John M. Amatruda

Total and resting energy expenditure in obese women reduced to ideal body weight.

Obesity could be due to excess energy intake or decreased energy expenditure (EE). To evaluate this, we studied 18 obese females (148 +/- 8% of ideal body weight [IBW], mean +/- SD) before and after achieving and stabilizing at IBW for at least 2 mo and a control group of 14 never obese females (< 110% of IBW or < 30% fat). In the obese, reduced obese, and never obese groups, the percent of body fat was 41 +/- 4%, 27 +/- 4%, and 25 +/- 3%; total energy expenditure (TEE) was 2704 +/- 449, 2473 +/- 495, and 2259 +/- 192 kcal/24 h; while resting metabolic rate was 1496 +/- 169, 1317 +/- 159, and 1341 +/- 103 kcal/24 h, respectively. 15 obese subjects who withdrew from the study had a mean initial body composition and EE similar to the subjects who were successful in achieving IBW. In 10 subjects followed for at least one year after stabilizing at IBW there was no significant relationship between the deviation from predicted TEE at IBW and weight regain. These studies indicate that, in a genetically heterogeneous female population, neither the propensity to become obese nor to maintain the obese state are due to an inherent metabolic abnormality characterized by a low EE.

Insulin effects on apolipoprotein B lipoprotein synthesis and secretion by primary cultures of rat hepatocytes

Lipoprotein synthesis and secretion were examined in primary cultures of rat hepatocytes cultured on collagen-coated plates and incubated with pharmacologic and physiologic concentrations of insulin. Media insulin concentration declined rapidly over the course of incubation indicating that hepatocytes rapidly degrade insulin. When insulin was present in the media, cellular triglyceride accumulated while lipid secretion declined. Insulin inhibited the incorporation of labeled amino acids into total secretory lipoprotein apoproteins and apolipoprotein B (apo B) as well as apo B mass as measured by monoclonal radioimmunoassay. The effect of insulin on apo B secretion occurred as early as three hours after the addition of insulin to the culture media and both apo B of higher molecular weight (apo BH) and apo B of lower molecular weight (apo BL) were affected. Cellular apo B did not accumulate within cells. The majority of secretory lipid radioactivity synthesized from acetate was in VLDL density lipoproteins. The composition of newly synthesized lipids as assessed by thin layer chromatography was not significantly altered with insulin. These studies support the finding that insulin inhibits VLDL secretion by hepatocytes while at the same time stimulating overall triglyceride synthesis. A suggested mechanism is that insulin uncouples triglyceride and apo B synthesis, which influences subsequent lipoprotein assembly and secretory pathways. These results are consistent with the concept that postprandial insulin release inhibits hepatic lipoprotein secretion while intestinal lipoprotein metabolic pathways are most active.

Potentiation of Insulin Action by a Sulfonylurea in Primary Cultures of Hepatocytes from Normal and Diabetic Rats

Although sulfonylureas have been used extensively in the treatment of non-insulin-dependent (type II) diabetes, controversy exists as to whether these agents act primarily by increasing insulin secretion or by enhancing insulin action. To determine whether sulfonylureas potentiate insulin action in the liver, we evaluated the ability of the sulfonylurea tolazamide to affect insulinsensitive lipogenesis utilizing primary cultures of hepatocytes prepared from both normal and nonketotic streptozotocin-diabetic rats. Hepatocytes were cultured for 16 h in serum-free media with no additions, tolazamide alone (0.3 mg/ml), or insulin (10−10 to 10−7 M) in the absence and presence of tolazamide. Following culture, lipogenesis and specific insulin binding were assessed. Dose-dependent increases in lipogenesis were found in hepatocytes from both normal and diabetic rats after the chronic exposure to insulin. In hepatocytes from diabetic rats, the basal and the maximal insulin-stimulated rates of lipogenesis were only 27% and 13% of normal, respectively, establishing this as a model of hepatic insulin resistance. In the presence of tolazamide, significant potentiation of insulin action was found in hepatocytes from normal and diabetic rats although hepatocytes from diabetic animals remained relatively resistant to insulin when compared with those from nondiabetic animals. While exposure to tolazamide increased insulin responsiveness in both groups of cells, no changes in insulin sensitivity (ED50) were observed. Tolazamide significantly increased insulin binding (12%) in hepatocytes from normal rats cultured in the absence of insulin, but no alterations in insulin binding were found under incubation conditions in which tolazamide potentiated insulin action. These results give the first direct evidence for an insulin-dependent action of a sulfonylurea on the liver from both normal and diabetic rats and indicate that the enhancement of insulin responsiveness occurs through postbinding mechanisms.

Insulin-Mimetic Effects of Vanadate in Primary Cultures of Rat Hepatocytes

To evaluate possible mechanisms by which insulin inhibits hepatic apolipoprotein B (apoB) secretion, we incubated primary cultures of rat hepatocytes with sodium ort ho vanadate, a phosphotyrosine phosphatase inhibitor and insulin-mimetic agent. Vanadate (10 μM) and insulin (10 nM) inhibited the medium accumulation of apoB (secretion) by 21 and 37%, respectively, without increasing intracellular apoB. The effects of insulin and vanadate together were not additive. Both insulin and vanadate enhanced intracellular glycogen accumulation by 82 and 37%, respectively. Unlike insulin, vanadate, at a concentration that inhibited apoB secretion (10 μM), had no effect on intracellular lipogenesis, inhibited the secretion of newly synthesized hepatic proteins, and had a delayed onset and termination of action on inhibition of apoB secretion. At higher concentrations (40 and 80 μM), vanadate stimulated intracellular lipogenesis. In conclusion, our data indicate that vanadate mimics insulin action in hepatocytes with regard to the inhibition of medium accumulation of apoB. These data are consistent with the hypothesis that inhibition of apoB secretion may be secondary to an increase in phosphotyrosine content at its site of synthesis. The kinases responsible for this effect have not been identified. Several effects of vanadate, however, are different from those of insulin, suggesting a differential sensitivity to vanadate, a divergence of the signal transfer by insulin and vanadate at the insulin-receptor or postreceptor level, or both.

Insulin regulates apolipoprotein B turnover and phosphorylation in rat hepatocytes

Our laboratory has previously shown that insulin inhibits the secretion of newly-synthesized and immunoreactive apo B from rat hepatocytes. We have also shown that apo B is secreted as a phosphoprotein and that phosphorylation is increased in hypoinsulinemic nonketotic diabetes. The present studies were conducted to determine whether the ability of insulin to inhibit apo B secretion is related to alterations in apo B turnover and whether insulin itself affects apo B phosphorylation. Pulse-chase studies with [35S]methionine in primary cultures of hepatocytes from normal rats in the absence and presence of insulin show that the secretion of apo B100 and apo B48 are inhibited by insulin and that this inhibition may be due in part to enhanced intracellular degradation. In addition, there is a second intracellular apo B48 pool which is not insulin regulated or degraded. In experiments in which hepatocytes were incubated with [32P]orthophosphate, insulin decreased 32P incorporation into apo B100 (42%) with only small effects on apo B48 (11%). The small insulin effect on apo B48 may relate to an insulin-insensitive apo B48 intracellular pool. These studies show that insulin can affect the intracellular turnover, secretion, degradation, and phosphorylation of apo B and emphasize the differential regulation of apo B100 and apo B48 with regard to these parameters in rat liver.

Increased protein turnover in obese women

Some previous studies have indicated that rates of proteolysis and protein synthesis are greater in obese than in lean subjects, whereas others have not supported this finding. In the present study, we have measured postabsorptive protein turnover in a large group (n = 24) of obese women to establish more conclusively whether obese women have higher rates of protein turnover than lean women (n = 12), and to determine whether obese subjects with the greatest abdominal fat accumulation or those with the most severe insulin resistance (as determined by oral glucose tolerance testing) have the highest rates of protein turnover. Leucine appearance rate (Ra) was used as an index of whole-body proteolysis, and the fraction of Ra not oxidized was used as an index of whole-body protein synthesis. Leu Ra, oxidation, and incorporation into protein after an overnight fast were approximately 25% greater in obese than in lean women, and were approximately 10% to 15% greater after dividing by lean body mass (LBM) or adjusting for LBM by analysis of covariance. Among obese women, the degree of obesity (over the range of 30% to 47% fat) was not a significant determinant of protein turnover, nor were degree of insulin resistance, visceral fat accumulation (determined by magnetic resonance imaging [MRI]), or subcutaneous abdominal fat accumulation (also determined by MRI). However, the women with the highest rates of protein turnover also had higher waist to hip circumference ratios (WHR). We conclude that even moderate obesity is associated with increased protein turnover, and that this effect is not completely explained by the higher LBM in obese subjects.

Insulin inhibits apolipoprotein B secretion in isolated human hepatocytes

The effect of insulin on apolipoprotein (apo) B secretion was investigated in human hepatocytes. Freshly isolated hepatocytes, prepared by collagenase dispersion of liver specimens, were incubated in serum-free media in the absence and presence of 100 nmol/L insulin for 2 hours. The media was then assayed for apo B content by radioimmunoassay. In hepatocytes incubated without insulin, the secretion of apo B (relative to human low-density lipoprotein [LDL]) was 125 +/- 37 ng/10(6) cells/2 hours. In the presence of insulin, apo B secretion was reduced to 83 +/- 29 ng/10(6) cells/2 hours (34% inhibition, P less than .05). These results using human hepatocytes are consistent with previous data from our laboratory describing insulin-dependent inhibition of apo B secretion in primary cultures of rat hepatocytes and studies by others employing the human-derived hepatoma cell line, Hep G2. We conclude that human hepatic apo B secretion is under insulin control. The role of more chronic insulin exposure requires further investigation.

Vigorous supplementation of a hypocaloric diet prevents cardiac arrhythmias and mineral depletion

We have previously demonstrated that a hypocaloric, nutritionally deficient, liquid protein diet is associated with potentially life-threatening cardiac arrhythmias, which increased in frequency and complexity over the duration of the study. The present investigation was designed to evaluate the metabolic and cardiac changes associated with a hypocaloric, but otherwise nutritionally complete, diet. Six healthy, obese females from 154 to 182 percent of ideal body weight were evaluated in a metabolic ward for 48 days. The subjects ingested a weight maintenance diet during an eight-day period, which was followed by 40 days of an experimental diet containing 472 kcal of a mixture of protein (60 percent of calories), carbohydrate (25 percent), and fat (15 percent). This diet equaled or exceeded the recommended daily allowances for minerals, trace elements, vitamins, and essential fatty acids. The subjects were monitored for balances of nitrogen and minerals, as well as for the appearance of cardiac arrhythmias by 24-hour electrocardiographic recordings. Nitrogen balance was positive, and the previously demonstrated negative balances for potassium, sodium, calcium, magnesium, and phosphorus were either reversed or markedly decreased. In contrast to our previous study, no arrhythmias were observed in subjects ingesting the present experimental diet, and no significant change in cardiac rhythm was found in 13 obese, but otherwise healthy, outpatients. The data, based on a limited number of subjects, suggest that a hypocaloric diet vigorously supplemented with essential elements, micronutrients, and vitamins appears to be safer than the once popular, incomplete liquid protein preparation.

Insulin-Induced Alterations in Insulin Binding and Insulin Action in Primary Cultures of Rat Hepatocytes

The exposure of primary cultures of hepatocytes to insulin. 10−8 M, for 16 h results in a decrease in high affinity insulin binding with no alterations in lower affinity binding. This is reflected in a decrease in the sensitivity, but normal responsiveness, of cultured hepatocytes to the acute effect of insulin on the uptake of aminoisobutyric acid. The shift in sensitivity, however, can only be partially explained by the decrease in insulin binding. With regard to lipid synthesis, hepatocytes cultured in the presence of insulin, 10−8 M, are normally sensitive and hyperresponsive to the acute effects of insulin. These data indicate that the insensitivity or resistance of a given tissue to insulin may be specific for the biologic response being evaluated, that postbinding events may be more important than alterations in insulin binding in determining both the sensitivity and responsiveness of a tissue to insulin, and that generalizations concerning the sensitivity or responsiveness of a tissue to insulin based on binding data alone may be unwarranted.

Differential effect of insulin on whole-body proteolysis and glucose metabolism in normal-weight, obese, and reduced-obese women

The whole-body rate of proteolysis, as indicated by the postabsorptive appearance rate (Ra) of leucine, is increased in obese women. The present study was conducted to examine the hypothesis that the increased proteolysis is explained by insulin resistance, and to determine if proteolysis returns to normal when obese women reduce to normal weight. The mean basal leucine Ra was 21% higher in 31 obese women (> 135% ideal weight) than in 17 normal-weight women, and 9% higher per kilogram lean body mass ([LBM] P > .05). When 17 of the obese women reduced and stabilized at 100% to 116% of ideal weight, their mean basal leucine Ra decreased 17% (7%/kg LBM) and was not significantly different from that of the normal-weight control group. Insulin (40 mU/m2/min) was infused for 2 hours while maintaining euglycemia in eight normal-weight, 14 obese, and eight reduced-obese subjects. Glucose disposal per kilogram LBM was 29% lower in obese than in normal-weight subjects (P < .05) and was normal in the reduced-obese subjects. Insulin suppressed the leucine Ra an average of 18.4% in the control group, 20.4% in the obese group, and 24.1% in the reduced-obese group. Suppression of the leucine Ra by insulin did not correlate with the waist to hip ratio (WHR), glucose disposal rate, or basal leucine Ra. We conclude that the increased basal proteolysis of obese women is reversed by weight loss, and is not caused by insulin resistance.