Oxidation and ketogenesis in hepatocytes of lean and obese Zucker rats

Ketone body production and oxidation of ¹⁴C fatty acids to CO₂ were measured in hepatocytes isolated from lean and obese Zucker rats. The oxidation of [1-¹⁴C]octanoate, [1-¹⁴C]palmitate and [1-¹⁴C]palmitoyl carnitine to ¹⁴CO₂ was 50%–70% less in obese than in lean rats. Although ketone body production in hepatocytes from both lean and obese rats was increased by fasting, there was a significantly lower rate of ketone body production in hepatocytes from obese rats. Ketone body production was reduced to a comparable extent by increasing the glucose concentration in the incubation media of hepatocytes from both lean and obese rats. Glucagon and carnitine increased ketogenesis and the effects were additive and similar in lean and obese rats. These data suggest that β-oxidation and ketogenesis are suppressed in the obese Zucker rat, and further that ketone bodies can be modulated similarly in hepatocytes from lean and obese rats by nutritional and hormonal intervention. It is postulated that the decreased β-oxidation and ketone body production may play a role in the development or maintenance of obesity in the Zucker rat.     

Carbohydrate metabolism in lean and obese Zucker rats

Carbohydrate metabolism was evaluated in lean and obese Zucker rats. Plasma glucose concentration, renal and hepatic gluconeogenesis, and hepatic glycogen content and rates of synthesis were investigated in 2-mo and 8-mo-old animals. Mild hyperglycemia was observed in obese Zucker rats compared to lean rats and was more pronounced in males than in females. Rates of glucose disappearance were normal in both female and male rats, although there was a trend toward decreased clearance in the male. Total organ hepatic and kidney PEPCK activity and kidney glucose production were elevated in obese compared to lean rats. Total organ hepatic glycogen levels and rates of glycogen synthesis were increased significantly in obese compared to lean, the increase being greater in males than females. The mild hyperglycemia present in obese Zucker rats is not associated with delayed disappearance of intravenously administered glucose, but may be due to the increased production of glucose by whole kidney and liver.     

Metabolic consequences of fasting in old lean and obese Zucker rats

The effects of fasting on lipid and carbohydrate metabolism and plasma insulin and glucagon levels were compared in lean and obese Zucker rats. Sixteen-month-old female and male rats were fasted for periods of 2, 4, 6 and 12 days. Fasting produced significant decreases in hepatic rates of lipid, cholesterol, and glycogen synthesis, as well as circulating levels of triglycerides, cholesterol, phospholipids, and insulin. Significant increases in hepatic lipid levels and serum free fatty acids were noted. When compared to lean rats, obese rats had elevated rates of hepatic lipid and glycogen synthesis, hepatic lipid and glycogen stores, serum triglycerides, cholesterol, phospholipids, and plasma insulin. Lean rats had higher plasma glucagon levels. Sex differences in several parameters were observed. Females demonstrated higher levels of lipid and cholesterol synthesis and serum free fatty acids, whereas serum cholesterol levels and hepatic glycogen stores were higher in males. Following a 12-day fast, carcass fat and protein content were decreased in both lean and obese rats, but the obese animals maintained an obese body composition. It is concluded that fasting results in qualitatively similar metabolic and hormonal changes in both lean and obese rats, but that abnormalities in carbohydrate and lipid metabolism persist in obese rats even after a 12-day fast.     

Effect of brief bursts of carotid sinus stimuli on heart rate and atrioventricular conduction

The effect of a single brief stimulus burst applied simultaneously to both carotid sinus nerves on atrioventricular conduction (PR) was examined in paced and unpaced preparations of anesthetized open-chest dogs. The relative timing of the stimulus burst was varied to encompass the complete cardiac cycle. Carotid sinus/vagal effect curves were constructed to identify the time course of the response. In paced preparations the maximum increase in PR was 20.3 +/- 2.7 msec and this occurred 458.0 +/- 22.8 msec after the stimulus. There was a latency of 246.0 +/- 12.4 msec after the electrical stimulus before the PR began to increase. In unpaced heart preparations the effect of single carotid sinus nerve stimuli on heart period (PP) and PR was also determined. PP was maximally lengthened by 251.4 +/- 49.4 msec at 644.2 +/- 50.9 msec after the stimulus. There was a latency of 251.4 +/- 10.5 msec before the first noticeable change in PP occurred. The PR response was biphasic. The maximum lengthening of the PR interval was 16.2 +/- 4.2 msec. This response occurred at 361 +/- 22.0 msec after the electrical stimulus. The PR decreased to a minimum value of 13.6 +/- 2.2 msec below control values at 767.1 +/- 44.0 msec after the stimulus. The overall effect of carotid sinus activity on atrioventricular conduction depends not only on the direct nodal effect of acetylcholine but also on the indirect heart rate changes. We conclude that brief bursts of carotid sinus nerve stimulation produce cardiac electrophysiological effects qualitatively similar to the effects of direct vagal stimulation, differing only in having a lower amplitude, a longer latency and a somewhat wider time dispersion.     

Study on lipid metabolism in obesity diabetes

Knowing the relationship between obesity and diabetes, the purpose of our work was to study the alterations in lipid metabolism as measured by continuous indirect calorimetry in the course of a 100-g oral glucose-tolerance test in groups of obese patients without and with diabetes, respectively. Seventy-nine obese patients participated in the study. They were divided into four groups according to the degree of carbohydrate intolerance: group A, normal glucose tolerance; group B, impaired glucose tolerance; group C, diabetes with hyperinsulinemic response to the load; group D, diabetes with impaired insulin response. All four groups of patients presented an increase in lipid oxidation, both in the fasting state and during the three-hour glucose tolerance test, when compared to the control group. The lipid oxidation rate was roughly parallel to plasma free fatty acid (FFA) levels. The contribution of lipids to energy expenditure was higher in obese as compared to control subjects. These observations suggest that the larger part taken by lipids in the energy metabolism of both nondiabetic and diabetic obese humans is a consequence of their increased fat stores and that the resulting decrease in carbohydrate metabolism may lead, as a late consequence, to alterations in glucose tolerance. The latter may result in delayed glucose storage and oxidation in the obese patient.     

Age-related changes in lipid and carbohydrate metabolism of the genetically obese mouse

Obese mice (C57BL/6J ob/ob) and their lean controls were studied longitudinally from immediately post-weaning until 62 wk of age, at which time the experiment was terminated. The dynamic nature of the metabolic aberrations of the obese mouse syndrome was clearly demonstrated. Obese mice were hyperinsulinemic at all ages yet the concentration of glucose in plasma was elevated only at 5-20 wk and 63 wk of age, but was similar to that of lean mice at 20-60 wk of age. Triacylglycerols accumulated in the liver of obese mice between 5 and 18 wk of age to a level that was 20-fold greater than that found in the age-matched lean control. A decreased concentration of DNA/g of liver was also found in 5-18 wk-old obese mice, indicative of an enlarged hepatocyte. With the exception of 5-wk-old animals, total DNA per liver was increased in obese mice when compared to the lean control throughout the profile. Following the peak in 18-wk-old mice, the hepatic content of triacylglycerols precipitously fell so that at 45 wk of age its concentration in obese mice was similar to that of the lean control. Plasma free fatty acid levels as well as liver glycogen content were comparable in obese mice and their lean controls throughout the profile. In obese mice older than 45 wk of age, the content of triacylglycerols in plasma was significantly lower than that of the age-matched lean control while an accumulation of liver triacylglycerols was again found in obese mice. Myocardial triacylglycerols were elevated in obese mice when compared to the lean control at all ages. The longitudinal metabolic profile of the obese mouse developed in the present study clearly demonstrates the dynamic nature of the deviations in carbohydrate and lipid metabolism in this animal model of human obesity and insulin resistance.     

The contribution of hepatic metabolism to diet-induced thermogenesis

In order to examine the possible contribution of the liver to diet-induced thermogenesis, we examined the metabolism of hepatocytes from rats that had been fed a varied choice of highly palatable human food items ("cafeteria feeding"). Liver cells derived from cafeteria-fed rats that had been fasted for 20 hours showed marked increases in rates of respiration and gluconeogenesis in the presence of glycerol or sorbitol. These cells were also much less sensitive to the inhibitory effects of rotenone than were hepatocytes of control animals. hepatocytes from fasted cafeteria-fed rats also demonstrated a substantially enhanced rate of fatty acid oxidation and ketogenesis, which did not appear to be correlated with cellular demands for adenosine triphosphate (ATP). This apparent fall in metabolic efficiency was confirmed by calorimetric studies, which indicated augmented cellular heat production. These changes in hepatic metabolism, associated with cafeteria-feeding, suggest that the liver may have a significant role in diet-induced thermogenesis.     

Hepatic lipid metabolism in severe human obesity

The rate of incorporation of glycerolcarbon into triglyceride (TG) in liver slices obtained from 17 severely obese normolipoproteinemic patients during jejunoileal bypass was significantly elevated compared to normal controls. The obese patients had an almost sevenfold increase in hepatic TG content as well. None of the patients had manifest diabetes, though increased basal levels of insulin and impaired glucose tolerance during oral glucose tolerance tests (OGTT) were common. There were statistically significant positive Spearman rank correlations between plasma insulin levels and TG synthesis (r = 0.57; p < 0.01), TG content (r = 0.54; p < 0.01), and serum fatty acids (r = 0.52; p < 0.05). The basal insulin levels were also positively correlated to the incorporation rate of fructose-carbon into fatty acids (r = 0.47; p < 0.05). The sum of insulin values during OGTT showed a trend toward positive correlation with the hepatic cholesterol content (r = 0.34; p < 0.10). The hepatic protein content as well as the rate of leucinecarbon incorporation into proteins did not differ from controls. However, there was a trend toward a negative correlation between protein synthesis and basal plasma insulin levels (r = ?0.33; p < 0.10). It is suggested that liver steatosis in severely obese patients is due to significantly increased hepatic lipid synthesis in the face of elevated levels of serum fatty acids. The possible importance of plasma insulin is also emphasized.     

The metabolism of oleic acid by the perfused rat liver in experimental diabetes induced by antiinsulin serum

The metabolism of varying quantities of oleic acid was examined in isolated perfused livers from normal fed rats and from animals made diabetic by pretreatment with guinea pig antiinsulin serum (AIS). The data presented reemphasize the fact that the quantity of free fatty acid (FFA) coming to the liver is a necessary, but not the most important, factor affecting the subsequent metabolism of the FFA. Rates of ketogenesis and output of triglyceride and the terminal concentration of hepatic triglyceride were proportional to uptake of FFA in certain concentration ranges. For equal rates of uptake of FFA, ketogenesis was greater, and the quantity of triglyceride secreted or accumulated within the liver was less, with livers from diabetic animals than with livers from normal animals. In confirmation of previous data, the liver was observed to have a maximal capacity to secrete triglyceride. Triglyceride accumulated in livers from normal-fed and diabetic animals only when uptake of FFA was more than sufficient to saturate the secretory process. Since proportionately more FFA was catabolized by livers from AIS treated animals, greater uptake of FFA was required to produce maximal rates of output of triglyceride and accumulation in livers from diabetic than from normal animals. Rates of ketogenesis by livers from normal fed animals increased minimally with increasing uptake of FFA (up to 1.0 mM free fatty acid). Even when uptake increased considerably with FFA concentrations of approximately 2.5 mM, rates of ketogenesis by livers from normal animals were less than half those of livers from diabetic rats, and maximal rates were not achieved by the normal controls. It is evident that changes in hepatic metabolism of FFA in the intact diabetic animal result from simultaneous alterations of supply of FFA and hormonally induced metabolic changes in the liver. Moreover, although hepatic secretion and accumulation of triglyceride is greater in isolated perfused livers from normal rats than from diabetic animals when the livers are exposed to equal quantities of FFA, the diabetic livers can accumulate more triglyceride, secrete more triglyceride, and oxidize more FFA to ketone bodies than can the normal under conditions in which considerably more substrate is available to the diabetic rather than to the normal livers. These differences might also be expected to occur in the acutely insulin deficient intact animal, in which changes in hormonal status and substrate (FFA) availability occur simultaneously, and might, in part, explain the ketonemia, hypertriglyceridemia, and hepatic steatosis often observed in vivo.     

Adrenal phasing of a diurnal rhythm of testicular responsiveness to FSH in chickens

The adrenal steroids have been implicated in phasing a diurnal rhythm of testicular responsiveness to follicle stimulating hormone (FSH) in sexually immature white Leghom cockerels. FSH injected at the end of a 16-hr photoperiod stimulated a significantly greater weight increase by the testes than the same dose given at 0 or 8 hr. This diurnal rhythm of response to FSH did not occur in chicks receiving Metopirone, an inhibitor of adrenal steroid synthesis. This rhythm of response was absent also in chicks maintained in continuous light. However, among chicks in continuous light, injections of corticosterone phased a period of increased responsiveness to FSH 16 hr later. This diurnal rhythm of response to FSH by the testes indicates the probable occurrence of a rhythmic internal factor capable of mediating this temporally based differential response. The data suggest that in white Leghorn cockerels this factor is corticosterone.     

Effect of gemfibrozil on biliary lipid metabolism in normolipemic subjects

The mechanisms of the lipid-lowering agent gemfibrozil on biliary lipid metabolism were studied in eight normolipemic male volunteers. These measurements were performed before and after 3 months of administration. During administration of gemfibrozil, plasma cholesterol decreased by 19% (P less than 0.01) and triglycerides by 46% (P less than 0.01), and HDL cholesterol increased by 10% (P less than 0.01). The lithogenic index in gallbladder bile increased from 0.73 to 1.37 (P less than 0.05) and in hepatic bile from 0.86 to 1.42 (P less than 0.01). The increase in lithogenicity of gallbladder bile and hepatic bile was due to an increased biliary output of cholesterol from 47 to 70 mg/h (P less than 0.01) and a decreased output of bile acids from 943 to 694 mg/hr (P less than 0.01), whereas phospholipid output was not altered. The reduction in bile acid output was a result of a significant decrease in chenodeoxycholic acid secretion (r = 0.852; P less than 0.01). Cholic acid output was not affected by gemfibrozil. These results suggest that administration of gemfibrozil enhances the possible risk of gallstone formation like clofibrate.     

The effect of weight reduction on biliary lipid metabolism in patients with hyperlipoproteinemia type IV

Nine slightly obese patients with hyperlipoproteinemia type IV were studied, before and after a mean weight reduction of about 15 kg, with respect to bile acid kinetics, cholesterol balance and biliary lipid composition. The bile acid pool size was not consistently changed. The synthesis of cholic acid and chenodeoxycholic acid was decreased by about 65% and 50%, respectively. The net steroid balance, calculated as bile acid synthesis plus fecal excretion of neutral steroids minus dietary cholesterol intake, was reduced by about 50%. In all but one of the patients bile was supersaturated with cholesterol but weight reduction was not associated with any change in cholesterol saturation. The results indicate that hyperlipoproteinemia type IV may be associated with some metabolic defects which are not corrected for by weight reduction.     

Changes of vasopressin- and oxytocin-immunoreactive neurons after hypophysectomy in young and old mice

Changes of magnocellular neurons after hypophysectomy were immunohistochemically studied using antisera to arginine vasopressin (AVP) and oxytocin (OXT) in young and old female mice of the C57BL/Tw strain. AVP-immunoreactive neurons in the supraoptic and paraventricular nuclei of intact 19-month-old mice showed a marked reduction in number and immunoreactivity as compared with those of intact 3-month-old mice. Age difference of OXT-immunoreactive neurons was less pronounced than that of AVP-immunoreactive neurons. After hypophysectomy, both AVP- and OXT-immunoreactive neurons showed an intense stainability 10 days after the operation regardless of ages. However, the rate of reduction in number of immunoreactive neurons after hypophysectomy was less marked in 19-month-old than 3-month-old mice.     

Effect of colestipol and clofibrate on plasma lipid and lipoproteins in Type IIa hyperlipoproteinemia

The effects of 2.0 g of clofibrate and 15, 20 and 30 g of colestipol on plasma lipid and lipoprotein levels were evaluated in adult patients with Type IIa hyperlipoproteinemia. Clofibrate treatment was associated with decreases of 11.0% in plasma cholesterol, 15.2% in LDL cholesterol, 26.1% in triglycerides, and an 11.3% increase in HDL cholesterol. The reductions in total cholesterol with the various doses of colestipol ranged from 11.9 to 17.8% and reductions in LDL cholesterol ranged from 16.1 to 27.3%. Colestipol treatment was not associated with any significant change in HDL cholesterol levels and minor increases in triglycerides. The addition of clofibrate to patients receiving colestipol resulted in a significant increase in HDL cholesterol and a decrease in triglycerides, but no additional reduction in total or LDL cholesterol.     

Differential effects of insulin, antibodies against rat adipocyte plasma membranes, and other agents that mimic insulin action in rat adipocytes

The effects of insulin and several insulin-mimetic agents on rat adipocyte D-glucose metabolism were studied in an effort to determine if any of the insulin-mimetic agents could be used to define the mechanism of insulin action. Antibodies against rat adipocyte plasma membranes have been characterized as having insulin-mimetic effects on glucose transport and these effects may be caused by divalent clustering of cell surface antigens. In contrast to insulin, antimembrane antibodies had little stimulatory effect on D-glucose conversion to lipids in isolated rat adipocytes, under conditions where both reagents stimulated D-glucose oxidation. Among other insulin-mimetic agents tested, the reagents hydrogen peroxide and concanavalin A most closely resembled insulin in their ability to increase both [14C]-CO2 and [14C]-lipid formation from [14C]D-glucose in rat adipocytes. Vitamin K5 and diamide had the unusual effect of inhibiting [1-14C]D-glucose conversion to [14C]-lipids at a concentration that gave maximal stimulation of glucose oxidation by rat adipocytes. Analysis of the lipid components into which glucose derivatives were incorporated revealed that insulin increased D-glucose incorporation into both nonesterified fatty acids and triglycerides and H2O2 and concanavalin A had similar effects. These findings argue against the possibility that insulin and the antimembrane antibodies or the insulin-mimetic agents other than H2O2 and concanavalin A share the same mechanism of action.     

In vitro induction of Xenopus oocyte maturation by 4-pregnene-17 alpha, 20 beta-diol-3-one

Maturation in Xenopus oocyte is initiated by progesterone and other steroids. The possibility that a metabolite of progesterone is the active agent was explored. In the present study the 20 beta-dihydro metabolite of progesterone, 4-pregnene-17 alpha, 20 beta-diol-3-one was found to be as potent as progesterone at concentrations of 0.5 to 3.0 microM in inducing germinal vesicle breakdown (GVBD) in Xenopus oocytes. The order of relative potencies of the steroids tested were progesterone approximately equal to 17 alpha-hydroxy-20 beta-dihydroprogesterone greater than 17 alpha-hydroxy-20 alpha-dihydroprogesterone greater than 17 alpha-hydroxyprogesterone greater than 11-deoxycorticosterone. 17 alpha-hydroxy-20 beta-dihydroprogesterone induces the production of maturation-promoting factor leading to GVBD. The 20 beta-dihydro metabolite may be the active metabolite under in vivo condition.     

The effects of a new aldose reductase inhibitor (tolrestat) in galactosemic and diabetic rats

Tolrestat(N-[[5-(trifluoromethyl)-6-methoxy-1-naphthalenyl] thioxomethyl]-N-methylglycine; AY-27,773; Alredase) is a potent, structurally novel inhibitor of aldose reductase (AR). In vitro, tolrestat inhibited in dose-dependent fashion the AR from bovine lenses (IC50, 3.5 X 10(-8) mol/L) and the formation of sorbitol in human RBC incubated with glucose (IC50, 3 X 10(-8) mol/L). Upon administration with the diet to rats made galactosemic or diabetic, tolrestat decreased, in a dose-related fashion, the accumulation of galactitol or sorbitol in the sciatic nerve and lens. The effectiveness of tolrestat depended upon the experimental conditions and tended to be higher in less severe galactosemia and after suitable pretreatment, particularly in galactosemic rats, resulting in ID50 of 5 mg/kg/d in the sciatic nerve and 12-15 mg/kg/d in the lens. Tolrestat also decreased, in dose-related manner, the RBC sorbitol levels in normal and in streptozotocin diabetic rats; in the latter, at less than 2 mg/kg/d, the RBC sorbitol was reduced to control levels.     

Cafeteria feeding promotes diet-induced thermogenesis in monosodium glutamate-treated mice

We studied diet-induced thermogenesis (DIT) in cafeteria fed monosodium glutamate (MSG) and saline-treated mice. From 12 weeks of age MSG and saline-treated mice were fed a diet of either standard chow or a cafeteria diet of standard chow supplemented with chocolate or biscuits on alternate days for six weeks. There was a significant weight gain in cafeteria fed MSG-treated mice but not in cafeteria fed saline-treated mice. In cafeteria fed MSG-treated mice there was a significant increase in resting oxygen consumption. The response to exogenous norepinephrine was significantly increased in cafeteria fed saline-treated mice. The level of specific tritiated guanosine 5'-diphosphate binding to isolated mitochondrial fractions was significantly increased in both cafeteria fed MSG and saline-treated mice. It is concluded that (1) cafeteria feeding is capable of promoting DIT, within brown adipose tissue (BAT), in MSG-treated mice and (2) the mechanisms for the induction of thermoregulatory thermogenesis (TRT) and DIT are distinct since cold-induced TRT has previously been shown to be defective in MSG-treated mice.     

Apolipoprotein B metabolism in normal,type IV,and type V hyperlipoproteinemic subjects

Apolipoprotein B (apoB) metabolism was investigated in four normal, three type IV, and three type V hyperlipoproteinemic subjects. Following injection of autologous radioiodinated very low density lipoprotein (VLDL) the rate of clearance of the apoprotein from this particle and its subsequent appearance in low density lipoprotein (LDL) was measured by frequent apoB specific activity determinations over an 11-day period. The resultant data were analyzed using the SAAM 27 computer program. In the normal subjects, more than 95% of the injected VLDL apoB was rapidly transferred to the LDL density range and accounted for all LDL apoB synthesis in that group. The plasma VLDL apoB concentration in the type IV group was, on average, five times the normal level. This resulted primarily from a doubling of the VLDL apoB synthetic rate associated with a defective or saturated catabolic mechanism. Only 60% of this material subsequently appeared in LDL, while the remainder was catabolized via an LDL-independent pathway. The turnover parameters of LDL apoB were normal in the type IV patients. Type V hyperlipoproteinemic subjects exhibited a 12- to 35-fold increase in plasma VLDL apoB concentration over normal. This again derived from increased VLDL apoB synthesis in the presence of defective removal of the apoprotein; the fractional catabolic rate of VLDL apoB in this group was 14% of the normal value. However, in contrast to the type IV patient data, more than 85% of the apoB in type V VLDL eventually appeared in LDL whose turnover rate was raised as a result of an increase in its catabolism; the fractional catabolic rate of LDL apoB in type V patients was four-fold above normal. The plasma LDL apoB pool size was substantially reduced in these subjects. This study shows that in hyperlipoproteinemic pheno-types IV and V there exist multiple anomalies of apoB metabolism affecting both VLDL and LDL.     

A role for insulin in the diet-induced thermogenesis of cafeteria-fed rats

Rats fed a varied and palatable “cafeteria” diet exhibited hyperphagia, increases in resting metabolic rate (VO₂) and the thermogenic response to noradrenaline as well as hypertrophy of brown adipose tissue (BAT). In streptozotocin-diabetic rats, cafeteria feeding failed to produce increases in VO₂ or the response to noradrenaline, although BAT mass was greater than in their respective stockfed controls. Replacement doses of insulin (protamine-zinc-insulin, PZI) at two levels (2 and 4 units/rat every alternate day) failed to restore the thermogenic response of diabetic rats to the cafeteria diet. Acute replacement (8 units PZI) 12hr before the measurements resulted in resting and noradrenaline-stimulated values for VO₂ that were similar to those of non-diabetic cafeteria rats. These findings suggest an insulin requirement for diet-induced thermogenesis and the failure of diabetic rats to maintain body temperature when exposed to cold (5°C) suggests a further insulin requirement for cold-induced thermogenesis. In non-diabetic cafeteria rats, plasma insulin levels were significantly lower than those of stock fed controls in spite of a high carbohydrate intake and normal blood glucose.