Influence of alterations in meal frequency on lipogenesis and body fat content in the rat.

Rats were allowed to eat only 2 hr per day (meal-fed) or were fed ad libitum (nibbler) for 12 wk; another group of animals was meal-fed for 3 wk and then fed ad libitum (converted I) while the fourth group of rats (converted II) was meal-fed for 3 wk, allowed to nibble for the next 3 wk, meal-fed from the 6th to 9th wk and then returned to ad libitum feeding for the last 3 wk. Body fat gain and food efficiency was increased in converted I rats. The lipogenic capacity of adipose tissue from meal-fed rats was greater than observen meal-fed rats were reverted to ad libitum feeding whereas lipogenic activity increased rapidly when ad libitum fed rats were switched to meal-feeding.     

Dietary protein and the control of fatty acid synthesis in rat adipose tissue

Fatty acid synthesis in adipose tissue normally proceeds at a high rate when fasted animals are refed a diet containing carbohydrate, protein, and low levels of fat. This study investigated the effect of omitting protein from the refeeding diet. Rats were fasted for 48 hr and refed either a protein-free diet or a balanced diet, and the rate of fatty acid synthesis from glucose, pyruvate, lactate, and aspartate was measured. Refeeding the animals a diet devoid of protein resulted in a low rate of fatty acid synthesis from each of these substrates as well as a reduction in carbon flow over the citrate cleavage pathway. The activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-malate dehydrogenase, and ATP-citrate lyase were also reduced in epididymal fat pads from these rats. On the other hand, adipose tissue phosphoenolpyruvate carboxykinase activity was five times as great as that in tissue from animals refed a balanced diet. This difference could be eliminated if actinomycin D was injected coincident with refeeding. Refeeding rats diets high in carbohydrate is not, therefore, capable of inducing high rates of fatty acid synthesis in adipose tissue in the absence of dietary proteins. Thus, liver and adipose tissue respond differently to dietary protein.     

Meal-feeding studies in mice: effects of diet on blood lipids and energy expenditure

To identify optimal study-design conditions to investigate lipid metabolism, male, C57BL/6J mice (age, 59 +/- 3 days) were allotted to eight groups, with six animals per group that were stratified by three factors: diet type (high fat [HF]: 60% of energy from fat versus that of a standard rodent diet, 14% fat, fed for 7 weeks), feeding regimen (ad libitum [ad lib] versus meal fed), and metabolic state (data collected in fasted or fed states). Serum free fatty acids (FFA) and triacylglycerols (TAG) concentrations, and energy expenditure (EE) were assessed. Mice gained 0.30 +/- 0.11 g of body weight/day when allowed ad lib access to HF diet, similar weight when meal-fed the HF or ad lib-fed the standard diet (0.10 +/- 0.03 g/day), and no weight when meal-fed the standard diet (0.01 +/- 0.02 g/day). Fed-state TAG concentration was 88 to 100% higher (P < 0.02) than that of the fasted state, except when animals were ad lib-fed the HF diet. When the standard diet was meal fed, FFA concentration was 30% higher in the fasted compared with the fed state (P = 0.003). Mice had 33% higher postprandial EE when either diet was meal fed (P = 0.01). Mice adapted to meal feeding developed transitions in metabolism consistent with known physiologic changes that occur from fasting to feeding. When fed the standard diet, a 6-h per day meal-feeding regimen was restrictive for normal growth. These data support use of a meal-feeding regimen when HF diets are used and research is focused on metabolic differences between fasted and fed states. This protocol allows study of the metabolic effects of an HF diet without the confounding effects of over-consumption of food and excess body weight gain.     

Lipoprotein lipase and lipogenic enzyme activities in adipose tissue from rats fed different lipid sources

This work was designed to study the effect of different lipid sources on the activities of lipoprotein lipase and lipogenic enzymes in adipose tissue from rats fedad libitum or energy-controlled diets. Male Wistar rats were fed diets containing 40% of energy as fat (olive oil, sunflower oil, palm oil or beef tallow), for 4 wk. Underad libitum feeding no differences were found among dietary fat groups in final body weight, adipose tissue weights and total body fat. Under energy-controlled feeding, despite isoenergetic intake, rats fed the beef tallow diet gained significantly less weight than rats fed the other three diets. Beef tallow fed rats showed the lowest values for adipose tissue weights and total body fat. When rats had free access to food no effect of dietary lipid source on lipogenic enzyme activities was found. In contrast, under energy-controlled feeding rats fed the beef tallow diet showed significantly higher activities of glucose-6-phosphate dehydrogenase and fatty acid synthase than rats fed the other three diets. Heparin-releasable lipoprotein lipase activity in perirenal and subcutaneous adipose tissues was not different among rats fed olive oil, safflower oil, palm oil or beef tallow. When comparing both adipose tissue anatomical locations, significantly higher activities were found in subcutaneous than in perirenal fat pad independently of dietary fat. In conclusion, under our experimental protocol, lipogenesis in rat adipose tissue does not seem to be affected by dietary fat type.     

The significance of hyperphagia and diet composition on the metabolism in ventromedial hypothalamic lesioned male rats

The metabolic consequences of ventromedial hypothalamic lesion were studied in a group of aged male rats which were obese and had decreased response to insulin. The effects of hyperphagia and ventromedial hypothalamic lesion per se were separated by comparing experimental animals fed isocalorically with controls and animals fed ad libitum. Ventromedial hypothalamic lesion as such led to increases in the glucose conversion to fatty acid and in lipoprotein lipase activity in adipose tissue. Protein catabolism as reflected by plasma urea levels, was enhanced. The lipoprotein lipase activity in heart tended to be lower after VMH lesion. These metabolic changes were amplified in the VMH lesioned rats fed ad libitum. The liver glycogen content was lowered by VMH lesion, but this effect was abolished by hyperphagia. In parallel experiments the influence of diet composition was studied by feeding similar groups with diet of high fat content. The glucose incorporation in fatty acids was in all groups markedly and similarly inhibited by the high fat diet. The increase in lipoprotein lipase activity in heart and adipose tissue of control rats with high fat intake could not be demonstrated in any of the groups with ventromedial hypothalamic lesion. The plasma urea level in the control group was not affected by the diet, but tended to increase in the ventromedial hypothalamic lesioned groups on high fat intake. These findings demonstrate that the well known metabolic effects of ventromedial hypothalamic lesions are also manifest in obese insulin resistant male rats. Furthermore, the responses to changes in diet composition are different from those of the control rats.     

Decreased serum leptin and muscle oxidative enzyme activity with a dietary loss of intra-abdominal fat in rats

The purpose of the present study was to investigate the relationship among intra-abdominal adipose storage, adaptation in the serum leptin concentration and skeletal muscle enzyme activity after a 4-week energy restriction (ER). Thirty-one male Wistar rats were divided into 40% energy restricted (n=24) or ad libitum-fed control (CL) rats (n=7). The energy-restricted rats were grouped into the most fat (MF, n=7), medium (n 10) and the least fat (LF, n=7) by their intra-abdominal fat pads mass (epididymal, mesenteric, and perirenal) after ER. A superficial portion of M. gastrocnemius tissue obtained before and after the diet period were analyzed to determine the activities of hexokinase (HK), beta-hydroxyacyl CoA dehydrogenase (beta-HAD) and citrate synthase (CS). Blood samples were also collected for a serum leptin assay. At the baseline, no difference was found in either the leptin concentration or the enzyme activities among LF, MF and CL. The serum leptin concentration was positively correlated with the muscle activities of beta-HAD and CS, while it negatively correlated with HK/beta-HAD. After ER, the activities of HK, beta-HAD and CS were all significantly lower in LF than in CL. Among the energy-restricted rats, the intra-abdominal fat pad weight, leptin concentration and the activities of beta-HAD, CS, beta-HAD/CS all significantly correlated with one another. The changes in leptin and the activity of beta-HAD were also positively correlated. These findings indicate that parallel decreases in the serum leptin and skeletal muscle enzyme activities with the energy restriction-induced intra-abdominal adipose reduction, thus may suggest the leptin to have a regulative effect on the muscle enzyme activity during ER.     

Effects of Proportions of Dietary Macronutrients on Glucocorticoid Metabolism in Diet-Induced Obesity in Rats

Tissue glucocorticoid levels in the liver and adipose tissue are regulated by regeneration of inactive glucocorticoid by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and inactivation by 5α- and 5β-reductases. A low carbohydrate diet increases hepatic 11β-HSD1 and reduces glucocorticoid metabolism during weight loss in obese humans. We hypothesized that similar variations in macronutrient proportions regulate glucocorticoid metabolism in obese rats. Male Lister Hooded rats were fed an obesity-inducing ad libitum ‘Western’ diet (37% fat, n = 36) for 22 weeks, then randomised to continue this diet (n = 12) or to switch to either a low carbohydrate (n = 12) or a moderate carbohydrate (n = 12) diet for the final 8 weeks. A parallel lean control group were fed an ad libitum control diet (10% fat, n = 12) throughout. The low and moderate carbohydrate diets decreased hepatic 11β-HSD1 mRNA compared with the Western diet (both 0.7±0.0 vs 0.9±0.1 AU; p<0.01), but did not alter 11β-HSD1 in adipose tissue. 5α-Reductase mRNA was increased on the low carbohydrate compared with the moderate carbohydrate diet. Compared with lean controls, the Western diet decreased 11β-HSD1 activity (1.6±0.1 vs 2.8±0.1 nmol/mcg protein/hr; p<0.001) and increased 5α-reductase and 5β-reductase mRNAs (1.9±0.3 vs 1.0±0.2 and 1.6±0.1 vs 1.0±0.1 AU respectively; p<0.01) in the liver, and reduced 11β-HSD1 mRNA and activity (both p<0.01) in adipose tissue. Although an obesity-inducing high fat diet in rats recapitulates the abnormal glucocorticoid metabolism associated with human obesity in liver (but not in adipose tissue), a low carbohydrate diet does not increase hepatic 11β-HSD1 in obese rats as occurs in humans.     

Organ specific regulation of malic enzyme and hexosemonophosphate shunt dehydrogenases activity by high carbohydrate diet

The effect of starvation-refeeding transitions on the activity of malic enzyme and hexosemonophosphate shunt dehydrogenases in lipogenic and non-lipogenic tissues from rats was investigated. Starvation of the rats caused a decrease of malic enzyme activity in the liver, white and brown adipose tissue. Refeeding of the animals with high carbohydrate diet caused a several fold increase of malic enzyme activity in these tissues. Substitution of high fat for high carbohydrate diet resulted in only a slight increase of malic enzyme activity in the liver, white and brown adipose tissues. In the same rats, no significant effect of starvation-refeeding transition on malic enzyme activity in the kidney cortex, brain, heart, skeletal muscle and spleen was observed. The changes of the activity of hexosemonophosphate shunt dehydrogenases during starvation-refeeding transition essentially paralleled those of malic enzyme in all the tissues examined.     

The concentration of cyclic AMP and the activity of cyclic AMP dependent protein kinase and an inhibitor in the adipose tissue of rats fed lard or glucose diets.

Measurements of tissue cyclic AMP (cAMP) concentration, the activity of cAMP-dependent protein kinase and the level of the enzyme's thermostable, macromolecular inhibitor were made on preparations of rat epididymal fat pad from animals fed high fat or high carbohydrate diets. The cAMP concentration from rats adapted to a high lard diet for 14-15 days was 153 +/- 17.8 pmoles/mg protein as opposed to 76 +/- 6.0 found with high glucose diet. No significant difference in total cAMP-dependent protein kinase activity was observed among rats fed high glucose, high lard or laboratory chow, although the enzyme's activity ratio (-cAMP)(+cAMP) was significantly elevated with lard feeding (0.49 +/- 0.02) as opposed to glucose feeding (0.43 +/- 0.01). Crude preparations from lard and glucose fed animals were equivalent in inhibitory activity when tested with enzyme from chow fed animals. Agarose column chromatography separated holoenzyme and C subunit forms of the protein kinase when 500 mM NaCl was present in the elution buffer. Absence of the salt allowed subunit reassociation to occur. Direct addition of NaCl greater than or equal to 75 mM significantly inhibited protein kinase activity. The results indicate that the adipose tissue of rats fed a high lard diet has a higher concentration of cAMP and an increased protein kinase activity ratio than tissue from rats fed a fat free, high glucose diet. Total cAMP-dependent protein kinase activity and the level of a thermostable macromolecular inhibitor remained unchanged.     

Diurnal response in endogenous amino acid oxidation of meal-fed rats.

A model has been developed to measure the effects of dietary protein on daily fluctuations in the rate of endogenous amino acid oxidation in meal-fed and starved rats. In addition, N tau-methylhistidine and hydroxyproline were utilized to determine changes in the rate of degradation of myofibrillar and collagen proteins. In rats meal-fed a normal diet of 18% (w/w) casein, a diurnal response was observed in rate of oxidation of radioactive amino acids contained in endogenous labelled body protein, with a nadir 16--20 h and maximum 4--8 h after beginning the feeding. This observation in part may be related to alterations in flux of amino acids from non-hepatic tissues to site of oxidation in liver, as well as alterations in rates of amino acid oxidation after a protein meal. When meal-fed a 70% protein diet, the maximal rates of endogenous amino acid oxidation were significantly increased by 4--8 h after meal-feeding, with no change in fractional rates of degradation of myofibrillar- or collagen-protein breakdown. This could suggest increases in activities of enzymes involved in amino acid oxidation, in rats meal-fed 70% compared with 18% dietary protein. In contrast, meal-feeding of a protein-free diet muted the diurnal response in the rate of oxidation of endogenously labelled amino acids, which correlated with a decrease in the fractional rate of degradation of myofibrillar or collagen protein. Thus dietary protein is apparently responsible for the observed diurnal rhythm rhythms in the rate of amino acid oxidation, whereas carbohydrates tend to mute the response.     

Effects of carbohydrate availability on lipogenesis in sheep

1. Lipogenesis in sheep liver and adipose tissue was investigated by incorporation studies in vitro with radioactive glucose and acetate and by assays of key enzymes. 2. Carbohydrate availability to sheep was increased by feeding on a diet containing 70% soluble carbohydrate, by infusing glucose into the abomasum or by direct intravenous infusion of glucose. 3. Under these conditions lipogenesis from glucose and acetate was increased from very low values in lìver and adipose tissue, especially in those animals where rumen fermentation was by-passed by glucose infusion. 4. Large increases in the activities of ATP citrate lyase (EC 4.1.3.8) and NADP-malate dehydrogenase (EC 1.1.1.40) occurred in both tissues when lipogenesis was increased. 5. No adaptations were found in the activities of pyruvate carboxylase (EC 6.4.1.1) in adipose tissue, glucokinase (EC 2.7.1.2) in liver or 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in liver. It is proposed that the absence of these enzymes is not related to glucose availability. 6. The effect of glucose on liver lipogenesis was to increase conversion of acetate into lipid. 7. This effect also occurred in adipose tissue, but in this tissue glucose also became a quantitatively important precursor of triglyceride fatty acid.     

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.     

Influence of restricted food intake on brown adipose tissue function in genetically obese mice (genotype, ob/ob)

Measurements were made of cytochrome c oxidase activity and the GDP-binding capacity of mitochondria in brown adipose tissue of genetically obese mice and wild-type siblings, to estimate the thermogenic capacity of the tissue. The binding capacity was decreased in ad libitum fed obese animals compared with wild-type animals. Limited feeding of obese animals to restrict their body weight caused a large increase in the binding capacity of the tissue, which was greater than that in wild-type animals fed either ad limitum or on a limited diet. The decreased binding capacity of brown adipose tissue mitochondria in obese mice appears to be a consequence of ad libitum feeding and therefore not a cause of the obesity. Limit feeding of obese animals also corrected their characteristic hypothermia at low ambient temperature. The large increase in the thermogenic capacity of brown adipose tissue in obese animals, induced by limited feeding, may account for the vital improvement of their thermoregulation. However, close similarities were found between obesity hypothermia and hypothermia induced in wild-type animals by restraint. It is suggested that changes in posture caused by obesity, resulting in increased loss of body heat, may be important in the development of obesity hypothermia. Obese animals fed less than wild-type grained more weight than wild-type animals, indicating that the high thermogenic capacity of their brown adipose tissue did not function to regulate their calorie intake.     

Comparison of tissue pyruvate dehydrogenase activities on re-feeding rats fed ad libitum or meal-fed rats with a chow-diet meal.

Meal-fed rats and rats fed ad libitum had similar rates of hepatic glycogenesis at 60 min after the initiation of re-feeding a chow meal after 22 h starvation, but hepatic PDHa (active form of pyruvate dehydrogenase) activities were 4-fold higher in the meal-fed group. In heart, PDHa activities were 3-fold higher before re-feeding and 2-fold higher after re-feeding in the meal-fed group compared with the group fed ad lib. The blood metabolite profile suggested diminished fat oxidation in starved meal-fed rats and accelerated flux through PDH in meal-fed re-fed rats compared with the group fed ad lib.     

Effects of dietary nutrients on substrate and effector levels of lipogenic enzymes, and lipogenesis from tritiated water in rat liver

When fasted rats were refed for 4 days with a carbohydrate and protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the effects of dietary nutrients on the fatty acid synthesis from injected tritiated water, the substrate and effector levels of lipogenic enzymes and the enzyme activities were compared in the livers. In the carbohydrate diet group, although acetyl-CoA carboxylase was much induced and citrate was much increased, the activity of acetyl-CoA carboxylase extracted with phosphatase inhibitor and activated with 0.5 mM citrate was low in comparison to the carbohydrate and protein diet group. The physiological activity of acetyl-CoA carboxylase seems to be low. In the protein diet group, the concentrations of glucose 6-phosphate, acetyl-CoA and malonyl-CoA were markedly higher than in the carbohydrate and protein group, whereas the concentrations of oxaloacetate and citrate were lower. The levels of hepatic cAMP and plasma glucagon were high. The activities of acetyl-CoA carboxylase and also fatty acid synthetase were low in the protein group. By feeding fat, the citrate level was not decreased as much as the lipogenic enzyme inductions. Comparing the substrate and effector levels with the Km and Ka values, the activities of acetyl-CoA carboxylase and fatty acid synthetase could be limited by the levels. The fatty acid synthesis from tritiated water corresponded more closely to the acetyl-CoA carboxylase activity (activated 0.5 mM citrate) than to other lipogenic enzyme activities. On the other hand, neither the activities of glucose-6-phosphate dehydrogenase and malic enzyme (even though markedly lowered by diet) nor the levels of their substrates appeared to limit fatty acid synthesis of any of the dietary groups. Thus, it is suggested that under the dietary nutrient manipulation, acetyl-CoA carboxylase activity would be the first candidate of the rate-limiting factor for fatty acid synthesis with the regulations of the enzyme quantity, the substrate and effector levels and the enzyme modification.     

Scheduled feeding results in adipogenesis and increased acylated ghrelin

Ghrelin, known to stimulate adipogenesis, displays an endogenous secretory rhythmicity closely related to meal patterns. Therefore, a chronic imposed feeding schedule might induce modified ghrelin levels and consequently adiposity. Growing Wistar rats were schedule-fed by imposing a particular fixed feeding schedule of 3 meals/day without caloric restriction compared with total daily control intake. After 14 days, their body composition was measured by DEXA and compared with ad libitum-fed controls and to rats daily intraperitoneal injection with ghrelin. Feeding patterns, circadian activity, and pulsatile acylated ghrelin variations were monitored. After 14 days, rats on the imposed feeding schedule displayed, despite an equal daily calorie intake, a slower growth rate compared with ad libitum-fed controls. Moreover, schedule-fed rats exhibiting a feeding pattern with intermittent fasting periods had a higher fat/lean ratio compared with ad libitum-fed controls. Interestingly, ghrelin-treated rats also showed an increase in fat mass, but the fat/lean ratio was not significantly increased compared with controls. In the schedule-fed rats, spontaneous activity and acylated ghrelin levels were increased and associated with the scheduled meals, indicating anticipatory effects. Our results suggest that scheduled feeding, associated with intermittent fasting periods, even without nutrient/calorie restriction on a daily basis, results in adipogenesis. This repartitioning effect is associated with increased endogenous acylated ghrelin levels. This schedule-fed model points out the delicate role of meal frequency in adipogenesis and provides an investigative tool to clarify any effects of endogenous ghrelin without the need for ghrelin administration.     

PVN galanin increases fat storage and promotes obesity by causing muscle to utilize carbohydrate more than fat

To understand the function of the feeding-stimulatory peptide, galanin (GAL), in eating and body weight regulation, the present experiments tested the effects of both acute and chronic injections of this peptide into the paraventricular nucleus (PVN) of rats. With food absent during the test, acute injection of GAL (300 pmol/0.3 microl) significantly increased phosphofructokinase activity in muscle, suggesting enhanced capacity to metabolize carbohydrate, and reduced circulating glucose levels. It also decreased beta-hydroxyacyl-CoA dehydrogenase activity in muscle, indicating reduced fat oxidation, while increasing circulating non-esterified fatty acids (NEFA) and lipoprotein lipase activity in adipose tissue (aLPL). Chronic PVN injections of GAL (300 pmol/0.3 microl/injection) versus saline over 7-10 days significantly stimulated daily caloric intake and increased the weight of four dissected fat depots by 30-40%. These effects, accompanied by elevated levels of leptin, triglycerides, NEFA and aLPL activity, were evident only in rats on a diet with at least 35% fat. Thus, by favoring carbohydrate over fat metabolism in muscle and reversing hyperglycemia, PVN GAL may have a function in counteracting the metabolic disturbances induced by a high-fat diet. As a consequence of these actions, GAL can promote the partitioning of lipids away from oxidation in muscle towards storage in adipose tissue.     

The effects of meal-feeding and the diurnal cycle on lipogenesis in brown adipose tissue of rats

A significant diurnal variation in the rates of lipogenesisin vivo in brown adipose tissue occurred in both virgin and lactating rats. On a meal-feeding regime of either a chow, high-sucrose, or high-lipid diet, there was a very large increase in BAT lipogenesis following the meal. The rates observed after the sucrose meal are the highest so far reported. There was no significant difference in BAT lipogenesis between lactating and virgin rats, contrary to previous reports by others. The pattern of stimulation of BAT lipogenesis by these feeding regimes was different from that for white adipose tissue and liver and was not correlated with plasma insulin levels.