Body weight and composition in laboratory rats: effects of diets with high or low protein concentrations.

Adult rats fed high concentrations of dietary protein for 9 weeks gained more weight than rats fed isoenergetic diets containing less protein. There were no significant differences in tail and body lengths among several groups of rats on diets containing different amounts of protein; however, total body fat was significantly greater in the rats fed on diets containing 25 percent protein compared to the rats fed 5 percent protein diets. These findings suggest that the role of dietary protein in obesity and other conditions deserves further scrutiny.

Glucagon-sensitive adenyl cylase in plasma membrane of hepatic parenchymal cells.

The plasma membrane of hepatic parenchymal cells contains an adenyl cyclase system that is stimulated by glucagon. Adrenocorticotropin and epinephrine do not stimulate this adenyl cyclase, and very little cyclic phospho-diesterase activity is present in the membrane. These findings support the concept that glucagon exerts its regulatory action in the liver by stimulating adenyl cyclase activity in the plasma membrane.

Evaluating clinical accuracy of systems for self-monitoring of blood glucose.

Although the scientific literature contains numerous reports of the statistical accuracy of systems for self-monitoring of blood glucose (SMBG), most of these studies determine accuracy in ways that may not be clinically useful. We have developed an error grid analysis (EGA), which describes the clinical accuracy of SMBG systems over the entire range of blood glucose values, taking into account 1) the absolute value of the system-generated glucose value, 2) the absolute value of the reference blood glucose value, 3) the relative difference between these two values, and 4) the clinical significance of this difference. The EGA of accuracy of five different reflectance meters (Eyetone, Dextrometer, Glucometer I, Glucometer II, Memory Glucometer II), a visually interpretable glucose reagent strip (Glucostix), and filter-paper spot glucose determinations is presented. In addition, reanalyses of a laboratory comparison of three reflectance meters (Accucheck II, Glucometer II, Glucoscan 9000) and of two previously published studies comparing the accuracy of five different reflectance meters with EGA is described. EGA provides the practitioner and the researcher with a clinically meaningful method for evaluating the accuracy of blood glucose values generated with various monitoring systems and for analyzing the clinical implications of previously published data.

Effects of thyroid hormones and thyrotropin-releasing hormone on thyrotropin biosynthesis by mouse pituitary tumor cells in vitro.

Mouse thyrotropic tumor cells grown in primary culture were shown to synthesize TSH and proteins, as determined by the incorporation of radioactive proline into immunoprecipitable TSH and trichloroacetic acid-precipitable proteins. The net TSH content of the cells and medium determined by RIA is also increased during 24 h of incubation, and newly formed hormone is detected in the medium within 1 h after the addition of proline tracer. To study the effect of T4 and T3 on TSH synthesis, cultures were pulse-labeled with [3H]proline after they had been exposed to either T3 or T4. After 48 but not 24 h, exposure to either T3 or T4 was followed by inhibition. When studied after 48 h of incubation, T4, (10(-13) M) or T3 (10(-11) M) at the lowest concentration tested, was inhibitory to TSH synthesis. At concentrations of T4 and T3 greater than 10(-9) M, the inhibitory effects on TSH synthesis were partially reversed, suggesting a biphasic response. Incubation in TRH (10(-7) M) for 24 h led to a significant increase in TSH synthesis, total protein, acid-precipitable protein, and total DNA. The effect of TRH on TSH biosynthesis was a function of the logarithm of its concentration over the range of 10(-11)-10(-7) M. The inhibitory action of 10(-6) M T3 on TSH synthesis was reversed by exposure to 10(-10) or 10(-7) M TRH.

Insulin-induced insulin resistance of lipolysis in human adipocytes in organ culture.

Adipose tissue derived from open biopsies was used to develop a system for studying insulin resistance in human tissue in vitro. Subcutaneous adipose tissue obtained from obese donors was incubated in Parker's medium 199 in the absence or presence of insulin for 24 h under sterile conditions. Adipocytes were then isolated by collagenase digestion, washed thoroughly, and incubated for 2 h with multiple insulin concentrations in Krebs-Ringer phosphate buffer with 4% bovine serum albumin. Lipolysis was estimated by measuring glycerol. Basal lipolysis in adipocytes cultured with insulin did not differ significantly from that of adipocytes cultured without insulin (2.49 +/- 0.18 vs. 2.67+/- 0.58 mumol glycerol/mmol triglyceride). The maximum acute response in adipocytes prepared from tissue exposed to insulin during culture was 55% inhibition of basal lipolysis, whereas the maximum response in cells prepared from tissue not exposed to insulin chronically was 80%. Statistical analysis by paired t test showed a significant difference (P < 0.01) in the reaction of the two groups of cells to acute exposure to insulin. The insulin dose required to produce the half-maximal effect was increased from 3 to 24 microU/ml. Thus, after chronic exposure to insulin, adipocytes were not as responsive to the acute antilipolytic action of the hormone. We conclude that chronic exposure to insulin induces insulin resistance in human adipocytes.

Effect of very-low-calorie diets with weight loss on obstructive sleep apnea.

To determine the effect of very-low-calorie diets (VLCDs) with weight loss on obstructive sleep apnea (OSA), we studied eight obese subjects with OSA, five males and three females. Subjects consumed a VLCD of 1760 kJ (420 kcal) (67% protein, 4% fat, 29% carbohydrate) or 3350 kJ (800 cal) (20% protein, 30% fat, 50% carbohydrate) with 100% of the recommended daily allowance of vitamins and minerals. Mean (+/- SD) values of weight and respiration before and after weight loss were, for weight, 153 +/- 37 and 132 +/- 29 kg (P less than 0.05); for BMI (kg/m2), 54 +/- 13 and 46 +/- 10 (P less than 0.05); for desaturations/h sleep, 106 +/- 50 and 52 +/- 45 (P less than 0.05); for apneas + hypopneas/h sleep, 90 +/- 32 and 62 +/- 49; for Pco2, 48 +/- 10 and 42 +/- 4 torr (P less than 0.05). Desaturation episodes/h and apnea + hypopneas/h improved in six patients. The most obese subject (female, BMI 81) who lost the most weight (47 kg) did not improve, nor did the subject who lost the least weight, 7 kg. The number of movements + arousals from sleep decreased in all patients (P less than 0.05). We conclude that VLCD with weight loss can produce improvement in OSA; subjects who lose a small amount of weight or subjects who are extraordinarily obese before and after weight loss may not improve.

Psyllium fiber reduces rise in postprandial glucose and insulin concentrations in patients with non-insulin-dependent diabetes.

The ability of psyllium fiber to reduce postprandial serum glucose and insulin concentrations was studied in 18 non-insulin-dependent diabetic patients in a crossover design. Psyllium fiber or placebo was administered twice during each 15-h crossover phase, immediately before breakfast and dinner. No psyllium fiber or placebo was given at lunch, which allowed measurement of residual or second-meal effects. For meals eaten immediately after psyllium ingestion, maximum postprandial glucose elevation was reduced by 14% at breakfast and 20% at dinner relative to placebo. Postprandial serum insulin concentrations measured after breakfast were reduced by 12% relative to placebo. Second-meal effects after lunch showed a 31% reduction in postprandial glucose elevation relative to placebo. No significant differences in effects were noted between patients whose diabetes was controlled by diet alone and those whose diabetes was controlled by oral hypoglycemic drugs. Results indicate that psyllium as a meal supplement reduces proximate and second-meal postprandial glucose and insulin concentrations in non-insulin-dependent diabetics.

An evaluation of diabetic and pseudodiabetic glomerulosclerosis.

Diabetic glomerulosclerosis must be either a primary manifestation or a secondary consequence of the metabolic abnormalities of diabetes. Several earlier reports have attempted to support the former hypothesis by describing cases of pathognomonic renal lesions in nondiabetic subjects; however, the clinical and pathologic data in these reports are inconclusive. We have reviewed our experience at the University of Virginia Hospital with 447 percutaneous renal biopsies performed over a period of four years from July 1973 through July 1977. Of these cases, only two appeared to represent diabetic glomerulosclerosis occurring in nondiabetic subjects. Upon further investigation, one case provided to be light chain disease demonstrated by immunofluorescence staining. The other case, on repeat renal biopsy, proved to be membranoproliferative glomerulonephritis. We conclude that a diagnosis of diabetic glomerulosclerosis must be viewed with suspicion in nondiabetic subjects. Suspected cases should be labeled pseudodiabetic glomerulosclerosis and investigated further.

Determinants of glucose and ketoacid concentrations in acutely hyperglycemic diabetic patients.

Diabetic hyperosmolar coma is a syndrome of marked hyperglycemia and minimal ketoacidosis. In general, the serum glucose concentrations are not predictive of the serum ketoacid concentrations in acutely decompensated diabetes. The endocrine factors that modulate glucose concentrations may be different from those that modulate ketoacid concentrations in patients with acutely decompensated diabetes. To test this hypothesis, regression analysis was used to determine the endocrine and metabolic characteristics that correlated with serum concentrations of glucose and ketoacids in 26 diabetic patients with spontaneous, acute hyperglycemia. All patients had a serum glucose level greater than 390 mg/dl, and ketoacid levels were from 0.17 to 25.5 mM. Multiple regression analysis showed that increased serum glucose concentrations correlated with increased plasma glucagon levels (p = 0.0007, r2 = 0.45), but with no other factors. Increased total ketoacid levels (acetoacetate plus 3-hydroxybutyrate) correlated with increased free fatty acid levels (p = 0.0001), decreased C-peptide levels (p = 0.002), and increased body mass index (p = 0.002) (r2 = 0.72). Body mass index only correlated with ketoacid levels, when it was analyzed with C-peptide and free fatty acid levels. A model is proposed that predicts the serum glucose and ketoacid concentrations in patients with acutely decompensated diabetes. Glucagon modulates the serum glucose concentration in these patients with an absolute or relative insulin deficiency. Total serum ketoacid levels are determined by the serum free fatty acid concentration, residual pancreatic insulin secretion (as reflected by C-peptide), and the patient's body habitus. This model allows for the marked hyperglycemia and minimal ketosis of diabetic nonketotic hyperosmolar coma, as well as the glucose and ketoacid concentrations in other presentations of acutely decompensated diabetes.

Changes in breathing and the pharynx after weight loss in obstructive sleep apnea.

The effect of weight loss following dietary restriction on disordered breathing on the pharyngeal airway is controversial in patients with obstructive sleep apnea (OSA). We therefore prospectively studied eight patients before and after dietary-induced weight loss. Mean weight loss was 20.6 kg +/- 12.8 SD. After weight loss there were significant improvements in PO2 and PCO2 measured during wakefulness, and in the number of desaturation episodes per hour of sleep, average desaturation per episode, and number of movement arousals. The number of apneas and hypopneas significantly decreased in six of eight patients. There was a significant correlation between body mass index and number of disordered breathing events. Nasopharyngeal collapsibility and pulse flow resistance decreased in awake patients after weight loss. We conclude that moderate weight loss in obese patients with OSA improves oxygenation during both sleep and wakefulness, decreases the number of disordered breathing events in many patients, decreases the collapsibility of the nasopharyngeal airway.

beta-Adrenergic desensitization by chronic insulin exposure in 3T3-L1 cultured adipocytes.

Cultured 3T3-L1 cells provide a model system for studies of the long-term regulation of lipolysis. Insulin acutely inhibits isoproterenol-stimulated lipolysis primarily by decreasing the apparent affinity apparent Km for isoproterenol. In contrast, chronic insulin exposure inhibits lipolysis by a reduction in the maximal effect of isoproterenol Vmax. The decrease in Vmax can be observed with insulin concentrations that are as low as 10(-9) mol/L at the time of addition. The effect is stable to washing, and the cells' responsiveness to isoproterenol returns partially with continued culture. Chronic insulin exposure also markedly reduced dibutyryl-cAMP-stimulated lipolysis indicating an insulin-induced change distal to cAMP concentration in the cascade of reactions controlling lipolysis in these cells. Time course and insulin dose-response experiments indicate an additional proximal alteration. These results indicate that: (1) 3T3-L1 cells are a useful model for studying the long-term regulation of lipolysis. (2) Chronic insulin exposure inhibits lipolysis by a mechanism that differs from the acute effect of insulin. (3) The chronic effects of insulin may be mediated through changes at multiple levels in the lipolytic cascade.

The relationship between psychological stress and insulin-dependent diabetic blood glucose control: preliminary investigations.

Clinical literature has frequently alluded to the role of psychological stress in diabetic blood glucose fluctuations. Past research in the area has been minimal and inconsistent. Recent methodological and measurement advances have made it possible to more accurately assess the impact of psychological stress on long-term diabetic control. Study 1 of this report found a significant positive correlation between the Hassles Scale and Hemoglobin A1 levels in a group of 59 adult insulin-dependent diabetic patients. Social Supports, Type A behavior, and reported therapeutic compliance neither correlated with hemoglobin A1 nor influenced the Hassles-Hemoglobin A1 relationship. In a separate sample of 123 subjects, Study II revealed that diabetic patients generally perceive stress as a very potent factor in blood glucose control, but that different stressors may have differential effects for different diabetic patients. A factor analysis of these data reveals three different stress dimensions of the perceived stress-blood glucose relationship: fight/flight, passive/ruminative, and positive affect.

Measurement of glycosylated hemoglobins using boronate affinity chromatography.

A boronate affinity column method for the measurement of glycosylated hemoglobins was evaluated. In the procedure the glycosylated hemoglobins were bound by immobilized boronic acid to separate them from nonglycosylated hemoglobins. Elution of bound glycosylated hemoglobins was carried out with sorbitol buffer, and the absorbance was read at 414 nm. The method was linear to a glycosylated hemoglobin concentration of at least 20 percent. The precision of the method ranged from 1.2 to 2.8 percent (C.V.) within-run, and 3.4 to 5.3 percent day-to-day. The reference interval was 4.8 to 6.4 percent. The method correlated with a cation exchange resin mini-column method (r = 0.94) and a colorimetric method (r = 0.93) but results from the boronate affinity method were higher in diabetic patients. The measured glycosylated hemoglobin was significantly correlated with estimated one-day-mean plasma glucose in diabetic patients (r = 0.54, n = 52, p less than 0.002). The affinity method provides an attractive alternative to earlier methods for measuring glycosylated hemoglobins.

Cyclic nucleotides and lipolysis.

The recent literature regarding the mechanisms of regulation of lipolysis with emphasis on the role of cyclic nucleotides is reviewed. The following conclusions appear warranted at present. (1) Cyclic AMP (cAMP) is a major regulator of lipolysis. However, mechanisms other than the production and catabolism of cAMP also exist. (2) Insulin can lower adipocyte cyclic AMP levels, but this effect cannot explain all aspects of the antilipolytic effect of insulin. (3) Insulin stimulates cyclic AMP phosphodiesterase and inhibits adenylate cyclase in adipocytes. In addition, there are probably other targets of insulin action. The possibilities include cAMP dependent protein kinase, phosphoprotein phosphatase, and triacylglycerol lipase. (4) Cyclic GMP is probably not directly involved in the regulation of lipolysis. (5) Cytosolic Ca2+ probably plays an important role in the regulation of lipolysis. The nature of such a role for Ca2+ and the potential role of calmodulin in the regulation of lipolysis remain to be explored.

Insulin-induced insulin resistance of alpha-aminoisobutyric acid transport in cultured human skin fibroblasts.

Cultured fibroblasts derived from skin biopsies were used to develop a system for studying insulin resistance in human tissue in vitro. Uptake of alpha-aminoisobutyric acid by cultured human skin fibroblasts was found to occur by a combination of saturable and nonsaturable processes. Insulin stimulated uptake by decreasing the Km of the saturable transport system from 0.58 mM to 0.26 mM. The maximal velocity of saturable uptake was 16.6 nmol/10(7) cells/min in both the presence and absence of insulin. Uptake of alpha-aminoisobutyric acid at 0.2 mM was studied in human skin fibroblasts with and without chronic exposure to insulin for 4 days at an initial concentration of 10 micrograms/ml. Unstimulated uptake was increased from 17 to 20 nmol/10(8) cells/min, and the increase in uptake due to maximal stimulation by insulin was unchanged at 16 nmol/10(8) cells/min in the cells exposed chronically to insulin. The apparent Km for insulin was increased from 80 microunits/ml to 2400 microunits/ml in the insulin-exposed cells. Thus, chronic exposure to insulin induces resistance of alpha-aminoisobutyric acid uptake by decreasing the apparent affinity for insulin.

Home glucose monitoring for insulin-dependent diabetics: preliminary results.

Home glucose monitoring is one of several new and aggressive approaches to improving blood glucose homeostasis in the insulin-deficient diabetic. The advent of a successful long-term pancreatic transplant or a reliable miniaturized artificial pancreas may, soon, supercede it as a treatment modality, but for the time it would appear that home glucose monitoring is a viable alternative to the more conventional and less successful methods of control and should at least be considered for use in patients with insulin-deficient diabetes mellitus.

Effects of chronic exposure to insulin on lipid synthesis in 3T3-L1 fatty fibroblasts.

The acute effects of insulin on 3H incorporation into lipid from glucose was measured in 3T3-L1 fatty fibroblasts cultured with and without insulin at 10 microgram/ml for 7 days. Basal lipid synthesis did not differ between control cells and cells treated chronically with insulin. There was no insulin stimulation in treated cells while 3H incorporation into lipid in control cells increased from a basal level of 1.39 to 3.85 nmol/dish/90 min with a maximally-stimulating concentration of insulin. This is the first study of 3T3-L1 fatty fibroblasts which describes a lack of acute insulin responsiveness in cells exposed chronically to insulin as compared to control cells.

The reaction of glucagon with its receptor: evidence for discrete regions of activity and binding in the glucagon molecule.

Des-histidine-glucagon (DH-glucagon, glucagon(2-29)) does not activate the glucagon-sensitive adenylate cyclase system present in either liver plasma membranes or in fat-cell "ghosts", but inhibits the response of these systems to submaximal concentrations of glucagon. DH-glucagon also inhibits, competitively, the binding of [(125)I]glucagon to its receptor in liver plasma membranes. Amino-terminal fragments of glucagon (glucagon(1-21), glucagon(1-23)) and carboxy-terminal fragments (glucagon(20-29), glucagon(22-29)) failed to activate adenylate cyclase, to inhibit the response of the enzyme to glucagon, or to compete with labeled glucagon at its receptor. It is concluded that the amino-terminal histidine residue of glucagon is essential for biological activity and that a hydrophobic near-carboxy-terminal region (residues 22-27) is essential for binding of glucagon to its receptor. Amino-terminal histidine may also contribute to the binding of glucagon, since the apparent affinity of DH-glucagon for the receptor is only about one-sixth that of glucagon. Thus, essentially the entire molecule of glucagon must be considered to be the biologically active species.Because, as shown elsewhere, the binding of glucagon to its receptor shows characteristics of hydrophobic bonding, and because certain detergents induce conformational changes in the carboxy-terminal binding region of glucagon, the binding is probably of a lipophilic type.