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.     

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.     

The actions of dichloroacetic acid on blood glucose, liver glycogen and fatty acid synthesis in obese-hyperglycaemic (ob/ob) and lean mice

Obese-hyperglycaemic mice and lean mice were injected with dichloroacetate to determine the significance of gluconeogenesis in maintaining the hyperglycaemia of obese mice and to investigate the effects of a fall in blood glucose on fatty acid synthesis. One hour after the second of two, hourly, injections of dichloroacetate the blood glucose concentrations in fed and starved lean mice were decreased, whereas in obese mice they were sharply increased. In obese and lean mice, both fed and starved, dichloroacetate decreased plasma lactate but insulin was unchanged. The quantity of liver glycogen was decreased in all dichloroacetate treated mice, with the largest falls in fed and starved obese mice, which had much larger glycogen stores than lean mice. Dichloroacetate treatment decreased the concentration of plasma non-esterified fatty acids in fed and starved obese mice and fed lean mice but not in starved lean mice. Fatty acid synthesis in white (inguinal, subcutaneous) adipose tissue was stimulated by dichloroacetate in fed obese mice and inhibited in fed lean mice. Fatty acid synthesis in brown adipose tissue (scapular) was faster than in white adipose tissue and was less affected by dichloroacetate although the changes were in the same direction as in white adipose tissue. We attribute the increased hyperglycaemia of obese mice treated with dichloroacetate to increased glycogenolysis coupled with a failure to secrete additional insulin in response to the raised blood glucose. This high blood glucose concentration in dichloroacetate treated obese mice may in turn explain the increased fatty acid synthesis in their white adipose tissue.     

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.     

Serum hormone levels and tissue metabolism in pair-fed lean and obese Zucker rats.

Obese Zucker rats were either pair-fed to their lean litter-mates or fed ad lib, to determine the effect of hyperphagia on serum hormone levels and tissue metabolism as indicated by enzyme activities and in vitro metabolite flux. Hyperphagia was shown to be non-essential for the elevation in serum insulin and suppression in serum growth hormone and prolactin in the genetically obese rat. It was also shown that the increased liver cell lipogenic rate was not dependent on hyperphagia in the obese rat and that adipose cell lipogenesis was not significantly altered in the pair-fed obese rat. The utilization of alanine for glucose synthesis in vitro was similar for both lean and obese rats, but its utilization for fatty acid synthesis was higher in the obese rat. Data is presented which suggest that the inhibitory effect of glucagon on liver lipogenesis is blunted in the obese rat.     

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.     

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.     

A comparison of the enzyme levels and the in vitro utilization of various substrates for lipogenesis in pair-fed lean and obese pigs.

In this study of spontaneous obesity of pigs, specific metabolic shifts were observed, which explain an increase in fat deposition. Liver tissue utilization of pyruvate and glucose for oxidation and lipogenesis showed no significant difference between lean and obese pigs. Adipose tissue utilization of glucose, acetate and glycerol for triglyceride and fatty acid synthesis was greater in obese pigs than lean pigs (P less than 0.01). No significant difference in leucine incorporation into lipid fractions was found. Of the substrates utilized, glucose supplied 86 and 94% of the glyceride-glycerol synthesized in lean and obese pigs, respectively. Glycerol was not a major contributor to glyceride-glycerol synthesis (3.5 to 5.5%), in spite of the presence of adipose tissue glycerokinase. An increase (P less than 0.05) in alanine incorporation into glucose was observed in liver tissue from obese pigs. In general, the levels of enzymes activities associated with gluconeogenesis, glycolysis, and lipogenesis supported the findings of in vitro utilization of these substrates.     

Development of fatty acid-synthetic capacity in interscapular brown adipose tissue during suckling in genetically obese Zucker rats.

The development of the lipogenic capacity in brown adipose tissue was studied in suckling lean (Fa/fa) and obese (fa/fa) Zucker pups aged from 7 to 22 days. In both lean and obese pups, activities of the two key lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase, and of citrate cleavage enzyme rose from the early to the late suckling period. Compared with lean pups, 7-day-old fa/fa pups showed a 35% increase in fat accumulation in interscapular brown adipose tissue and a 25% increase in fatty acid synthetase activity. By 10 days of age, fat deposition, lipogenesis in vivo (assessed by the incorporation of 3H from 3H2O into fatty acids) and fatty acid synthetase activity were 1.5-2-fold higher in pre-obese than in lean pups. Compared with lean pups, the increased lipogenesis in vivo observed in brown adipose tissue of 10-day-old pre-obese pups could not entirely account for the difference in fat deposition observed in this tissue, suggesting that additional mechanisms are at play to explain the increased fat content of this tissue.     

Dissociation between adipose tissue fluxes and lipogenic gene expression in ob/ob mice

Recent evidence has been presented that expression of lipogenic genes is downregulated in adipose tissue of ob/ob mice as well as in human obesity, suggesting a functionally lipoatrophic state. Using (2)H(2)O labeling, we measured three adipose tissue biosynthetic processes concurrently: triglyceride (TG) synthesis, palmitate de novo lipogenesis (DNL), and cell proliferation (adipogenesis). To determine the effect of the ob/ob mutation (leptin deficiency) on these parameters, adipose dynamics were compared in ob/ob, leptin-treated ob/ob, food-restricted ob/ob, and lean control mice. Adipose tissue fluxes for TG synthesis, de novo lipogenesis (DNL), and adipogenesis were dramatically increased in ob/ob mice compared with lean controls. Low-dose leptin treatment (2 microg/day) via miniosmotic pump suppressed all fluxes to control levels or below. Food restriction in ob/ob mice only modestly reduced DNL, with no change in TG synthesis or adipogenesis. Measurement of mRNA levels in age-matched ob/ob mice showed generally normal expression levels for most of the selected lipid anabolic genes, and leptin treatment had, with few exceptions, only modest effects on their expression. We conclude that leptin deficiency per se results in marked elevations in flux through diverse lipid anabolic pathways in adipose tissue (DNL, TG synthesis, and cell proliferation), independent of food intake, but that gene expression fails to reflect these changes in flux.     

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.     

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.     

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.     

Genetically obese C57BL/6 ob/ob mice respond normally to sympathomimetic compounds

The suggestion that defective thermoregulatory thermogenesis in the genetically obese (ob/ob) mouse is due to a low thermic response to noradrenaline has been investigated using both noradrenaline and the longer-acting sympathomimetic compounds, ephedrine and BRL 26830A. Below thermoneutrality (23.5°C) the metabolic rate of obese mice was lower than that of their lean littermates, whereas at a thermoneutral temperature (31°C) the metabolic rate of the obese nice was as high as that of lean mice. This confirms the view that the ob/ob mouse has defective thermoregulatory thermogenesis. However, in C57BL/6 mice, this defect is not due to a failure to respond to noradrenaline, because at 31°C the maximum thermic effects of noradrenaline, ephedrine and BRL 26830A were as high in obese as in lean mice and at 23.5°C they were higher in obese than in lean mice. Furthermore, the response of brown adipose tissue to β-adrenoceptor stimulation appears normal since noradrenaline caused a normal rise in brown adipose tissue temperature, and treatment with noradrenaline or BRL 26830A $\text{in}$$\text{vivo}$ caused a normal increase in GDP binding by brown adipose tissue mtiochondria. At 31°C propranolol depressed metabolic rate equally in lean and obese C57BL/6 mice, whereas at 23.5°C it depressed metabolic rate more in lean than obese mice. In contrast to C57BL/6 mice, Aston ob/ob mice showed a reduced thermic response to noradrenaline. These results suggest that defective thermoregulatory thermogenesis in the ob/ob mouse is primarily due to a reduced ability to raise sympathetic tone, but in some strains an additional failure in the thermic response to noradrenaline may develop.     

Pyruvate dehydrogenase activity in several tissues of genetically obese hyperglycemic mice

The active form (PDHa) and total activity of pyruvate dehydrogenase (PDH) were measured in homogenates from heart muscle, epididymal fat pads and liver of genetically obese hyperglycemic mice and compared with similar data derived from lean controls or Swiss albino mice. Both PDHa and total PDH activities were similar in heart muscle from all mice with a precipitous decrease in the PDHa upon fasting. Adipose tissue and liver of obese mice had a PDHa level that was almost two-fold higher than either lean control or Swiss albino mice. Fasting for 24 hours decreased the elevated activity of PDHa in adipose tissue and liver in obese mice to a value that was comparable to lean control or Swiss albino mice, fasted similarly. The elevation in both the active form and total activity of pyruvate dehydrogenase in livers from obese mice could explain the increased provision of acetyl-CoA units necessary for the accelerated hepatic lipogenesis observed with this mouse, a model for human obesity and insulin resistance.     

Coordinated stimulation by triiodothyronine of fatty acid synthesis and isoproterenol-sensitive fatty acid release in two preadipocyte cell lines of lean or genetically obese mice

The development and thyroid hormone sensitivity of fatty acid and triacylglycerol synthesis from 14C-acetate and of the isoproterenol-sensitive fatty acid release, were studied in two preadipocyte cell lines during the adipose differentiation: the Ob 17 and the HGFu cell lines cloned from the periepididymal adipose tissue of adult mice genetically obese and phenotypically lean respectively. Both parameters increased and peaked in the same time-period during the second week of culture after confluence. Both parameters were also amplified when T3 was added to the culture medium at confluence. The increment due to T3 was concentration dependent: it peaked at the physiological concentration of 1.5 nM and declined thereafter with the same pattern. This shows that some steps of two opposite pathways of lipid metabolism in differentiating preadipose cells can be stimulated by triiodothyronine in a similar manner and suggests a coordinated regulation. No significant difference could be detected between cells from lean or genetically obese mice.     

Plasma lipoproteins and the synthesis and turnover of plasma triglyceride in normal and genetically obese mice

1. Lipoproteins in the plasma of mice were characterized by agarose-gel chromatography and polyacrylamide-gel electrophoresis: genetically obese (ob/ob) mice exhibited hyperlipoproteinaemia (compared with lean mice), largely owing to an increase in the concentration of cholesterol in high-density lipoprotein. Plasma concentrations of triglyceride and phospholipid were not markedly increased in genetically obese mice. 2. The formation of glycerolipids in liver and plasma was investigated with (14)C-labelled precursors. The synthesis of hepatic triglyceride and phospholipid from glucose or palmitate was enhanced in ob/ob mice, compared with lean mice. The rate of entry of triglyceride into plasma, calculated from the time-course of incorporation of (14)C from [(14)C]palmitate into plasma triglyceride, was increased in ob/ob mice (0.5mumol of fatty acid/min, compared with 0.2 in lean mice). 3. The removal from plasma of murine lipoprotein triglyceride-[(14)C]fatty acid was increased in ob/ob mice (half-time 2.2min, compared with 7.2min in lean mice). Similar results were obtained with an injected lipid emulsion (Intralipid). 4. From these measurements, estimates of the rates of turnover of plasma triglyceride in mice (fed on a mixed diet, female, 3 months old) are about 1.0mumol of fatty acid/min in ob/ob mice, and 0.25 in lean mice. 5. The major precursor of hepatic and plasma triglyceride in lean and ob/ob mice was calculated to be plasma free fatty acid. 6. These results are discussed, in connexion with the role of the liver in triglyceride metabolism in mice, especially in relation to genetic obesity.     

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.     

Early development of adipose cell lipogenesis and glycerol utilization in Zucker obese rats.

A study of adipose cell metabolism was made at ages 5, 7, 10, and 14 wk of age in genetically obese Zucker rats. Adipose samples were surgically removed and used for in vitro adipose cell incubations and for characterization of enzyme patterns. Lipogenic capacity from glucose and enzymes normally associated with lipogenesis (malic enzyme, citrate cleavage enzyme and glucose-6-PO4 dehydrogenase) followed the same pattern of development. At 5 wk of age, the adipose cells of obese animals had a greater capacity for fat synthesis than the lean rats. At all other ages lipogenic activity and enzyme levels were either similar or less than the pair-fed lean littermates. Glycerol utilization by isolated fat cells was similar; however, adipose tissue glycerokinase was elevated in obese rats at 14 wk of age. It was concluded that there was no apparent change in specific lipogenic capacity of fat cells from the obese rat when compared to its lean littermate. It was also concluded that increased adipose glycerokinase activity in obese rats represented a secondary shift in metabolism.     

In vivo lipogenesis in genetically fat and lean chickens of various ages

Lipogenesis was studied in vivo in genetically fat and lean male chickens at 2, 5 and 15 weeks of age using tritiated water. At 2 and 5 weeks of age, the liver was the major site of de novo fatty acid synthesis rather than the carcass. Per 100 g of tissue lipogenesis was greater in the carcass at 15 weeks of age than at 2 or 5 weeks. Lipogenesis in the liver did not vary according to age. At 15 weeks of age there was no observable exaggeration in the degree of fattening (as in genetically obese rodents) of fat line chickens. The regulation of fattening in fat line chickens, at least at the onset of sexual maturity, appears to be different to that in genetically obese rodents.