Effects of aging on contributions of dietary fat and triiodothyronine treatment to lipogenic enzyme induction

Although lipogenic enzyme inductions are reduced by fat feeding, this reduction decreases with aging and is particularly detectable in the case of acetyl-CoA carboxylase and fatty acid synthetase activities. On the other hand, the fat-dependent reductions of malic enzyme and acetyl-CoA carboxylase were consistently relieved by triiodothyronine (T3) treatment. The effects of T3 treatment on these enzyme inductions were greater in 10-month-old rats than in 1-month-old rats, while the carbohydrate-dependent induction and the fat-dependent reduction of the enzymes decreased with aging. In these animals, alterations in malic enzyme mRNA translational activities were roughly in parallel to the enzyme activities. Therefore, the age-dependent alterations in effects of T3 treatment and fat on malic enzyme induction do not appear to occur in post-translation.     

Effects of dietary nutrients on lipogenic enzyme and mRNA activities in rat liver during induction

By feeding a carbohydrate diet (without protein) to fasted rats, malic enzyme mRNA activity in the liver was increased to the level in rats fed a carbohydrate and protein diet, whereas the enzyme activity itself was increased to 60% of that level. It appears that malic enzyme mRNA activity was increased by dietary carbohydrate, while dietary protein contributed to an increase in the translation of mRNA. In the animals fed carbohydrate without protein, glucose-6-phosphate dehydrogenase mRNA activity increased to 50% of the level in rats fed the carbohydrate and protein diet, whereas the enzyme activity increased to only 25%. By feeding a protein diet (without carbohydrate), glucose-6-phosphate dehydrogenase activity increased to 65% of the level in rats fed both carbohydrate and protein. This enzyme induction appears to be more dependent on protein than carbohydrate. With the carbohydrate diet, acetyl-CoA carboxylase was induced up to the level in the carbohydrate and protein diet group, whereas fatty acid synthetase was induced to only 33%. Acetyl-CoA carboxylase induction appears to be carbohydrate dependent. On the other hand, isotopic leucine incorporation studies showed that the magnitudes of the enzyme inductions caused by the dietary nutrients should be ascribed to the enzyme synthesis rates rather than the degradation. By fat feeding, the mRNA activities of malic enzyme and glucose-6-phosphate dehydrogenase were markedly decreased along with the enzyme induction. Fat appears to reduce these enzyme inductions before the translation of mRNA.     

Effect of clofibrate feeding on some lipogenic enzyme activities induced by high carbohydrate diet

Clofibrate administration by stomach tube or intraperitoneally for 3 successive days to rats fed standard diet or starved for 72 hr caused about 2-fold increase of malic enzyme activity in the liver and adipose tissue. The drug administered by stomach tube (but not intraperitoneally) to the rats fed fat free-high carbohydrate diet significantly blocked the inducing effect of the diet on malic enzyme activity in both tissues. Clofibrate blocked the induction by fat free-high carbohydrate diet of hexose monophosphate shunt dehydrogenases and ATP-citrate lyase in the liver. The amount of fat free-high carbohydrate diet consumed by rats received clofibrate by stomach tube was much less than by untreated animals. It is concluded therefore that the significant decrease of food consumption by rats receiving clofibrate by stomach tube is responsible for the inhibitory effect of the drug on some lipogenic enzymes activity induced by fat free-high carbohydrate diet.     

Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes

To elucidate the physiological role of sterol regulatory element-binding protein-1 (SREBP-1), the hepatic mRNA levels of genes encoding various lipogenic enzymes were estimated in SREBP-1 gene knockout mice after a fasting-refeeding treatment, which is an established dietary manipulation for the induction of lipogenic enzymes. In the fasted state, the mRNA levels of all lipogenic enzymes were consistently low in both wild-type and SREBP-1(-/-) mice. However, the absence of SREBP-1 severely impaired the marked induction of hepatic mRNAs of fatty acid synthetic genes, such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase, that was observed upon refeeding in the wild-type mice. Furthermore, the refeeding responses of other lipogenic enzymes, glycerol-3-phosphate acyltransferase, ATP citrate lyase, malic enzyme, glucose-6-phosphate dehydrogenase, and S14 mRNAs, were completely abolished in SREBP-1(-/-) mice. In contrast, mRNA levels for cholesterol biosynthetic genes were elevated in the refed SREBP-1(-/-) livers accompanied by an increase in nuclear SREBP-2 protein. When fed a high carbohydrate diet for 14 days, the mRNA levels for these lipogenic enzymes were also strikingly lower in SREBP-1(-/-) mice than those in wild-type mice. These data demonstrate that SREBP-1 plays a crucial role in the induction of lipogenesis but not cholesterol biosynthesis in liver when excess energy by carbohydrates is consumed.     

Effect of dimethylnitrosamine on enzyme induction in rat liver

1. The effects of various doses of dimethylnitrosamine on the hydrocortisone induction of tryptophan pyrrolase were studied. A single LD(50) dose of dimethylnitrosamine inhibits the synthesis of the enzyme if given within the first 5hr. of the induction. A quarter of this dose also inhibits the synthesis of the enzyme, but, in addition, retards the rate of decay of the enzyme. 2. A single small dose of dimethylnitrosamine significantly inhibits the hydrocortisone induction of tryptophan pyrrolase for at least 14 days without causing widespread damage to liver substructure. 3. The inhibition by dimethylnitrosamine of induced tryptophan pyrrolase synthesis is probably independent of any action of the toxin on the synthesis of cofactors necessary for full expression of the enzyme's activity. Dimethylnitrosamine appears to act directly on the synthesis of the enzyme protein. 4. The synthesis of that form of the enzyme which is most sensitive to hydrocortisone is apparently also most susceptible to the action of dimethylnitrosamine. 5. It is suggested that the inhibition of protein synthesis by dimethylnitrosamine may be a result of methylation of messenger RNA, which then is unable to code effectively for amino acid polymerization.     

Effect of temperature on lipoprotein lipase and lipogenic enzyme activities in brown adipose tissue of hypophysectomized rats

It has been shown that the same modifications on the composition of brown adipose tissue (BAT) which are normally induced following cold stimulation are also observed in hypophysectomized rats acclimated either at 28 degrees C or 15 degrees C. To test the possibility of BAT stimulation in hypophysectomized rats, we have determined some enzymatic activities known to modulate the energy supply to that organ. Seven week old Long-Evans rats were hypophysectomized. Three weeks later, they were exposed to either 28 degrees C or 15 degrees C ambient temperature for five or six weeks. Hypophysectomized rats were compared to age matched or weight matched controls. Total lipoprotein lipase activity (LPL) (triglyceride uptake) was enhanced in BAT of 28 degrees C hypophysectomized rats compared to controls. Cold acclimation led to a large increased activity. Total LPL activity was comparable in BAT of hypophysectomized and control rats. Total malic enzyme and glucose-6-phosphate dehydrogenase activities (in situ lipogenesis) were doubled in BAT of 28 degrees C hypophysectomized compared to controls. A large enhancement was observed in BAT of either 15 degrees C control or 15 degrees C hypophysectomized rats. Among the studied organs (liver, white adipose tissue, heart, BAT) hypophysectomy promotes the three enzyme activities only in BAT. These variations were discussed with relation to the effect of hypophysectomy on brown adipose tissue at 15 degrees C and 28 degrees C.     

Effect of aging on changes in substrate and effector levels of rat-liver glycolytic and lipogenic enzymes during induction

The uptake and subcellular processing of radiolabelled prolactin has been studied in male and female rats. Analytical subcellular fractionation of liver homogenates from rats injected with 125I-prolactin showed that in female rats the prolactin was primarily internalised to low density (1.12 g X cm-3) membranes. Approx. 10-15% of the total label was found in high density membranes, similar in distribution to lysosomal marker enzymes. In the normal male rat, prolactin was internalised solely to lysosomal type membranes. However, in male rats treated with estrogen, the distribution of prolactin was very similar to that seem in the female, indicating that internalisation to low density membrane is dependent on the presence of prolactin receptors. Gel exclusion chromatography showed that the prolactin internalised to the lysosomal membranes was extensively degraded whereas that associated with the low density membrane remained intact. Use of digitonin, to establish the identity of the low density membrane gave inconclusive results, but suggested that the prolactin was associated with membrane bearing NADH pyrophosphatase rather than the classical Golgi marker, galactosyltransferase.     

Effect of 2-chloroethylphosphonic acid on enzyme induction in barley endosperm

The effect of 2-chloroethylphosphonic acid (CEPA) on GA induction of α-amylase in the barley endosperm test was studied. No amylolytic activity could be detected when CEPA was substituted for GA. When CEPA was added simultaneously with GA it inhibited GA induction of α-amylase. Inhibition increased as increasing concentrations of CEPA were added. This inhibition is partially due to a direct influence of CEPA on the enzyme-starch reaction.     

Initiation of lipogenic enzyme activities in rat mammary glands.

The activities of acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthetase remained low until parturition at 22 days of gestation and increased significantly within 1 day post partum. Administration of progesterone on days 20 and 21 and at parturition abolished the increases for at least 48 h after parturition. Removal of the pups of normal rats prevented the increases in activities of acetyl-CoA carboxylase and ATP citrate-lyase, but not of fatty acid synthetase, and administration of prolactin corticosterone or insulin did not stimulate activity. Tissue from suckled glands in which the ducts had been ligated at parturition showed no increase in the activities of acetyl-CoA carboxylase and ATP citrate-lyase within 24 h, whereas fatty acid synthetase activity was similar to that in the sham-operated contralateral glands. Foetoplacentectomy on day 18 increased the activity of fatty acid synthetase but not of acetyl-CoA carboxylase and ATP citrate-lyase; suckling of these dams by foster pups increased both acetyl-CoA carboxylase and ATP citrate-lyase.     

Diurnal variations of lipogenic enzyme mRNA quantities in rat liver.

The diurnal variations in mRNA quantities of lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, malic enzyme and glucose-6-phosphate dehydrogenase) in rat livers were detected. When the rats began feeding actively after lights out at 1900 h, the mRNA quantities were high from 0500 h to 0900 h in the morning. The variation in fatty acid synthase mRNA quantities was the most dramatic. However, no measurable variation in any enzyme levels including fatty acid synthase was detected. It may be because the half-lives of the enzymes are too long to be effected by the mRNAs which were high for several hours.     

Comparative studies on lipogenic enzyme activities in the liver of human and some animal species

1. The activities of enzymes involved in fatty acid synthesis in the human liver (sample taken during abdominal surgery) and in the livers of some animals were studied. 2. Fatty acid synthase, ATP-citrate lyase and malic enzyme activities were found to be from 4 to 70-fold lower in human liver than in rat or bird livers. 3. The activities of hexose monophosphate shunt dehydrogenases in human liver were from half to almost equal to the corresponding activities in birds, but much lower than in rat liver. 4. The activities of all enzymes listed above in human and beef liver were very similar (except fatty acid synthase which was undetectable in the beef liver). 5. Very high activity of NADP-linked isocitrate dehydrogenase was found in livers of all species tested. 6. These results are discussed in relation to the role of the human liver in lipogenesis. 7. The activities of the enzymes generating NADPH in human liver taken during abdominal surgery were similar to the activities observed in the tissue obtained post mortem. 8. This suggested that post mortem tissue may be used as a reliable human material for some enzyme assays. 9. Thus we also examined the activity of malic enzyme in post mortem human kidney cortex, heart, skeletal muscle and brain. 10. Relatively high activity of NADP-linked malic enzyme has been observed in human brain.     

Effect of aging on induction of rat liver messenger RNA activity for malic enzyme

Rats were fasted and then refed a high carbohydrate-fat free diet, and the activities of the mRNA coding for liver malic enzyme [EC 1.1.1.40] in 6-week-old and 10-month-old male rats were determined by in vitro translation of the liver cytoplasmic poly(A)-containing RNA in a rabbit reticulocyte lysate. After refeeding the mRNA activities of the young rats were about 7-fold of those of the aged rats, and roughly parallel to the enzyme activities. This suggests that the age-dependent impairment of the enzyme induction [Iritani, N. et al. (1981) Biochim. Biophys. Acta 665, 636] can be ascribed to the decrease of mRNA activity.     

Deletion of insulin-degrading enzyme elicits antipodal, age-dependent effects on glucose and insulin tolerance

Background

Insulin-degrading enzyme (IDE) is widely recognized as the principal protease responsible for the clearance and inactivation of insulin, but its role in glycemic control in vivo is poorly understood. We present here the first longitudinal characterization, to our knowledge, of glucose regulation in mice with pancellular deletion of the IDE gene (IDE-KO mice).

Methodology

IDE-KO mice and wild-type (WT) littermates were characterized at 2, 4, and 6 months of age in terms of body weight, basal glucose and insulin levels, and insulin and glucose tolerance. Consistent with a functional role for IDE in insulin clearance, fasting serum insulin levels in IDE-KO mice were found to be ∼3-fold higher than those in wild-type (WT) controls at all ages examined. In agreement with previous observations, 6-mo-old IDE-KO mice exhibited a severe diabetic phenotype characterized by increased body weight and pronounced glucose and insulin intolerance. In marked contrast, 2-mo-old IDE-KO mice exhibited multiple signs of improved glycemic control, including lower fasting glucose levels, lower body mass, and modestly enhanced insulin and glucose tolerance relative to WT controls. Biochemically, the emergence of the diabetic phenotype in IDE-KO mice correlated with age-dependent reductions in insulin receptor (IR) levels in muscle, adipose, and liver tissue. Primary adipocytes harvested from 6-mo-old IDE-KO mice also showed functional impairments in insulin-stimulated glucose uptake.

Conclusions

Our results indicate that the diabetic phenotype in IDE-KO mice is not a primary consequence of IDE deficiency, but is instead an emergent compensatory response to chronic hyperinsulinemia resulting from complete deletion of IDE in all tissues throughout life. Significantly, our findings provide new evidence to support the idea that partial and/or transient inhibition of IDE may constitute a valid approach to the treatment of diabetes.