Glucoregulatory effects of bombesin in lean and genetically obese hyperglycaemic (ob/ob) mice

1. Plasma glucose and insulin responses to bombesin were examined in 12-15-week-old 12 hr fasted lean and genetically obese hyperglycaemic (ob/ob) mice. 2. Bombesin (1 mg/kg ip) produced a prompt but transient increase of plasma insulin in lean mice (maximum increase of 50% at 5 min), and a more slowly generated but protracted insulin response in ob/ob mice (maximum increase of 80% at 30 min). Plasma glucose concentrations of both groups of mice were increased by bombesin (maximum increases of 40 and 48% respectively in lean and ob/ob mice at 15 min). 3. When administered with glucose (2 g/kg ip), bombesin (1 mg/kg ip) rapidly increased insulin concentrations of lean and ob/ob mice (maximum increases of 39 and 63% respectively at 5 min). Bombesin did not significantly alter the rise of plasma glucose after exogenous glucose administration to these mice. 4. The results indicate that bombesin exerts an insulin-releasing effect in lean and ob/ob mice. The greater insulin-releasing effect in ob/ob mice renders bombesin a possible component of the overactive entero-insular axis in the ob/ob mutant, especially if it acts within the islets as a neurotransmitter or paracrine agent.     

Hepatic delta 6-desaturase activity in lean and genetically obese ob/ob mice.

Hepatic delta 6-desaturase activity is primarily located in the mitochondrial fraction in mice. Both delta 6- and delta 5-desaturase activities are increased in the liver of young (6-week-old) obese mice. The increase in hepatic delta 6-desaturase activity in obese mice does not occur until weaning. Neither restriction of food intake nor hyperinsulinaemia normalize hepatic delta 6-desaturase activity of obese mice. Both cold acclimation and tri-iodothyronine (30 micrograms/day per kg) decreased hepatic delta 6-desaturase activity of obese mice to levels observed in lean mice, whereas the increase in activity in obese mice was still maintained after the induction of hypothyroidism.     

Differential response of adenylate cyclase to beta-adrenergic agonists in white adipocyte membranes from lean and obese (ob/ob) mice

The quality of the beta-adrenergic response, as measured by the activation of adenylate cyclase, was found to differ in adipocyte membranes from lean and obese mice. In the tissue from lean mice, the response was similar to that in rat adipose tissue and could, by analogy, be classified as beta 1-receptor response. In the tissue preparations from the obese mice, the rank order of potency of the three classical agonists (isoproterenol, epinephrine, and norepinephrine) was more typical of a beta 2-receptor response.     

Mitochondrial Ca2+ transport in lean and genetically obese (ob/ob) mice.

Isolated mitochondria from liver or brown adipose tissue of obese ob/ob mice demonstrated increased rates of Ca2+ uptake and release compared with those of lean mice. This enhanced transport activity was not found in mitochondria from kidney or skeletal muscle. Respiration-induced membrane potential was the same in mitochondria from lean and ob/ob mice. It is therefore concluded that the increased Ca2+ uptake rates reflect an activation of the Ca2+ uniporter rather than a change in the electrophoretic driving force. As mitochondria from pre-obese ob/ob mice did not show elevated rates of Ca2+ transport, the activated transport in the obese animals was thus a consequence of the state of obesity rather than being a direct effect of the ob/ob genotype. It is suggested that the enhanced activity of the Ca2+-transport pathways in liver and brown adipose tissue may alter metabolic functions in these tissues by modifying cytoplasmic or intramitochondrial Ca2+ concentrations.     

Membrane fluidity and adenylate cyclase activity in genetically obese mice

Adipocyte plasma membranes of genetically obese $\text{ob}\text{ob}$ mice are more fluid than their lean littermates but the fluidity was normalised in mice maintained at high environmental temperatures. The defective response of adenylate cyclase to isoproterenol was improved after normalisation of membrane fluidity. No major changes in the phospholipid composition of $\text{ob}\text{ob}$ membranes were detected.     

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.     

Adenylate cyclase in lean and obese (ob/ob) mouse epididymal white adipocytes

The results presented in this study indicate that the defect in catecholamine-stimulated adenylate-cyclase which is characteristic of the ob/ob mouse is associated with a decrease in the sensitivity of the system to guanine nucleotides (guanosine 5'-[beta gamma-imido]triphosphate and guanosine 5'-triphosphate). No difference in the beta-adrenergic receptor activity was found between the lean and obese mice on the basis of their capacity to bind the beta-adrenergic antagonist [3H]dihydroalprenolol. The data suggest that a defect in the activation of the adenylate cyclase by beta-adrenergic agents may reside in the guanyl nucleotide binding site(s).     

Glucose tolerance in aging obese (ob/ob) and lean mice

Examination of the glucose tolerance in younger (3 month) and older (6 month) obese mice revealed that most of their postinjection hyperglycemia arises from the disproportionately large glucose responses to the injection/bleeding procedures rather than from the added glucose. Simultaneous measurements of circulating glucagon, corticosterone and insulin indicated that simple differences in the levels of these hormones, in their circulating ratios, or in the magnitude of the hormone responses to stimulation did not fully account for the "stress"-induced hyperglycemia.     

An impaired response of adenylate cyclase to stimulation by epinephrine in adipocyte plasma membranes from genetically obese mice (ob/ob).

The present studies have established that there is an impaired response to epinephrine of the adenylate system in adipocyte preparations from obese hyperglycemic mice as compared to their thin littermates. In contrast, membrane preparations from both groups of animals were found to exhibit a similar response to fluoride ion. The response of adenylate cyclase to epinephrine was enhanced to a similar extent by increasing the ATP concentration in adipocyte plasma membranes from the two groups of animals. While GTP (0.1 muM) elicited an ATP-like response of similar magnitude in adenylate cyclase activity in both membrane preparations, it did not therefore abolish the impaired response to epinephrine of adenylate cyclase activity in membranes of obese mice. The response of adenylate cyclase activity to (--)-epinephrine in membrane preparations from obese mice progressively diminished with the age of these animals. In contrast, the concentration of (--)-epinephrine required for half-maximal stimulation of adenylate cyclase was similar and remained unchanged with the age for both membrane preparations. These data suggest that a perturbation may occur in the coupling step between the hormone receptor and the catalytic site of the adenylate cyclase system in obese mice. While a 15-day restrictive diet or a 72-h period of fasting was found to normalize the hyperinsulinemia of obese animals, neither affected the response of adenylate cyclase to epinephrine in preparations of adipocyte membranes from these mice. These results suggest that the observed defect in the response of plasma membrane adenylate cyclase activity to epinephrine in obese mice does not result from their hyperinsulinism.     

Islet cell surface antibodies in genetically obese hyperglycaemic (ob/ob) mice

A quantitative method for circulating islet cell surface antibodies (ICSA), based on the binding of¹²⁵I-protein A to insulin-producing RINm5F cells, was used to evaluate ICSA in plasma of 4- to 40-week-old Aston obese hyperglycaemic (ob/ob) mice and normal control (+/+) mice. RINm5F cells bound 2502±l196 c.p.m.¹²⁵I-protein A per l0⁵ cells (mean±S.D.,n=54) after incubation with +/+ plasma. ICSA positive plasma (defined as¹²⁵I-protein A binding, mean±2 S.D. of +/+ plasma) was detected in 3 out of 54+/+ mice and 3 out of 54 ob/ob mice. ICSA were not observed in ob/ob mice before the onset of diabetes (7 weeks of age), but were detected at 9, 20 and 40 weeks. At 20 weeks¹²⁵I-protein A binding produced by ob/ob plasma was 35% greater than +/+ plasma (P<0.05). The low occurrence of ICSA in ob/ob mice (6%) suggests that factors other than ICSA are responsible for B-cell dysfunction and eventual islet degeneration observed in Aston ob/ob mice.     

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.     

Quantification of the alpha and beta subunits of the transducing elements (Gs and Gi) of adenylate cyclase in adipocyte membranes from lean and obese (ob/ob) mice.

The abundance of the alpha and beta subunits of the GTP-binding proteins (G-proteins) that transduce hormonal messages to adenylate cyclase was assessed in adipocyte membranes from lean (+/+) and obese (ob/ob) mice, using ADP-ribosylation with bacterial toxin and immunodetection. Both methods revealed two Gs alpha species (48 and 42 kDa) in the membranes. Compared with those of lean mice, the membranes from obese mice contained substantially less of the 48 kDa species of Gs alpha, as assessed by both methods. ADP-ribosylation by pertussis toxin showed that only half as much ADP-ribose was incorporated into Gi alpha in the membranes from obese as compared with lean mice. Immunodetection revealed two separate Gi alpha peptides (39 and 40 kDa) and showed that the 40 kDa species was less abundant in the membranes from obese mice, whereas the amount of the 39 kDa species was similar in membranes from both lean and obese animals. Based on ADP-ribosylation assays, in membranes from lean mice the ratio Gs alpha/Gi alpha was 1:16, whereas in the membranes from obese mice it was 1:10. Similar amounts of immunodetectable beta peptide were found in both types of membranes. On the basis of the currently accepted dissociation model of adenylate cyclase activation, the decrease in the abundance of the Gi alpha subunit in adipocyte membranes from obese mice could account for the abnormal kinetics of the enzyme in these membranes.     

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.     

Plasma B-cell tropin (ACTH22-39) concentration in lean and obese (ob/ob) mice and lean and obese (fa/fa) Zucker rats

A method has been developed for the measurement of plasma concentrations of Beta-cell tropin (BCT), which is a potent insulinotropic and lipogenic peptide secreted by the pituitary. The method was employed to compare plasma Beta-cell tropin concentrations between lean and genetically obese (ob/ob) mice and between lean and genetically obese (fa/fa) Zucker rats. The plasma concentration in lean mice was 0.17 +/- 0.02 (5)nmole/l (mean +/- SEM, n = 5), while that in obese (ob/ob) mice was significantly higher, being 2.88 +/- 1.13 (5)nmole/l. The plasma BCT concentration in Zucker rats was 0.14 +/- 0.02 (15)nmole/l, while that in obese Zucker (fa/fa) rats was significantly higher, being 1.69 +/- 0.72 (16)nmole/l. These results explain previously observed differences in the Beta-cell tropin-like biological activity in plasma from lean and obese animals, and support the hypothesis that the peptide has a role in the development of hyperinsulinaemia and obesity.     

Dietary copper supplementation increases the catecholamine levels in genetically obese (ob/ob) mice

The interactive relationship between Cu deficiency and depressed synthesis of certain neurotransmitters has been recognized. To investigate the effects of dietary Cu supplementation on the catecholamine levels in genetically obese mice, male obese (ob/ob) mice and their lean (+/?) counterparts were administered either a control diet (4.0 mg/kg) or a Cu-supplemented diet (50 mg/kg) for 4 wk. The ob/ob mice that were fed a control diet showed lower liver and higher plasma levels of Cu. Depressed levels of plasma and brain catecholamines were also found in ob/ob mice that were fed the control diet. The ob/ob mice that received a Cu-supplemented diet showed significant increases in the levels of catecholamine in the plasma and brain. This study showed that catecholamine levels in ob/ob mice can be increased by dietary Cu supplementation. However, the interaction between Cu and sympathetic nervous activity in obesity was not elucidated in this study.     

Anorectic effect of fenfluramine, cholecystokinin and neurotensin in genetically obese (ob/ob) mice

To investigate the satiety defect of hyperphagic genetically obese (ob/ob) mice, acute feeding responses to three differently acting anorectic agents were determined in 7-9 weeks old lean (+/+) and ob/ob mice habituated to a restricted (0900-1230 hr) daily feeding routine. Fenfluramine (10 mg/kg), cholecystokinin (100 U/kg) and neurotensin (500 micrograms/kg), administered intraperitoneally 15 min before feeding, each produced a rapid but transient suppression of food consumption in ob/ob mice, similar to lean controls. The results suggest that neural satiety mechanisms triggered via serotoninergic pathways (fenfluramine), vagal afferents (cholecystokinin) and the hypothalamic paraventricular nucleus (neurotensin) are functional in ob/ob mice, supporting the view that the satiety defect of ob/ob mice resides outside of the nervous system.     

Effects of zinc supplementation on the plasma glucose level and insulin activity in genetically obese (ob/ob) mice

The effects of zinc supplementation (20 mM ZnCl₂ from the drinking water for eight weeks) on plasma glucose and insulin levels, as well as its in vitro effect on lipogenesis and lipolysis in adipocytes were studied in genetically obese (ob/ob) mice and their lean controls (+/?). Zinc supplementation reduced the fasting plasma glucose levels in both obese and lean mice by 21 and 25%, respectively (p < 0.05). Fasting plasma insulin levels were significantly decreased by 42% in obese mice after zinc treatment. In obese mice, zinc supplementation also attenuated the glycemic response by 34% after the glucose load. The insulin-like effect of zinc on lipogenesis in adipocytes was significantly increased by 80% in lean mice. However, the increment of 74% on lipogenesis in obese mice was observed only when the zinc plus insulin treatment was given. This study reveals that zinc supplementation alleviated the hyperglycemia of ob/ob mice, which may be related to its effect on the enhancement of insulin activity.     

Effect of metformin on nitric oxide synthase in genetically obese (ob/ob) mice.

Genetically obese (ob/ob) mice were employed for the study of the effect of metformin on activity and expression of nitric oxide synthase (NOS ) in vitro and in vivo. For in vitro analysis, mouse liver extracts were used. For the in vivo study, (ob/ob) and their control litter mates (ob/c) mice were injected with specified amounts of metformin and the expression of NOS in the adipose tissue and hypothalamus was measured by Western blotting. Results show that metformin exhibited a biphasic effect on NOS activity in vitro. Expression of metformin was differentially altered in the hypothalamus and adipose tissues of the normal and ob/ob animals that were treated with metformin. Further, a significant decrease in food intake occurred in the (ob/ob) mice that received metformin. This decrease in food intake was not accompanied by changes in serum glucose. At inhibitory concentrations, hypothalamic NOS expression changes differentially in normal and ob/ob mice. In normal mice, metformin stimulated NOS expression, while in ob/ob mice there was an inhibition. NOS expression increased in brown adipose tissue of metformin treated control mice, while no such increase was observed in ob/ob mice. No effect of metformin was observed in white adipose tissue of control or obese mice. Thus, metformin may produce anorectic effects through modulation of NOS.