipolysis occupies a key position in the metabolic concert of carbohydrates and lipids, and insulin plays the role of the conductor. Insulin favours the storage of fat in adipose tissue by 3 mechanisms: — 1. it inhibits lipolysis; — 2. increases the glucose uptake, lipogenesis and reësterification of free fatty acids; — 3. enhances the activity of lipoprotein-lipase, which is responsible for the uptake and storage of blood lipoproteins in adipose tissue. — On the other hand, basal lipolytic activity of adipose tissue is very much elevated in rats which are refed after prolonged starvation. It appears that insulin induces a lipase, which, in its presence, is nonfunctional. This dual role of insulin — induction of a lipase activity which it inhibits — would provide animals with a very efficient mechanism of switching from glucose utilization and lipid storage to lipid mobilization and oxidation. — High plasma levels of free fatty acids decrease glucose utilization of muscle and the effects of insulin thereon. Therefore, pharmacologic inhibition of lipolysis has been investigated in order to find new and better ways to adjust the metabolic situation in diabetes mellitus. We have used 5-methylpyrazole-3-carboxylic acid as a model antilipolytic compound. This drug, nicotinic acid and prostaglandin E₁ not only inhibit lipolysis, but they also markedly enhance glucose uptake of adipose tissuein vivo. 5-methylpyrazole-3-carboxylic acid prevents the rise of the free fatty acids and of blood glucose acutely after the administration of anti-insulin serum. When this drug is administered over a period of several days, adipose tissue develops an escape mechanism; 5-methylpyrazole-3-carboxylic acid still effectively inhibits lipolysis and stimulates lipogenesis from glucose, but only for a short period of time. Basal lipolysis is activated and the newly synthesized glycerides are rapidly split and released as free fatty acids into the blood, so that soon after its administration the plasma level of free fatty acids is increased above normal. These findings shed doubt on the potential usefulness of antilipolytic drugs in the treatment of diabetes mellitus. — Purified nonsuppressible ILA of serum is mentioned as an antidiabetic substance of potential therapeutic interest since it increases glucose uptake of muscle more markedly than that of adipose tissue compared with insulin.The studies reported here were supported by the US Public Health Service (grant No. A 5387) and by the Schweizerische Nationalfonds (grant No. 3854).     

Assay of insulin-like activity by the isolated fat cell method

Summary Human sera prepared in different ways were assayed for insulin-like activity (ILA) by the isolated fat cell method, with and without insulin antibodies. The lowest values of non-suppressible ILA (equivalent to 20–40 U of insulin per ml) were found when sera were prepared in an atmosphere of 5% CO₂. The non-suppressible ILA increased when sera were prepared with air as the gas phase or when heparin was added to the blood samples, whereas the suppressible ILA remained unchanged. Haemoglobin (0.1 mg per ml or more) depressed the activity of suppressible ILA but not of non-suppressible ILA. The non-suppressible ILA of rat serum varied with the nutritional state of the animals. It declined during fasting and when the animals were given a high fat diet. The activity of crystalline insulin and of nonsuppressible ILA was quantitatively recovered when added to sera from fasted or fat-fed rats. — A variety of different treatments of serum increased the ILA. Acid-alcohol extraction, incubation with heparin, warm dia-lysis, and electrophoretic fractionation caused a rise of the non-suppressible ILA but not of the suppressible ILA. — The results indicated that non-suppressible ILA is present in native serum and is likely to be physiologically significant. The concept of a form of insulin in serum different from suppressible ILA was not supported.Supported in part by: Nordisk Insulinfond.     

Measurements of insulin activities in pancreas and serum of mice with spontaneous (“obese” and “New Zealand obese”) and induced (goldthioglucose) obesity and hyperglycemia,with considerations on the pathogenesis of the spontaneous syndrome

Summary 1. Insulin-like activity (ILA) and immunoreactive insulin (IRI) of pancreatic extracts and of serum have been measured in lean and obese (obob) mice from the Jackson Memorial Laboratory in Bar Harbor, Maine; in New Zealand obese mice; and in goldthioglucose-obese Swiss mice, and their lean controls. In all three types of mice the relative amounts of suppressible and nonsuppressible insulin activities of serum have also been established. — 2. Pancreatic content of IRI in (obob) mice is much greater than in their lean sibblings at five months of age, but smaller at 5 weeks, whereas serum IRI and ILA of (obob) mice are greatly in excess of the same activities of serum from their lean littermates at both ages. It is suggested that increased peripheral demand for insulin preceeds increased insulin synthesis and storage in this type of obesity. — 3. In all three strains of mice, obese or lean, the ratio of biologic to immunologic activity of pancreatic extracts was constant and close to unity. In serum, this ratio was also constant but approximately five times greater than unity. These findings do not support the notion that differences in insulin structure might account for differences in the biological activity of insulin in obese and non-obese animals; they are in keeping with the concept of a potentiation of serum insulin activity by some component of serum in all mice. — 4. Approximately 90% of serum ILA in all three types of obese mice studied was suppressed in the presence of anti-insulin serum, although the absolute levels of non-suppressible ILA were similar to those reported for other species. It is unlikely, therefore, that bound insulin accounts for a significant part of the increased ILA of serum of any of the three types of obese animals studied. — 5. Present data support the concept of a marked tissue-bound muscular resistance to insulin action as an important, possibly primary pathogenetic feature in (obob) mice. A similar, but much less marked muscular resistance can be demonstrated in NZO mice, whereas muscular resistance to insulin action is totally absent in goldthioglucose obese Swiss mice after a 24 hour fast.This work was supported by the Fonds National Suisse de la Recherche Scientifique (Grant No. 3618), Berne, Switzerland, the Fondation Emil Barell pour le développement de la recherche médico-scientifique, Basel, Switzerland, and the Diabetes Foundation, Inc., Boston, Massachusetts. Dr.Lambert is aspirant of the Fonds National Belge de la Recherche Scientifique.     

Insulin-like activity and immunoreactive insulin content of gel-filtered protein fractions of bovine serum

Summary Serum globulin fractions isolated by gel filtration from fasting bovine sera with simultaneous exclusion of low molecular endogenous immunoreactive insulin (IRI), were found to contain immunoreactive insulin-like material in quantities which equal or surpass the directly assayable IRI content of native fasting sera. In one additional and clearly aberrant serum, independently isolated globulin fractions were found to contain 306 U of IRI per ml of serum, and appeared to exert an equivalent antibody-suppressible insulin-like activity (ILA) on rat adipose tissuein vitro. The IRI content of the native serum amounted to 15 U/ml and its suppressible ILA content to 46 U/ml. The presence of insulin-binding antibodies and accidental contamination with crystalline insulin could be excluded. It is suggested that in addition to low molecular IRI which reflects the insulin response to glucose loading, normal bovine serum may contain small basal levels of a high molecular globulin-associated form of IRI, which may be subject to pathologic elevation.This work was supported by a grant from the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.).     

Einfluß der Gewichtsreduktion auf Metabolite des Kohlenhydrat- und Fettstoffwechsels und auf das Verhalten des Seruminsulins bei Adipositas

Zusammenfassung Um Anhaltspunkte dafür zu gewinnen, welche der bei der Fettsucht beobachteten Stoffwechselanomalien durch eine Reduktionsdiät rückbildungsfähig sind, untersuchten wir unter ambulanten Bedingungen 37 adipöse Patienten mit einem mittleren Übergewicht von 71% nachBroca. Ein klinisch-manifester Diabetes und endokrine Erkrankungen wurden ausgeschlossen. 20 der Probanden zeigten Störungen der Kohlenhydrattoleranz. Bei 17 lagen der i.v. und der orale Glucosetoleranz-Test im Normbereich. Beide Teilkollektive behandelten wir mit einer 1000 Cal.-Mischkost und sahen darunter eine Gewichtsabnahme von 21.2 bzw. 17.5 kg, entsprechend 34 bzw. 28% nachBroca, d.h. im Durchschnitt etwas weniger als die Hälfte des bestehenden Übergewichtes. — Beim Teilkollektiv mit gestörter Kohlenhydrattoleranz traten dabei folgende Veränderungen ein: — 1. Signifikante Besserung der Glucosetoleranz mit weitgehender Normalisierung des Nüchternblutzuckers und des 120 Minuten-Wertes nach oraler Glucosegabe, sowie derk-Werte für die i.v. und oralen Glucosetoleranz-Teste. — 2. Ein hochsignifikanter Abfall der stark erhöhten Nüchternwerte für das freie Serumglycerin mit Normalisierung des Quotienten: Freie Fettsäuren/ Glycerin im Nüchternblut. — 3. Signifikanter Rückgang der überhöhten Werte für die insulinähnliche Aktivität und für das immunologisch reagierende Insulin. — Unter den gleichen Bedingungen fanden wir im Teilkollektiv mit normaler Kohlenhydrattoleranz lediglich einen mäßigen Anstieg der freien Fettsäuren 90 und 120 nach Glucose. — Im Gesamtkollektiv der Fettsüchtigen war unter der Reduktionsdiät ein signifikanter Rückgang der Esterfettsäuren festzustellen. — Die unter 1–3 geschilderten Veränderungen traten unter dieser Diätbehandlung deutlicher hervor als bei den bisher beschriebenen Fastenkuren und betreffen vorwiegend das Teilkollektiv mit gestörter Kohlenhydrattoleranz. Die Befunde deuten daraufhin, daß der Hyperinsulinismus, der von einigen Autoren als ursächlich für die Fettsucht angesehen wird, eher adaptiver Natur ist.

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Influence of weight reduction on carbohydrate and fat metabolism and on serum insulin response in obesity

Summary To find out which of the metabolic changes observed in obesity are reversible by a reducing diet, we examined 37 obese out-patients with a mean overweight of 71%Broca. A manifest diabetes mellitus and endocrine diseases were excluded by clinical means. 20 subjects showed disturbances of carbohydrate-tolerance. Oral and i.v. glucose-tolerance tests gave normal results in 17. Both subgroups were put on a 1000 cal. mixed diet and had mean weight-losses of 21.2, vs. 17.5 kg, corresponding to 34% vs. 28%Broca, i.e. less than half of their overweight. — The following changes were observed in the subgroup with impaired carbohydrate-tolerance: — 1. Significant improvement of glucose-tolerance with beginning normalization of fasting blood sugar, 120 value after oral glucose and of thek-values for i.v. and oral glucose-tolerance tests. — 2. Highly significant reduction of the elevated fasting values for free serum glycerol with normalization of the quotient: free fatty acids/glycerol in fasting serum. — 3. Significant fall of the high levels for insulin-like activity and for immunologically reacting insulin. — Under identical conditions the subgroup with normal carbohydrate-tolerance showed only a moderate increase of free fatty acids 90 and 120 after glucose. In the obese group as a whole, we found a reduction of serum esterified fatty acids under low calorie diet. — The changes described under 1–3 were more pronounced with this dietary treatment than in fasting periods generally described until now, and occurred predominantly in the subgroup with impaired carbohydrate-tolerance. Our findings indicate that hyperinsulinism held responsible for obesity up to now by some authors is probably adaptive.

Mit dankenswerter Unterstützung der Deutschen Forschungsgemeinschaft und des Landesamtes für Forschung des Landes Nordrhein-Westfalen. — Die Befunde wurden auszugsweise auf dem 6. Kongreß der Internationalen Diabetes Föderation vom 30. 7. — 4. 8. 1967 in Stockholm vorgetragen.     

Relationships between insulin-like growth factor-1 levels and growth hormone concentrations during diurnal profiles and following oral glucose in acromegaly

OBJECTIVE The aim of this study was to refine the biochemical definition of disease activity in acromegaly by comparing serum growth hormone (GH) measurements during a 10-hour day profile with serum GH values during an oral glucose tolerance test. DESIGN Using plasma insulin-like growth factor-1 (IGF-1) levels as a measure of disease activity, serum GH data from a day profile and from an oral glucose tolerance test were compared. PATIENTS Thirty-five acromegalic patients were studied, 13 of whom had serum GH measured during a day profile and 22 during an oral glucose tolerance test. In addition, basal plasma IGF-1 levels were estimated in all acromegalic patients, and in 24 normal subjects. MEASUREMENTS Following acid-ethanol extraction of the plasma samples, IGF-1 levels were measured by radioimmunoassay using a polyclonal antibody. In a day profile, six to eight blood samples for serum GH estimation were taken at hourly intervals during the day; during an oral glucose tolerance test samples for serum GH estimation were taken in the fasting state and every 30 minutes for 2 hours and measured by a two-site IRMA for GH. RESULTS Ninety-four per cent of acromegalic patients with raised plasma IGF-1 levels had serum GH concentrations <10 mU/l whilst 98% of acromegalic patients with plasma IGF-1 levels in the normal range had serum GH concentrations lt;6 mU/l. A highly significant positive correlation was found between the mean serum GH concentrations (r=0 67), the minimum serum GH concentration (r = 0 65) and the area under the GH curve (r = 0–66) estimated during an oral glucose tolerance test and plasma IGF-1 concentrations. The relations between identical indices of serum GH concentration measured during a day profile and plasma IGF-1 levels, although significant, show a less powerful correlation. The relation between serum GH and plasma IGF-1 levels describes a curvilinear model, plasma IGF-1 levels exhibiting a plateau at serum GH concentrations >40 mU/l but maintaining a linear relationship with serum GH levels <20 mU/l. CONCLUSIONS A highly significant correlation exists between plasma IGF-1 levels and various parameters of serum GH levels in acromegalic patients. Hormonal assessment of disease activity in acromegaly is more accurately reflected by the serum GH concentration during an oral glucose tolerance test rather than by the serum GH level during a day profile. Normalization of plasma IGF-1 levels is rarely achieved unless the mean serum GH level is reduced to <6 mU/l.     

In vivo metabolic action of insulin-like growth factor I in adult rats

Summary The acute metabolic actions of insulin-like growth factor I were studied in anaesthetized adult rats and its potency was compared to that of insulin. Following an i. v. bolus injection of insulin-like growth factor I a dose-dependent decrease of blood glucose and serum non-esterified fatty acid concentrations was noted with a potency of about 2% that of insulin. Stimulation of total body glucose disposal during euglycaemic clamping required 50times higher insulin-like growth factor I serum concentrations to achieve an identical half-maximal response. A similar difference in potency was observed for the stimulatory action on 2-de-oxyglucose uptake and on glycogen formation in skeletal muscle. Lipogenesis in epididymal fat pads was increased dose-dependently by both hormones requiring approximately 30 times higher half-maximally effective serum concentrations of insulin-like growth factor I. These data demonstrate that insulin-like growth factor I exerted acute insulin-like metabolic actions in vivo with low potency. These effects were probably mediated via insulin receptors. A preferential stimulation of glucose metabolism in skeletal muscle was not observed.     

Untersuchungen des Kohlenhydrat- und Fettstoffwechsels bei Prädiabetes

Zusammenfassung Wegen der bekannten Veränderungen der Metabolite des Fettstoffwechsels und der erhöhten Seruminsulinwerte bei Adipositas können nur Untersuchungen normgewichtiger Prädiabetiker charakteristische Befunde für den genetisch determinierten Prädiabetes erbringen. Bei 66 Nachkommen zweier manifest diabetischer Elternteile wurden die insulinähnliche Aktivität im Serum (ILA) und das immunologisch erfaßbare Seruminsulin (IRI) im oralen GTT, die freien Fettsäuren (FFS) im i.v. GTT und bei einem Teil Cholesterin, Triglycéride und Esterfettsäuren untersucht und mit einem Normalkollektiv verglichen. — Probanden mit pathologischen oralen und/oder i.v. GTT und mehr als 10% Übergewichtige wurden ausgesondert und getrennt bewertet. — Signifikant erhöhte ILA-Nüehternwerte ließen sich nur bei der Gruppe der übergewichtigen, nicht der normgewichtigen Prädiabetiker nachweisen, die Nüchternwerte des IRI lagen in allen Gruppen eher niedrig. Bei normgewichtigen Prädiabetikern war der Anstieg von IRI und ILA im or. GTT geringer und verzögert, die Insulinreserve bei beiden Meßmethoden signifikant vermindert, der Abfall der FFS im i. v. GTT signifikant geringer. Darüberhinaus bestanden keine nachweisbaren Fettstoffwechselstörungen; — Folgende Schlußfolgerungen wurden gezogen: — 1. Erhöhte ILA-Nüchternwerte sind bei normgewichtigen Prädiabetikern nicht die Regel und sind auf eine gleichzeitig bestehende Übergewiehtigkeit bzw. Fettsucht zurückzuführen. Sie können nicht als charakteristisch für den genetisch determinierten Prädiabetes angesehen werden. — 2. Da auch das IRI nicht erhöht ist, besteht keine Veranlassung, insulinantagonistische Mechanismen im Stadium des Prädiabetes anzunehmen. — 3. Der mit beiden Insulinbestimmungsmethoden übereinstimmend gefundene verzögerte und verringerte Anstieg des Insulins nach Glucosegabe, die verringerte Insulinreserve und der verzögerte Abfall der FFS nach Glucosegabe lassen vielmehr eine frühe und primäre Störung der Insulinsekretion vermuten. — 4. Da die Untersuchungen an nichtdiabetischen monozygoten Zwillingen diabetischer Partner die geschilderten Ergebnisse bestätigen, erscheint die biochemische Erfaßbarkeit der prädiabetischen Phase auch im Einzelfall denkbar.

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Investigations of carbohydrate and fat metabolism in pre-diabetes

Summary Because of the known changes of fat metabolites and elevated serum insulin levels in obesity, only investigations of normal-weight prediabetics can result in findings that are characteristic for genetically determined prediabetes. Serum insulin-like activity (ILA) and immunoreactive serum insulin (IRI) during oral GTT, free fatty acids (FFA) during i.v. GTT, and in some of the subjects cholesterol, triglycerides and esterified fatty acids were examined in 66 persons where both parents were diabetics and compared with a normal control group. — Subjects with pathological oral and/or i.v. GTT and those more than 10% overweight were excluded and evaluated separately. — A significant elevation of fasting ILA-levels was observed only in the overweight group, but not in normal-weight prediabetics; fasting IRI-levels were rather low in all groups. In normal-weight prediabetics the rise in IRI and ILA during oral GTT was less than that in normal subjects and was delayed, a diminished insulin-reserve was found with both methods, and the decrease in FFA levels during i.v. GTT was significantly smaller. No other disturbance of fat metabolism was observed. — The following conclusions were reached: — 1. Elevated fasting ILA levels are not the rule in normal-weight prediabetics and must be attributed to concomitant overweight or obesity. They cannot be considered characteristic of genetically determined prediabetes. — 2. Since IRI is also not increased, there is no reason to assume insulinantagonistic mechanisms in the prediabetic state. — 3. The delayed and diminished rise of insulin after glucose which we found in good accordance with both methods for insulin-determination, as well as the diminished insulin-reserve and the delayed decrease of FFA after glucose, suggests an early and primary impairment of insulin secretion. — 4. Since investigations in nondiabetic and monozygotic twins of diabetics have confirmed the results described above, the possibility of detecting the prediabetic state in individual cases is conceivable.

Teilweise vorgetragen auf dem Second Annual Meeting European Association for the Study of Diabetes, 6.-8. 7. 1966 in Aarhus, Dänemark, dem 72. Kongreß der Deutschen Gesellschaft für Innere Medizin in Wiesbaden, April 1966 und dem 6. Kongreß der Internationalen Diabetes Federation, 30. 7. – 4. 8. 1967 in Stockholm, Schweden. — Die Untersuchungen wurden mit dankenswerter Unterstützung der Deutschen Forschungsgemeinschaft und des Landesamtes für Forschung des Landes Nordrhein-Westfalen durchgeführt.     

Surrénale et insulinémie chez le rat II. Corticosurrénale et insuline sérique

Résumé La surrénalectomie diminue l'insuline «supprimable» et l'insuline «non supprimable» mises en évidence par la méthode du diaphragme ainsi que celle que mesure la technique immunologique. — Nous n'avons pas pu mettre en évidence d'interaction périphérique entre hormones corticosurrénaliennes et insuline. Par contre, nous avons précisé l'influence de la corticosurrénale sur la sécrétion insulinique de trois façons. — La relation glycémieinsulinémie, qui n'existe pas chez le Rat surrénalectomisé, est rétablie après un traitement par la corticosterone ou l'hydrocortisone. On l'observe chez le Rat à surrénale énucléée si son sérum contient de la corticostérone. —Lors d'une surcharge en glucose le pancréas du Rat surrénalectomisé répond mal à l'augmentation de la glycémie, alors que le Rat à surrénale énucléée répond aussi bien que le Rat normal. — Enfin, les expériences de sécrétion pancréatique «_{in vitro}» dans un milieu tampon ou dans du sérum ont montré que les fragments de pancréas prélevés sur des rats surrénalectomisés sécrètent moins d'insuline que ceux de rats normaux ou de rats à surrénale énucléée, si la concentration en glucose est dans des limites physiologiques. Pour des concentrations en glucose élevées (7.5 mg/ml) la sécrétion d'insuline du pancréas de rats surrénalectomisés s'accroît, mais n'atteint pas les valeurs observées dans les deux autres groupes de rats. — Par conséquent, la présence d'hormones corticosurrénaliennes dans le sérum est nécessaire pour que la sécrétion insulinique réponde normalement aux variations du glucose sanguin.

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Adrenals and insulinemia in the rat II. Adrenal cortex and serum insulin

Summary Adrenalectomy decreased suppressible and nonsuppressible serum insulin-like activities, which were shown by the rat diaphragm method, and insulin which was measured by an immunological procedure. —We were not able to find any peripheral correlation between eorticosteroid hormones and insulin. On the other hand, we have shown the influence of the adrenal cortex on insuline by 3 means. — The relation between serum glucose and serum insulin concentration, which was not present in the adrenalectomized rat, was reestablished after corticosterone or hydrocortisone treatment. It was also seen in the demedullated rat when corticosterone was present in the serum. — During a glucose tolerance test, the pancreatic insulin response was poor in the adrenalectomized rat, but was as good in the demedullated as in the normal rat. — Finally, in vitro experiments demonstrated that pancreas fragments obtained from adrenalectomized rats secreted less insulin than pancreas fragments obtained from demedullated or normal animals, when the incubation was carried out in buffer or serum, at physiological glucose levels. When the buffer or serum glucose concentration was raised to 7.5 mg/ml, the amount of insulin secreted by the pancreas obtained from adrenalec tomized rats was increased, but it still remained less than the amount of insulin secreted in the other groups. — Therefore, the presence in the serum of corticosteroid hormones was necessary to permit a normal pancreatic insulin secretion in response to changes in blood glucose concentration.

D.R.M.E.     

Impaired insulin-like growth factor I-mediated stimulation of glucose incorporation into glycogen in vivo in the ob/ob mouse

Summary The ability of insulin to modulate glucose metabolism is impaired in insulin resistant ob/ob mice. It has been shown that insulin-like growth factor I stimulates the uptake and metabolism of glucose in muscle through the insulin-like growth factor receptor not the insulin receptor. Thus, we have compared the abilities of insulin-like growth factor I and insulin to stimulate the in vivo incorporation of [¹⁴C]-glucose into glycogen in the diaphragm of ob/ob mice and their lean littermates. The animals used in these studies were 12–14 weeks old and the serum insulin levels of the ob/ob mice were 16-fold higher than in their lean littermates. There were no differences in the serum levels of glucose or insulin-like growth factor I. Both insulin and insulin-like growth factor I stimulate the incorporation of [¹⁴C]-glucose into glycogen in lean mice. Significant stimulation occurs at doses as low as 1 g/kg of either peptide. The effective doses of insulin and insulin-like growth factor I are quite similar, which indicates that the effect of insulin-like growth factor I is mediated by the insulin-like growth factor receptor and not the insulin receptor. In contrast, greater than 100 g/kg of insulin-like growth factor I is required to stimulate [¹⁴C]-glucose incorporation into glycogen in the diaphragm of ob/ob mice. Thus, ob/ob mice are resistant to the action of both insulin and insulin-like growth factor I. In contrast to the decrease in the number of insulin receptors which occurs in ob/ob mice, there is no significant difference in the number of type 1 insulin-like growth factor receptors or in their affinity for insulin-like growth factor I in muscle membranes prepared from lean and ob/ob mice. In addition, the ability of insulin-like growth factor I to stimulate the catalysis of Val⁵-angiotensin II phosphorylation by the partially purified muscle type 1 insulin-like growth factor receptor is not decreased in ob/ob mice as compared with their lean littermates. These data indicate that the loss in sensitivity of the ob/ob mouse of both insulin and insulin-like growth factor I is most likely mediated by a post-receptor defect in metabolism and not by receptor down-regulation or desensitisation.     

A serum inhibitor of insulin action on muscle. II. Quantitative assessement of inhibitory activity

Summary 1. The intraperitoneal assay of insulin has been used to compare the effectiveness of insulin injected in serum with that of insulin injected in 5% albumin. The difference was interpreted as being due to a serum inhibitor of insulin action on muscle. — 2. To obtain a quantitative assessment of the inhibitory activity, the insulin effectiveness was estimated by the arithmetic subtraction of the effect of the serum alone from that of the insulin added to the serum. Since the effect of insulin is related to the logarithm of the dose, such a subtraction requires that the two forms of insulin-like activity are essentially different. — 3. Dose-response curves for insulin injected in albumin and in serum were parallel, despite the considerable ILA of the serum; and analysis of the phenomenon clearly shows that the effects of the serum ILA and the added insulin were arithmetically additive. — 4. The ILA of the serum was distinguishable from that of the added insulin in not being suppressed by potent anti-insulin serum in the intraperitoneal assay system used. — 5. The consistency in the results obtained by the application of this procedure to the data of a variety of experiments provides effective support for the procedure.Supported by United States Public Health Service, Grant No. F2, AM-23, 661.     

Effect of chronic octreotide treatment on intestinal absorption in patients with acromegaly

The adverse gastrointestinal effects of octreotide, a synthetic analog of somatostatin, have not been fully elucidated. Low-dose octreotide frequently causes adverse gastrointestinal symptoms in normal individuals. We investigated the adverse gastrointestinal effects of high-dose octreotide, which is required for the normalization of growth hormone hypersecretion in some patients with acromegaly. Patients with acromegaly (N=8) were treated with octreotide, 450 g/day, then 1500 g/day for two months at each dosage. Carbohydrate absorption was assessed using thed-xylose test, and fat absorption using fecal fat excretion and serum carotene concentrations, at baseline, at each dosage of octreotide, and after one month of washout. Ultrasonography was used to monitor for cholelithiasis. Growth hormone and insulin-like growth factor-I concentrations were significantly suppressed at both dosages. Adverse gastrointestinal symptoms were mild and transient.d-Xylose absorption remained normal at each dosage and after one month of washout. Fecal fat excretion increased from 7±2 to 12±2 g/24 hr (P<0.05) after the higher dosage and resumed baseline levels after the washout. Mean fasting serum carotene levels remained normal, and carotene loading test (15,000 units three times a day for three days) was unreliable in identifying patients with high fecal fat. No new cholelithiasis was detected by ultrasonography. One of two patients with preexisting cholelithiasis developed biliary colic several days after the treatment period. Although steatorrhea was common, small intestinal absorptive capacity was otherwise unchanged by four months of high-dose octreotide treatment, which significantly suppressed growth hormone secretion in acromegalic patients.This work was supported by Sandoz Pharmaceuticals Corp., East Hanover, New Jersey.     

Mice lacking ANGPTL8 (Betatrophin) manifest disrupted triglyceride metabolism without impaired glucose homeostasis

Angiopoietin-like protein (ANGPTL)8 (alternatively called TD26, RIFL, Lipasin, and Betatrophin) is a newly recognized ANGPTL family member that has been implicated in both triglyceride (TG) and glucose metabolism. Hepatic overexpression of ANGPTL8 causes hypertriglyceridemia and increased insulin secretion. Here we examined the effects of inactivating Angptl8 on TG and glucose metabolism in mice. Angptl8 knockout (Angptl8^−/−) mice gained weight more slowly than wild-type littermates due to a selective reduction in adipose tissue accretion. Plasma levels of TGs of the Angptl8^−/− mice were similar to wild-type animals in the fasted state but paradoxically decreased after refeeding. The lower TG levels were associated with both a reduction in very low density lipoprotein secretion and an increase in lipoprotein lipase (LPL) activity. Despite the increase in LPL activity, the uptake of very low density lipoprotein-TG is markedly reduced in adipose tissue but preserved in hearts of fed Angptl8^−/− mice. Taken together, these data indicate that ANGPTL8 plays a key role in the metabolic transition between fasting and refeeding; it is required to direct fatty acids to adipose tissue for storage in the fed state. Finally, glucose and insulin tolerance testing revealed no alterations in glucose homeostasis in mice fed either a chow or high fat diet. Thus, although absence of ANGPTL8 profoundly disrupts TG metabolism, we found no evidence that it is required for maintenance of glucose homeostasis.Adipose tissue is the major energy reservoir in the body and serves to buffer metabolically active tissues against diurnal changes in food intake by releasing free fatty acids into the circulation during fasting. Upon refeeding, adipose tissue is replenished with fatty acids obtained from circulating triglyceride (TG)-rich lipoproteins produced in the gut and liver. Fatty acids are cleaved from lipoprotein TGs by the enzyme lipoprotein lipase (LPL), which is anchored to the capillary endothelial surfaces of peripheral tissues (1). The uptake of TG-fatty acids is controlled by regulating LPL activity in accordance with nutritional status (2). In fasting, LPL activity is reduced in adipose tissue and increased in heart and skeletal muscle, thereby directing TG-fatty acids to muscle for oxidation (3). In the fed state, LPL activity declines in muscle and increases in adipose tissue, directing TG-fatty acids to adipose tissue for storage.Two angiopoietin-like proteins, ANGPTL3 and ANGPTL4 (4, 5), play roles in the partitioning of TGs in fasting and refeeding. Both ANGPTLs inhibit LPL (6–8). Overexpression of either protein inhibits LPL activity and causes hypertriglyceridemia, whereas loss-of-function mutations lead to very low TG levels (7, 8). ANGPTL4 is expressed at high levels in liver and adipose tissue, primarily in the fasted state (5). It suppresses LPL activity selectively in adipose tissue and redirects TG to muscle (9). ANGPTL3 is expressed almost exclusively in liver and its levels are only modestly altered by fasting and refeeding (10). It has been suggested that ANGPTL3 is involved in the redirection of TGs to adipose tissue during feeding (9), but direct evidence is lacking.Previously, we showed that ANGPTL8 [also known as TD26, RIFL (11), Lipasin (12), and Betatrophin (13)] acts together with ANGPTL3 (14). ANGPTL8 is expressed at the highest levels in liver and adipose tissue, and is markedly up-regulated by feeding and suppressed by fasting (11, 12, 14). Adenovirus-mediated hepatic overexpression of ANGPTL8 increases plasma TG levels in wild-type mice but not in mice lacking ANGPTL3 (12). Coexpression of ANGPTL8 and ANGPTL3 increased plasma TG levels more than 10-fold, suggesting that the two proteins act together (14). Consistent with this hypothesis, ANGPTL3 and ANGPTL8 can be coimmunoprecipitated in the plasma of mice or in the medium of cells expressing both proteins (14). These data suggest that ANGPTL8 acts together with ANGPTL3 to coordinate the trafficking of TGs to tissues in response to food intake.Recently, Melton and his colleagues (13) reported that hepatic overexpression of ANGPTL8, which they called Betatrophin, promotes proliferation of pancreatic β-cells and increases insulin secretion. Thus, ANGPTL8 may also contribute to glucose homeostasis. To determine the physiological role of ANGPTL8 in lipid and glucose metabolism, we generated Angptl8 knockout (Angptl8^−/−) mice and compared their responses to fasting and refeeding with those of their wild-type littermates. Here we show that disruption of Angptl8 profoundly alters TG metabolism in fed animals. The knockout mice have decreased adipose tissue mass and a pronounced reduction in plasma TG levels in the fed state. Despite an increase in LPL activity, the uptake of very low density lipoprotein (VLDL)-TG into adipose tissue was markedly reduced in fed Angptl8^−/− mice. These changes in TG metabolism were not associated with significant alterations in glucose homeostasis or insulin levels.     

Serum IGF-I and IGFBP-3 levels for the assessment of disease activity of acromegaly

To assess the disease activity of acromegaly in patients, we measured the changes in serum growth hormone (GH) levels during oral glucose tolerance test and the basal serum levels of insulin-like growth factor I (IGF-I) and insulin-like growth factor-binding protein 3 (IGFBP-3) in 29 acromegalic patients and 30 health persons served as normal controls. Based on the clinical and laboratory criteria, acromegaly was in an active state of disease in 18 patients and was inactive in the other 11 patients. Basal serum IGF-I levels were 177+/-116 ng/ml (mean+/-SD), 250+/-135 ng/ml and 810+/-297 ng/ml in the normal subjects, the inactive and active acromegalic patients, respectively. Basal serum IGFBP-3 levels were 1.71+/-1.29 microg/ml, 2.98+/-0.96 microg/ml and 6.82+/-1.31 microg/ml in the normal controls, the inactive and active acromegalic patients, respectively. Serum levels of IGF-I and IGFBP-3 significantly correlated with each other in the normal subjects as well as the patients. Both IGF-I and IGFBP-3 levels were significantly higher in the group of patients with active acromegaly than inactive acromegalic patients and the normal subjects but there was not statistically difference between the normal controls and the inactive acromegalics. While serum IGF-I levels presented considerable overlapping instances among the three groups, the serum IGFBP-3 of inactive patients and the normal controls could rarely reach 4.44 ng/ml, the lowest value of the active acromegalics. The serum IGF-I and IGFBP-3 levels declined with increased age in normal controls, but not in the patients with acromegaly. There was no sex predilection of serum IGF-I and IGFBP-3 found in our study. The results of this study indicated that the serum IGFBP-3 level is an important laboratory parameter for assessing growth hormone function in humans, and might be a more reliable discrimination for the disease activity of acromegaly than the serum IGF-I is.     

Thyrotropin secretion during oral glucose tolerance test in acromegalic patients and control subjects

It has been suggested that acute hyperglycemia stimulates somatostatin release from the hypothalamus, thus causing inhibition of growth hormone and thyrotropin secretion. Abnormal growth hormone secretory pattern to glucose load is characteristic of acromegaly, and it might reflect alterations in somatostatin release. We evaluated the sensitivity of serum thyrotropin response to presumed somatostatin inhibition during oral glucose tolerance test in 29 patients with active acromegaly, in 13 patients with inactive disease, and in 19 control persons suspected of impaired glucose tolerance. Both the acromegalic patients and the control subjects were euthyroid. Serum insulin, growth hormone, thyrotropin, free triiodthyronine, free thyroxine, and glucose were collected before and 30, 60, 90, and 120 min after the ingestion of 75 g glucose. While the free triiodthyronine and free thyroxine values did not change during the glucose test, the thyrotropin levels progressively and significantly declined in all groups. The basal to nadir thyrotropin ratio was higher in active acromegaly than in inactive disease and in control subjects (p<0.01), suggesting that the glucose load inhibited thyrotropin stronger in active acromegalic patients. These data suggest that there is a possible strong somatostatin response to glucose load in acromegalic patients, which inhibits thyrotropin secretion. These data do not support the concept of decreased somatostatin drive in acromegaly.     

The role of IGF binding protein-3 as a parameter of activity in acromegalic patients.

OBJECTIVE: The production of insulin-like growth factor binding protein-3 (IGFBP-3), the main IGF-I binding protein, is regulated by GH, and its serum levels are increased in acromegaly. We investigated its potential value as a parameter of acromegaly activity or remission in comparison with IGF-I, taking GH suppression below 2 microg/l after glucose load as the normal standard. METHODS: Data from 40 acromegalic patients (12 males and 28 females, aged 28 to 79 years) were obtained retrospectively from stored samples. From these, 145 pairs of IGF-I/IGFBP-3 values were collected; in 67 of them, simultaneous measurement of GH after glucose loading allowed their classification as active or inactive acromegaly. Relationships between IGF-I, IGFBP-3 and GH after glucose load were assessed, as well as differences between IGF-I and IGFBP-3 levels in active and inactive acromegaly. RESULTS: Significant positive correlation between IGF-I and IGFBP-3 in 145 samples was observed (r=0.49, P<0. 0001). As for the 67 samples in which activity or remission could be defined in terms of GH after glucose load, 50 were active and 17 inactive. Both IGF-I and IGFBP-3 significantly correlated with minimum GH (r=0.53, P<0.0001 and r=0.41, P<0.001 respectively). For both parameters, significant differences of means between active and inactive cases were observed (623+/-296 vs 300+/-108 ng/ml, P<0.0001 for IGF-I, and 4.1+/-1.3 vs 3.2+/-0.9 microg/ml, P<0.006 for IGFBP-3). Yet, when comparing in individual cases their classification as active or inactive with the finding of normal or increased IGF-I and IGFBP-3, among active cases 16% appeared as normal according to IGF-I, and 50% appeared as normal in terms of IGFBP-3. Among inactive cases, 23.5% appeared as active according to IGF-I, while 17.5% appeared as active in terms of IGFBP-3. CONCLUSION: Even though IGFBP-3 reflects GH secretion, it offers no advantage over IGF-I in the assessment of acromegaly, and it may underestimate disease activity in acromegalic patients.     

Surrénale et insulinémie chez le rat I. Médullosurrénale et insuline sérique

Résumé L'insuline «totale», l'insuline «non supprimable» et l'insuline «supprimable», dosées par la méthode du diaphragme de Rat, ainsi que l'insuline immunoréactive, ont été mesurées dans le sérum de rats normaux, de rats surrénalectomisés traités par des hormones corticosurrénaliennes et de rats à surrénale énucléée. — Les différentes formes d'activité insulinique sont plus élevées chez le Rat médulloprive que chez le Rat normal. L'insuline immunoreactive est, par contre, identique dans les deux groupes d'animaux. Ceci s'explique par le fait déjà démontré que l'adrénaline est un inhibiteur (et non un antagoniste) de l'effet de l'insuline sur le muscle et le tissu adipeux. — L'influence du niveau glycémique sur la sécrétion pancréatique se traduit, chez le Rat normal, par une relation linéaire entre la glycémie et linsulinémie. En l'absence de la médullosurrénale, les pentes des droites correspondant aux trois formes d'insuline mesurées par la méthode du diaphragme sont augmentées. Il ne s'agit cependant pas d'un effet de l'adrénaline sur la sécrétion insulinique mais de l'absence de l'effet inhibiteur de l'adrénaline dans le sérum des rats médulloprives, comme l'ont montré des expériences«in vitro». — Les réponses pancréatiques à des surcharges en glucose et la sécrétion d'insuline «invitro» dans du tampon ou du sérum de rats surrénalectomisés glucoses à 1 mg ou 7.5 mg/ml, n'ont pas permis de mettre en évidence une influence de l'adrénaline endogène sur la sécrétion d'insuline par le pancréas.

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Adrenals and insulinemia in the rat. I. Adrenal medulla and serum insulin

Summary Total, non-suppressible and suppressible serum insulin-like activities in normal rats, in adrenalectomized rats treated with corticosteroids and in rats with demedullated adrenals were measured using the rat diaphragm in vitro and the immunoreactive insulin determined by a radio-immunological procedure. — The three forms of insulin-like activities were higher in the demedullated than in the normal rat. In contrast, the levels of immunoreactive insulin were identical in the two groups of animals. These results can be explained by the fact that epinephrine is an inhibitor (and not an antagonist) of insulin action on muscle and adipose tissue. —The influence of the basal serum glucose level on insulin secretion was seen in the normal rat, by a linear relationship between the serum glucose levels and the serum insulin concentrations. In the absence of the adrenal medulla, the slopes corresponding to the three forms of insulin-like activities measured by the rat diaphragm were increased. However, this phenomenon was not related to an influence of epinephrine on insulin secretion: in vitro experiments showed that the inhibitory effect of physiological doses of epinephrine was greater when the concentration of insulin in the medium was elevated, which explains the observations madein vivo. — The serum response in vivo to glucose loads, and the insulin secretion in vitro into a medium or into sera taken from adrenalectomized rats and brought to a glucose concentration of 1 or 7.5 mg/ml, have not led to the demonstration of an influence of basal concentrations of endogenous epinephrine on the pancreatic secretion of insulin.

C.N.R.S.     

Relationship between fat and ketone body metabolism in obese and nonobese diabetics and nondiabetics during norepinephrine infusion

Summary The effects of an intravenous infusion of norepinephrine, 0.08 g/kg.min on lipolysis (as measured by an increase of free glycerol and nonesterified fatty acids (NEFA)), on the blood concentration of ketone bodies and on the serum concentrations of immunoreactive insulin (IRI) and insulin-like activity (ILA) were studied in normal weight and obese nondiabetics and diabetics. Normal weight diabetics and nondiabetics showed the same increase in lipolysis. A significantly higher rate of lipolysis occurred in obese persons, irrespective of whether they were diabetic or not. Even the maximum absolute concentrations of free glycerol and NEFA during the infusion were higher in obese persons than in insulindependent diabetics, who showed the highest values before the beginning of the infusion. — In obese subjects, the infusion of norepinephrine according to the theoretical normal weight was still sufficient to produce a higher rate of lipolysis than in normal weight subjects. This probably reflects the greater mass of adipose tissue in obese subjects. — In diabetic and nondiabetic obese persons, the concentration of ketone bodies rose higher than in control subjects, which is in agreement with the higher rate of lipolysis in the obese groups. On the other hand, the normal weight insulin-dependent diabetics showed a significantly higher increase in the concentration of ketone bodies than the obese persons. This demonstrates that the degree of ketonaemia in man is not exclusively determined by the plasma level of NEFA. — The higher increase in the-hydroxybutyrate/acetoacetate ratio in insulindependent diabetics points to a higher rate of oxidation of fatty acids in the liver. —ILA and IRI responded in a different way to norepinephrine infusion, demonstrating again, that changes in ILA can, but may not always reflect changes in immunoreactive insulin. According to these results, changes in the rate of lipolysis and in ketonaemia in obese diabetics are determined by the factor obesity, whereas changes in these parameters in insulindependent diabetics are determined by the factor insulin deficiency.A part of this work has been presented at the 12. Symposion der Deutschen Gesellschaft für Endokrinologie, Wiesbaden, Deutschland, 21.–23. 4. 1966.     

Effect of aging on muscle mitochondrial substrate utilization in humans

Previous studies have implicated age-associated reductions in mitochondrial oxidative phosphorylation activity in skeletal muscle as a predisposing factor for intramyocellular lipid (IMCL) accumulation and muscle insulin resistance (IR) in the elderly. To further investigate potential alterations in muscle mitochondrial function associated with aging, we assessed basal and insulin-stimulated rates of muscle pyruvate dehydrogenase (V_PDH) flux relative to citrate synthase flux (V_CS) in healthy lean, elderly subjects and healthy young body mass index- and activity-matched subjects. V_PDH/V_CS flux was assessed from the ¹³C incorporation from of infused [1-¹³C] glucose into glutamate [4-¹³C] relative to alanine [3-¹³C] assessed by LC-tandem MS in muscle biopsies. Insulin-stimulated rates of muscle glucose uptake were reduced by 25% (P < 0.01) in the elderly subjects and were associated with ∼70% (P < 0.04) increase in IMCL, assessed by ¹H magnetic resonance spectroscopy. Basal V_PDH/V_CS fluxes were similar between the groups (young: 0.20 ± 0.03; elderly: 0.14 ± 0.03) and increased approximately threefold in the young subjects following insulin stimulation. However, this increase was severely blunted in the elderly subjects (young: 0.55 ± 0.04; elderly: 0.18 ± 0.02, P = 0.0002) and was associated with an ∼40% (P = 0.004) reduction in insulin activation of Akt. These results provide new insights into acquired mitochondrial abnormalities associated with aging and demonstrate that age-associated reductions in muscle mitochondrial function and increased IMCL are associated with a marked inability of mitochondria to switch from lipid to glucose oxidation during insulin stimulation.Impaired glucose tolerance and type 2 diabetes affect ∼40% of all Americans over the age of 65, yet the pathogenesis of this age-associated deterioration in glucose metabolism is poorly understood (1–4). In this regard, previous studies demonstrated that aging is associated with muscle insulin resistance, increased intramyocellular lipid (IMCL) content, and reductions in basal rates of muscle mitochondrial oxidative and phosphorylation activity (5). These results led to the hypothesis that age-associated reductions in muscle mitochondrial function may be an important predisposing factor in the elderly predisposing them to develop increased ectopic lipid deposition in skeletal muscle, muscle insulin resistance, and type 2 diabetes (5–9).To further examine the impact of aging on basal and insulin-stimulated muscle mitochondrial function, we developed and applied a novel LC-tandem MS (LC-MS/MS) method to directly estimate relative rates of muscle mitochondrial pyruvate dehydrogenase flux (V_PDH) and citrate synthase flux (V_CS) in vivo under both basal and insulin-stimulated conditions. Relative rates of mitochondrial V_PDH to V_CS fluxes were assessed by measuring the relative ¹³C enrichments of [4-¹³C] glutamate to [3-¹³C] alanine in muscle biopsies obtained before and after a hyperinsulinemic-euglycemic clamp using [1-¹³C] glucose (10). Using this approach, we observed that aging is associated with a marked inability for muscle mitochondria to switch from lipid to glucose oxidation during insulin stimulation.     

Aorta and muscle metabolism in pigs with peripheral hyperinsulinaemia

Summary Peripheral hyperinsulinaemia usually found in conventionally treated Type 1 (insulin-dependent) diabetic patients may have deleterious metabolic effects. We have used a hyperinsulinaemic model to examine intermediary metabolism in two key peripheral tissues, aorta and muscle. Nine pigs were immunized with crystalline insulin. Subsequently, they showed an insulin-binding capacity of 86.2±25.0 pmol/l and fasting total serum insulin of 3.9±3.1 nmol/l (control range 0.034–0.072 nmol/l), impaired glucose tolerance after oral glucose tolerance testing, significantly elevated levels of peripheral venous serum free insulin and C-peptide, and increased mean post-prandial free insulin/glucose ratios. The immunized pigs showed marked elevation of aorta and muscle triglycerides compared with control pigs (n = 15) but similar levels of non-esterified fatty acids. The glucose-6-phosphate-dehydrogenase, malic enzyme and 3-hydroxyacyl-CoA-dehydrogenase activities were all increased significantly (by 50%–300%) in both aorta and muscle. Phosphofructokinase was decreased in both tissues. Hexokinase was increased in muscle alone whereas pyruvate kinase was significantly decreased in aorta. Glyceraldehyde-3-phosphate dehydrogenase activity was not significantly different in aorta and muscle. Thus in insulin immunized pigs with normal -cell function and pronounced peripheral hyperinsulinaemia there was increased peripheral lipogenic activity. These findings have potentially important implications with regard to macrovascular disease in diabetes.