Cold acclimation of obese (ob/ob) mice: effects on skeletal muscle and bone

Obese (ob/ob) and lean mice at 4 weeks of age were housed at 23 degrees C or 14 degrees C for 4 or 8 weeks to determine effects of acclimation to mild cold on the growth of skeletal muscle, bone, and fat. Body weights at 12 weeks of age averaged 48 +/- 0.6 g and 27 +/- 1.9 g for obese mice housed at 23 degrees C and 14 degrees C and 29 +/- 0.5 g and 26 +/- 0.6 g and 26 +/- 0.6 g for lean mice housed at 23 degrees C and 14 degrees C, respectively. At 23 degrees C, muscle weights of obese mice were approximately 60% of those in lean mice. Muscles of obese mice did not grow during the first 4 weeks at 14 degrees C (4 to 8 weeks of age) but did show a small gain during the second 4 weeks (9 to 12 weeks of age) at 14 degrees C. As a result, by the end of 8 weeks at 14 degrees C, muscles of obese mice weighed only 35% to 45% as much as muscles of lean mice. Growth of the tibia and femur followed the same pattern as the muscles. Obese mice housed at 23 degrees C from 4 to 12 weeks of age contained about six times as much fat as lean mice at this age. Although exposure to 14 degrees C for 8 weeks depressed the accumulation of fat in obese mice, they still contained approximately three times the percentage body fat as lean counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a warm environment on energy balance in obese (ob/ob) mice

Low rates of thermoregulatory heat production associated with low metabolic activity of brown adipose tissue, the primary site of thermoregulatory heat production, contribute substantially to the high efficiency of energy retention in obese (ob/ob) mice housed at 20 degrees C to 28 degrees C. To eliminate the need for thermoregulatory heat production lean and ob/ob mice were housed at 34.5 degrees C. At this temperature ob/ob mice still retained energy with a greater efficiency than lean littermates. Next, we investigated the possibility that the high efficiency of energy retention in ob/ob mice housed at 34.5 degrees C was related to depressed dietary-induced thermogenesis associated with low metabolic activity of brown adipose tissue. The sympathetic nervous system is a primary regulator of brown adipose tissue metabolism. Therefore, rates of norepinephrine (NE) turnover in brown adipose tissue, as an indicator of sympathetic nervous system stimulation of the tissue, were measured. Lean and ob/ob mice housed at 34.5 degrees C had equally low rates of NE turnover in their brown adipose tissue. Thus, the high efficiency of energy retention in ob/ob mice maintained at 34.5 degrees C is caused by factors other than low sympathetic stimulation of brown adipose tissue.     

Functional correlates of Na+,K+-ATPase in lean and obese (ob/ob) mice

Concentrations of Na+,K+-ATPase enzyme units are lower in skeletal muscle and liver of adult obese (ob/ob) mice than in their lean counterparts. The present studies were designed to provide information on functional correlates of Na+,K+-ATPase in ob/ob mice. Obese mice had lower potassium (K+) content in muscle and liver and higher sodium (Na+) content in muscle and liver and higher sodium (Na+) content in muscle than lean counterparts. The calculated intracellular Na+/K+ ratio in muscle of obese mice was approximately twice as high as in muscle of lean mice. Oxygen consumption was measured in mice maintained at 14 degrees, 25 degrees, or 33 degrees for 40 min and injected with 0.3 or 0.9 microgram ouabain per g body weight. Ouabain, a specific inhibitor of Na+,K+-ATPase, decreased oxygen consumption less in obese mice (12%--25%) than in lean mice (19%--38%). These results suggest that Na+ pump activity may be reduced in obese mice.     

Skeletal muscle accretion and turnover in lean and obese (ob/ob) mice.

Skeletal muscle growth in obese (ob/ob) and lean mice was estimated from changes in fat-free carcass weight during development. No differences were observed at 2 wk of age, but fat-free carcasses of obese mice weighed less than those of lean mice at 5 wk of age. Fat-free carcasses of the adult obese mice were only 78% as heavy as those of lean mice, although the obese mice weighed up to twice as much as the lean mice. Even though obese mice accumulated less muscle than lean mice, urinary creatinine output of the obese mice was equal to that of the lean mice. The amount of 3-methylhistidine (3MH) in the carcasses of the mice and urinary output of 3MH were used to calculate the fractional breakdown rate (FBR) of myofibrillar proteins. Accumulation of 3MH in the carcasses paralleled carcass weight gain; thus, obese mice accumulated less 3MH than lean mice. Urinary output of 3MH was measured from 5 to 37 wk of age and was as great in the obese as in the lean mice; consequently, the FBR of the myofibrillar proteins was faster in the obese than in the lean mice. The FBR averaged 8.5 ± 0.6%/day for obese mice and 5.0 ± 0.1%/day for lean mice. The rate of 3MH accumulation in the carcass and the FBR were subsequently utilized to calculate a fractional synthesis rate (FSR) for the myofibrillar proteins. The FSR was not reduced in obese mice. The results suggest that obese mice accumulate less muscle because the rate of muscle degradation is increased.     

Effects of thyroxine on Na+,K+-ATPase and norepinephrine turnover in brown adipose tissue of obese (ob/ob) mice

Thyroxine treatment improves some of the defective thermogenic properties of obese (ob/ob) mice. Because brown adipose tissue (BAT) is an important thermogenic organ in mice, effects of thyroxine treatment on Na+, K+-ATPase, a thyroid-hormone responsive enzyme, and on rates of norepinephrine (NE) turnover, an indicator of sympathetic nervous system activity, in BAT of lean and obese mice were evaluated. Female mice, six weeks old, were injected with approximately 4 micrograms thyroxine daily for 2 weeks. Numbers of Na+, K+-ATPase enzyme units in BAT were similar in control lean and obese mice. Thyroxine treatment increased numbers of Na+, K+-ATPase enzyme units by 60% and 100% in lean and obese mice, respectively, indicating that the BAT of obese mice was responsive to thyroxine treatment. Fractional rates of NE turnover were 75% faster in BAT of control lean mice than in obese mice. Thyroxine treatment decreased functional rates of NE turnover in BAT of lean mice by 35%, but had no effect on NE turnover in BAT of obese mice. Rates of NE turnover in heart and pancreas of control lean and obese mice were unaffected by phenotype. Although the decreases in fractional rates of NE turnover in heart (-23%) and pancreas (-11%) of lean mice in response to thyroxine injections were not statistically significant, the calculated rates of NE turnover (fractional rate of NE turnover times the NE content of the organ) in these organs of lean mice were decreased 25% to 30% (P less than 0.05) in response to thyroxine. Thyroxine injections did not affect NE turnover in either heart or pancreas of obese mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dietary carbohydrate, fat, and protein on norepinephrine turnover in rats

To investigate effects of diet composition on rates of norepinephrine (NE) turnover in sympathetically innervated organs, weaning rats were fed for 2 to 21/2 weeks diets varying in carbohydrate (74.2% to 7.4% of total metabolizable energy) and fat (5.2% to 72.0%), or diets varying in protein (9.9% to 39.6%) and carbohydrate (77.8% to 48.1%). Changing the proportions of carbohydrate and fat in the diet, while maintaining similar intakes of energy and all other essential nutrients did not affect rates of NE turnover in heart, white adipose tissue (WAT), liver or pancreas and only minimally affected NE turnover in interscapular brown adipose tissue (IBAT). Decreasing the proportion of protein in the diet from 39.6% to 9.9% accelerated rats of NE turnover in heart (52%), IBAT (20%), WAT (42%), and liver (37%). When rats fed a diet containing 19.8% protein were also given a 10%(wt/vol) sucrose solution to drink for three days, their rates of NE turnover increased in heart (45%), IBAT (17%), liver (71%), and pancreas (55%). This response to sucrose depended on the protein content of the diet, since rats fed a 9.9% protein diet in which rates of NE turnover was already accelerated had no further increase in NE turnover when given the sucrose solution to drink. These data demonstrate that diet composition can affect activity of the sympathetic nervous system, as indicated by changes in rates of NE turnover. Changing the proportion of protein in the diet was more effective in altering NE turnover than changing the proportion of carbohydrate or fat.     

Formation of β-hydroxyisovalerate by an α-ketoisocaproate oxygenase in human liver

Previously we demonstrated the occurrence of a soluble dioxygenase in rat liver which converts α-ketoisocaproic acid (the keto acid analog of leucine) to β-hydroxyisovaleric acid. Herein we show that human liver contains a similar soluble enzyme which converts α-ketoisocaproate to β-hydroxisovaleric acid. We suggest this enzyme functions as a “safety valve” in liver to help prevent excessive accumulation of α-ketoisocaproate.     

Comparisons of prostaglandin vasoactive effects and interactions in the in vivo microcirculation of the rat urinary bladder

A combination of techniques for in vivo transillumination, topical application of vasoactive agents, and direct microscopic observation of microcirculatory responses was utilized to evaluate the vasomotor actions of prostaglandins (PGs) E₁, E₂, F_1α, F_2α, A₁, and A₂ on rat urinary bladder arterioles and venules. The effects of PGE₁ and histamine (HIS) on arteriolar responsiveness to norepinephrine (NE), serotonin (5-HT), and PGF_2α were measured. Histochemical studies were completed to determine the primary site of prostaglandin (PG) metabolic deactivation in the urinary bladder. Arteriolar dilatation occurred with HIS, PGE₁, PGE₂, PGA₁, PGA₂, and PGF_1α, all of which (with the exception of HIS) demonstrated significant dose-related responses. Overall, PGE₁ and PGE₂ were of greatest potency. Significant dose-related arteriolar constriction occurred with NE > PGF_2α > 5-HT (in order of decreasing potency). HIS, PGE₁, PGE₂, and PGA₁ produced significant venular dilatation; PGE₁ and HIS were dose related. Only NE resulted in significant venoconstriction. Arteriolar responsiveness to NE and PGF_2α decreased after pretreatment with PGE₁ but was unchanged by HIS pretreatment, whereas application of 5-HT following pretreatment with PGE₁ or HIS produced equivalent levels of arteriolar constriction. The primary site of deactivation of PGE₁ was histochemically localized to bundles of smooth muscle fibers in the muscular coat of the rat urinary bladder wall.     

Effect of adrenalectomy on the metabolism of glucose in obese (C57 B1/6J ob/ob) mice

The genetically obese mouse, C57 B1/6J ob/ob, has been suggested as an appropriate model for the study of obesity associated with diabetes mellitus. Employing glucose ¹⁴C(μl) as a tracer, the data presented here indicate that obese mice are able to clear glucose from the blood compartment at the same rate as their lean littermates. This was demonstrated with or without an associated cold glucose load. The abnormal glucose tolerance curves observed in the obese animals may be a result of secretion of glucose into the blood. Removal of the adrenal glands from the obese mice and their lean littermate does not impair their ability to clear a glucose load from the vascular compartment. The capacity for endogenous glucose secretion of ob/ob mice is severely curtailed by adrenalectomy, in that the glucose tolerance curves of these adrenalectomized animals become similar to those of sham-operated lean littermates. Thus, it appears that a considerable component of the hyperglycemia in ob/ob mice reflects major adrenal involvement that is activated by stress, ie, ether anesthesia and blood sampling. The hyperglycemia in ob/ob mice may reflect glucocorticoid-dependent gluconeogenesis.     

Effects of diphenylhydantoin on coronary resistance and myocardial contractile force before and after ouabain

These studies suggest that the direct local effect of DPH is to relax coronary smooth muscle and depress myocardial muscle. Further, the results indicate that neither the vascular nor myocardial effects of DPH are mediated through a stimulating action of the drug on the cell membrane Na⁺, K⁺ ATPase. In contrast, the studies support the hypothesis that the vasodilator but not the cardiac depressant action of potassium results from a stimulating action of this ion on the sarcolemmal sodium-potassium pump.     

Na+ pump activity and nuclear T3 receptors in tissues of genetically obese (ob/ob) mice.

A dramatic reduction in ouabain-sensitive tissue respiration of obese mouse muscle and liver was observed, suggesting that Na+-transport-dependent calorigenesis is impaired in these animals. Additionally, a significantly depressed nuclear triiodothyronine binding capacity in liver and lung tissue was exhibited by obese mice. These data support the suggestion that the hypometabolism and hypothermia of the genetically obese mouse is a result of reduced Na+-pump-related thermogenesis; and further, provides evidence that this may be the consequence of reduced nuclear binding of triiodothyronine.     

The effect of different types of anesthesia on digitalis toxicity

This investigation was undertaken in order to determine whether or not the type of anesthetic agent used modified the cardiotoxicity of either ouabain or acetylstrophanthidin. Ouabain toxicity was greater in animals anesthetized with chloralose and urethane than it was in animals anesthetized with pentobarbital. The toxicity of acetylstrophanthidin was the same under both types of anesthesia. It is suggested that these results can be explained by an interplay between differences in the pharmacological actions of the digitaloid substances studied and differences in the pharmacological actions of the anesthetics under question.The results of this study are significant for two reasons. They reinforce the importance of carefully choosing an anesthetic agent(s). All anesthetics do not produce identical pharmacological actions. Drugs that produce anesthesia can modify an animal's response to other drugs. There is a need for more information on the interaction between anesthetics and other drugs. This has clinical as well as experimental applicability. The differences between anesthetic agents with regard to interactions with catecholamines have been long recognized.^26,27 However, there appears to be little information on such differences between anesthetics with regard to other drugs.This study also points out the need for further study of the specific pharmacological actions of different digitaloid substances. It appears as though different cardenolides may have different potentials for increasing sympathetic activity. With further study, other differences may be found. The existence of differences in the pharmacological actions of different cardiac glycosides should increase the chances of finding a therapeutically useful cardioactive steroid that produces less toxicity than the agents that are currently in use. For example, the finding of a difference between digitaloid substances in ability to increase sympathetic outflow would improve the possibility of the development of a cardiotonic digitaloid substance that has little ability to increase sympathetic outflow. The new compound should have less potential for producing cardiac arrhythmias. Such a substance might be expected to be very useful clinically because the margin of safety should be larger than that of a similar compound that produces a significant increase in sympathetic outflow in addition to its direct positive inotropic effects.     

Postweaning development of diabetes in ob/ob mice.

In association with moderate diabetes, fully mature obese mice ($\text{C57BL}\text{6J}\text{ob}\text{ob}$) display several disturbances of metabolism, behavior, and growth. As yet, no precise developmental relationships have been established between the major abnormalities of these mice, although obesity, hypometabolism, and reduced activity have been reported as early as 7 days of age and appear to precede the development of diabetes. In the present studies, examination of glucose regulation revealed that preweanling obese mice, which are already characterized by many metabolic and growth deficits, had significantly lower fasting glucose levels when compared with lean controls, and displayed subnormal glycemia in response to glucose or control stimulation. In contrast, 28-day-old mutant mice had mildly elevated fasting glucose levels and maintained severe hyperglycemia after either glucose or distilled water administration. In response to exogenous insulin (1 U/kg), obese mice at each age displayed significant reductions of serum glucose, but appeared to be mildly insulin-insensitive when their glucose responses were compared with those of lean mice. The present results suggest that several factors are likely to be responsible for the rapid postweaning development of diabetes in $\text{ob}\text{ob}$ mice, including insulin resistance, dietary changes, and, possibly, an increased capacity for diabetogenic hormone production; however, the appearance of diabetes substantially later than several other metabolic disturbances indicates that abnormal glycoregulation is a relatively indirect consequence of the $\text{ob}\text{ob}$ mutation.     

The effects of a high-carbohydrate low-fat cholesterol-restricted diet on plasma lipid, lipoprotein, and apoprotein concentrations in insulin-dependent (type I) diabetes mellitus

Six women with well-defined insulin-dependent diabetes mellitus (IDDM) were studied for 4 weeks during a control diet containing 45% of the calories as carbohydrate, 40% fat (P/S ratio 0.14), 15% protein, and 580 mg of cholesterol, and for 6 weeks during a high-carbohydrate low-fat cholesterol-restricted diet with 65% carbohydrate, 20% fat (P/S ratio 1.40), 15% protein, and 62 mg cholesterol. All subjects completed both dietary periods in a crossover experimental design. Individual menus were subject-selected from a calculated exchange list containing conventional food items consistent with current American dietary patterns. The diets were well-tolerated by all subjects. Total plasma cholesterol decreased from 201 to 156 mg/100 mL (P less than 0.05) during the cholesterol-restricted diet, while total plasma triglyceride (TG) increased from 96 to 115 mg/100 mL (P less than 0.01). During this same period, very low-density lipoprotein cholesterol (VLDL-C) and VLDL-TG increased from 17 to 21 mg/100 mL (P less than 0.05) and from 59 to 76 mg/100 mL (P less than 0.001), respectively, while low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) decreased from 126 to 90 mg/100 mL (P less than 0.05) and from 50 to 39 mg/100 mL (P less than 0.05), respectively. LDL-C/HDL-C and total-C/HDL-C ratios were lower but not significantly different, and LDL-TG and HDL-TG were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brain deviations in adult obese-hyperglycemic mice (ob/ob)

In the brain of adult obese-hyperglycemic mice (ob/ob) deviations, such as reduced brain weight, diminished myelination, and reduced amount of DNA were found. These findings cannot be explained by hypothyroidism in adults since above mentioned deviations could only have been caused by a reduced thyroidal activity in the first weeks of postnatal life. Therefore, our data are in support of the earlier hypothesis of congenital hypothyroidism.     

Successful correction of total anomalous pulmonary venous connection in a 2.5 kilogram premature neonate.

A 16 day old 2.5 kg premature infant with total anomalous pulmonary venous drainage to the coronary sinus was successfully treated with use of profound hypothermia and total circulatory arrest. To the best of our knowledge, this neonate is the smallest to successfully undergo correction of this anomaly. A plea is made for a more aggressive approach in early recognition and assessment of these critically ill infants, followed by early surgical correction.     

Effects of hemodialysis-induced hypokalemia and hypomagnesemia on blood pressure in dogs

Experiments were completed in dogs to test the hypothesis that mild hypokalemia or the combination of hypokalemia and hypomagnesemia cause an increase in systemic arterial blood pressure. Results indicate that under the conditions of hypokalemia and hypomagnesemia does not produce changes in blood pressure different than those observed in control dogs, in which blood pressure consistently fell. Additionally, hypokalemia alone did not affect systemic blood pressure in dogs with their neurologic barostatic system rendered inoperable. The significant finding of the study is that the blood pressure fall was not as marked in dogs with a spinal block in which hypomagnesemia and hypokalemia were created simultaneously.     

Glucose and insulin effects on the novo amino acid synthesis in young men: studies with stable isotope labeled alanine, glycine, leucine, and lysine

We have explored interrelationships between te dynamic aspects of whole body glucose and alanine and glycine metabolism in adult humans. Using a primed, continuous intravenous infusion of [1-13C] leucine or lysine given simultaneously with [2H3] or [15N]alanine or [15N]glycine, respectively, whole body alanine and glycine fluxes and their rates of de novo synthesis were determined in three experiments with healthy young men. Subjects were studied in the post-absorptive state and during a 150 min period of an intravenous infusion with unlabeled glucose, at a rate of 4 mg.kg-1 min-1. In one experiment, insulin was given together with the glucose infusion to maintain normoglycemia. In the other two studies, subjects received glucose alone. For the post-absorptive state, alanine flux (mean +/- SEM) was 381 +/- 26 and 317 +/- 18 mumole.kg-1 hr-1 in two separate experiments and glycine flux was 240 +/- 22 mumole.kg-1 hr-1. De novo synthesis of alanine and glycine accounted for 75%-81% and 81% of flux, respectively. Infusion with glucose alone raised plasma glucose to a mean level of 152 mg/dl and increased alanine flux, due to a rise in alanine synthesis of 98 mumole.kg-1 hr-1 (p less than 0.01). Glycine flux and synthesis rate were unaffected by the glucose infusion. When insulin was given with glucose to maintain normoglycemia, the rate of alanine synthesis was unchanged. Because glucose uptake rate, measured with [6,6-2H2] glucose was the same whether glucose was infused along or with exogenous insulin, these results support the view that the circulating plasma glucose level itself may affect alanine synthesis and that the hyperglycemic state is an important factor in regulating interorgan nitrogen transfer, via alanine, in various pathophysiologic states.     

Dynamic aspects of whole body glycine metabolism: Influence of protein intake in young adult and elderly males

The influence of adult age and adequacy of dietary protein intake on whole body glycine metabolism was studied in human subjects. Five healthy young adult males (19–25 yr) and six elderly males (64–78 yr) were given an adequate-protein diet (1.5 g protein/kg/day) for 7 days and a low-protein diet (0.4 g protein/kg/day) for 14 days. At the end of each dietary period, whole body glycine flux and rates of glycine synthesis were estimated with the use of a continuous 60 hr oral administration of ¹⁵N-glycine and determination of ¹⁵N enrichment of plasma glycine by gas chromatography-mass spectrometry with selected ion monitoring. Mean whole body glycine flux and the rate of endogenous glycine synthesis were 458 and 351 μmole/kg body weight/hr, respectively, for young adults receiving the diet adequate in protein; similar values were obtained in the elderly group. Feeding the diet low in protein resulted in an extensive and significant reduction in both parameters in young adults and also in elderly subjects to a similar extent. Measurement of ¹⁵N enrichment in plasma serine gave a constant ratio of ¹⁵n enrichment in plasma free serine relative to glycine for both age groups and at the two protein intake levels. It is concluded that aging of adults has little impact on the quantitative aspects of whole body glycine metabolism, but that it responds extensively to changes in protein intake. Thus, it appears that glycine synthesis and flux are integrated with the body's total nitrogen metabolism and requirement for dietary nitrogen.     

Mechanism of the acute pressor action of hypokalemia, hypomagnesemia, and hypo-osmolality

The hemodynamic mechanism of the acute pressor action of hypokalemia, hypomagnesemia, and hypo-osmolality was studied in the anesthetized dog. The electrolyte changes were first produced rapidly (within 5 minutes) by a dilutional technique and then more slowly (within 50 minutes) utilizing a potent diuretic and modified Ringer's solution. Blood pressure and cardiac output were measured and total peripheral resistance was calculated. The data suggest that the blood pressure rises because of effects on the periphery when the electrolyte changes are produced rapidly by the dilutional technique and because of effects on both the heart and periphery when the electrolyte changes are produced more slowly by the diuretic technique.