Developmental changes in fatty acid synthesis in interscapular brown adipose tissue of lean and genetically obese (ob/ob) mice.

Fatty acid synthesis was measured in vivo with 3H2O in interscapular brown adipose tissue of lean and genetically obese (ob/ob) mice. At 26 days of age, before the development of hyperphagia, synthesis in brown adipose tissue was higher in the obese than in the lean mice; synthesis was also elevated in the liver, white adipose tissue and carcass of the obese mice. At 8 weeks of age, when hyperphagia was well established, synthesis remained elevated in all tissues of the obese mice, with the exception of brown adipose tissue. Elevated synthesis rates were not apparent in brown adipose tissue of the obese mice at 14 days of age, nor at 35 days of age. These results demonstrate that brown adipose tissue in ob/ob mice has a transitory hyperlipogenesis at, and just after, weaning on to a low-fat/high-carbohydrate diet. Once hyperphagia has developed, by week 5 of life, brown adipose tissue is the only major lipogenic tissue in the obese mice not to exhibit elevated rates of fatty acid synthesis; this suggests that insulin resistance develops much more rapidly in brown adipose tissue than in other lipogenic tissues of the ob/ob mouse.     

Fatty acid synthesis in liver and adipose tissue of normal and genetically obese (ob/ob) mice during the 24-hour cycle.

1. The synthesis of long-chain fatty acids de novo was measured in the liver and in regions of adipose tissue in intact normal and genetically obses mice throughout the daily 24h cycle. 2. The total rate of synthesis, as measured by the rate of incorporation of 3H from 3H2O into fatty acid, was highest during the dark period, in liver and adipose tissue of lean or obese mice. 3. The rate of incorporation of 14C from [U-14C]glucose into fatty acid was also followed (in the same mice). The 14C/3H ratios were higher by a factor of 5-20 in parametrial and scapular fat than that in liver. This difference was less marked during the dark period (of maximum fatty acid synthesis). 4. In normal mice, the total rate of fatty acid synthesis in the liver was about twofold greater than that in all adipose tissue regions combined. 5. In obese mice, the rate of fatty acid synthesis was more rapid than in lean mice, in both liver and adipose tissue. Most of the extra lipogenesis occurred in adipose tissue. The extra hepatic fatty acids synthesized in obese mice were located in triglyceride rather than phospholipid. 6. In adipose tissue of normal mice, the rate of fatty acid synthesis was most rapid in the intra-abdominal areas and in brown fat. In obese mice, all regions exhibited rapid rates of fatty acid synthesis. 7. These results shed light on the relative significance of liver and adipose tissue (i.e. the adipose 'organ') in fatty acid synthesis in mice, on the mino importance of glucose in hepatic lipogenesis, and on the alterations in the rate of fatty acid synthesis in genetically obese mice.     

Desaturation of stearic acid by liver and adipose tissue from obese-hyperglycaemic mice (ob/ob).

Stearic acid desaturase activity was assayed in preparations from perigenital adipose tissue and liver from lean and genetically obese female mice (ob/ob). The total activity in the perigenital adipose tissue from obese mice was threefold greater than in the tissue from lean mice, but per g of adipose tissue the activity was twofold greater in tissue from lean mice. In liver, the activity in obese mice was elevated at 8 weeks of age, remained elevated up to 24 weeks and then decreased by half at 48 weeks, but at all ages was higher than that in lean mice. The decrease in desaturase activity of liver from obese mice at 48 weeks corresponded to a change in the fatty acid composition of liver lipids toward that found in lean mice. Whereas in adipose tissue much of the increased enzyme activity may be due to tissue hyperplasia, in liver it is mainly an increased activity per cell.     

Resistance to hepatic action of vasopressin in genetically obese (ob/ob) mice.

1. Fatty acid synthesis, measured in the perfused liver of genetically obese (ob/ob) mice with 3H2O or [14C]actate, did not show the inhibition by [8-arginine]vasopressin (antidiuretic hormone) that is observed in livers from normal mice. 2. Hepatic glycogen breakdown in obese mice was stimuulated by vasopressin, but not as extensively as in lean mice. 3. If obese mice received a restricted amount of food, then fatty acid synthesis still did not respond to vasopressin, but glycogen breakdown was fully stimulated. 4. Cholesterol synthesis was not inhibited by vasopressin in livers from obese mice. 5. Vasopressin inhibited fatty acid synthesis in intact lean mice, but not in obese animals. 6. These results suggest that genetic obesity could be due to an inborn error within the mechanisms (other than adenylate cyclase) which mediate responses to extracellular effectors.     

Carbohydrate metabolism of the isolated perfused liver of normal and genetically obese–hyperglycaemic (ob/ob) mice

1. A technique for perfusion of the mouse liver has been developed, and aspects of carbohydrate metabolism have been investigated in the perfused liver of normal and genetically obese mice, homozygous for the recessive gene ob. 2. Rates of gluconeogenesis in perfused mouse liver were faster than those reported for slices of mouse liver, particularly from lactate and pyruvate. 3. The rate of glycogen breakdown to glucose, but not to lactate, was faster in liver from fed obese mice. 4. The capacity for glycogen synthesis from glucose was enhanced in liver from 20h-starved obese mice. 5. The capacity for gluconeogenesis from a number of substrates was not significantly altered in livers from fed or starved obese mice when compared with that of lean mice. 6. These results suggest that the liver contributes to the hyperglycaemia of the obese mice by increased glycogenolysis, and that liver glycogen in obese mice is maintained by synthesis from dietary glucose.     

High-energy diets produce different effects on fatty acid synthesis in brown adipose tissue, white adipose tissue and liver in the rat

The influence of feeding rats a high-energy diet for 7 days on fatty acid synthesis in brown adipose tissue, white adipose tissue and liver of the rat was investigated. The incorporation of 3H2O and [U-14C]glucose into fatty acid was measured in vivo. The rats fed the high-energy diets had higher rates of fatty acid synthesis in white adipose tissue than the controls fed on chow, while fatty acid synthesis in brown adipose tissue and liver was either decreased or unchanged relative to that of controls fed on chow. After an oral load of [U-14C]glucose the incorporation of radioactivity into tissue fatty acid was several-fold higher in brown adipose tissue than in white adipose tissue in rats fed on chow. In rats fed the high-energy diets, incorporation of radioactivity into fatty acid in brown adipose tissue was decreased while that into white adipose tissue was either increased (Wistar rats) or unchanged (Lister rats).     

Continuous flow microcalorimetric measurement of heat production in white adipose tissue from obese (ob/ob) mice and their lean littermates

Heat production, free fatty acid and glycerol release from white adipose tissue fat pads from obese (ob/ob) mice and their lean littermates are determined. Heat production was significantly lower in obese mice compared to lean mice when expressed on wet weight basis but not when expressed on DNA basis. Noradrenaline significantly increased the heat production in fat pads from both groups of animals. However, the increase in heat production due to noradrenaline addition in fat pads from lean mice was significantly higher than in fat pads from obese mice. The release of free fatty acids and glycerol before incubation with noradrenaline was similar from fat pads from both groups of animals. Addition of noradrenaline to the fat pads increased the release of free fatty acids and glycerol in both groups of animals, but the increase was significantly larger from fat pads from lean mice. In the absence of noradrenaline the free fatty acid/glycerol ratio (mol/mol) in the effluent was 7.9:1 and 4.8:1 for lean mice and obese mice, respectively. In the presence of noradrenaline the ratio decreased to 3:1 for both groups of animals.     

The regulation of hepatic stearoyl-coenzyme A desaturase in obese-hyperglycaemic (ob/ob) mice by food intake and the fatty acid composition of the diet.

1. The effects of food intake and the fatty acid composition of the diet on the hepatic stearoyl-CoA desaturase activity of obese-hyperglycaemic (ob/ob) mice were investigated. 2. Obese mice fed on a commercial mouse diet, ad libitum, had 6.5-fold more activity per liver cell than had lean mice. 3. On a diet containing 14% corn oil the activity was 65% less in obese mice and 62% less in lean mice compared with animals fed on the commercial diet. 4. Feeding with 14% saturated fat in the diet doubled the activity in lean mice compared with those on the commercial diet, but had no effect on the activity in obese mice. 5. Obese mice fed on the corn-oil diet contained a higher proportion of linoleic acid in the liver lipids than did lean mice fed on the commercial diet, but the acyl-CoA desaturase activity was 125% higher than in the lean mice. 6. Limiting the food intake of obese mice by pair-feeding with lean mice decreased their acyl-CoA desaturase activity when the animals were fed on the saturated-fat diet, but the activity remained 75% higher than in lean mice, whereas in obese mice pair-fed on the corn-oil diet the activity was the same as in lean mice. 7. During starvation the acyl-CoA desaturase activity in livers from obese mice decreased more slowly and proportionately less than in livers from lean mice. 8. It is concluded that increased substrate supply as a result of hyperphagia and not low concentration of linoleic acid is the main factor causing high acyl-CoA desaturase activity in obese mice.     

Conversion of glucose, acetate and lactate to CO2 and fatty acids in liver and adipose tissue of prairie voles (Microtus ochrogaster)

Production of CO2 and fatty acids from acetate, glucose and lactate was determined in slices of liver and adipose tissue from prairie voles fed either a high-starch or a high-cellulose diet. Acetate and lactate were oxidized to CO2 and converted to fatty acids at greater rates than was glucose in both liver and adipose tissue. Fatty acid synthesis occurred at greater rates in adipose tissue than in liver. Fatty acid synthesis per adipocyte increased with increased adipocyte diameter. Fiber content of diets had only minimal effect on metabolic activities of liver and adipose tissue.     

Effects of insulin on lipolysis and lipogenesis in adipocytes from genetically obese (ob/ob) mice.

A method for the preparation of isolated adipocytes from obese mice is described. Similar yields of adipocytes (50--60%), as judged by several criteria, are obtained from obese mice and lean controls. Few fat-globules and no free nuclei were observed in cell preparations, which are metabolically active, respond to hormonal control and appear to be representative of intact adipose tissue. Noradrenaline-stimulated lipolysis was inhibited by insulin, equally in adipocytes from lean and obese mice. Inhibition in obese cells required exogenous glucose, and the insulin dose--response curve was shifted to the right. Basal lipogenesis from glucose was higher in adipocytes from obese mice, and the stimulatory effect of insulin was greater in cells from obese mice compared with lean controls. A rightward shift in the insulin dose--response curve was again observed with cells from obese animals. This suggests that adipose tissue from obese mice is insulin-sensitive at the high blood insulin concentrations found in vivo. The resistance of obese mice to the hypoglycaemic effect of exogenous insulin and their impaired tolerance to glucose loading appear to be associated with an impaired insulin response by muscle rather than by adipose tissue.     

Adrenalectomy increases norepinephrine turnover in brown adipose tissue of obese (ob/ob) mice

The hyperphagia and rapid body weight gain normally observed in young obese (ob/ob) mice were abolished by removal of their adrenal glands, whereas food intake and weight gain of lean mice were not significantly affected by adrenalectomy. Adrenalectomy lowered body energy density (kcal/g carcass) in obese mice more than could be attributed to reduced food intake per se, suggesting that their energy expenditure was also increased. In control obese mice, low stimulation of brown adipose tissue by the sympathetic nervous system, as indicated by the low fractional rates of norepinephrine (NE) turnover in their brown adipose tissue may have contributed to the reduced energy expenditure in these animals. Adrenalectomy increased the rates of NE turnover in brown adipose tissue of obese mice to rates nearly equal to those observed in lean mice without affecting NE turnover in this tissue of lean mice. Likewise, removal of the adrenals normalized the low rates of NE turnover in hearts of obese mice without affecting lean mice. Rates of NE turnover in two other organs, white adipose tissue and pancreas, of control and adrenalectomized obese mice were similar to rates observed in lean counterparts. The adrenal may thus contribute to both the hyperphagia and the low energy expenditure by brown adipose tissue that together cause gross obesity in ob/ob mice.     

Alterations in Oral [1-14C] 18:1n-9 Distribution in Lean Wild-Type and Genetically Obese (ob/ob) Mice

Obesity may result from altered fatty acid (FA) disposal. Altered FA distribution in obese individuals is poorly understood. Lean wild-type C57BL/6J and obese C57BL/6J^ob/ob mice received an oral dose of [1-¹⁴C]18:1n-9 (oleic acid), and the radioactivity in tissues was evaluated at various time points. The ¹⁴C concentration decreased rapidly in gastrointestinal tract but gradually increased and peaked at 96 h in adipose tissue, muscle and skin in lean mice. The ¹⁴C concentration was constant in adipose tissue and muscle of obese mice from 4h to 168h. ¹⁴C-label content in adipose tissue was significantly affected by genotype, whereas muscle ¹⁴C-label content was affected by genotype, time and the interaction between genotype and time. There was higher total ¹⁴C retention (47.7%) in obese mice than in lean mice (9.0%) at 168 h (P<0.05). The ¹⁴C concentrations in the soleus and gastrocnemius muscle were higher in obese mice than in lean mice (P<0.05). Perirenal adipose tissue contained the highest ¹⁴C content in lean mice, whereas subcutaneous adipose tissue (SAT) had the highest ¹⁴C content and accounted for the largest proportion of total radioactivity among fat depots in obese mice. More lipid radioactivity was recovered as TAG in SAT from obese mice than from lean mice (P<0.05). Gene expression suggested acyl CoA binding protein and fatty acid binding protein are important for FA distribution in adipose tissue and muscle. The FA distribution in major tissues was altered in ob/ob mice, perhaps contributing to obesity. Understanding the disparity in FA disposal between lean and obese mice may reveal novel targets for the treatment and prevention of obesity.     

The role of the cytoplasmic redox potential in the control of fatty acid synthesis from glucose, pyruvate and lactate in white adipose tissue

The metabolism of lactate, pyruvate and glucose was studied in epididymal adipose tissue of starved, normally fed and starved-re-fed rats. Lactate conversion into fatty acid occurred at an appreciable rate only in the adipocyte of starved-re-fed animals. NNN'N'-Tetramethyl-p-phenylenediamine, an agent that transports reducing power from the cytoplasm to the mitochondria, caused large increments of fatty acid synthesis from lactate and a smaller one from glucose but a decrease in that from pyruvate. Glucose (1.0mm) increased fatty acid synthesis from lactate 4.3-fold but only 1.67-fold from pyruvate in adipocytes from normally fed animals. 2-Deoxyglucose decreased fatty acid synthesis from lactate to a greater degree (threefold) compared to that from pyruvate in adipocytes from starved-re-fed animals. l-Glycerol 3-phosphate contents were approximately equal in epididymal fat-pads, incubated in the presence of lactate or pyruvate, from normally fed animals, whereas the addition of 1mm-glucose resulted in a tenfold increase in l-glycerol 3-phosphate content only in the presence of lactate. The l-glycerol 3-phosphate content was tenfold higher in adipose tissue from starved-re-fed animals incubated in the presence of lactate than in the presence of pyruvate. 2-Deoxyglucose caused these values to be slightly lowered in the presence of lactate. We suggest that lactate metabolism is limited by the rate of NADH removal from the cytoplasm. In the starved-re-fed state, this occurs by reduction of dihydroxyacetone phosphate formed from glycogen to produce l-glycerol 3-phosphate, thus permitting lactate conversion into fatty acid. When glucose is the substrate, and rates of transport are not limiting, the rate of removal of cytoplasmic NADH limits glucose conversion into fatty acid.     

Long-term effects of a fatty acid synthase inhibitor on obese mice: food intake, hypothalamic neuropeptides, and UCP3

Short-term treatment of lean and obese mice with the fatty acid synthase (FAS) inhibitor, C75, alters expression of hypothalamic neuropeptides thereby reducing food intake, body weight, and body fat. Here we report the long-term effects of C75 on obese (Ob/Ob) mice. A low dose of C75 administered every third day for 30 days reduced food intake by 62% and body weight by 43% whereas body weight of ad lib-fed controls increased by 11%. Loss of body weight correlated with decreased adipose and liver tissue mass. Decreased food intake correlated with decreased expression of hypothalamic neuropeptide mRNAs for NPY, AgRP, and MCH and an increased expression of neuropeptide mRNAs for alphaMSH (i.e., POMC) and CART. Consistent with increased energy expenditure, C75 treatment caused greater weight loss than pair-fed controls and increased expression of skeletal muscle UCP-3 mRNA. Lowered blood glucose was due largely to restriction of food intake. C75 blocked the normal fasting-induced rise in blood free fatty acids and ketones due either to decreased adipose tissue lipolysis and hepatic ketogenesis or increased fatty acid and ketone utilization by peripheral tissues, notably skeletal muscle.     

Regulation of fructose-2,6-bisphosphate and glycogen synthesis by dichloroacetate and phenazine methosulphate in rat adipose tissue

The effects of dichloroacetate and phenazine methosulphate on the content of fructose-2,6-bisphosphate and glycogenesis in incubated epididymal adipose tissue were examined. Both agents stimulated the synthesis of fructose-2,6-bisphosphate in the presence of glucose, the effect being higher in tissue from fasted-refed rats than in normal fed rats. Additions of dichloroacetate to the incubation medium also increased the incorporation of [U-14C]glucose into glycogen and this effect was additive with that of insulin. However phenazine methosulphate strongly depressed the insulin-dependent glycogen synthesis. These data are considered in relation to the increased rate of glucose metabolism known to occur in the presence of dichloroacetate and the stimulation of pentose phosphate pathway with phenazine methosulphate.     

Changes in the lipogenic response to feeding of liver, white adipose tissue and brown adipose tissue during the development of obesity in the gold-thioglucose-injected mouse.

Lipogenic response to feeding was measured in vivo in liver, epididymal white adipose tissue (WAT) and interscapular brown adipose tissue (BAT), during the development of obesity in gold-thioglucose (GTG)-injected mice. The fatty acid synthesis after a meal was higher in all tissues of GTG-treated mice on a total-tissue basis, but the magnitude of this increase varied, depending on the tissue and the time after the initiation of obesity. Lipogenesis in BAT from GTG mice was double that of control mice for the first 2 weeks, but subsequently decreased to near control values. In WAT, lipogenesis after feeding was highest 2-4 weeks after GTG injection, and in liver, lipid synthesis in fed obese mice was greatest at 7-12 weeks after the induction of obesity. The post-prandial insulin concentration was increased after 2 weeks of obesity, and serum glucose concentration was higher in fed obese mice after 4 weeks. These results indicate that increased lipogenesis in GTG-injected mice may be due to an increase in insulin concentration after feeding and that insulin resistance (assessed by lipogenic response to insulin release) is apparent in BAT before WAT and liver.     

Studies on lipogenesis in vivo: Fatty acid and cholesterol synthesis in hyperglycaemic-obese mice

1. Lipogenesis has been studied in intact genetically obese mice by measuring the incorporation of a single oral dose of 250mg. of [U-¹⁴C]glucose into fatty acid and cholesterol in the liver and extrahepatic tissues. Studies were also carried out with [U-¹⁴C]glucose added to the diet and fed for 24hr. With either method of isotope administration, the conversion of [U-¹⁴C]glucose into fatty acid was greatly elevated in the livers of the obese mice. In contrast, conversion of the single dose of [¹⁴C]glucose into fatty acid in extrahepatic tissues of obese mice was only half that occurring in the non-obese litter mates. When [¹⁴C]glucose was given in the diet for 24hr. the total accumulation of labelled fatty acid in extrahepatic tissues of obese mice was slightly less than in the non-obese. Uptake of labelled glucose and conversion into fatty acid in adipose tissue of the obese mice decreased with age. 2. Conversion of the single dose of [¹⁴C]glucose into liver cholesterol was comparable in obese and non-obese mice fed on a purified low-fat diet. However, obese mice given this diet for 12 weeks accumulated 1·54% of cholesterol in the liver compared with 0·29% in the non-obese litter mates. This accumulation apparently resulted from a decrease in removal of cholesterol from the liver, rather than an increased synthesis. 3. Conversion of the single dose of [¹⁴C]glucose into extrahepatic fatty acid was decreased by 18hr. starvation proportionally as much in obese as in non-obese mice. The decrease in liver fatty acid synthesis caused by starvation also was considerable in obese mice, although somewhat less marked than in the non-obese. 4. The metabolic derangements in the liver could be more fundamental to the development of the obesity than the changes seen in extrahepatic tissues.     

Extracellular superoxide dismutase in tissues from obese (ob/ob) mice

We have examined the protein content and gene expression of three superoxide dismutase (SOD) isoenzymes in eight tissues from obese ob/ob mice, particularly placing the focus on extracellular-SOD (EC-SOD) in the white adipose tissue (WAT). Obesity significantly increased EC-SOD level in liver, kidney, testis, gastrocnemius muscle, WAT, brown adipose tissue (BAT), and plasma, but significantly decreased the isoenzyme level in lung. Tumor necrosis factor-alpha and interleukin-1beta contents in WAT were significantly higher in obese mice than in lean control mice. Immunohistochemically, both WAT and BAT from obese mice could be stained deeply with anti-mouse EC-SOD antibody compared with those from lean mice. Each primary culture per se almost time-dependently enhanced EC-SOD production, and overtly expressed its mRNA. The loss of heparin-binding affinity of EC-SOD type C with high affinity for heparin occurred in kidney of obese mice. These results suggest that the physiological importance of this SOD isoenzyme in WAT may be a compensatory adaptation to oxidative stress.     

Maternal Obesity Reduces Milk Lipid Production in Lactating Mice by Inhibiting Acetyl-CoA Carboxylase and Impairing Fatty Acid Synthesis

Maternal metabolic and nutrient trafficking adaptations to lactation differ among lean and obese mice fed a high fat (HF) diet. Obesity is thought to impair milk lipid production, in part, by decreasing trafficking of dietary and de novo synthesized lipids to the mammary gland. Here, we report that de novo lipogenesis regulatory mechanisms are disrupted in mammary glands of lactating HF-fed obese (HF-Ob) mice. HF feeding decreased the total levels of acetyl-CoA carboxylase-1 (ACC), and this effect was exacerbated in obese mice. The relative levels of phosphorylated (inactive) ACC, were elevated in the epithelium, and decreased in the adipose stroma, of mammary tissue from HF-Ob mice compared to those of HF-fed lean (HF-Ln) mice. Mammary gland levels of AMP-activated protein kinase (AMPK), which catalyzes formation of inactive ACC, were also selectively elevated in mammary glands of HF-Ob relative to HF-Ln dams or to low fat fed dams. These responses correlated with evidence of increased lipid retention in mammary adipose, and decreased lipid levels in mammary epithelial cells, of HF-Ob dams. Collectively, our data suggests that maternal obesity impairs milk lipid production, in part, by disrupting the balance of de novo lipid synthesis in the epithelial and adipose stromal compartments of mammary tissue through processes that appear to be related to increased mammary gland AMPK activity, ACC inhibition, and decreased fatty acid synthesis.     

Adenylate cyclase activity in brown adipose tissue of the genetically obese (ob/ob) mouse

The activation of brown adipose tissue adenylate cyclase by catecholamines was studied in genetically obese (ob/ob) and lean mice. In obese mice, the maximum activation of the enzyme by several beta-adrenergic agonists was only two-thirds that in lean mice and, as an activator, noradrenaline was only one-eighth as potent. The adenylate cyclase was also less responsive to guanine nucleotides. In these respects, the defect in catecholamine-stimulated adenylate cyclase was similar in both white and brown adipose tissue of the obese mouse. The enzyme in brown adipose tissue differed from that in white adipose tissue in its sensitivity to other beta-adrenergic agonists and in its requirement for Mg2+. It is suggested that this abnormal catecholamine-activated adenylate cyclase in brown adipose tissue may be relate to the thermoregulatory defect of the obese mouse and hence may contribute to the obesity syndrome.