Long-term maintenance of efficacy of dapagliflozin in patients with type 2 diabetes mellitus and cardiovascular disease.

AIM: To evaluate the long-term efficacy, safety and tolerability of dapagliflozin versus placebo added to usual care in patients with type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). METHODS: Data were pooled from two phase III studies (NCT01031680 and NCT01042977) in high-risk patients (N = 1887) with T2DM and CVD treated with dapagliflozin (10 mg/day) or placebo. Patients completing the double-blind treatment studies (24 weeks) entered one or two sequential double-blind, long-term (LT) extensions of 28 (LT1; n = 1649) and 52 (LT2; n = 568) weeks. RESULTS: Baseline and CVD characteristics were similar in the two groups. Patients entering LT1 and LT2 on dapagliflozin maintained a greater mean reduction in glycated haemoglobin (HbA1c) versus placebo at 52 weeks [LT1, -0.58% (95% confidence interval -0.68, -0.49)] and 104 weeks [LT2, -0.35% (95% confidence interval -0.59, -0.12)]. Mean body weight and systolic blood pressure (SBP) reductions versus placebo were maintained in patients entering LT1 (52 weeks; -2.23 kg and -3.25 mmHg, respectively) and LT2 (104 weeks; -3.16 kg and -2.03 mmHg, respectively). Patients on dapagliflozin had a better three-item composite endpoint of clinical benefit (glycaemia, weight and SBP) compared with placebo at week 24 (LT1, 10.1% vs. 1.1%) and week 104 (LT2, 6.7% vs. 1.4%). Genital and urinary tract infections were more frequent with dapagliflozin than with placebo. Events of hypoglycaemia, renal impairment/failure and volume depletion were similar between groups. CONCLUSIONS: The long-term efficacy of dapagliflozin to maintain reductions in HbA1c, SBP and body weight over 2 years, together with its tolerability profile, make dapagliflozin an appropriate option in high-risk patients with T2DM and CVD.

Inverse correlation of serum inflammatory markers with metabolic parameters in healthy, Black and White prepubertal youth.

OBJECTIVE: To examine for the first time the associations between pro-inflammatory cytokines and obesity-related metabolic biomarkers in, exclusively prepubertal, otherwise healthy obese and non-obese Black and White children, 7-9 years of age. DESIGN AND METHODS: Body mass index (BMI), homeostasis model assessment-estimated insulin resistance, visceral adipose tissue and subcutaneous adipose tissue (SAT (magnetic resonance imaging)); total body fat (dual-energy X-ray absorptiometry), ectopic, intrahepatic lipid (IHL) and intramyocellular lipid (IMCL) fat (proton magnetic resonance spectroscopy) and serum levels of interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1 were measured in 40 obese and non-obese children. Relationships between inflammatory cytokines and obesity were assessed by analysis of variance and Spearman's rank correlation. RESULTS: Significant inverse correlations were found between BMI z-score, SAT, total BF, and IHL and levels of TNF-α (Spearman's ρ=-0.36, -0.39, -0.43 and -0.39, respectively; P<0.05). Levels of IL-8 were significantly and inversely correlated with IMCL (-0.39; P=0.03) and remained significant after adjusting for race. IMCL was inversely associated with TNF-α only after adjusting for race (-0.37; P=0.04). CONCLUSIONS: Relationships between pro-inflammatory and metabolic markers commonly observed in adults are reversed in healthy, Black and White children before puberty. Prospective studies are warranted to determine how these inverse relationships modify chronic disease risk later in life.

Artemisia dracunculus L. extract ameliorates insulin sensitivity by attenuating inflammatory signalling in human skeletal muscle culture.

AIMS: Bioactives of Artemisia dracunculus L. (termed PMI 5011) have been shown to improve insulin action by increasing insulin signalling in skeletal muscle. However, it was not known if PMI 5011's effects are retained during an inflammatory condition. We examined the attenuation of insulin action and whether PMI 5011 enhances insulin signalling in the inflammatory environment with elevated cytokines. METHODS: Muscle cell cultures derived from lean, overweight and diabetic-obese subjects were used. Expression of pro-inflammatory genes and inflammatory response of human myotubes were evaluated by real-time polymerase chain reaction (RT-PCR). Insulin signalling and activation of inflammatory pathways in human myotubes were evaluated by multiplex protein assays. RESULTS: We found increased gene expression of monocyte chemoattractant protein 1 (MCP1) and TNFα (tumour necrosis factor alpha), and basal activity of the NFkB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway in myotubes derived from diabetic-obese subjects as compared with myotubes derived from normal-lean subjects. In line with this, basal Akt phosphorylation (Ser473) was significantly higher, while insulin-stimulated phosphorylation of Akt (Ser473) was lower in myotubes from normal-overweight and diabetic-obese subjects compared with normal-lean subjects. PMI 5011 treatment reduced basal phosphorylation of Akt and enhanced insulin-stimulated phosphorylation of Akt in the presence of cytokines in human myotubes. PMI 5011 treatment led to an inhibition of cytokine-induced activation of inflammatory signalling pathways such as Erk1/2 and IkBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha)-NFkB and moreover, NFkB target gene expression, possibly by preventing further propagation of the inflammatory response within muscle tissue. CONCLUSIONS: PMI 5011 improved insulin sensitivity in diabetic-obese myotubes to the level of normal-lean myotubes despite the presence of pro-inflammatory cytokines.

Efficacy and safety of canagliflozin monotherapy in subjects with type 2 diabetes mellitus inadequately controlled with diet and exercise.

AIMS: Canagliflozin is a sodium glucose co-transporter 2 inhibitor in development for type 2 diabetes mellitus (T2DM). The efficacy and safety of canagliflozin were evaluated in subjects with T2DM inadequately controlled with diet and exercise. METHODS: In this 26-week, randomized, double-blind, placebo-controlled, phase 3 trial, subjects (N = 584) received canagliflozin 100 or 300 mg or placebo once daily. Primary endpoint was the change from baseline in haemoglobin A1c (HbA1c) at week 26. Secondary endpoints included the proportion of subjects achieving HbA1c < 7.0%; change from baseline in fasting plasma glucose (FPG), 2-h postprandial glucose (PPG) and systolic blood pressure (BP); and percent change in body weight, high-density lipoprotein cholesterol (HDL-C) and triglycerides. Adverse events (AEs) were recorded throughout the study. RESULTS: At week 26, HbA1c was significantly reduced from baseline with canagliflozin 100 and 300 mg compared with placebo (-0.77, -1.03 and 0.14%, respectively; p < 0.001 for both). Both canagliflozin doses significantly decreased FPG, 2-h PPG, body weight and systolic BP (p < 0.001 for all), and increased HDL-C compared with placebo (p < 0.01 for both). Overall incidences of AEs were modestly higher with canagliflozin versus placebo; rates of serious AEs and AE-related discontinuations were low and similar across groups. Incidences of genital mycotic infections, urinary tract infections and osmotic diuresis-related AEs were higher with canagliflozin; these led to few discontinuations. The incidence of hypoglycaemia was low across groups. CONCLUSION: Canagliflozin treatment improved glycaemic control, reduced body weight and was generally well tolerated in subjects with T2DM inadequately controlled with diet and exercise.

Intrahepatic and intramyocellular lipids are determinants of insulin resistance in prepubertal children.

AIMS/HYPOTHESIS: We hypothesised that ectopic fat deposition is present in liver and skeletal muscle before puberty and that both are potentially important factors in the early pathogenesis of insulin resistance. METHODS: Proton magnetic resonance spectroscopy was used to evaluate intramyocellular and intrahepatic lipids in 50 male and 42 female multi-ethnic, prepubertal (Tanner < 2) children (8.1 ± 0.8 years; 35.4 ± 10.7 kg; 27.9 ± 8.3% body fat; means ± SD). Intramyocellular lipid was measured in soleus muscle and intrahepatic lipid in the middle right lobe. Abdominal fat was measured by magnetic resonance imaging, body fat by dual energy X-ray absorptiometry, and insulin resistance using homeostatic model assessment. RESULTS: Intrahepatic lipid ranged from 0.11% to 4.6% relative to the liver water signal (mean 0.79 ± 0.79%) whereas intramyocellular lipid ranged from 0.13% to 1.86% relative to the muscle water signal (mean 0.51 ± 0.28%). Intramyocellular and intrahepatic lipids were significantly correlated with total adiposity (r = 0.49 and 0.59), abdominal adiposity (r = 0.44 and 0.54), and each other (r = 0.39, p < 0.05, Spearman). Both intramyocellular and intrahepatic lipid were positively correlated with fasting insulin (r = 0.37 and 0.38 respectively) and insulin resistance (r = 0.37 and 0.37; p < 0.01). After adjustment for race and sex, the relations between ectopic fat and insulin resistance remained, whereas both disappeared when further adjusted for body fat or BMI z scores. CONCLUSIONS/INTERPRETATIONS: These results suggest that typical relations between body composition, ectopic fat and insulin resistance are present in children before puberty. Thus, interventions aimed at reducing adiposity have the potential to decrease ectopic fat accumulation, delay the onset of insulin resistance and decrease the risk for development of type 2 diabetes in children.

Template to improve glycemic control without reducing adiposity or dietary fat.

Drugs that improve chronic hyperglycemia independently of insulin signaling or reduction of adiposity or dietary fat intake may be highly desirable. Ad36, a human adenovirus, promotes glucose uptake in vitro independently of adiposity or proximal insulin signaling. We tested the ability of Ad36 to improve glycemic control in vivo and determined if the natural Ad36 infection in humans is associated with better glycemic control. C57BL/6J mice fed a chow diet or made diabetic with a high-fat (HF) diet were mock infected or infected with Ad36 or adenovirus Ad2 as a control for infection. Postinfection (pi), systemic glycemic control, hepatic lipid content, and cell signaling in tissues pertinent to glucose metabolism were determined. Next, sera of 1,507 adults and children were screened for Ad36 antibodies as an indicator of past natural infection. In chow-fed mice, Ad36 significantly improved glycemic control for 12 wk pi. In HF-fed mice, Ad36 improved glycemic control and hepatic steatosis up to 20 wk pi. In adipose tissue (AT), skeletal muscle (SM), and liver, Ad36 upregulated distal insulin signaling without recruiting the proximal insulin signaling. Cell signaling suggested that Ad36 increases AT and SM glucose uptake and reduces hepatic glucose release. In humans, Ad36 infection predicted better glycemic control and lower hepatic lipid content independently of age, sex, or adiposity. We conclude that Ad36 offers a novel tool to understand the pathways to improve hyperglycemia and hepatic steatosis independently of proximal insulin signaling, and despite a HF diet. This metabolic engineering by Ad36 appears relevant to humans for developing more practical and effective antidiabetic approaches.

Human adenovirus 36 decreases fatty acid oxidation and increases de novo lipogenesis in primary cultured human skeletal muscle cells by promoting Cidec/FSP27 expression.

BACKGROUND: It has been well documented that human adenovirus type 36 (Ad-36) is associated with obesity. However, the underlying molecular mechanism of Ad-36 inducing obesity remains unknown. We sought to investigate the effect of Ad-36 infection on Cidec, AMPK pathway and lipid metabolism in primary cultured human skeletal muscle cells. METHODS: Cidec/fat-specific protein 27 (FSP27), fatty acid oxidation, AMPK signaling and the abundance of proteins involved in lipid synthesis were determined in muscle cells infected with various doses (1.9-7.6 MOI) of Ad-36 and non-lipogenic adenovirus type 2 (Ad-2) as a negative control as well as an uninfected control. Cidec/FSP27 siRNA transfection was performed in Ad-36-infected muscle cells. RESULTS: Our data show that Ad-36 significantly reduced fatty acid oxidation in a dose-dependent manner (all P values are <0.01), but Ad-2 did not affect fatty acid oxidation. Ad-36 substantially increased Cidec/FSP27, ACC, sterol regulatory element-binding protein 1c (SREBP-1c), SREBP-2 and 3-hydroxy-3-methylglutaryl-CoA reductase protein abundance, but significantly reduced AMPK activity, mitochondrial mass and uncoupling protein 3 (UCP3) abundance in comparison with control cells (all P values are <0.01). Oil Red O staining revealed that there was substantial fat accumulation in the Ad-36-infected muscle cells. Furthermore, Cidec/FSP27 siRNA transfection significantly reduced FSP27 expression and partially restored AMPK signaling, increased UCP3 and decreased SERBP 1c and perilipin proteins in Ad-36-infected muscle cells. Interestingly, neither Ad-36 nor Ad-2 affected peroxisome proliferator-activated receptor γ protein expression in muscle cells. CONCLUSION: This study suggests that Ad-36 induced lipid droplets in the cultured skeletal muscle cells and this process may be mediated by promoting Cidec/FSP27 expression.

Efficacy and safety of sitagliptin when added to insulin therapy in patients with type 2 diabetes.

OBJECTIVE: To evaluate the efficacy and tolerability of sitagliptin when added to insulin therapy alone or in combination with metformin in patients with type 2 diabetes. METHODS: After a 2 week placebo run-in period, eligible patients inadequately controlled on long-acting, intermediate-acting or premixed insulin (HbA1c > or = 7.5% and < or = 11%), were randomised 1:1 to the addition of once-daily sitagliptin 100 mg or matching placebo over a 24-week study period. The study capped the proportion of randomised patients on insulin plus metformin at 75%. Further, the study capped the proportion of randomised patients on premixed insulin at 25%. The metformin dose and the insulin dose were to remain stable throughout the study. The primary endpoint was HbA1c change from baseline at week 24. RESULTS: Mean baseline characteristics were similar between the sitagliptin (n = 322) and placebo (n = 319) groups, including HbA1c (8.7 vs. 8.6%), diabetes duration (13 vs. 12 years), body mass index (31.4 vs. 31.4 kg/m(2)), and total daily insulin dose (51 vs. 52 IU), respectively. At 24 weeks, the addition of sitagliptin significantly (p < 0.001) reduced HbA1c by 0.6% compared with placebo (0.0%). A greater proportion of patients achieved an HbA1c level < 7% while randomised to sitagliptin as compared with placebo (13 vs. 5% respectively; p < 0.001). Similar HbA1c reductions were observed in the patient strata defined by insulin type (long-acting and intermediate-acting insulins or premixed insulins) and by baseline metformin treatment. The addition of sitagliptin significantly (p < 0.001) reduced fasting plasma glucose by 15.0 mg/dl (0.8 mmol/l) and 2-h postmeal glucose by 36.1 mg/dl (2.0 mmol/l) relative to placebo. A higher incidence of adverse experiences was reported with sitagliptin (52%) compared with placebo (43%), due mainly to the increased incidence of hypoglycaemia (sitagliptin, 16% vs. placebo, 8%). The number of hypoglycaemic events meeting the protocol-specified criteria for severity was low with sitagliptin (n = 2) and placebo (n = 1). No significant change from baseline in body weight was observed in either group. CONCLUSION: In this 24-week study, the addition of sitagliptin to ongoing, stable-dose insulin therapy with or without concomitant metformin improved glycaemic control and was generally well tolerated in patients with type 2 diabetes.

Failure of dietary quercetin to alter the temporal progression of insulin resistance among tissues of C57BL/6J mice during the development of diet-induced obesity.

AIMS/HYPOTHESES: High-fat diets produce obesity and glucose intolerance by promoting the development of insulin resistance in peripheral tissues and liver. The present studies sought to identify the initial site(s) where insulin resistance develops using a moderately high-fat diet and to assess whether the bioflavonoid, quercetin, ameliorates progression of this sequence. METHODS: Four cohorts of male C57BL/6J mice were placed on diets formulated to be low-fat (10% of energy from fat), high-fat (45% of energy from fat) or high-fat plus 1.2% quercetin (wt/wt). After 3 and 8 weeks, cohorts were evaluated using euglycaemic-hyperinsulinaemic clamps, metabolomic analysis of fatty acylcarnitines and acute in vitro assessments of insulin signalling among tissues. RESULTS: After 3 and 8 weeks, the high-fat diet produced whole-body insulin resistance without altering insulin-dependent glucose uptake in peripheral tissues. The primary defect was impaired suppression of hepatic glucose production by insulin at both times. Quercetin initially exacerbated the effect of high-fat diet by further increasing hepatic insulin resistance, but by 8 weeks insulin resistance and hepatic responsiveness to insulin were similarly compromised in both high-fat groups. The high-fat diet, irrespective of quercetin, increased short-chain fatty acylcarnitines in liver but not in muscle, while reciprocally reducing hepatic long-chain fatty acylcarnitines and increasing them in muscle. CONCLUSIONS/INTERPRETATION: Failure of insulin to suppress hepatic glucose output is the initial defect that accounts for the insulin resistance that develops after short-term consumption of a high-fat (45% of energy) diet. Hepatic insulin resistance is associated with accumulation of short- and medium-, but not long-chain fatty acylcarnitines. Dietary quercetin does not ameliorate the progression of this sequence.

Pharmacotherapy for the treatment of patients with type 2 diabetes mellitus: rationale and specific agents.

Type 2 diabetes, the most common form of diabetes, is characterized by abnormalities in hepatic glucose production, insulin resistance, and a progressive decline in beta-cell function over time. To treat effectively the individual with type 2 diabetes, the provider must have a thorough understanding of the underlying pathophysiology to provide treatment that precisely addresses the metabolic abnormalities. Currently, the provider who cares for subjects with type 2 diabetes can choose an antidiabetic agent from no less than eight pharmacologic classes. These classes include agents that increase insulin secretion, improve insulin action, and delay absorption of carbohydrates. The newer treatments available, specifically incretin therapy, address a previously unmet need in diabetes by modulating glucose supply. The currently available agents can be combined and combination therapy markedly improves glycemic control. This allows the provider to design regimens to specifically address underlying abnormalities. A review of all currently available agents is provided.

Liver and kidney tissue membranes as tissue markers for nonenzymatic glycosylation.

We investigated the relationship of serum protein glycosylation to peripheral tissue membrane glycosylation. We studied 27 Sprague-Dawley rats and induced diabetes in 20 of them. Blood glucose levels were treated in 10 of the diabetic animals with daily subcutaneous insulin. After 8 wk, liver and kidney tissue was removed, purified membranes were prepared, and the percentage of glycosylated membrane protein was determined for the liver and kidney membranes by boronate-affinity methods. The percentage of glycosylated membrane protein for both liver and kidney tissue was found to correlate significantly with the glycemic state of the animal as assessed with glycosylated serum albumin, total glycosylated serum proteins, and glycosylated hemoglobin determinations (P less than 0.001 for each glycosylated protein parameter). In addition, the percentage of glycosylated membrane protein in the liver tissue correlated significantly with the measured level in the corresponding kidney tissue (r = 0.78, P less than 0.001). To identify the nature of the glycosylated membrane proteins, boronate-affinity methods were used to separate the glycosylated and nonglycosylated membrane proteins. It was determined that two major glycosylated protein bands exist for the liver membrane (78,000 and 58,700 Mr) and four for the kidney membranes (ranging from 48,700 to 74,000 Mr). The ultrastructural location and identification of these glycosylated membrane proteins are not known. This study demonstrates that measurement of clinical glycemic state, as reflected in glycosylated blood protein parameters such as glycosylated serum albumin and glycosylated hemoglobin, correlates significantly with ongoing tissue membrane accumulation of glucose.

Effect on glycemic control of the addition of 2.5 mg glipizide GITS to metformin in patients with T2DM.

AIMS: This study evaluated the effects on glycemic control of the addition of 2.5 mg glipizide GITS to metformin in patients with mild-to-moderate, but suboptimally controlled type 2 diabetes. METHODS: In this multicenter, double-blind, placebo-controlled study, 122 patients with type 2 diabetes inadequately controlled (A1c 7-8.5%) on metformin (> or =1000 mg/day for > or =3 months) were randomized to 16 weeks treatment with 2.5 mg/day glipizide GITS (n=61) or placebo (n=61), in addition to their current metformin dose. The primary efficacy variable was the change in A1c from baseline to endpoint. Changes in fasting plasma glucose (FPG), insulin concentrations, lipid profile and safety variables were also measured. RESULTS: The addition of glipizide GITS to metformin gave significantly greater improvements in mean A1c and FPG from baseline to endpoint than placebo addition (p<0.0002). Significantly more patients in the glipizide GITS group than in the placebo group achieved the target A1c level of A1c<7.0% (p<0.0001) and an A1c<6.5% (p<0.0033). Fasting insulin concentrations were similar in both groups and unchanged by treatment. Addition of glipizide GITS to metformin did not produce any significant or clinically relevant weight gain or changes in BMI. Both treatment regimens were well tolerated. CONCLUSIONS: This study showed that the addition of 2.5 mg glipizide GITS to metformin significantly improved glucose control in patients with type 2 diabetes inadequately controlled by metformin monotherapy.

Comparative efficacy of a once-daily controlled-release formulation of glipizide and immediate-release glipizide in patients with NIDDM.

OBJECTIVE: To compare the efficacy and safety of controlled-release glipizide (glipizide-GITS [gastrointestinal therapeutic system]) and immediate-release glipizide in patients with non-insulin-dependent diabetes mellitus (NIDDM). RESEARCH DESIGN AND METHODS: In a multicenter, open-label, randomized, two-way crossover study, 132 patients with NIDDM received daily doses of 5, 20, or 40 mg of either glipizide-GITS or immediate-release glipizide for 8 weeks followed by 8 weeks of the alternate formulation. Plasma glucose, serum insulin, C-peptide, and plasma glipizide levels were measured at fasting and post-Sustacal challenge at the end of 1 and 8 weeks of each treatment phase. HbA1c was measured at the end of weeks 7 and 8 of each treatment phase. RESULTS: Both formulations of glipizide yielded similar mean HbA1c values. However, mean fasting plasma glucose (FPG) levels were significantly lower with glipizide-GITS treatment than with immediate-release glipizide at the end of week 1 (11.0 vs. 11.6 mmol/l; P < 0.01) and at the end of the 8-week treatment phase (10.9 vs. 11.7 mmol/l; P < 0.001). Fasting insulin and C-peptide levels were lower after 5 mg glipizide-GITS vs. immediate-release glipizide. Glucose responses to Sustacal were similar after both formulations of glipizide; however, serum insulin (P < 0.01) and C-peptide responses (P < 0.05) were lower with glipizide-GITS than with immediate-release glipizide treatment at the end of the 8-week treatment phase. Mean plasma glipizide concentrations were stable by the end of week 1, and the concentrations increased proportionately with dose. Once-daily Glipizide-GITS provided effective mean glipizide concentrations (> 50 ng/ml) 24 h after dosing, even at the lowest (5 mg) dose level. Both formulations were well tolerated. CONCLUSIONS: Glipizide-GITS was significantly more effective than immediate-release glipizide in reducing FPG levels. Both formulations reduced postprandial plasma glucose levels equally; however, glipizide-GITS exerted its control in the presence of lower plasma glipizide concentrations in addition to significantly lower insulin and C-peptide levels. This suggests that glipizide-GITS improves insulin sensitivity.

Total serum glycosylated proteins in detection and monitoring of gestational diabetes.

The goal of this study was to determine whether serum glycosylated protein levels (i.e., fructosamine) can reliably screen for gestational diabetes and whether these levels are valid markers of short-term glycemic control in the third trimester of pregnancy. Ninety-seven pregnant women at 26-28 wk gestation were evaluated over 9 mo. HbA1c and serum glycosylated protein (serum fructosamine) were determined at the baseline venipuncture of the 100-g oral glucose tolerance test performed to detect gestational diabetes. Of the 97 women studied, 13 tested positive for gestational diabetes (National Diabetes Data Group criteria). There were significant differences in the fasting and 1-, 2-, and 3-h glucose values between nondiabetic and diabetic patients (P less than 0.005 at each time point). No difference was noted in the baseline serum glycosylated protein level (2.02 +/- 0.08 vs. 1.98 +/- 0.02 mM, NS) or HbA1c level (4.42 +/- 0.2 vs. 4.6 +/- 0.3%, NS) between gestational and nondiabetic patients. Diabetic patients were followed at 2-wk intervals, with serum glycosylated protein analysis, HbA1c, fasting glucose, and mean glucose determined by outpatient monitoring. Serum glycosylated protein correlated significantly to fasting blood glucose (r = 0.81, P less than 0.001) and mean outpatient glucose (r = 0.62, P less than 0.001) at the 2-wk follow-up visits. No correlation was found between HbA1c and fasting blood glucose (r = 0.11, NS) or mean outpatient glucose (r = -0.12, NS) during the follow-up period. The serum glycosylated protein level (serum fructosamine) is not a useful screening test for gestational diabetes. However, this assay shows potential as an objective marker of short-term control in evaluating the maternal glycemic state.

In vivo effects of dietary quercetin and quercetin-rich red onion extract on skeletal muscle mitochondria, metabolism, and insulin sensitivity.

Red onions and low doses of the flavonoid, quercetin, increase insulin sensitivity and improve glucose tolerance. We hypothesized that dietary supplementation with red onion extract (RO) would attenuate high fat diet (HFD)-induced obesity and insulin resistance similar to quercetin supplementation by increasing energy expenditure through a mechanism involving skeletal muscle mitochondrial adaptations. To test this hypothesis, C57BL/6J mice were randomized into four groups and fed either a low fat diet (LF), HFD (HF), HFD + quercetin (HF + Q), or HFD + RO (HF + RO) for 9 weeks. Food consumption and body weight and composition were measured weekly. Insulin sensitivity was assessed by insulin and glucose tolerance tests. Energy expenditure and physical activity were measured by indirect calorimetry. Skeletal muscle incomplete beta oxidation, mitochondrial number, and mtDNA-encoded gene expression were measured. Quercetin and RO supplementation decreased HFD-induced fat mass accumulation and insulin resistance (measured by insulin tolerance test) and increased energy expenditure; however, only HF + Q showed an increase in physical activity levels. Although quercetin and RO similarly increased skeletal muscle mitochondrial number and decreased incomplete beta oxidation, establishing mitochondrial function similar to that seen in LF, only HF + Q exhibited consistently lower mRNA levels of mtDNA-encoded genes necessary for complexes IV and V compared to LF. Quercetin- and RO-induced improvements in adiposity, insulin resistance, and energy expenditure occur through differential mechanisms, with quercetin-but not RO-induced energy expenditure being related to increases in physical activity. While both treatments improved skeletal muscle mitochondrial number and function, mtDNA-encoded transcript levels suggest that the antiobesogenic, insulin-sensitizing effects of purified quercetin aglycone, and RO may occur through differential mechanisms.

Augmented transport and metabolism of sex steroids in lymphoid neoplasia in the rat.

Sex steroid hormones have been shown to influence a number of biological properties of lymphoid neoplastic tissue. Since receptor occupancy is a function of the pool size of cellular exchangeable hormone, it is important to understand the mechanisms regulating hormone transport from the microcirculation and hormone metabolism, since these two pathways are the dominant factors controlling cellular exchangeable hormone. In the present studies, steroid hormone transport and metabolism were investigated in control and neoplastic lymph nodes after transplanting control rats with the WR-6 leukemic line. Steroid hormone transport and metabolism were studied after pulse labeling the nodal tissue in vivo with arterial bolus injections of [3H]testosterone. Residual vascular radioactivity was monitored by simultaneously injecting 113mindium chelated to bovine transferrin. Both testosterone and estradiol were partially available for transport through the capillary barriers of control and neoplastic lymph nodes from the circulating albumin-bound pool. Estradiol was readily available for transport from the circulating sex hormone-binding globulin-bound pool in both control and neoplastic lymph nodes. Testosterone was not available for transport from the sex hormone-binding globulin-bound pool in control lymph nodes, but was readily available for transport in metastatic lymph nodes. Thaw-mount autoradiography and physiological measurements showed that plasma proteins such as albumin or transferrin were confined to the microcirculation compartment. Therefore, the transport of protein-bound hormones into lymph node represents a mechanism of enhanced steroid hormone dissociation from the binding protein without the plasma protein per se significantly exiting the microcirculation compartment. Metabolic studies showed no measurable metabolism of [3H]testosterone in the control lymph nodes by 60 sec after arterial injection. However, testosterone was extensively metabolized in metastatic lymph nodes; the major metabolites formed from testosterone comigrated on a two-dimensional TLC system with epiandrosterone/androsterone and dihydrotestosterone. In conclusion, these studies indicate that both steroid hormone transport and metabolism are augmented in the lymphoid neoplastic state, and both processes may alter the concentration of cellular exchangeable hormone and, thus, steroid hormone receptor occupancy in lymphoid neoplasms.

Serum bioavailability and tissue metabolism of testosterone and estradiol in rat salivary gland.

The concentration of testosterone in saliva is probably in equilibrium with the concentration of cellular exchangeable testosterone in salivary gland, and these pools are a function of hormone transplant from the plasma compartment, and hormone metabolism in salivary gland cells. Both of these processes were examined in the present study using the carotid injection technique in normal and pilocarpine-stimulated ketamine-anesthetized rats. Both testosterone and estradiol were rapidly transported across salivary gland capillaries in vivo from the circulating albumin-bound pool. Estradiol, but not testosterone, was also rapidly transported into salivary gland from the circulating human sex hormone-binding globulin-bound pool. Hormone transport was several-fold greater than the capillary transport of [3H]bovine albumin, indicating that bound hormone was available for transport across salivary gland capillaries via an enhanced dissociation mechanism, with the plasma protein primarily residing in the plasma compartment. This result was confirmed by thaw-mount autoradiography, which showed diffuse distribution of [3H]testosterone in salivary gland, but vascular retention of [3H]bovine albumin. The concentration of exchangeable cellular testosterone in rat saliva was less than 4% of the total or plasma exchangeable testosterone in the rat. This marked discrepancy between the concentration of plasma and cellular exchangeable hormone suggested that there was rapid metabolism of androgen by salivary gland in vivo. This was confirmed by chromatographic separation of [3H] testosterone and labeled metabolites in homogenates of salivary gland. By 60 sec after injection, approximately 30% of the radioactivity in the salivary gland was in the form of androgen metabolites, which primarily comigrated with an androstenedione standard. The data indicate that albumin-bound testosterone, albumin-bound estradiol, and sex hormone-binding globulin-bound estradiol are all exchangeable in salivary gland capillaries. The low concentration of cellular exchangeable testosterone in salivary gland appears to be due to rapid tissue metabolism of this hormone. Thus, changes in androgen metabolism may alter salivary gland hormone concentrations independent of any change in the concentration of biologically active hormone in plasma.

Hepatic bioavailability of thyroxine and testosterone in familial dysalbuminemic hyperthyroxinemia.

The bioavailability of [125I]T4 or [3H]testosterone in serum obtained from normal subjects and from subjects with familial dysalbuminemic hyperthyroxinemia (FDH) was studied with a portal vein injection technique in ketamine-anesthetized rats. In the present studies this technique was modified by performing uptake measurements in the presence of serum loaded with either 25 microM T4 or 1 microM testosterone. Loading of serum with these high concentrations displaced the labeled hormone from the lower capacity globulin or prealbumin-binding sites to the high capacity albumin or dysalbumin-binding sites, and allowed for the analysis of hormone availability in liver when the labeled hormone was delivered to the tissue bound either to albumin or to dysalbumin binding sites. In the presence of normal serum, 33 +/- 3% (SE) of T4 was available to rat liver, as opposed to 20 +/- 2% for FDH serum. When normal serum was loaded with 25 microM T4, the bioavailable T4 increased to 97 +/- 2%, consistent with the availability of T4 bound to albumin. However, the hepatic bioavailability of T4 in the presence of 25 microM T4 in FDH serum was only 33 +/- 4%. Testosterone bioavailability was similar in normal and in FDH sera, and was 49 +/- 7% in the absence and 99 +/- 4% in the presence of 1 microM testosterone. These studies suggest that T4 bound to the FDH albumin binding site is not readily available for entry into liver, whereas T4 bound to the normal albumin binding site is freely available for uptake in vivo. The differential bioavailability of T4 is compatible with the model that the normal and FDH binding sites are situated on different parts of the albumin molecule, and that only T4 bound to the normal binding site is freely available for delivery to the liver.

Enhanced hepatic extraction of estrogens used for replacement therapy.

Oral estrogen administration elicits greater effects on hepatic than on nonhepatic markers of estrogen action. This has important clinical implications, since the hepatic actions of estrogens are believed to account for several complications of this form of therapy. To date, the mechanism responsible has been attributed to the so-called first pass effect of the orally administered hormones. The present study was undertaken to examine the in vivo extraction from the circulation of all commercially available classes of estrogens used for replacement therapy. Influxes from the microcirculation into three important target organs of estrogen action, i.e. the brain, uterus, and liver, were assessed. This was accomplished by the use of previously described double isotope, single injection, timed tissue-sampling techniques. For the brain, two patterns of influx were found using different injection vehicles. The first was characterized by high extraction (80-100%) in the absence of plasma proteins. The only inhibitory effects on influx were exerted by plasma proteins. Estrogens displaying this pattern were estradiol, estrone, and ethinyl estradiol. The second pattern was characterized by very restricted influx in the absence of plasma proteins, and plasma protein binding had little or no additional effect. In the absence of plasma proteins, the percentages extracted of estrone sulfate (E1S) and diethylstilbestrol were 6.5% and 38.5%, respectively. For the uterus, the patterns of extraction of all five estrogens were similar to those found for the brain. In contrast, the hepatic microvasculature was freely permeable to all estrogens including E1S and diethylstilbestrol. Albumin binding had little or no effect on hepatic uptake. Significant reductions in the influx of estradiol and E1S were found only when the injection vehicle was human pregnancy serum (high sex hormone-binding globulin concentration). In the presence of plasma proteins, the hepatic extraction of all of the estrogens studied significantly exceeded that in the brain and uterus. We conclude that enhanced delivery of circulating estrogens to the liver compared to that to the brain and uterus provides a further explanation for the enhanced hepatic actions of these preparations when used for oral replacement therapy.