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).     

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.     

Serum lipoprotein and apolipoprotein profiles of the genetically obese ob/ob mouse

The lipid transport system of 3-month-old male C57BL/6J obese (ob/ob) mice was investigated. Serum lipoproteins were separated by density gradient ultracentrifugation and characterized by their chemical and electrophoretic properties as well as their relative apolipoprotein contents, defined according to molecular weight and charge. Obese, ob/ob mice exhibited a marked hyperlipoproteinemia resulting from large increases in low-density lipoproteins (LDL, d 1.021-1.058 g/ml) and high-density lipoproteins (HDL, d 1.058-1.137 g/ml), particularly, the HDL2 subclass (d 1.058-1.109 g/ml). This increase in lipoproteins was entirely responsible for their hypercholesterolemia and hyperphospholipidemia. By contrast, these obese mice had a net decrease in very-low-density lipoproteins (VLDL, d less than 1.016 g/ml) and intermediate-density lipoproteins (IDL, d 1.016-1.021 g/ml), which accounted for their moderate hypotriglyceridemia. The chemical composition of heterogeneous light LDL (d 1.021-1.040 g/ml and dense LDL (d 1.040-1.058 g/ml) overlapped by HDL-like particles was highly modified. These modifications consisted of increases in the percentages of cholesteryl ester and phospholipid and decreases in that of triacylglycerol. There were also marked changes in the relative values of the apolipoproteins of VLDL, but principally, IDL and LDL. IDL and light LDL were poorer in apolipoproteins BH (Mr 340,000-320,000) and eventually in apolipoprotein BL (Mr 220,000-200,000) and enriched in apolipoproteins E (Mr 37,000-35,000) and C-A-II (Mr approximately equal to 12,000). A similar and very significant change occurred in VLDL for both the apolipoproteins BL and C-A-II. Dense LDL, mainly poorer in apolipoprotein BH and enriched in apolipoprotein A-I (Mr 28,000-27,000), closely resembled HDL2 in all the groups, and were enriched in apolipoproteins C-A-II in only the obese mice. We suggest that ob/ob mice are probably protected against atheromata because of the low VLDL and IDL levels, and the increase in HDL2.     

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

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

Defective cold-induced stimulation of thyroxine 5'-deiodinase in brown adipose tissue of the genetically obese (ob/ob) mouse

Exposure of a normal lean mouse to cold (14 degrees C) for 12 h increases the activity of thyroxine 5'-deiodinase in brown adipose tissue 26-fold. In contrast, exposure of the genetically obese, ob/ob, mouse to cold results in little more than a doubling of thyroxine 5'-deiodinase activity. The physiological significance of endogenous 3,5,3'-triiodothyronine production in brown adipose tissue is not understood. However, it seems likely that defective cold-induced stimulation of the 5'-deiodinase in brown adipose tissue of the ob/ob mouse might cause a relatively hypothyroid state of the tissue. Thyroid hormone is known to be required for a normal thermogenic response of brown adipose tissue to noradrenaline. It is suggested that the defect in the response of the 5'-deiodinase in the ob/ob mouse could contribute to the defective thermogenic response of brown adipose tissue to cold-exposure and to noradrenaline.     

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.     

Reduced levels of peroxisomal enzymes in the kidney of the genetically obese (ob/ob) mouse. Contrast with liver

Kidney post-nuclear supernatants from genetically lean and obese mice were subjected to subcellular fractionation by dual centrifugation through sucrose gradients in B XIV zonal rotors. Considerable purification of peroxisomes was achieved which allowed the demonstration of acyl-CoA beta-oxidation enzymes and carnitine acyltransferases in these organelles. Comparison of kidney peroxisome-enriched fractions from obese and lean mice indicated a likely relative depression in beta-oxidation enzymes in the obese animal. Measurement of catalase, acyl-CoA oxidase and carnitine octanoyltransferase in whole homogenate of liver and kidney of obese and lean mice revealed significantly reduced levels (to approximately 2/3) of these peroxisomal enzymes in the kidney of ob/ob mice. In contrast the specific activity of catalase and acyl-CoA oxidase was significantly raised in the liver of obese mice.     

Insulin degradation. XVIII. On the regulation of glutathione-insulin transhydrogenase in the hyperglycemic obese (ob/ob) mouse.

The occurrence of insulin-degrading activity in the liver of the obese hyperglycemic mouse (ob/ob) and its litter mate has been studied. The trichloroacetic acid-soluble product formed from insulin upon incubation with liver homogenate was identified as the A chain of insulin. In Ouchterlony double-diffusion experiments with antibody to purified rat liver glutathione-insulin transhydrogenase, mouse liver homogenate and the microsomal fraction each gave a single precipitation band of identity with the purified rat liver enzyme. These results indicate that the insulin-degrading activity present in the mouse liver is, in fact, glutathione-insulin transhydrogenase. Subcellular distribution studies of glutathione-insulin transhydrogenase and marker enzymes indicate that the transhydrogenase is located primarily in the microsomal fraction of mouse liver homogenate. The ob/ob mouse, which is a genetic mutant characterized by obesity, hyperinsulinism and resistance to the hypoglycemic action of insulin, contains hepatic glutathione-insulin transhydrogenase activity (per mg microsomal protein) markedly higher (40--60%) than its lean litter mates. However, a major portion of the increased hepatic enzyme in the ob/ob mouse occurs in a latent state; the increased amount of enzyme either is unavailable or is nonfunctional, although the ob/ob mouse still contains more of the functional form than the lean mouse. Thus, the results are consistent with the suggestion that the hepatic glutathione-insulin transhydrogenase is probably under a feedback control by circulating insulin.     

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-α and interleukin-1β 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.     

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.     

Liver monoamine oxidase in the obese-hyperglycaemic (ob/ob) mouse.

1. The specific activity of monoamine oxidase was found to be greater in liver mitochondria from ob/ob mice than from lean mice. The activities of marker enzymes were similar in both tissues. 2. Experiments with various substrates (5-hydroxytryptamine, benzylamine and tyramine) and inhibitors (clorgyline and deprenyl) indicated that, unlike rat liver mitochondria, mouse liver mitochondria contain a predominance of the B-form of monoamine oxidase. 3. The Km values for lean and ob/ob mice were the same for any given substrate and were in the increasing order 5-hydroxytryptamine less than tyramine less than benzylamine. Vmax. was approximately 50% greater in obese than in lean mice. 4. Extraction of liver mitochondria with acetone/water or acetone/water/NH3 to remove lipids decreased the enzyme activity relatively more in obese- than in lean-mice preparations, but residual activity was the same in both preparations.     

Adrenalectomy and steroid treatment in obese (ob/ob) and diabetic (db/db) mice

Adrenalectomy in young obese (ob/ob) and the diabetic (db/db) mouse slowed body weight gain. Treatment of adrenalectomized ob/ob mice with cortisone or deoxycorticosterone acetate (DOCA) significantly increased weight gain in a dose-related manner. Cortisone had no effect on weight gain on lean mice and treatment with dehydroepiandrosterone sulfate was without effect on either ob/ob or lean mice. The increment in body weight of adrenalectomized ob/ob mice treated with corticosterone and DOCA was associated with an increase in body weight and an increase in food intake. When adrenalectomy was performed at twenty-three days of age (five days before weaning), animals carrying the (db/db) genotype remained lighter than their normal littermates. These data document the importance of the adrenal gland and its steroids for the development and maintenance of many features of the obese or diabetes mouse.     

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.     

Glucagon-like peptide-1 and the entero-insular axis in obese hyperglycaemic (ob/ob) mice

The intestines of obese hyperglycaemic (ob/ob) mice contain greatly increased amounts of glucagon-like immunoreactive peptides. To investigate their role in the increased activity of the entero-insular axis of these mice, the insulin-releasing effect of glucagon-like peptide-1 (GLP-1) was examined in 24 hour fasted 12-15 weeks old ob/ob mice under conditions of basal and elevated glycaemia. Compared with glucagon (100 micrograms/kg ip), which produce an approximately 3-fold increase in basal plasma glucose and insulin concentrations, GLP-1 (100 micrograms/kg ip) produce a very small (less than 1 fold) increase in plasma insulin, with no significant change in plasma glucose. The insulin-releasing effect of glucagon, but not GLP-1 was increased by administration in combination with glucose (2 g/kg ip). The results indicate that GLP-1, which exhibits considerable sequence homology with glucagon, exerts only a weak insulin-releasing effect without a significant hyperglycaemic effect in ob/ob mice. Thus GLP-1 is unlikely to be an important endocrine component of the two over-active entero-insular axis in ob/ob mice.     

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.     

Ultrastructural analysis of progressive endometrial hypercytolipidemia induced by obese (ob/ob) and diabetes (db/db) genotype mutations: structural basis of female reproductive tract involution

The diabetes (db/db) and obese (ob/ob) genotype mutations induce a progressive, hypercytolipidemic condition within the endometrium of the female reproductive tract that promotes sterility and premature organ involution in C57BL/KsJ mice. The current studies focus on the ultrastructural changes that occur within the epithelial and stromal layers of the uterine endometrium during the progressive expression of these mutations, which induce a hyperglycemic-hyperinsulinemic metabolic state and promote tissue cytolipidemia and organoinvolution. Control (normal: +/-), diabetes (db/db) and obese (ob/ob) genotype groups were prepared for high resolution light (LM) and transmission (TEM) microscopic analysis of endometrial tissue samples collected from 4 (young)- to 20 (aged)-week-old mice, allowing for the progressive influences of the mutational aberrations on uterine structure to be evaluated. Compared to controls, both (ob/ob) and (db/db) mutations induced a dramatic increase in endometrial epithelial cytolipid vacuole accumulation, which increased in density between 4 and 20 weeks of age. Lipid vacuoles aggregated at the baso-polar regions of epithelial cells in response to the hyperglycemic-hyperlipidemic conditions typical of both (ob/ob) and (db/db) groups. Progressive cytoplasmic movement of the lipid pools induced a perinuclear isolation from surrounding cytoplasmic organelles. Apical lipid accumulations forced cytoplasmic organelles into peripheral cell compartments and altered the periepithelial stromal cell profile relative to controls. These studies define the progressive, intracellular accumulation of hypercytolipidemic pools which induce a transformation of normal endometrial cell types into adipocyte-like entities. The lipidemia-induced alterations in cell structure disrupt normal tissue continuity and function, culminating in organoinvolution and overt female reproductive sterility.     

Isolated adipocytes from growth hormone-treated obese (ob/ob) mice exhibit insulin resistance

The genetically obese (ob/ob) mouse is a useful model for the study of the diabetogenic action of growth hormone (GH), because treatment of these animals with GH results in decreased responsiveness of their adipose tissue to insulin in vitro. Studies of the mechanisms involved in GH-induced insulin resistance using isolated adipocytes of ob/ob mice have not been possible, however, because of their extreme fragility and the lack of an adequate system for the maintenance of these cells. This study describes a new method for the isolation of ob/ob mouse adipocytes. The isolated cells are stable, viable and metabolically responsive to insulin. In addition, these adipocytes have been maintained in primary culture, in serum-free medium, for up to 3 days. During culture, the cells exhibit large increases in 125I-hGH binding (10-20-fold) and porcine 125I-insulin binding (5-10-fold). The induction of insulin resistance by GH has also been demonstrated in these freshly isolated ob/ob mouse adipocytes. The studies to date indicate that the ob/ob mouse adipocyte system should provide a useful model for detailed studies of the cellular and molecular mechanisms of GH induced insulin resistance.