Ovarian hypercytolipidemia induced by obese (ob/ob) and diabetes (db/db) mutations: basis of female reproductive tract involution II

The diabetes (db/db) and obese (ob/ob) genotype mutations induce a progressive, hypercytolipidemic condition within the ovarian compartments of the female reproductive tract that results in sterility and premature organ involution in C57BL/KsJ mice. The current studies focus on the ultrastructural changes that occur within the ovarian interstitial, thecal, and follicular granulosa cell layers during the progressive expression of these mutations which promote tissue cytolipidemia-induced organoinvolution. Control (normal: +/?), diabetes (db/db), and obese (ob/ob) genotype groups were prepared for high resolution light (HRLM) and transmission electron microscopic (TEM) analysis of ovarian tissue samples collected from 4 (young)- to 20 (aged)-week-old mice, allowing for the progressive influences of the mutational aberrations on tissue structure to be evaluated. Compared to controls, both (ob/ob) and (db/db) mutations induced a dramatic increase in ovarian interstitial, thecal and follicular granulosa cytolipid vacuole accumulations, which increased in density between 4 and 20 weeks of age. Initially, lipid vacuoles aggregated in the interstitial and thecal regions of ovarian follicles in response to the hyperglycemic-hypertriglyceridemic metabolic conditions typical of both (ob/ob) and (db/db) groups. Progressive cytoplasmic movement of the lipid pools established a perinuclear isolation from associated cytoplasmic organelles. Progressive lipid accumulations forced cytoplasmic organelles to peripheral cell compartments and altered the follicular cell profile towards that of adipocyte-like entities relative to controls. The progressive hypercytolipidemia-induced alterations in cell structure disrupted normal tissue continuity, which culminated in premature ovarian organo-involution and female reproductive sterility.     

Genomic modulation of diabetes (<Emphasis Type="Italic">db/db)</Emphasis> and obese (<Emphasis Type="Italic">ob/ob</Emphasis>) mutation-induced hypercytolipidemia: cytochemical basis of female reproductive tract involution

Infertility and hypercytolipidemic utero-ovarian involution are recognized consequences of the diabetes-obesity syndrome (DOS) in C57BL mice with either obese (ob/ob) or diabetes (db/db) single gene mutations. We have evaluated the interdependent deleterious influences of both mutation types and differences in the genomic background on utero-ovarian dysfunction in C57BL mice. Control (+/?) C57BL mice were matched with littermate ob/ob and db/db mutants expressed on either the /KsJ or /6 background. Both ob/ob and db/db mutations increased body weights of /KsJ and /6 background strains relative to +/? groups. In contrast, uterine and ovarian weights were depressed by ob/ob and db/dbmutations relative to +/?, regardless of the background strain, but especially when expressed on the /KsJ background. Functionally, both ob/ob and db/db mutations induced hyperglycemic-hyperinsulinemic states coupled with depressed serum estradiol-17-β and progesterone concentrations when expressed on a /KsJ background. Microscopic analysis of utero-ovarian tissue samples revealed marked hypercytolipidemia in the follicular granulosa and endometrial epithelial tissue layers of both ob/oband db/db mutant groups relative to normal +/? cytoarchitecture. The db/db mutation consistently promoted more severe hypercytolipidemic profiles than the ob/obmutation, regardless of background strain. Thus, the severity of utero-ovarian hypercytolipidemia following the expression ofob/ob and db/db mutations in C57BL mice is influenced, or moderated, by the genomic background on which the mutation is expressed.     

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.     

Hypercytolipidemia-induced cellular lipoapoptosis: cytostructural and endometabolic basis of progressive organo-involution following expression of diabetes (db/db) and obese (ob/ob) mutation syndromes

Onset expression of Type 2 (NIDDM) diabetes and obesity metabolic syndromes (DOS) are characterized by premature, progressive cytoatrophy and organo-involution induced by dysregulated cellular gluco- and lipo-metabolic cascades. The consequential systemic, interstitial and intracellular hyperlipidemia disrupts normal cytointegrity and metabolic responsivity to the established hypercaloric pericellular environments. The sequential cytostructural, metabolic and endocrine disturbances associated with the development of progressive DOS-associated hypercytolipidemia compromises cellular metabolic response cascades and promotes cytochemical disturbances which culminate with nuclear lipoapoptosis and cytoatrophy. The dramatic alterations in interstitial glucose and lipid (free fatty acids/triglycerides) concentrations are recognized to influence interstitial and cytoplasmic microchemical environments, which markedly alter cellular nutrient diffusion and active trans-membrane flux rates. The progressive exacerbation of interstitial and cytoplasmic lipid imbibition has been demonstrated to be associated with DNA fragmentation by lipo-infiltration into the chromatin matrix, inducing structural disruption and physical dissolution, indexed as nuclear lipoapoptosis. Therapeutic reduction of the severity of hypercytolipidemia-induced structural and cytochemical compromise promotes the restoration of homeostatic metabolic support for normalized cytostructural indices and supportive cellular gluco- and lipo-metabolic cascades. The re-establishment of a homeostatic interstitial microenvironment moderates the severity of cytolipidemic compromise within affected cell types, reduces nuclear lipo-infiltration and DNA lipo-dissolution, resulting in the preservation of cytostructural integrity. Through the therapeutic restoration of extra- and intra-cellular microchemical environments in genetically dysregulated metabolic syndrome models, the coincident cytochemical, endocrine and metabolic disturbances associated with progressive hypercytolipidemia, resulting from the expressed systemic hypercaloric environmental and hepato-pancreatic endometabolic disturbances which characterize Type 2 (NIDDM) diabetes–obesity and metabolic (X) syndromes, may be ameliorated.     

Oleoyl-estrone lowers the body weight of both ob/ob and db/db mice.

Homozygous obese db/db (BKS-Lepr(db) and ob/ob (B6-Lep(ob)) mice were treated for 14 days with a continuous infusion of a fat emulsion (controls) or loaded with oleoyl-estrone at doses of 12.5 and 50 nmol/g x d using surgically inserted osmotic minipumps. Treatment with oleoyl-estrone resulted in a marked decrease in body weight in both strains, compared with the unchecked growth of controls. In db/db mice, plasma urea and insulin, as well as liver lipid decreased with treatment. In ob/ob mice, the effect on insulin was more marked, in parallel with higher plasma lipids pointing to increased fat mobilisation. The results suggest that oleoyl-estrone effects on body fat reserves and insulin resistance are not mediated by leptin, since ob/ob mice lack this hormone and in the db/db it is present but cannot induce effects because of defective leptin receptors; in both cases oleoyl-estrone treatment lowers body weight.     

Adrenalectomy in genetically obese ob/ob and db/db mice increases the proton conductance pathway

Adrenalectomy (ADX) prevents the excessive weight gain in the genetically obese ob/ob and db/db mice. To test the possibility that this results from increased energy expenditure due to increased thermogenesis in brown adipose tissue (BAT), we measured GDP binding to mitochondria from interscapular brown adipose tissue (BAT) in db/db and ob/ob mice and their lean controls after adrenalectomy, with and without corticosterone replacement. Both the vehicle treated and corticosterone treated db/db and ob/ob mice had lower body weights than the sham-operated mice GDP binding to mitochondria from IBAT was significantly lower in both the db/db and ob/ob mice than in their lean controls. Adrenalectomy significantly increased GDP binding in all mice compared to the respective sham-operated mice, but, the percentage increase was always greater in the db/db and ob/ob mice. Corticosterone treatment of adrenalectomized db/db, ob/ob or lean mice lowered GDP binding to sham levels. Our data confirm previous findings that adrenalectomy results in increased GDP binding to mitochondria from IBAT. Injections of corticosterone into adrenalectomized mice results in a decrease in GDP binding to values which are similar to values in sham-operated mice. Thus adrenalectomy may inhibit the development of obesity by increasing the thermic activity in IBAT.     

Diabetes (<Emphasis Type="Italic">db/db</Emphasis>) mutation-induced endometrial epithelial lipoapoptosis: Ultrastructural and cytochemical analysis of reproductive tract atrophy

Background  

The diabetes (db/db) mutation in C57BL/KsJ mice promotes a progressive cytolipidemia within the endometrial epithelial (EE) layer of the female reproductive tract which results in premature cellular and organ atrophy. The current studies focus on the ultrastructural and cytochemical changes which promote nuclear apoptosis and cytostructural disruption following the expression of endometrial hypercytolipidemia which promotes diabetes-associated organoinvolution and manifest infertility.     

Leptin effect on intestinal galactose absorption in ob/ob and db/db mice

Our previous works demonstrated that leptin inhibits galactose absorption in rat and mice intestinal rings. Here, we have studied the effect of exogenous leptin on intestinal galactose absorption in the genetically obese db/db (leptin-resistant) and ob/ob (leptin-deficient) mice. Assays were performed by incubating the intestinal rings in saline solution containing 5 mM galactose in the absence or presence of 0.2 or 0.4 nM leptin. Basal galactose uptake was similar in the wild-type and the two obese groups. Contrarily to what happens in wild-type mice, leptin increased galactose uptake in db/db animals; since these mice lack the functional long leptin receptor, the measured effect may be due to the short receptor signaling. In the ob/ob mice, 0.2 nM leptin also increased galactose absorption whereas 0.4 nM did not have any effect, suggesting that in the genetically obese animals the expression and regulation of leptin receptors may be altered.     

Association of monoamine oxidase and malate dehydrogenase with liver peroxisomes of genetically obese (ob/ob and db/db) mice.

1. Liver post-nuclear supernatants (PNS) from genetically obese (ob/ob and db/db), lean (+/?), and albino mice were fractionated by dual centrifugation in B-XIV zonal rotors and subcellular fractions were analysed by marker-enzyme estimations and by electron microscopy. 2. Rate-dependent banding of PNS yielded a peroxisome-enriched region (PER) well-separated from mitochondria. 3. Density-dependent banding of PER in ob/ob and db/db mice only, yielded purified peroxisomes which were associated with malate dehydrogenase (cytosolic) and monoamine oxidase. 4. Markers for the mitochondrial matrix, intermembrane space and inner membrane compartments were absent from the peroxisomes. 5. The experimental results are interpreted as indicating that peroxisomes of genetically obese mice are either altered so that protein import is imprecise or so that their attachment to mitochondria is more extensive.     

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.     

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.     

Zinc attenuation of GDP binding to brown adipocytes mitochondria in genetically obese (ob/ob) mice

In this study, we investigate the in vitro effect of zinc addition on guanosine diphosphate (GDP) binding to mitochondria in brown adipocytes of genetically obese (ob/ob) mice. Interscapular brown adipocytes of male mice (obese; lean) at 4 and 12 wk of age were incubated with 0, 50, 100, or 200 μM zinc sulfate. Mitochondria were then isolated and their GDP binding capacities were measured. The GDP-binding capacities of ob/ob mice were lower than lean mice, with or without zinc addition, in both age groups (p<0.05). Zinc addition did not have any significant effect on GDP binding in lean mice. GDP binding decreased with increasing zinc addition in ob/ob mice, and this attenuation was more predominant in 12-wk old ob/ob mice. Moreover, we found that high magnesium addition (5 mM) increased GDP binding in lean mice, but this effect was not significant in ob/ob mice. This study reveals that brown adipose tissue thermogenesis in ob/ob mice could be greatly attenuated by zinc addition, suggesting that zinc may play a regulatory role in obesity.     

Variable onset determinants and consequences of diabetes (db/db) obesity mutation expression: adrenergic promotion of utero-ovarian dysfunction.

Expression of the diabetes ( db/db) genotype mutation in female C57BL/KsJ mice induces a complex diabetes-obesity syndrome (DOS) responsible for reproductive tract involution promoted by hypercytolipidemia (HCL). Current studies define the complex and influences of the endometabolic variables that promote reproductive tract involution at the time of initial db/db mutation expression onset in female C57BL/KsJ mice. Littermate-paired, normal ( +/?) and db/db groups were isolated between 2 - 4 weeks of age and tissue samples analyzed for utero-ovarian alterations induced by the systemic, tissue, cellular and structural consequences of mutation expression. Significantly elevated body weights, blood glucose concentrations and serum insulin levels contrasted with atrophic utero-ovarian indices in db/db mutants compared to +/? groups. The onset of the db/db-expression promoted obesity and a mild hyperglycemic-hyperinsulinemic state. Initial db/db expression was characterized by significantly increased utero-ovarian insulin binding without variation in membrane insulin receptor concentrations. However, significant elevations in tissue glucose sequestration rates, norepinephrine (NE) concentrations and triacylglyceride lipase activity in db/db groups indicated that a complex of endometabolic counter-regulatory influences promoted the metabolic shunting of excess glucose and triglyceride moieties towards hypercytolipidemic storage. The resulting DOS-promoted accumulation of utero-ovarian cytolipidemic pools compromised reproductive tract cytoarchitecture in db/db mice. The results of these studies indicate that the inability of utero-ovarian tissue compartments to exhibit metabolic adaptation to the enhanced availability, transport and cellular imbibition of extracellular glucose-lipid pools promotes the initial cellular compromise recognized to induce reproductive failure in db/db mutants.     

Diabetes (db/db) mutation-induced ovarian involution: progressive hypercytolipidemia

Ovarian atrophy and reproductive tract incompetence are recognized consequences of the progressive expression of the overt, diabetes-obesity syndrome (DOS) in C57BL/KsJ (db/db) mutant mice. The present studies evaluated the progressive changes in ovarian cytoarchitecture, endocrine expression, and reproductive tract cytolipidemic parameters that promote reproductive failure and ovarian involution during the pre-onset, initial, progressive, and chronic expression stages of the DOS. Paired littermate control (normal: +/?) and diabetic (mutant: db/db) C57BL/KsJ females were selected for analysis of ovarian parameters at 2 weeks (pre-onset expression of DOS), 4 weeks (initial DOS expression), 8 weeks (progressive DOS: hyper-glycemic/lipidemic), and 16 weeks (overt/chronic DOS expression) of age. All 4- to 16-week-old (db/db) groups were obese, hyperglycemic, and hyperinsulinemic as compared with age-matched (+/?) controls. Prior to phenotypic expression of the DOS (2 week groups), ovarian interstitial cytolipidemia characterized the perifollicular and cortical regions of db/db tissue samples relative to +/? indices, while comparable body weight, blood glucose, as well as serum insulin and ovarian steroid hormone concentrations characterized both the +/? and db/db groups. Overt DOS expression in the 4-week-old db/db groups was characterized by body obesity, systemic hyperglycemia-hyperinsulinemia, and extensive hypercytolipidemia of ovarian folliculothecal compartments, as well as enhanced tissue lipase activities. By 8 weeks of age, progressive hypercytolipidemia characterized interstitial, thecal, and follicular granulosa cell layers of db/db tissue samples concurrent with suppressed ovarian steroid hormone production, enhanced lipid sequestration, and exacerbation of systemic hyper-glycemia/insulinemia. By 16 weeks of age, the chronic-DOS was characterized by extensive ovarian follicular involution, cortical perivascular hyperlipidemic infiltration, thecal cell atrophy, and follicular granulosa lipid imbibition. These data indicate that db/db mutation-induced ovarian structural and functional involution is a direct reflection of the cellular metabolic shift towards lipogenesis, indicated by the progressive cytoarchitectural transformation into adipocyte-like entities. The cytological indications of cellular metabolic compromise, which precede the phenotypic expression of the DOS indices, suggests that correction of these abnormal shifts in ovarian endocrine and cellular metabolism may restore, delay, or prevent the further compromise of ovarian function by db/db mutation expression.     

Leptin receptor-deficient MMTV-TGF-alpha/Lepr(db)Lepr(db) female mice do not develop oncogene-induced mammary tumors

Being overweight is a risk factor for postmenopausal breast cancer and is associated with an increased incidence and shortened latency of spontaneous and chemically induced mammary tumors in rodents. However, leptin-deficient obese Lep(ob)Lep(ob) female mice have reduced incidences of spontaneous and oncogene-induced mammary tumors. Of interest, leptin enhances the proliferation of human breast cancer cell lines in which leptin receptors are expressed, which suggests that leptin signaling plays a role in tumor development. We evaluated oncogene-induced mammary tumor development in obese MMTV-TGF-alpha/Lepr(db)Lepr(db) mice that exhibit a defect in OB-Rb, which is considered to be the major signaling isoform of the leptin receptor. Lepr and MMTV-TGF-alpha mice were crossed, and the offspring were genotyped for oncogene expression and the determination of Lepr status. Lean MMTV-TGF-alpha/Lepr(+)Lepr(+) (homozygous) and MMTV-TGF-alpha/Lepr(+)Lepr(db) (heterozygous) mice and obese MMTV-TGF-alpha/Lepr(db)Lepr(db) mice were monitored until age 104 weeks. Body weights of MMTV-TGF-alpha/ Lepr(db)Lepr(db) mice were significantly heavier than those of the lean groups. No mammary tumors were detected in MMTV-TGF-alpha/Lepr(db)Lepr(db) mice, whereas the incidence of mammary tumors in MMTV-TGF-alpha/Lepr(+)Lepr(+) and MMTV-TGF-alpha/ Lepr(+)Lepr(db) mice was 69% and 82%, respectively. Examination of mammary tissue whole mounts indicated an absence of duct formation and branching for MMTV-TGF-alpha/Lepr(db)Lepr(db) mice. Both age at mammary tumor detection and tumor burden (tumors/mouse and tumor weights) were similar for the lean genotypes. Serum leptin levels of MMTV-TGF-alpha/Lepr(db)Lepr(db) mice were 12-20-fold higher than levels of lean mice. Thus, despite elevated serum leptin levels, leptin receptor-deficient MMTV-TGF-alpha/Lepr(db)Lepr(db) mice do not develop mammary tumors. This study provides additional evidence that leptin and its cognate receptor may be involved in mammary tumorigenesis.     

Hepatic glycogen metabolism in the db/db mouse

Summary Knowledge of the metabolic changes that occur in insulin-resistant type 2 diabetes is relatively lacking compared to insulin-deficient type 1 diabetes. This paper summarizes the importance of the C57BL/KsJ-db/db mouse as a model of type 2 diabetes, and illustrates the effects that insulin-deficient and insulin-resistant states have on hepatic glycogen metabolism. A longitudinal study of db/db mice of ages 2–15 weeks revealed that significant changes in certain parameters of hepatic glycogen metabolism occur during this period. The liver glycogen levels were similar between diabetic and control mice. However, glycogen particles from db/db mice were on average smaller in mass and had shorter exterior and interior chain lengths. Total phosphorylase and phosphorylase a activities were elevated in the genetically diabetic mice. This was primarily due to an increase in the amount of enzymic protein apparently the result of a decreased rate of degradation. It was not possible to find a consistent alteration in glycogen synthase activity in the db/db mice. Glycogen synthase and phosphorylase from diabetic liver revealed some changes in kinetic properties in the form of a decrease in V_max, and altered sensitivity to inhibitors like ATP. The altered glycogen structure in db/db mice may have contributed to changes in the activities and properties of glycogen synthase and phosphorylase. The exact role played by hormones (insulin and glucagon) in these changes is not clear but further studies should reveal their contributions. The db/db mouse provides a good model for type 2 diabetes and for fluctuating insulin and glucagon ratios. Its use should clarify the regulation of hepatic glycogen metabolism and other metabolic processes known to be controlled by these hormones. The other animal models of type 2 diabetes, ob/ob mouse and fatty Zucker (fa/fa) rat, show similar impairment of hepatic glycogen metabolism. The concentrations of glycogen metabolizing enzymes are high and in vitro studies indicate enhanced rate of glycogen synthesis and breakdown. However, streptozotocin-induced diabetic animals and BB rats which resemble insulin-deficient type 1 diabetes are characterized by decreased glycogen turnover as a result of reduction in the levels of glycogen metabolizing enzymes.     

Increased pulmonary responses to acute ozone exposure in obese db/db mice

Epidemiological studies indicate the incidence of asthma is increased in obese and overweight humans. Responses to ozone (O(3)), an asthma trigger, are increased in obese (ob/ob) mice lacking the satiety hormone leptin. The long form of leptin receptor (Ob-R(b)) is required for satiety; mice lacking this receptor (db/db mice) are also substantially obese. Here, wild-type (WT) and db/db mice were exposed to air or O(3) (2 ppm) for 3 h. Airway responsiveness, measured by the forced oscillation technique, was greater in db/db than WT mice after air exposure. O(3)-induced increases in pulmonary resistance and airway responsiveness were also greater in db/db mice. BALF eotaxin, IL-6, KC, and MIP-2 increased 4 h after O(3) exposure and subsided by 24 h, whereas protein and neutrophils continued to increase through 24 h. For each outcome, the effect of O(3) was significantly greater in db/db than WT mice. Previously published results obtained in ob/ob mice were similar except for O(3)-induced neutrophils and MIP-2, which were not different from WT mice. O(3) also induced pulmonary IL-1beta and TNF-alpha mRNA expression in db/db but not ob/ob mice. Leptin was increased in serum of db/db mice, and pulmonary mRNA expression of short form of leptin receptor (Ob-R(a)) was similar in db/db and WT mice. These data confirm obese mice have innate airway hyperresponsiveness and increased pulmonary responses to O(3). Differences between ob/ob mice, which lack leptin, and db/db mice, which lack Ob-R(b) but not Ob-R(a), suggest leptin, acting through Ob-R(a), can modify some pulmonary responses to O(3).     

Dehydroepiandrosterone decreases elevated hepatic glucose production in C57BL/KsJ-db/db mice

Dehydroepiandrosterone (DHEA) is known to improve hyperglycemia in diabetic db/db mice that are obese and insulin resistant. In a previous study, we reported that DHEA suppresses the elevated hepatic gluconeogenic glucose-6-phosphatase (G6Pase) activity and gene expression in C57BL/KsJ-db/db mice. In the present study, we evaluated the total amount of gluconeogenesis using NaH[(14)C]CO(3) and hepatic glucose production using fructose as a substrate in primary cultured hepatocytes. Despite hyperinsulinemia, the glucose production of db/db mice in the total body and hepatocytes was elevated as compared to their heterozygote littermate C57BL/KsJ-db/+m mice. Administration of DHEA significantly decreased the blood glucose level and increased the plasma insulin level in db/db mice. Administration of DHEA decreased the elevated total body and hepatic glucose production in db/db mice. In addition, the glucose production in the primary cultured hepatocytes of db/db mice was decreased significantly by the direct addition of DHEA or DHEA-S to the medium. These results suggest that administration of DHEA suppresses the elevated total body and hepatic glucose production in db/db mice, and this effect on the liver is considered to result from increased plasma insulin and DHEA or DHEA-S itself.