The perfused rat lung as a model for studies on the formation of surfactant and the effect of Ambroxol on this process

The isolated perfused rat lung was used as a model to investigate the synthesis of surfactant phospholipids from various radioactive precursors and the effect of Ambroxol, a bronchial secretolyticum, on this process. Lungs were ventilated and perfused for periods up to 5 h without detectable development of pulmonary edema. The lungs remained metabolically stable during the entire period of perfusion. Both in whole lung tissue and in the surfactant fraction the radioactive substrates incorporated predominantly into phosphatidylcholine and phosphatidylglycerol. The degree of saturation of labelled phosphatidylcholines synthesized during perfusion with [Me-¹⁴C]choline, D [U-¹⁴C]glucose, [1(3)-³H]glycerol and [1-¹⁴C]palmitate was higher in surfactant than in whole lung tissue. A delayed incorporation into surfactant phospholipids was observed for all precursors. Under the conditions employed, glucose carbon was recovered mainly in the glycerol backbone of phosphatidylcholine and phosphatidylglycerol. Compared to glucose, glycerol appeared to be a minor substrate for lung lipid formation. If the lungs were perfused after pretreatment of the rats with Ambroxol on three consecutive days, the incorporation of labelled choline and glycerol into pulmonary phospholipids was found to be enhanced. This stimulation was more pronounced in the surfactant fraction than in whole lung tissue. The stimulatory effect on the formation of surfactant lipids was smaller after pretreatment of the animals with Ambroxol for one day. The results of the present study suggest that Ambroxol may specifically stimulate the synthesis of phospholipids in the alveolar type II cells and that the drug may not only affect the formation but also the secretion of surfactant lipids by these cells.     

Comparative use of glucose and fructose in cultured fibroblasts from patients with hereditary fructose intolerance

The utilization of fructose and glucose by fibroblast cultures obtained from patients with hereditary fructose intolerance (HFI) was studied in comparison with fibroblast controls. The cell growth, the time course ofd-glucose ord-fructose uptake and the consumption of fructose were similar for both HFI and control cells. Some results showed significant differences between these two cell types: HFI cells consumed less glucose, produced less lactate and contained less glycogen than control cells. Furthermore, significantly less [U-¹⁴C]d-glucose and [U-¹⁴C]d-fructose was incorporated into lipids in HFI cells than in control cells. The mechanisms responsible for these differences observed between the two cell types are not known.     

Late results of combined percutaneous balloon valvuloplasty of mitral and tricuspid valves

Although combined mitral and tricuspid stenosis are rarely seen in patients with rheumatic heart disease, when both exist together, combined percutaneous balloon valvuloplasty can be an alternative to surgical treatment in suitable cases. We present the immediate and late follow up results of 12 patients with rheumatic tricuspid and mitral stenosis treated with combined percutaneous balloon valvuloplasty. Twelve patients (11 female, 91.7%; 1 male, 8.3%) with a mean age of 35.3 ± 6.4 years were enrolled in the study. The patients were followed up for 38.8 ± 12.6 months. The mitral valve area increased from 1.2 ± 0.2 cm² to 2.3 ± 0.2 cm² (P < 0.01) and on follow up the mitral valve area did not differ significantly (2.2 ± 0.2 cm²; P > 0.05). The tricuspid valve area increased from 1.6 ± 0.3 cm² to 3.2 ± 0.2 cm² (P < 0.01) and on follow up the tricuspid valve area did not differ significantly (3.1 ± 0.2 cm²; P > 0.05). Two patients (16.6%) had tricuspid restenosis and tricuspid re-valvuloplasty. One other patient (8.3%) was referred to surgery 14 months after the procedure secondary to severe tricuspid regurgitation. In conclusion, this study demonstrates a sustained benefit on late follow up after combined percutaneous balloon valvuloplasty of mitral and tricuspid valves and confirms the efficacy and safety of the procedure as an alternative to surgery in selected cases of combined mitral and tricuspid stenosis. Cathet. Cardiovasc. Diagn. 45:246–250, 1998. © 1998 Wiley-Liss, Inc.     

Glycogen accumulation in rat pancreatic islets

Under conditions of sustained hyperglycemia, glycogen accumulates in pancreatic islets, but not so in acinar pancreatic cells. Advantage conceivably could be taken from such a situation in the perspective of the noninvasive imaging of the endocrine pancreas. The present experiments aim, therefore, at characterizing the time course for glycogen accumulation in pancreatic islets cultured at a high concentration (30 mM) of d-glucose in the presence of tracer amounts of either d-[U-¹⁴C]glucose or 2-deoxy-2 [¹⁸F]fluoro-d-glucose. The ¹⁴C-labeled glycogen content of the cultured islets increased with time (150 min to 72 h), exceeded that found in acinar tumoral cells, and did not decrease over 60 min of incubation at 30 mM d-glucose in the absence of d-[U-¹⁴C]glucose. Glycogenolysis was observed, however, when the concentration of d-glucose was decreased to 2.8 mM and, in such a case, was further enhanced by forskolin and theophylline. Such a glycogenolysis concided with the generation of ¹⁴CO₂ from radioactive intracellular precursors and alteration of the B-cell secretory response to d-glucose. The radioactive glycogen content was higher in islets exposed to 2-deoxy-2-[¹⁸F]fluoro-d-glucose than d-[U-¹⁴C]glucose. Prior exposure of the islets to streptozotocin suppressed the accumulation of glycogen during their subsequent culture at high d-glucose concention. These findings may help to define the experimental conditions optimal for the labeling and accumulation of islet glycogen in vivo.     

Palmitate dependence of insulin secretion, “de novo” phospholipid synthesis and 45Ca2+-turnover in glucose stimulated rat islets

Summary Palmitate ability to modify D-[U-¹⁴C]glucose incorporation into different lipids (de novo synthesis), as well as sugar-stimulation of insulin release and ⁴⁵Ca²⁺-fluxes, was investigated in islets of fed and 48-h starved rats. The fatty-acid induced dose-dependent, correlative increments of insulin secretion, ⁴⁵Ca²⁺-influx and the de novo synthesis of each phospholipid fraction analysed at 20 mmol/l (but not 3 mmol/l) glucose. Omission of calcium reduced drastically (p<0.001) insulin release and the de novo synthesis of neutral glycerolipids, leaving unaltered that of acidic phospholipids (phosphatidate and phosphoinositides). The increased synthesis of the latter is therefore not the consequence of stimulated secretion. It could initiate or contribute to maintain an increased turnover of islet phosphoinositides, thus generating some mediators of the calcium signalling system (inositol phosphates). Starvation led to a drastic reduction (p<0.001) of both insulin secretion, de novo synthesis of each lipid fraction, and ⁴⁵Ca²⁺-influx in response to glucose and palmitate. The presence of a fatty-acid oxidation inhibitor (2-bromostearate or 2-tetradecylglycidate) prevented the effect of starvation on ⁴⁵Ca²⁺-influx, as it has been shown to do on insulin secretion and palmitate incorporation into islet lipids. It is finally suggested that palmitate might amplify the insulin secretory response of islets to glucose, through the stimulation of the de novo synthesis of phosphoinositides and the subsequent generation of inositol phosphates, which would contribute to accelerated calcium turnover.     

Significant tricuspid regurgitation is a marker for adverse outcome in patients undergoing percutaneous ballon mitral valvuloplasty

Objectives, This study examined the association between the presence of tricuspid regurgitation and immediate and late adverse outcomes in patients undergoing balloon mitral valvuloplasty.Background. Significant tricuspid regurgitation has an adverse impact on morbidity and mortality in patients undergoing mitral valve surgery for mitral stenosis.Methods. We studied 318 consecutive patients (mean [± SD] age 54 ± 15 years) who underwent ballon mitral valvuloplasty and had color Doppler ecnocardiographic studies before the procedure. Patients were classified into three groups: 221 with no or mild (69%), 60 with moderate (19%) and 37 with severe (12%) tricuspid regurgitation. Clinical follow-up ranged from 6 to 62 months.Results. Before mitral valvuloplasty, increasing degrees of tricuspid regurgitation were associated with a smaller initial mitral valve area (p < 0.05), higher echocardiographic score (p < 0.05), lower cardiac output (p < 0.01) and higher pulmonary vascular resistance (p < 0.01). Although the initial success rate did not differ significantly between groups, patients with a higher degree of tricuspid regurgitation had less optimal results, as reflected by a smaller absolute increase in mitral valve area (1.02 vs. 0.9 vs. 0.7 cm², p < 0.01). The estimated 4-year event-free survival rate (freedom from death, mitral valve surgery, repeat valvuloplasty and heart failure) was lower for the group with severe tricuspid regurgitation (68% vs. 58% vs. 35%, p < 0.0001). At 4 years, 94% of patients with mild tricuspid regurgitation were alive compared with 90% and 69%, respectively, of patients with moderate or severe tricuspid regurgitation (p < 0.0001). Cox proportional analysis identified tricuspid regurgitation as an independent predictor of late outcome (p < 0.001).Conclusions. Patients with mitral stenosis and severe tricuspid regurgitation undergoing mitral valvuloplasty have advanced mitral valve and pulmonary vascular disease, suboptimal immediate results and poor late outcome.     

Hexose metabolism in pancreatic islets

Islets from fed and 3-day-starved rats were incubated for 60 min in the presence of either 2.8 or 16.7 mM d-glucose, mixed with tracer amounts ofd-[5-³H]glucose,d-[3,4-¹⁴C]glucose,d-[6-¹⁴C]glucose andd-[2-¹⁴C]glucose. Starvation decreased the generation of³HOH fromd-[5-³H]glucose and the production of¹⁴CO₂ from¹⁴C-labelledd-glucose, both at low and high hexose concentrations. In islets from starved and fed rats, a rise ind-glucose concentration preferentially stimulated oxidative glycolysis, pyruvate decarboxylation and acetyl residue oxidation, relative tod-glucose utilization. At both low and high hexose concentrations and in both fed and starved rats, the decarboxylation of pyruvate exceeded the oxidation of glucose-derived acetyl residues, the C₁ of such residues being more efficiently converted to¹⁴CO₂ than their C₂. Starvation decreased oxidative glycolysis more severely than non-oxidative glycolysis, impaired the preferential stimulation ofd-[3,4-¹⁴C]glucose oxidation relative tod-[5-³H]glucose utilization as observed in response to a rise in hexose concentration, and lowered the ratio betweend-[6-¹⁴C]glucose oxidation and hexose utilization. It is proposed, therefore, that the short-term regulation of mitochondrial oxidative events byd-glucose is itself modulated in islet cells by the nutritional status.     

The effects of meal feeding on the incorporation of D-[U-14C]-glucose into tissue lipids and glycogen as a function of increasing intravenous glucose dose.

The in vivo incorporation of D-[U-¹⁴C]-glucose into lipids and glycogen of adipose tissues, muscle tissues, and liver was measured 1 hr after the i.v. injection of increasing glucose doses (0.75, 1.5 and 2.5 g glucose/kg of body weight) in meal-fed and ad libitum-fed rats. In both perirenal and epididymal fat tissue, the levels of ¹⁴C-label in the total lipid extract was significantly higher in meal-fed than in nibbling rats at all glucose doses. As the glucose dose increased, the ¹⁴C-label in the lipids of both adipose tissues in meal-fed rats increased more than would be expected, assuming a linear dose dependency. In adipose tissues of nibbling rats, glucose dose dependency was linear. The ¹⁴C radioactivity in heart muscle lipids was significantly higher in meal-fed rats at all three glucose doses. In the diaphragm, this effect was seen only at the two higher doses; in liver, only at the highest dose; in skeletal muscle, there was no difference at any of the dose levels. The incorporation of ¹⁴C-label into tissue glycogen exhibited an entirely different pattern. Muscle glycogen synthesis tended to reach a plateau at the middle glucose dose in meal-fed rats, whereas it increased sharply with increasing glucose dose in nibbling rats. Indeed, muscle glycogen synthesis was much greater in nibbling rats than would be expected, assuming linear dose dependency. It was concluded that the two groups of rats responded quite differently to the increasing glucose load. The excess glucose tended to be incorporated into lipid in meal-fed rats and to muscle glycogen in nibbling rats.     

HPRT-Deficiency associated with normal PRPP concentration and APRT activity

Deficiencies of HPRT are usually associated with increased concentrations of PRPP and increased levels of APRT activity in erythrocytes. We report the case of a male with a partial deficiency of HPRT in whom these two parameters were normal. The clinical features of this patient were those associated with severe hyperuricaemia and gout. Studies of intact erythrocytes showed rates of incorporation of [¹⁴C]hypoxanthine and of [¹⁴C]adenine into purine nucleotides which were almost indistinguishable from normal. However, HPRT activity in erythrocyte lysates was only 9% of normal. In cell extracts of cultured lymphoblasts, the HPRT activity was 20% of control values and the APRT activity was normal. The PRPP concentration and the rate ofde novo purine synthesis in cultured lymphoblasts were both intermediate between controls and lymphoblasts from patients with the Lesch-Nyhan syndrome.     

Intracellular activity of HPRTCape Town: Purine uptake and growth of cultured cells in selective media

The low activity of the human variant HPRT_{Cape Town} is associated with substrate inhibition by hypoxanthine and guaninein vitro. The intracellular activity of this variant was investigated by studying the relative uptake or radiolabelled purine nucleotide precursors and the growth in selective media of EBvirus-transformed lymphoblasts prepared from the proband (TK). These cells incorporated less than 10% of the [¹⁴C]hypoxanthine and [¹⁴C]guanine of the control cells; while their purinede novo synthesis was accelerated 8-fold. In selective media the HPRT_CapeTown cells grew in a similar manner to HPRT-ve cells. These results indicate that if substrate inhibition is responsible for the low intracellular activity of HPRT_{Cape Town}, the concentration of either hypoxanthine or guanine in the vicinity of the active site of the enzyme must be greater than theK _i(app) for these substrates, 118 and 28 µmol L^–1 respectively. Evidence is presented that the intracellular concentration of guanine, but not hypoxanthine, is well in excess of theK _i(app) in cultured lymphoblasts.     

Lipid metabolism of isolated oligodendrocytes maintained in long-term culture mimics events associated with myelinogenesis.

Oligodendrocytes isolated from ovine white matter according to a published procedure [Szuchet, S., Stefansson, K., Wollmann, R. L., Dawson, G. & Arnason, B. G. W. (1980) Brain Res. 200, 151-164] were cultured for up to 35 days and their capacity to incorporate precursors into lipids was investigated. At various times, cultures were double labeled with [3H]glycerol/[14C]acetate or [3H]galactose/35SO2-4. The cells were harvested 72 hr later and lipids were fractionated using standard procedures. The time course of incorporation for each precursor was distinct. In the days after attachment to substratum, oligodendrocytes preferentially incorporated [3H]glycerol into phospholipids and [14C]acetate into cholesterol while uptake of 35SO2-4 and [3H]galactose into glycolipids was modest. A switch in phospholipid metabolism from preferential incorporation into phosphatidylcholine to incorporation into phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol occurred at about the 10th day in vitro. After 20 days, uptake of [3H]glycerol into phospholipids and [14C]acetate into cholesterol had stabilized but incorporation of 35SO2-4 into glycolipids had increased. 35SO2-4 incorporation into glycolipids was even greater at 35 than at 20 days. Uptake of [3H]galactose did not change over time. An attempt was made to correlate changes in lipid metabolism with morphologic developments. High incorporation into phospholipids and cholesterol coincided in time with the extensive membrane synthesis required for cell attachment and process extension. Differentiation of these newly formed membranes, as assessed by the incorporation of myelin-characteristic glycolipids, galactocerebrosides, and sulfatides, occurred at a time when an intricate network of processes had already been established. The sequence of metabolic events observed in vitro parallels that observed at the onset of myelinogenesis in vivo. We postulate that mature oligodendrocytes can reenact those early events associated with myelinogenesis.     

Enhanced intestinal fat absorption in diabetic chinese hamsters

Summary Intestinal absorption of glyoeryl tri-[1-¹⁴C]-oleate and [1-¹⁴C]-oleic acid, measured by serial determinations of blood radioactivity after oral administration of the compounds in peanut oil, was significantly greater in non-ketotic diabetic Chinese hamsters than in nondiabetic controls. Incorporation of [1-¹⁴C]-oleic acid into tissue lipids by jejunal slices in vitro was equal in both groups on a unit tissue weight basis. The data suggest that intraluminal hydrolysis does not differ between diabetic and non-diabetic animals. Overall uptake of free fatty acids may be enhanced in diabetics as a consequence of increased small intestinal mass.     

Metabolic adaptations in the adipose tissue that underlie the body fat mass gain in middle-aged rats

Little is known about adipocyte metabolism during aging process and whether this can influence body fat redistribution and systemic metabolism. To better understand this phenomenon, two animal groups were studied: young—14 weeks old—and middle-aged—16 months old. Periepididymal (PE) and subcutaneous (SC) adipocytes were isolated and tested for their capacities to perform lipolysis and to incorporate D-[U-¹⁴C]-glucose, D-[U-¹⁴C]-lactate, and [9,10(n)-³H]-oleic acid into lipids. Additionally, the morphometric characteristics of the adipose tissues, glucose tolerance tests, and biochemical determinations (fasting glucose, triglycerides, insulin) in blood were performed. The middle-aged rats showed adipocyte (PE and SC) hypertrophy and glucose intolerance, although there were no significant changes in fasting glycemia and insulin. Furthermore, PE tissue revealed elevated rates (+50 %) of lipolysis during beta-adrenergic-stimulation. There was also an increase (+62 %) in the baseline rate of glucose incorporation into lipids in the PE adipocytes, while these PE cells were almost unresponsive to insulin stimulation and less responsive (a 34 % decrease) in the SC tissue. Also, the capacity of oleic acid esterification was elevated in baseline state and with insulin stimulus in the PE tissue (+90 and 82 %, respectively). Likewise, spontaneous incorporation of lactate into lipids in the PE and SC tissues was higher (+100 and 11 %, respectively) in middle-aged rats. We concluded that adipocyte metabolism of middle-aged animals seems to strongly favor cellular hypertrophy and increased adipose mass, particularly the intra-abdominal PE fat pad. In discussion, we have interpreted all these results as a metabolic adaptations to avoid the spreading of fat that can reach tissues beyond adipose protecting them against ectopic fat accumulation. However, these adaptations may have the potential to lead to future metabolic dysfunctions seen in the senescence.     

First heart sound: A phono-echocardiographic correlation with mitral,tricuspid, and aortic valvular events

We evaluated the relationship of S₁ recorded by phonocardiography at the mitral area with motion of the mitral, tricuspid, and aortic valves, recorded by simultaneous echocardiography in 20 cardiac patients with a normal PR interval. The first major component of S₁ coincided with mitral valve closure in 20 of 20 patients (100%) and also with tricuspid valve closure in 14 of 20 patients (70%). The second major component of S₁ coincided with aortic valve opening in 20 of 20 patients and also with tricuspid valve closure in six of 20 patients (30%). We conclude that the first major component of S₁ coincides with mitral valve closure in all patients but may also coincide with tricuspid valve closure in many patients, and the second major component of S₁ coincides with aortic valve opening in all patients but may also coincide with tricuspid valve closure in some patients.     

Inhibitory action of the anomers of 2-deoxy-d -glucose tetraacetate on the metabolism of d -glucose in rat pancreatic islets

Background. The tetra-acetate ester of 2-deoxy-d-glucose was recently found to either inhibit or augment insulin secretion, depending on the concentration of the ester. Both the positive and negative insulinotropic actions of the ester display anomeric specificity. Methods. The effects of the α- and β-anomer of 2-deoxy-d-glucose tetra-acetate (5.0 mM) on the metabolism of d-[5-³H]glucose and d-[U-¹⁴C]glucose (8.3 mM) were investigated in isolated rat pancreatic islets.     

Antiphospholipid Antibodies and Heart Valve Disease in Systemic Lupus Erythematosus

Evaluation of antiphospholipid antibodies (aPL) and correlation with heart valve abnormalities among patients with systemic lupus erythematosus (SLE). Nested case-control study was conducted with 70 patients with SLE selected from a longitudinal database based on levels of aPL and presence or absence of valve disease by echocardiogram. Valvular abnormalities observed were regurgitation (52), other (14), artificial valves (4), stenosis (2), thickening (2) and no Libman-Sacks endocarditis (0). The mitral valve was the most commonly affected (30 abnormalities), followed by the tricuspid (20 abnormalities). Multivariate logistic regression for those with and without an aPL value ≥20 units/mL, adjusted for disease duration and age, showed significant differences for any valve abnormality (odds ratio [OR] = 3.1; 95% CI: 1.0-8.9; P = 0.041) and individually for the tricuspid valve (OR = 3.3; 95% CI: 1.0-11.1; P = 0.052) but not for the mitral valve (OR = 2.1; 95% CI: 0.68-6.45; P = 0.195). Levels of aPL ≥20 units/mL showed no association with aortic (P = 0.253), pulmonic (P = 1.000), tricuspid (P = 0.127), or mitral (P = 0.249) valve abnormalities. Levels of aPL correlate with certain valvular abnormalities among patients with SLE.     

Pharmacokinetics and metabolism of a new antitumor semisynthetic ether phospholipid,14C-labeled plasmanyl-(N-acyl)ethanolamine,in mice bearing sarcoma Mc11

New natural and semisynthetic antitumor ether phospholipids PNAE and PNAE(s) [plasmanyl-(N-acyl)ethanolamines] and their selective antitumor activity in vivo have been described previously. We are now presenting the pharmacokinetics, in vivo metabolism and distribution of a [¹⁴C]PNAE(s) preparation (1-O-octadecyl-2-oleoyl-sn-glycero-3-phospho-(N-[U-¹⁴C]palmitoyl) ethanolamine in the intact or Mc11-tumor-bearing BDF₁ mice. Only partial degradation (about 50%–60%) of [¹⁴C]PNAE(s) was observed in vivo 24h after i.v. administration, as detected by TLC analysis of phospholipids extracted from the blood, liver, tumor and brain of animals. Pharmacokinetic curves of [¹⁴C]PNAE(s) and its metabolites were fitted with a two-compartment model (t ₁ ^2/ =2.5 h,t ₁ ^2/ =61.6 h). After repeated i.v. doses of [¹⁴C]PNAE(s) (administered on days 1, 2, 3, 4, and 5) accumulation of [¹⁴C]PNAE(s) and lyso-[¹⁴C]PNAE(s) in tumor tissue was detected. High levels of [¹⁴C]PNAE(s) were also detected in the liver, lung and spleen of animals. After i.v. administration of [¹⁴C]PNAE(s) the ether phospholipid was also detected in the brain tissue. The parmacokinetic data indicate that repeated parenteral doses of PNAE(s) are necessary to attain therapeutic concentrations in tumor tissue. The very high accumulation of [¹⁴C]PNAE(s) in the liver of animals after repeated i.v. doses, and the absence of toxic side-effects in vivo indicate a possible clinical therapeutic use of PNAE(s), especially in the treatment of tumor metastases in liver as well as in the prophylaxis of liver metastases after surgical removal of primary tumors.     

Studies in Lipogeiiesis by Red Cells of Mice

S ummary . The incorporation of[1-¹⁴C]acetate,[U-¹⁴C]glucose and [1-¹⁴C]palmitate into lipids of red cells from 5-, 30- and 180-day-old mice has been studied. The results show that the cells from 5- and 180-day-old mice have greater capacity for lipid synthesis than those from 30-day-old animals. Ghost preparations exhibit a similar pattern of incorporation but the levels of incorporation are higher than those of intact cells. There arc age-dependent differences in the incorporation of the precursors into various types of lipids. The results show that there are changes in the levels of free fatty acids and triglycerides of red cells as a function of animal age. No quantitative changes were observed in phospholipids and cholesterol.     

Effects of hagfish insulin in the atlantic hagfish, Myxine glutinosa. The in vivo metabolism of [14C]glucose and [14C]leucine and studies on starvation and glucose-loading

In hagfish, starved for 1 month at 4–6°, blood glucose decreased (1.9 to 0.8 mM) and serum insulin values diminished (2.2 to 1.1 nM). More than 90% of the glycogen in the liver and skeletal muscle was consumed, whereas protein and triglyceride contents were far more stable. The serum levels of amino nitrogen, triglycerides, and free fatty acids were all decreased after starvation. The results indicate that skeletal muscle glycogen was the prime source of energy. In starved hagfish, hagfish insulin (0.1 μg/g body weight) induced an approximate twofold stimulation of the synthesis of glycogen, protein, and neutral lipids from [¹⁴C]glucose after 33 hr at 4–6°. Most of the incorporation was detected in muscle glycogen. No insulin effects were seen in the liver. In analogous studies with [¹⁴C]leucine, hagfish insulin likewise stimulated the synthesis of glycogen and protein in muscle and protein synthesis in the liver. Despite the evidence of insulin-stimulated syntheses, the total glycogen and protein contents in muscle and liver were unaltered after 33 hr. Likewise, no insulin effects were seen on blood glucose, amino nitrogen, triglycerides, or free fatty acids. Only about 10% of the radioactive dose was incorporated into the muscle and liver, and the metabolic effects of insulin contributed to only about half of this fraction. Glucose-loading increased the serum insulin value from 0.7 to 1.9 nM. Pretreatment of hagfish with a mixture of glucose and amino acids for 3 days before and after injection of the above isotopes resulted in an increase of the serum insulin values. These endogenously elevated insulin levels were sufficient to stimulate the incorporation of the label into muscle glycogen and protein. The stimulations were similar to those obtained in experiments with exogenous insulin. It was concluded that the physiological role of insulin in skeletal muscle was similar to what has been observed in higher animals although the quantitative effects of insulin in the hagfish appeared smaller than in higher vertebrates.