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OBJECTIVE Obstructive sleep apnea (OSA) is associated with insulin resistance and has been described as a risk factor for type 2 diabetes. Whether OSA adversely impacts pancreatic islet β-cell function remains unclear. We aimed to investigate the association of OSA and short sleep duration with β-cell function in overweight/obese adults with prediabetes or recently diagnosed, treatment-naive type 2 diabetes. RESEARCH DESIGN AND METHODS Two hundred twenty-one adults (57.5% men, age 54.5 ± 8.7 years, BMI 35.1 ± 5.5 kg/m2) completed 1 week of wrist actigraphy and 1 night of polysomnography before undergoing a 3-h oral glucose tolerance test (OGTT) and a two-step hyperglycemic clamp. Associations of measures of OSA and actigraphy-derived sleep duration with HbA1c, OGTT-derived outcomes, and clamp-derived outcomes were evaluated with adjusted regression models. RESULTS Mean ± SD objective sleep duration by actigraphy was 6.6 ± 1.0 h/night. OSA, defined as an apnea-hypopnea index (AHI) of five or more events per hour, was present in 89% of the participants (20% mild, 28% moderate, 41% severe). Higher AHI was associated with higher HbA1c (P = 0.007). However, OSA severity, measured either by AHI as a continuous variable or by categories of OSA severity, and sleep duration (continuous or CONCLUSIONS In this baseline cross-sectional analysis of the RISE clinical trial of adults with prediabetes or recently diagnosed, untreated type 2 diabetes, the prevalence of OSA was high. Although some measures of OSA severity were associated with HbA1c, OSA severity and sleep duration were not associated with measures of insulin sensitivity or β-cell responses.

Background We evaluated whether diet quality is a predictor of weight loss and reduced diabetes risk, independent of caloric intake in the Diabetes Prevention Program (DPP) cohort, a randomized clinical trial of adults at risk for diabetes. Methods This secondary analysis included 2914 participants with available data (964 intensive lifestyle (ILS), 977 metformin, 973 placebo). Dietary intake was assessed using a 117-item food frequency questionnaire. Diet quality was quantified using the Alternative Healthy Eating Index 2010 (AHEI). AHEI ranges from 0 to 110, with higher scores corresponding to higher quality diets. ILS participants had greater improvement (p Results An increase in AHEI score was associated with weight loss in ILS [β per 10-point increase (SE) -1.2 kg (0.3, p p p = 0.01)]. However, AHEI change was not associated with incident diabetes in any group before or after adjustment for weight change. Conclusions Controlling for weight, diet quality was not associated with diabetes incidence but helps achieve weight loss, an important factor in diabetes prevention.

OBJECTIVE: Poor sleep may increase obesity and type 2 diabetes (T2D) risk in youth. We explored whether subjective sleep duration, sleep quality, or risk for obstructive sleep apnea (OSA) are associated with glycemia, body mass index (BMI), or blood pressure (BP) in overweight/obese youth. METHODS: Two-hundred and fourteen overweight/obese youth of 10 to 19 years of age at risk for or recently diagnosed with T2D who were screened for the Restoring Insulin Secretion (RISE) Study had a 2-hour oral glucose tolerance test (OGTT) and completed a Cleveland Adolescent Sleepiness questionnaire and a Sleep Disturbances Scale questionnaire. Independent associations between sleep variables and measures of glycemia, BMI, and BP were evaluated with regression models. RESULTS: The multiethnic cohort was 67% female, 14.1 ± 2.1 years, and BMI 35.9 ± 6.5 kg/m(2). Habitual sleep duration

Lifestyle changes may reduce the burdens of diabetes and cancer. We examined how changes in the 2018 WCRF/AICR Score, reflecting key cancer prevention recommendations, are associated with diabetes risk. The Diabetes Prevention Program (DPP) randomized adults with prediabetes to a lifestyle intervention (ILS), metformin (MET), or placebo (PLB) for a mean of 3.2 years, with follow-up in DPPOS for 11 years. The Score includes 7 components: weight, physical activity, plant-based diet, red/processed meat, sugar-sweetened drinks, fast foods, and alcohol (0-7 points, with 7 as the best total score). Scores were estimated at years 0, 1, 5, 6, 9, and 15 for participants with complete baseline data (N = 3,147). Covariates included sex, age, race/ethnicity, marital status, education, family history of diabetes, hormone therapy (females), and energy intake. Adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) stratified by treatment group estimated the association of Score changes with diabetes risk in DPP and DPPOS within and between groups. There were significant Score improvements within all groups over 15 years (p < 0.001); the differences between groups were only significant after 1 year (p < 0.001; 15 years: p = 0.137). For every 1-unit improvement in Score during DPP, there was a 29% and 16% lower diabetes risk in ILS (95% CI: 0.58 - 0.88) and PLB (95% CI: 0.72 - 0.99), respectively, and no significant association in MET. The time-dependent Score improvements in ILS and PLB were significantly associated with lower diabetes risk through year 15 (ILS HR: 0.86, 95% CI 0.77 - 0.96; PLB HR: 0.85, 95% CI 0.76 - 0.95). Changes in weight, physical activity, and plant-based diet appeared to be drivers of the Score’s effect. Findings suggest that Score improvements are associated with a long-term, lower risk for diabetes among adults with prediabetes. Future research can explore how this risk reduction impacts cancer risk. Disclosure M. M. M. Shams-white: None. B. M. Heckman-stoddard: None. D. Research group: None. A. H. Tjaden: None. S. Edelstein: None. S. Bassiouni: None. C. Kim: None. X. Pi-sunyer: None. K. A. Temple: None. E. M. Venditti: None. J. Reedy: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (U01DK048489)

Objective: Obstructive sleep apnea (OSA) is associated with insulin resistance and has been described as a risk factor for type 2 diabetes. Whether OSA adversely impacts pancreatic islet beta-cell function remains unclear. We aimed to investigate the association of OSA and short sleep duration with beta-cell function in overweight/obese adults with prediabetes or recently-diagnosed, treatment-naïve type 2 diabetes. Research Design and Methods: 221 adults (57.5% men, age 54.5±8.7 years, BMI 35.1±5.5 kg/m2) completed one week of wrist actigraphy and one night of polysomnography before undergoing a 3-h oral glucose tolerance test (OGTT) and a two-step hyperglycemic clamp. Associations of measures of OSA and actigraphy-derived sleep duration with HbA1c, OGTT-derived and clamp-derived outcomes were evaluated with adjusted regression models. Results: Mean±SD objective sleep duration by actigraphy was 6.6±1.0 hours/night. OSA defined as an apnea-hypopnea index (AHI) ≥5 events per hour was present in 89% of the participants; 20% mild, 28% moderate and 41% severe. Higher AHI was associated with higher HbA1c (p =0.007). However, OSA severity, measured by either AHI as a continuous variable or by categories of OSA severity, and sleep duration (continuous or Conclusion: In this baseline cross-sectional analysis of the RISE clinical trial of adults with prediabetes or recently-diagnosed, untreated type 2 diabetes, the prevalence of OSA was high. Although some measures of OSA severity were associated with HbA1c, OSA severity and sleep duration were not associated with measures of insulin sensitivity or beta-cell responses.

In cross-sectional studies, adolescents with T2D show poorer cognition than normoglycemic youth. In the Restoring Insulin Secretion (RISE) Study, we assessed longitudinal relationships between insulin sensitivity, β-cell function and cognition in 72 youth (mean age 14.3±2.0, 10-18 y) with IGT or recently diagnosed T2D, randomized to 12 months of metformin (MET) or 3 months of glargine followed by 9 months of metformin (G/M). Hyperglycemic clamps were used to measure insulin sensitivity and β-cell responses: acute (0-10 min) C-peptide response to glucose, steady-state C-peptide (SSCP) at glucose of 11.1 mmol/L, and arginine-stimulated maximum C-peptide response (ACPRmax) at glucose >25 mmol/L. A cognitive battery (CogStateTM and story recall) assessed 1) reaction time; 2) visual discrimination; 3) attention and working memory; and 4) learning and episodic memory. A composite measure of cognition was constructed by scaling the sum of the four domain scores. Linear regression models were fit to assess relationships between cognition, treatment and β-cell function. Treatment group models were adjusted for baseline cognitive function, HbA1c and fasting glucose. β-cell models were adjusted for baseline cognitive and β-cell function. Cognitive composite scores improved over the 12-month treatment period (p Disclosure S. Craft: Advisory Panel; Self; vTv Therapeutics. M. Tripputi: None. S. Edelstein: None. M. Espeland: Other Relationship; Self; Boehringer Ingelheim International GmbH, Ironwood Pharmaceuticals. A. Claxton: Employee; Self; Alkermes plc. S.A. Arslanian: Research Support; Self; National Institutes of Health. Other Relationship; Self; AstraZeneca, Eli Lilly and Company, Novo Nordisk Inc. E. Barengolts: None. T.S. Hannon: None. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Intarcia Therapeutics, Janssen Scientific Affairs, LLC., Merck & Co., Inc., Novo Nordisk A/S, Pfizer Inc. K.J. Mather: Research Support; Self; Abbott, Merck & Co., Inc., Novo Nordisk Inc., Sanofi. B. Mokhlesi: None. K.J. Nadeau: None. A. Sanderlin: None. K.A. Temple: None. P. Zeitler: Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo, Eli Lilly and Company, Merck & Co., Inc. T. Consortium: None. Funding American Diabetes Association (1-14-RISE-01 to S.E.K.); National Institute of Diabetes and Digestive and Kidney Diseases

The prevalence of prediabetes and diabetes is substantially higher in PCOS women with obstructive sleep apnea (OSA) compared to PCOS women without OSA1,2,3. Prior studies, however, did not examine the complex interaction between race and OSA on metabolic function in PCOS. We sought to determine if the impact of OSA on glucose and insulin metabolism is affected by race. We studied non-Hispanic white (NHW) (n=53) and African-American (AA) (n=48) women with PCOS. Following an overnight polysomnogram (PSG), PCOS women (NHW without OSA n=40; NHW with OSA n=13; AA without OSA n=36; AA with OSA n=12) had a 2-h 75-g oral glucose tolerance test (OGTT) with blood sampling every 30 minutes for measurement of glucose, insulin, and C-peptide concentrations. OSA severity was measured by the Apnea-Hypopnea Index (AHI). Only women without OSA (AHI < 5) or with moderate-to-severe OSA (AHI > 15) were included in these analyses; women with mild OSA were excluded. Insulin secretion rates (ISR) during the OGTT were derived by deconvolution of C-peptide levels 4. Area under the curve (AUC) response to the glucose challenge was calculated using the trapezoidal method. BMI and age did not differ between races in PCOS women without OSA (BMI [kg/m2]: 36.3±1.2 vs. 37.2±1.1, p=0.58; Age [yr]: 27.7±0.8 vs. 27.2±0.8, p=0.65; for NHW and AA respectively), or in PCOS women with OSA (BMI [kg/m2]: 42.8±1.7 vs. 44.7±2.0, p=0.50; Age [yr]: 31.4±1.6 vs. 28.6±1.6, p=0.18; for NHW and AA respectively). OSA severity was similar in NHW and AA PCOS women without OSA (AHI: 1.5±0.2 vs 2.1±0.2, p=0.076), and PCOS women with OSA (AHI: 32.0±4.9 vs. 28.3±4.4, p=0.26). Higher glucose responses during the OGTT were observed in NHW PCOS women with OSA compared to both NHW (AUC: 18,965±648 vs. 15,797±371, p=0.0004) and AA (AUC: 18,965±648 vs. 15,801±497, p=0.0005) PCOS women without OSA. Glucose responses did not differ significantly between AA PCOS women with OSA and AA PCOS women without OSA (AUC: 17,104±965 vs. 15,801±497, p=0.15). Similarly, ISR was higher in NHW PCOS women with OSA compared to both NHW (AUC: 5,648±488 vs. 3,907±231, p=0.0006) and AA (AUC: 5,648±488 vs. 3,981±235, p=0.0011) PCOS women without OSA. ISR did not differ significantly between AA PCOS women with OSA and AA PCOS women without OSA (AUC: 4,827±461 vs. 3,981±235, p=0.09). CONCLUSIONS: OSA has a greater impact on glucose and ISR during an oral glucose challenge in NHW compared to AA women with PCOS. Future studies would benefit from including race when evaluating metabolic outcomes in women with PCOS. References: 1Fogel et al., J Clin Endocrinol Metab. 2001: 86:1175–1180. 2Kapsimalis et al., Sleep. 2002; 25:499–506. 3Kapsimalis et al., Sleep. 2002; 25:412–419. 4Polonsky et al., J Clin Invest. 1986 Jan; 77(1):98–105.

Insulin resistance and impaired β-cell function are associated with risk of cognitive decline in adults over 65 y of age. Their impact on cognition in youth and adults 25 mmol/L. The cognitive battery (CogStateTM and story recall) assessed 1) psychomotor speed; 2) visual pattern separation; 3) visual attention and working memory; and 4) verbal learning and episodic memory. Linear regression models were fit to assess relationships between cognition and clamp measures by age group, adjusted for age, education (adults only), race, sex, and diabetes status. Models including β-cell responses were also adjusted for M/I to account for the role of insulin sensitivity to modulate β-cell function. M/I was not associated with cognitive outcomes for adults or children. For adults, reaction time was slower for participants with worse β-cell function assessed by ACPRg and ACPRmax (p Disclosure S. Craft: Advisory Panel; Self; vTv Therapeutics. Research Support; Self; Eli Lilly and Company. A. Claxton: Employee; Self; Alkermes. M. Tripputi: None. S. Edelstein: None. S.A. Arslanian: None. K.A. Temple: None. K.J. Nadeau: None. A. Sanderlin: None. M. Espeland: Research Support; Self; National Institutes of Health. Other Relationship; Self; Boehringer Ingelheim International GmbH, Ironwood Pharmaceuticals, Inc. B. Mokhlesi: None. K.J. Mather: Advisory Panel; Self; Roche Diabetes Care. Research Support; Self; Abbott, Merck & Co., Inc., Novo Nordisk A/S, Sanofi. P. Zeitler: Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Janssen Research & Development, Merck & Co., Inc., Novo Nordisk Inc. R. Consortium: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases

We examined the relationship between habitual daily activity and measures of GT, insulin sensitivity and β-cell responses in 230 adults (mean age 55 y; BMI 35 kg/m2) with IGT or drug-naïve, recently diagnosed T2D. Participants underwent a 3-h OGTT and hyperglycemic clamp. Wrist accelerometers worn for 7 days measured total activity counts (TAC; daily mean 233,461 {∼ 50th %ile for age}). We evaluated whether TAC predicted fasting plasma glucose (FPG); OGTT 2-h plasma glucose (2-h G) or glucose incremental area under the curve (G-iAUC); hyperglycemic clamp measures of insulin sensitivity (steady-state glucose infusion rate/insulin [M/I]) and β-cell responses (acute C-peptide response to glucose [ACPRg], steady-state C-peptide, maximal β-cell response [ACPRmax]); and OGTT C-peptide index (CPI, [ΔC-Peptide0-30/ΔGlucose0-30]). There was no association of TAC with FPG, 2-h G, or G-iAUC. Higher TAC was associated with higher insulin sensitivity. After adjusting for M/I, higher TAC was associated with lower CPI, but not with clamp-based β-cell responses. Despite similar post-load glucose values, adults with IGT or T2D who were more active demonstrated a greater than expected reduction in C-peptide response given their higher insulin sensitivity. Further studies are needed to understand this dissociation of activity effects on insulin sensitivity and β-cell function. Disclosure K.A. Temple: None. A.H. Tjaden: None. K.M. Atkinson: None. E. Barengolts: None. T.S. Hannon: Advisory Panel; Self; Eli Lilly and Company. K.J. Mather: Advisory Panel; Self; Roche Diabetes Care. Research Support; Self; Abbott, Merck & Co., Inc., Novo Nordisk A/S, Sanofi. K. Utzschneider: Consultant; Self; Novo Nordisk Inc. D.A. Ehrmann: None. B. Mokhlesi: None. R. Consortium: None. Funding National Heart, Lung, and Blood Institute; National Institute of Diabetes and Digestive and Kidney Diseases

We evaluated whether dietary quality is a predictor of reduced diabetes risk and/or weight loss, independent of caloric intake in the diverse DPP cohort, a randomized clinical trial of adults at risk for diabetes. This secondary analysis included the 2920 participants (90.3% of randomized) with complete baseline and 1-year data (967 intensive lifestyle (ILS), 979 metformin, 974 placebo). Dietary intake was assessed using a 117-item food frequency questionnaire. Dietary quality was quantified using the Alternative Healthy Eating Index 2010 (AHEI), which ranges from 0 to 110 with higher scores corresponding to healthier diets. Participants were 50.8 ±10.6 years, 67.5% female, 44.7% minority race/ethnicity and baseline AHEI was 44.2 ±10.4. ILS participants had greater improvement (p Disclosure B. Allaire: None. A.H. Tjaden: None. J.W. Apolzan: None. D. Dabelea: None. L.M. Delahanty: Advisory Panel; Self; Jana Care Inc., Omada Health, Inc., Weight Watchers International, Inc. S. Edelstein: None. M.A. Hoskin: None. L.M. Jaacks: None. K.A. Temple: None. E.M. Venditti: None. J. Wylie-Rosett: None. DPP Research Group: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases

OBJECTIVE Sleep disturbances and circadian misalignment (social jet lag, late chronotype, or shift work) have been associated with worse glycemic control in type 2 diabetes (T2D). Whether these findings apply to adults with prediabetes is yet unexplored. We hypothesized that self-reported short sleep, poor sleep quality, and/or circadian misalignment are associated with higher glycemia, BMI, and blood pressure (BP) in adults with prediabetes or recently diagnosed, untreated T2D. RESEARCH DESIGN AND METHODS Our cohort included 962 overweight/obese adults ages 20–65 years with prediabetes or recently diagnosed, untreated T2D who completed a 2-h oral glucose tolerance test and validated sleep questionnaires. Independent associations of sleep and circadian variables with glycemia, BMI, and BP were evaluated with regression models. RESULTS The multiethnic cohort was 55% men, with mean ± SD age 52.2 ± 9.5 years and BMI 34.7 ± 5.5 kg/m2. Mean sleep duration was 6.6 ± 1.3 h. Poor sleep quality was reported by 54% and high risk for obstructive sleep apnea by 64%. HbA1c was significantly higher in those reporting 8 h sleep per night. Sleep duration >8 h was also associated with higher fasting glucose and CONCLUSIONS In our cohort, self-reported short and long sleep were both associated with adverse measures of glycemia, and short sleep and shift work were associated with higher BMI. Further research using objective measures of sleep is needed to better delineate the relationship between sleep and glycemia in adults with prediabetes or T2D.

Poor sleep may be associated with an increased risk of obesity and T2D in youth. We explored whether subjective sleep duration, sleep quality or risk factors for obstructive sleep apnea (OSA) are associated with BMI, glycemia or blood pressure (BP) in youth. Between 2013 and 2016, 236 overweight and obese youth (10-19 years old) at risk for or with recently diagnosed T2D were screened for the Restoring Insulin Secretion (RISE) Study. Data from a 2 hour OGTT and validated Sleep Disturbances Scale and Cleveland Adolescent Sleepiness questionnaires were available for 214 youth. Regression models explored the independent association between sleep variables and outcomes of fasting glucose, 2-h glucose, HbA1c, BMI and BP. The cohort consisted of 67% females, 33% males; 26% white, 26% black, 33% hispanic; age 14.1±2.1 years and BMI 35.9±6.5 kg/m2 (mean±SD). Based on the ADA criteria, 20% had normal glucose tolerance, 60% had prediabetes and 20% had T2D. Sleep duration Disclosure B. Mokhlesi: None. K.A. Temple: None. A.N. Hogan: None. S. Edelstein: None. K.J. Nadeau: None. S. Sam: None. T. Hannon: Consultant; Self; Eli Lilly and Company. K. Utzschneider: Consultant; Self; Novo Nordisk Inc.. E. Barengolts: None. S. Manchanda: None. D.A. Ehrmann: None. E. Van Cauter: Research Support; Self; AstraZeneca. R. Consortium: None.

Abnormal sleep and circadian misalignment (e.g., social jetlag, late chronotype, or shift work) have been associated with abnormal glucose metabolism. These associations have not been well established in adults who meet the ADA criteria for prediabetes. We hypothesized that self-reported short sleep, poor sleep quality or circadian misalignment are associated with worse glycemia, BP and BMI in adults with prediabetes or early untreated T2D. We screened 1,355 overweight/obese men and women age 20-65 y for the Restoring Insulin Secretion (RISE) Study. Completed data from a 2-h OGTT, HbA1c and sleep questionnaires were available for 704 adults with prediabetes and 258 with early untreated T2D. We used a modified Pittsburgh Sleep Quality Index to assess usual bedtime, wake time, and sleep duration on workdays and days off, and the Berlin questionnaire to assess the risk of obstructive sleep apnea (OSA). Regression models explored the independent associations of sleep variables with glycemic variables, BMI and BP. The cohort was 55% men, 45% women; 42% white, 36% black, 16% Hispanic; age 52.2±9.5 years and BMI 34.7±5.5 kg/m2 (mean±SD). Sleep duration was 6.6±1.3 h, poor sleep quality was reported by 54%, high risk for OSA in 64% and 20% did shift work. After adjusting for age, sex, and race/ethnicity, shift work was associated with 1.32 kg/m2 higher BMI (p=0.0043). After adjusting for age, sex, race/ethnicity and BMI, no sleep or circadian variables (sleep duration, sleep quality, OSA risk, shift work, social jetlag and chronotype) were associated with HbA1c, fasting or 2-h glucose, or BP. In this cohort with prediabetes or early untreated T2D, subjective measures of sleep duration, quality and circadian misalignment were not associated with measures of glycemia or BP. Shift workers were more obese. Further research using objective measures of sleep is needed to better delineate the relationship between sleep and glycemia. Disclosure B. Mokhlesi: None. K.A. Temple: None. A.N. Hogan: None. S. Edelstein: None. K. Utzschneider: Consultant; Self; Novo Nordisk Inc.. K.J. Nadeau: None. T. Hannon: Consultant; Self; Eli Lilly and Company. S. Sam: None. E. Barengolts: None. S. Manchanda: None. D.A. Ehrmann: None. E. Van Cauter: Research Support; Self; AstraZeneca. R. Consortium: None.

Insulin-secreting pancreatic beta-cells play a key role in the pathogenesis of diabetes mellitus. Potential new treatments for this disease include cell-replacement therapies using embryonic stem cells (ESCs). We have generated ESCs from a transgenic mouse model, mouse insulin 1 promoter (MIP)-green fluorescent protein (GFP) mice, in which embryonic and adult beta-cells are genetically tagged with GFP. The aim of the present study is to examine the differentiation potential of MIP-GFP ESCs in the microenvironment of the kidney capsule. The ESCs grew rapidly and formed a teratoma with GFP-expressing beta-like cells present in clusters that formed a cord-like structure similar to what is seen in the embryonic pancreas. These structures also included glucagon-expressing alpha-cells and amylase-expressing acinar cells. Electron microscopic analysis showed insulin-like granules in columnar epithelium with microvilli adjacent to exocrine-like granule containing cells. The MIP-GFP ESCs should be a useful research tool to study the differentiation capacity of ESCs towards pancreatic lineages.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a central regulator of adipogenesis and recruits coactivator proteins in response to ligand. However, the role of another class of nuclear cofactors, the nuclear receptor corepressors, in modulating PPARgamma transcriptional activity is less clear. Such corepressors include the nuclear receptor corepressor (NCoR) and the silencing mediator of retinoid and thyroid hormone receptors (SMRT). Our data suggest that PPARgamma recruits SMRT and NCoR in the absence of ligand and that these corepressors are capable of down-regulating PPARgamma-mediated transcriptional activity. The addition of the PPARgamma ligand pioglitazone results in dissociation of the PPARgamma-corepressor complex. To define the role of SMRT and NCoR in PPARgamma action, 3T3-L1 cells deficient in SMRT or NCoR were generated by RNA interference. When these cells are exposed to differentiation media, they exhibit increased expression of adipocyte-specific genes and increased production of lipid droplets, as compared with control cells. These data suggest that the nuclear receptor corepressors decrease PPARgamma transcriptional activity and repress the adipogenic program in 3T3-L1 cells.

Estrogen-nonresponsive estrogen receptor-alpha (ERalpha) knock-in (ENERKI) mice were generated to distinguish between ligand-induced and ligand-independent ER-alpha actions in vivo. These mice have a mutation [glycine 525 to leucine (G525L)] in the ligand-binding domain of ERalpha, which significantly reduces ERalpha interaction with and response to endogenous estrogens, whereas not affecting growth factor activation of ligand-independent pathways. ENERKI mice had hypoplastic uterine tissues and rudimentary mammary gland ductal trees. Females were infertile due to anovulation, and their ovaries contained hemorrhagic cystic follicles because of chronically elevated levels of LH. The ENERKI phenotype confirmed that ligand-induced activation of ERalpha is crucial in the female reproductive tract and mammary gland development. Growth factor treatments induced uterine epithelial proliferation in ovariectomized ENERKI females, directly demonstrating that ERalpha ligand-independent pathways were active. In addition, the synthetic ERalpha selective agonist propyl pyrazole triol (PPT) and ER agonist diethylstilbestrol (DES) were still able to activate ligand-induced G525L ERalpha pathways in vitro. PPT treatments initiated at puberty stimulated ENERKI uterine development, whereas neonatal treatments were needed to restore mammary gland ductal elongation, indicating that neonatal ligand-induced ERalpha activation may prime mammary ducts to become more responsive to estrogens in adult tissues. This is a useful model for in vivo evaluation of ligand-induced ERalpha pathways and temporal patterns of response. DES did not stimulate an ENERKI uterotrophic response. Because ERbeta may modulate ERalpha activation and have an antiproliferative function in the uterus, we hypothesize that ENERKI animals were particularly sensitive to DES-induced inhibition of ERalpha due to up-regulated uterine ERbeta levels.

The localized activation of circulating glucocorticoids in vivo by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays a critical role in the development of the metabolic syndrome. However, the precise contribution of 11beta-HSD1 in the initiation of adipogenesis by inactive glucocorticoids is not fully understood. 3T3-L1 fibroblasts can be terminally differentiated to mature adipocytes in a glucocorticoid-dependent manner. Both inactive rodent dehydrocorticosterone and human cortisone were able to substitute for the synthetic glucocorticoid dexamethasone in 3T3-L1 adipogenesis, suggesting a potential role for 11beta-HSD1 in these effects. Differentiation of 3T3-L1 cells caused a strong increase in 11beta-HSD1 protein levels, which occurred late in the differentiation protocol. Reduction of 11beta-HSD1 activity in 3T3-L1 fibroblasts, achieved by pharmacological inhibition or adenovirally mediated delivery of short hairpin RNA constructs, specifically blocked the ability of inactive glucocorticoids to drive 3T3-L1 differentiation. However, even modest increases in exogenous 11beta-HSD1 expression in 3T3-L1 fibroblasts, to levels comparable with endogenous 11beta-HSD1 in differentiated 3T3-L1 adipocytes, were sufficient to block adipogenesis. Luciferase reporter assays indicated that overexpressed 11beta-HSD1 was catalyzing the inactivating dehydrogenase reaction, because the ability of both active and inactive glucocorticoids to activate the glucocorticoid receptor were largely suppressed. These results suggest that the temporal regulation of 11beta-HSD1 expression is tightly controlled in 3T3-L1 cells, so as to mediate the initiation of differentiation by inactive glucocorticoids and also to prevent the inhibitory activity of prematurely expressed 11beta-HSD1 during adipogenesis.

Peroxisome proliferator-activated receptor gamma (PPARgamma) interacts with retinoid X receptor (RXR) on PPAR response elements (PPREs) to regulate transcription of PPAR-responsive genes. To investigate the binding of PPARgamma and RXR to PPREs, three mutations were constructed in the DNA-binding domains of PPARgamma; two of the mutants maintained the structure of zinc finger I (PPARgamma-GS and PPARgamma-AA), and a third mutation disrupted the protein structure of zinc finger I (PPARgamma-CS). Results indicated that the mutations of PPARgamma that maintained intact zinc fingers were capable of binding to a variety of PPREs in the presence of RXR and could activate transcription on several PPREs. In parallel, a mutation was created in the DNA-binding domain of RXRalpha that maintained the structure of the zinc fingers (RXR-GS) but did not bind DNA and was transcriptionally inactive. Examination of the 3' half-site of several PPREs revealed that variations from the consensus sequence reduced or abolished transcriptional activity, but conversion to consensus improved transcriptional activity with PPARgamma-GS and PPARgamma-AA. Examination of the 5' half-site indicated that the upstream three nucleotides were more important for transcriptional activity than the downstream three nucleotides. Our data demonstrated that stringent binding of RXR to the 3' half-site of a PPRE is more influential on the binding of the PPARgamma/RXR heterodimer than the ability of PPARgamma to bind DNA. Thus, unlike RXR, PPARgamma exhibits promiscuity in binding on a PPRE, suggesting that the definition of a PPRE for PPARgamma may need to be expanded.

The interactions of transformed cells with the surrounding stromal cells are of importance for tumor progression and metastasis. The relevance of adipocyte-derived factors to breast cancer cell survival and growth is well established. However, it remains unknown which specific adipocyte-derived factors are most critical in this process. Collagen VI is abundantly expressed in adipocytes. Collagen(-/-) mice in the background of the mouse mammary tumor virus/polyoma virus middle T oncogene (MMTV-PyMT) mammary cancer model demonstrate dramatically reduced rates of early hyperplasia and primary tumor growth. Collagen VI promotes its growth-stimulatory and pro-survival effects in part by signaling through the NG2/chondroitin sulfate proteoglycan receptor expressed on the surface of malignant ductal epithelial cells to sequentially activate Akt and beta-catenin and stabilize cyclin D1. Levels of the carboxyterminal domain of collagen VIalpha3, a proteolytic product of the full-length molecule, are dramatically upregulated in murine and human breast cancer lesions. The same fragment exerts potent growth-stimulatory effects on MCF-7 cells in vitro. Therefore, adipocytes play a vital role in defining the ECM environment for normal and tumor-derived ductal epithelial cells and contribute significantly to tumor growth at early stages through secretion and processing of collagen VI.

Hepatic gluconeogenesis is essential for maintenance of normal blood glucose concentrations and is regulated by opposing stimulatory (cyclic adenosine monophosphate, cAMP) and inhibitory (insulin) signaling pathways. The cAMP signaling pathway leads to phosphorylation of cAMP response element-binding (CREB) protein, resulting in recruitment of the coactivators CREB-binding protein (CBP) and p300 and subsequent activation of gluconeogenesis. Insulin signaling leads to phosphorylation of CBP at serine 436, a residue near its CREB-interacting domain, but it is unknown whether this event modulates cAMP signaling. Here, we show in vitro and in 'knock-in' mice that a mutant CBP (S436A) is aberrantly recruited to CREB protein, resulting in inappropriate activation of gluconeogenesis in the fed state and glucose intolerance resulting from increased hepatic glucose production. We propose that insulin signaling may directly regulate many cAMP signaling pathways at the transcriptional level by controlling CBP recruitment.

The purpose of this study was to determine the short-term effect of feeding selenium-supplemented formulas on the selenium status of end-stage renal disease patients on hemodialysis.The prospective, randomized, single-blind study of parallel design was conducted at three hemodialysis clinics.A total of 79 hemodialysis patients were randomly assigned into one of three groups.Liquid nutritional formula supplemented with either selenite (28 microg Se/8 oz, n = 26), selenate (28 microg Se/8 oz, n = 26), or nonfortified (7 microg Se/8 oz, n = 27) was fed to hemodialysis patients as their sole source of nutrition for 14 days.Plasma and red blood cell (RBC) selenium and glutathione peroxidase (GPX) activities were measured in predialysis blood both before (day 1) and after (day 8) a 7-day baseline period, and after subjects received the formula as the sole source of nutrition (approximately 35 kcal/kg/d) for 14 days (day 22).Selenium intake (Mean +/- SEM, microg/d) was 134 +/- 9, 140 +/- 9, and 35 +/- 2 for patients receiving selenite-, selenate-, or non-supplemented formula, respectively. On day 22, plasma selenium (micromol/L) was greater (P.032) in the selenate-supplemented group (1.5 +/- 0.1) compared with the nonsupplemented group (1.2 +/- 0.1), but not compared with the selenite-supplemented group (1.4 +/- 0.1). Plasma GPX activity was 44% to 60% that of healthy controls and not different among groups. RBC selenium and GPX activities were within the normal range and were not different among groups.The results of this study indicate that a liquid formula supplemented with selenium as selenate is successful at maintaining selenium concentrations within normal range, as well as significantly increasing plasma selenium levels compared with nonsupplementation.

Adipocyte differentiation comprises altered gene expression and increased triglyceride storage. To investigate the interdependency of these two events, 3T3-L1 cells were differentiated in the presence of glucose or pyruvate. All adipocytic proteins examined were similarly increased between the two conditions. In contrast, 3T3-L1 adipocytes differentiated with glucose exhibited significant lipid accumulation, which was largely suppressed in the presence of pyruvate. Subsequent addition of glucose to the latter cells restored lipid accumulation and acute rates of insulin-stimulated lipogenesis. These data indicate that extracellular energy is required for induction of adipocytic proteins, while only glucose sustained the parallel increase in triglyceride storage.