Effects of hypothyroidism on mammary and liver lipid metabolism in virgin and late-pregnant rats

Untreated maternal hypothyroidism (hypoT) has serious consequences in offspring development that may result from the effect on lactation of maternal metabolism dysfunction. We studied the effects of prolonged propylthiouracil (PTU)-induced hypoT (0.1% PTU in drinking water starting 8 days before mating until day 21 of pregnancy or for 30 days in virgin rats) on liver and mammary lipid metabolism and serum lipid concentrations. In virgins, hypoT reduced hepatic mRNAs associated with triglyceride (TG) and cholesterol synthesis (including fatty acid synthase and 3-hydroxy-3-methylglutaryl coenzyme A reductase), and induced lobuloalveolar mammary development. Pregnancy increased hepatic mRNAs associated with TG and cholesterol synthesis and uptake (including LDL receptor) and with lipid oxidation, such as acyl CoA oxidase. HypoT decreased mRNAs and the activity of proteins associated with TG synthesis, and mRNAs associated with cholesterol uptake and lipid oxidation. Pregnancy increased mammary mRNAs related to lipid oxidation and decreased cholesterol synthesis, whereas hypoT decreased mRNAs and activities of proteins associated with TG synthesis and decreased epithelial mammary tissue. Virgin and pregnant hypoT rats had increased circulating VLDL + LDL cholesterol. HypoT decreased circulating TGs in pregnant rats. The observed effects of hypoT may result in decreased mammary lipid availability. This, along with the decreased epithelial mammary tissue during lactogenesis, may contribute to the future lactational deficit of hypoT mothers.     

Effect of lactation on insulin sensitivity of glucose metabolism in rat adipocytes

During lactation glucose metabolism in paraovarian adipocytes is characterized by a 40 and 80% decrease of glucose incorporation into CO2 and fatty acids in the presence of insulin. In contrast with the stimulation by insulin of glucose incorporation into lactate, glycerol remains unchanged. As a result, insulin sensitivity of total glucose metabolism (oxidation and lipid synthesis) is not altered in adipocytes from lactating rats.     

Effects of insulin and norepinephrine on glucose transport and metabolism in rat brown adipocytes. Potentiation by insulin of norepinephrine-induced glucose oxidation

Glucose is an important fuel for rat brown adipose tissue in vivo and its utilization is highly sensitive to insulin. In this study, the different glucose metabolic pathways and their regulation by insulin and norepinephrine were examined in isolated rat brown adipocytes, using [6-14C]glucose as a tracer. Glucose utilization was stimulated for insulin concentrations in the range of 40-1000 microU/ml. Furthermore, the addition of adenosine deaminase (200 mU/ml) or adenosine (10 microM) did not alter insulin sensitivity of glucose metabolism. The major effect of insulin (1 mU/ml) was a respective 7-fold and 5-fold stimulation of lipogenesis and lactate synthesis, whereas glucose oxidation remained very low. The 5-fold stimulation of total glucose metabolism by 1 mU/ml of insulin was accompanied by an 8-fold increase in glucose transport. In the presence of norepinephrine (8 microM), total glucose metabolism was increased 2-fold. This was linked to a 7-fold increase of glucose oxidation, whereas lipogenesis was greatly inhibited (by 72%). In addition, norepinephrine alone did not modify glucose transport. The addition of insulin to adipocytes incubated with norepinephrine, induced a potentiation of glucose oxidation, while lipogenesis remained very low. In conclusion, in the presence of insulin and norepinephrine glucose is a oxidative substrate for brown adipose tissue. However the quantitative importance of glucose as oxidative fuel remains to be determined.     

Reduced glucose transport and increased binding of insulin in adipocytes from diabetic and fasted rats

1. Animals made diabetic by injection of streptozotocin or animals after 3 days of fasting show decreased insulin levels and a decrease in mean cell diameter of adipocytes from epidydymal fat pads in comparison with cells from normal animals. 2. 14CO2 production from D-[U-14C]glucose is impaired in diabetic and fasted animals both in presence or in absence of a concentration of insulin stimulating 14CO2 production maximally. 3. Insulin binding is increased in adipocytes from diabetic and fasted animals due to changes in affinity. 4. Transport studies show that basal and insulin stimulated 2-deoxy[1-14C]-glucose transport is decreased in absolute terms due to a decrease in V and an increase in Km. 5. The relative stimulatory effect of insulin is impaired in adipocytes of diabetic and fasted animals. 6. A shift of the maximal effect of insulin to lower insulin levels is seen in these cells.     

Sulphonylureas-induced increase in insulin binding and glucose metabolism by isolated rat adipocytes

The effects of oral hypoglycaemic drugs, SPC-703 (n-/p-toluenesulphonyl/-5-methyl-2-pirazoline-1-carbonami de) and tolbutamide on insulin binding and glucose metabolism by isolated adipocytes were studied. After 10 days of administration of both sulphonylurea derivatives, no differences were observed in insulin concentration between both experimental and the control groups of animals, despite a significant fall in blood glucose level. SPC-703 and tolbutamide in concentrations of 1 mM added in vitro to the suspension of adipocytes had no effect on insulin binding or on basal and insulin simulated glucose metabolism. Daily administration of 300 mg/kg body weight of SPC-703 or tolbutamide for 10 days resulted in 48% and 34% increase of specific binding of insulin by adipocytes, respectively. From the Scatchard plot analysis we noted that the increase of binding resulted from increased affinity of insulin receptors for hormone. Simultaneous increase in basal and insulin stimulated glucose metabolism by adipocytes, as measured by 14CO2 production and 14C incorporation into cellular lipids, was observed. The results indicate that hypoglycaemic action of sulphonylureas may be explained by increased affinity of insulin receptors and the stimulating action of these compounds on peripheral glucose metabolism.     

Effects of insulin on glucose metabolism in isolated heart myocytes from adult rats

The study examined the effect of insulin on glucose metabolism in freshly isolated calcium-tolerant heart myocytes from adult rats. The uptake of 2-deoxyglucose demonstrated an initial lag in response to insulin and the maximal insulin effect was not attained until after 3 min preincubation with the hormone. A dose-response study of 14CO2 production from [14C]glucose revealed that the maximum insulin stimulation of glucose utilization occurred with 5 mU/ml. Both the uptake and the oxidation of glucose proceeded at a linear rate in the absence and presence of insulin. However, insulin exerted a greater effect on the uptake (42-54%) than on the oxidation (17-22%) of exogenous glucose. Incorporation of glucose into glycogen was markedly increased by insulin and resulted in the myocyte glycogen concentration returning to in vivo levels. In the absence of insulin, glucose incorporation plateaued within 10 min of incubation and the glycogen concentration was not altered. Our findings also indicate that at equilibrium, insulin-treated cells exhibited a higher glycogen turnover rate. It thus appears that insulin exerts a differential effect on the different pathways in glucose metabolism in the isolated cardiac cells. This may be related in part to their quiescent state and lower energy demand.     

Effect of adrenalectomy on in vivo glucose metabolism in insulin resistant Zucker obese rats

Lean (Fa/?) and obese (fa/fa) Zucker rats were adrenalectomized (ADX) in order to assess the contribution of adrenal hormones to insulin resistance of the obese Zucker rat. Glucose utilization was measured using an insulin suppression test. Sham-operated obese rats gained almost twice as much weight as sham-operated lean littermates. However, body weight gain of ADX animals was comparable in both genotypes. It was significantly less than that of the respective sham-operated controls. Body weight differences can be accounted for almost entirely by a marked loss of adipose tissue. Although insulin resistance may be attributable to obesity in part, steroid hormones are thought to be directly antagonistic to insulin for glucose metabolism. Adrenalectomy resulted in a decrease in serum glucose concentrations for both lean and obese Zucker rats compared with their respective sham-operated groups. Serum insulin concentration of lean ADX rats was 23% of sham-operated controls; in obese ADX rats, it was 9% of controls. Elevated levels of steady state serum glucose (SSSG) levels in sham-operated obese rats demonstrate a marked resistance to insulin induced glucose uptake compared with sham-operated lean animals. Adrenalectomy caused a marked improvement in insulin sensitivity of obese rats. The hyperglycemic SSSG levels of the obese rats were reduced 2.5 times by ADX. These results indicate that insulin resistance of Zucker obese rats can be ameliorated by ADX, suggesting adrenal hormones contribute to insulin resistance in these animals.     

Effect of insulin and adrenergic agonists on glucose transport of porcine adipocytes

1. Insulin increased basal 2-deoxyglucose uptake in isolated swine adipocytes by 75%. In the absence of insulin, isoproterenol did not inhibit basal 2-deoxyglucose transport. 2. Adenosine deaminase plus isoproterenol or theophylline alone reduced insulin effect by 10 and 40%, respectively. Isoproterenol alone or with 2-chloroadenosine did not inhibit insulin effect on glucose transport activity. 3. Insulin effect was inhibited by isoproterenol in the presence of theophylline but not in the presence of adenosine deaminase. 4. These results suggest that catecholamines do not counter-regulate basal and insulin-stimulated glucose transport in swine adipocytes.     

Transport and metabolism of D-glucose in human adipocytes. Studies of the dependence on medium glucose and insulin concentrations

Uptake and metabolism of the physiologically labelled D-glucose (D-[U-14C]glucose) has been characterized in human adipocytes at several unlabelled D-glucose concentrations in the absence and presence of insulin. Following a 90 min incubation, about 80% of the intracellular radioactivity was incorporated into total lipids at tracer glucose concentration, as well as at higher glucose concentrations in basal and insulin-stimulated cells, whereas 20% was recovered as hydrophilic metabolites. The only 14C-labelled metabolite escaping the cells in detectable amounts was CO2, which accounted about 4%. At trace glucose concentrations (5 mumol/l), the rate of glucose uptake was linear with time. Comparative studies of initial glucose uptake after 10 s and tracer D-glucose conversion to total lipids after 90 min showed high coefficients of correlation between basal rates (r = 0.87), maximal response above basal level to insulin (r = 0.92) and insulin sensitivity (r = 0.78). Thus, under these conditions glucose transport is rate-limiting for net glucose uptake, and measurements over long time intervals of rates for total cell-associated radioactivity or lipogenesis may serve as reliable estimates of initial glucose influx rates. However, the conversion rate of tracer glucose to metabolites decreased progressively with the glucose concentration and with an apparent Km of about 0.2 mmol/l. The three metabolic pathways exhibited similar percentage decreases in their activities, suggesting that a common enzymatic step is rate-limiting. In comparison, the Km for initial D-glucose uptake rate was about 7 mmol/l. Hence, the capacity for total glucose metabolism comprised only a small fraction of the glucose transport capacity at medium glucose concentrations above tracer concentrations. Both basal, half-maximal and maximal insulin-stimulated rates of adipocyte glucose utilization were dependent on the glucose concentration. Thus, comparing lipogenesis at tracer and at 0.5 mmol/l medium glucose concentration, it was shown that the higher medium glucose concentration was associated with a 60% lowering of the basal rate, a 35% reduction in the percentage response above baseline to maximal insulin stimulation and a 4-fold increase in the insulin sensitivity. Obviously, these findings reflect some intracellular step(s) being rate-limiting at medium glucose levels above tracer values.     

Adenosine effects on glucose oxidation of adipocytes isolated from streptozotocin-diabetic rats.

Streptozotocin-induced diabetes did not impair the response of adipocytes to adenosine effects in glucose oxidation. The greatest effect of adenosine in potentiating the action of insulin was in the physiological concentration range of insulin (10-100 mu units/ml). The desensitization of cells by diabetes to the effects of insulin is therefore probably not related to the response of cells to adenosine.     

Effect of exercise on insulin binding and glucose transport in adipocytes of normal humans

Acute exercise increases insulin binding to its receptors on blood cells. Whether the enhanced insulin binding explains the exercise-induced increase in glucose uptake is unclear, since insulin binding and glucose uptake have not been measured simultaneously in a target tissue of insulin. In this study, we determined insulin binding and the rate of glucose transport in adipocytes obtained by needle biopsy from 10 healthy men before and after 3 h of cycle-ergometric exercise. During the exercise, plasma glucose (P less than 0.01) and insulin (P less than 0.001) fell and serum free fatty acid level rose 4.3-fold (P less than 0.001). 125I-insulin binding to adipocytes remained unchanged during exercise. The rate of basal glucose transport clearance fell from 28.1 +/- 5.7 fl.cell-1.s-1 to 22.9 +/- 5.6 fl.cell-1.s-1 (P less than 0.005), and the insulin-stimulated increase in glucose transport rate rose from 196 +/- 26 to 279 +/- 33% (P less than 0.025) during the exercise. Thus, in the adipocytes during exercise, the basal glucose transport rate and the responsiveness of glucose transport to insulin changed in the absence of alterations in insulin binding. These data indicate that the exercise-induced changes in insulin binding show tissue specificity and do not always parallel alterations in glucose transport.     

Effect of insulin on glucose transport and metabolism in isolated fat-cells of gonadal adipose tissue from mature age-matched male and female rats.

Insulin action on glucose transport and metabolism was studied in paraovarian adipocytes from 3-month-old female rats and compared with insulin action in epididymal adipocytes from closely age-matched males. At maximal insulin concentrations the stimulations of 2-deoxyglucose uptake (4-fold the basal value) and of [U-14C]glucose incorporation into CO2 and total lipids (3- and 2-fold the basal values respectively) were similar in adipocytes from rats of both sexes. At submaximal insulin concentrations (less than 0.2 nM) the ability of paraovarian adipocytes to transport and to metabolize glucose was higher than that of epididymal adipocytes; accordingly an increase in insulin binding was observed in paraovarian adipocytes as compared with epididymal adipocytes. These results show that paraovarian adipocytes from mature female rats were highly responsive to insulin, and exhibited a higher sensitivity to the hormone than did epididymal adipocytes from male rats of the same age.     

Phosphoinositide metabolism in adipocytes from fed and fasted rats

Phosphoinositide turnover was investigated in adipocytes from fed and 48 hour fasted rats. Insulin stimulated phosphoinositide synthesis both in adipocytes from fed and fasted rats. Fasting enhanced this effect of insulin 2-fold. Hydrolysis of phosphoinositides to inositol phosphates was not activated by insulin, neither transient after 2 minutes nor after 60 minutes stimulation. Under similar conditions, alpha 1-adrenergic receptor stimulation induced a pronounced inositol phosphate production. Thus, it is suggested that phosphoinositide hydrolysis is not involved in insulin action. The alpha 1-adrenergic effect was similar in adipocytes from fed and fasting rats.     

Effect of experimentally-induced chronic hyperprolactinemia on insulin binding and antilipolytic response in adipocytes from male rats

Male Wistar rats with chronic hyperprolactinemia induced by grafting an anterior pituitary gland under the right kidney capsule were studied as experimental model. In these animals basal plasma glucose and insulin levels were unaltered. Epididymal adipocytes from hyperprolactinemic rats showed a significant increase in insulin binding at low unlabeled insulin concentrations. This increase in insulin binding can be principally attributed to an increase in the high affinity-low capacity binding sites, as demonstrated when Scatchard analysis was interpreted in terms of two types of insulin receptors. The dissociation constants (KD1 and KD2) were not different between the groups. The apparent insulin receptor affinity was also unchanged. Moreover, a decreased sensitivity to the antilipolytic effect of insulin was also obtained in adipocytes from hyperprolactinemic rats. These findings indicate that chronic hyperprolactinemia is able to increase high affinity insulin receptors in epididymal adipocytes, but tends to diminish the antilipolytic response, suggesting a lack of coupling between insulin binding and its biological activity in male adipose tissue. Several possible mechanisms involved in the process are suggested.