Sharon E. Fleming

Glucose and glutamine provide similar proportions of energy to mucosal cells of rat small intestine

The objectives of this study were to establish a reliable method for quantifying glycolytic flux in intestinal epithelial cells, to determine the proportion of energy provided to small intestine epithelial cells by glucose vs. glutamine, and to determine whether there was an energetic advantage to having both substrates present simultaneously. There was substantial retention of 3H in alanine and lactate when [2-(3)H]glucose was used as tracer for quantifying glycolysis, and the magnitude of the 3H retention was influenced by the presence of other substrates and metabolites. Detritiation was at least 99% complete, however, when [3-(3)H]glucose was used as tracer in this system and the tritium was recovered as 3H2O. Glycolytic flux was six- to sevenfold higher in cells of the proximal than distal small intestine but was not significantly different for young adult (4 mo) vs. aged adult (24 mo) rats. Net ATP production from exogenous substrates was higher when both glucose and glutamine were present simultaneously than when either substrate was present alone, and glucose was calculated to provide 50-60% of the net ATP produced from these two substrates. Most of the energy produced from glucose was produced via the anaerobic metabolic pathways (78% for glucose alone, 95% with glucose and glutamine). Net energy production was calculated to be 10% lower in cells from aged animals than in those from young animals, since CO2 production from these major substrates was lower in cells from aged animals.The objectives of this study were to establish a reliable method for quantifying glycolytic flux in intestinal epithelial cells, to determine the proportion of energy provided to small intestine epithelial cells by glucose vs. glutamine, and to determine whether there was an energetic advantage to having both substrates present simultaneously. There was substantial retention of 3H in alanine and lactate when [2-3H]glucose was used as tracer for quantifying glycolysis, and the magnitude of the3H retention was influenced by the presence of other substrates and metabolites. Detritiation was at least 99% complete, however, when [3-3H]glucose was used as tracer in this system and the tritium was recovered as3H2O. Glycolytic flux was six- to sevenfold higher in cells of the proximal than distal small intestine but was not significantly different for young adult (4 mo) vs. aged adult (24 mo) rats. Net ATP production from exogenous substrates was higher when both glucose and glutamine were present simultaneously than when either substrate was present alone, and glucose was calculated to provide 50-60% of the net ATP produced from these two substrates. Most of the energy produced from glucose was produced via the anaerobic metabolic pathways (78% for glucose alone, 95% with glucose and glutamine). Net energy production was calculated to be 10% lower in cells from aged animals than in those from young animals, since CO2 production from these major substrates was lower in cells from aged animals.

Acetylation of histones associated with the p21WAF1/CIP1 gene by butyrate is not sufficient for p21WAF1/CIP1 gene transcription in human colorectal adenocarcinoma cells.

Butyric acid is well recognized as a histone deacetylase (HDAC) inhibitor, and changes in histone acetylation are thought to alter gene expression. The mechanism by which sodium butyrate (NaB) induces p21WAF1/CIP1, a critical gene involved in the antiproliferative effect of NaB, was studied at the chromatin level. Using chromatin immunoprecipitation (ChIP) assay, acetylation of histone H3 was observed at the proximal region of the promoter within 30 min of NaB exposure and this extended to the distal region within 2 hr. By contrast, histone H4 was acetylated both at the proximal and the distal regions of the promoter within 30 min. NaB did not influence other histone modifications. NaB stimulated recruitment of the transcription factors ZBP89 and Sp1 as well as GCN5, but did not influence recruitment of Sp3, HDAC1, p300, or CBP. As recruitment of HDAC1 to the promoter appeared not to account for NaB‐induced changes in histone acetylation, we aimed to influence HDAC activity by altering its phosphorylation status. The kinase inhibitor, H7, suppressed p21WAF1/CIP1 mRNA in both the absence and the presence of NaB without influencing the butyrate‐induced hyperacetylation of H3 and H4 associated with the p21WAF1/CIP1 promoter. These results suggest that acetylation of histones at the p21WAF1/CIP1 promoter is not sufficient for NaB to exert antiproliferative effects via transcription of the p21WAF1/CIP1 gene. Induction of p21WAF1/CIP1 transcription by the phosphatase inhibitor, okadaic acid, in the absence of changes in association of acetylated histones with the p21WAF1/CIP1 promoter provides further evidence of the importance of phosphorylation to p21WAF1/CIP1 transcription.

Intestinal cell proliferation is influenced by intakes of protein and energy, aflatoxin, and whole-body radiation.

Intestinal epithelial cell proliferation in young male F344 rats was measured in response to dietary protein content (5%, 10%, and 20% casein diets), energy restriction (energy intake was 60% of ad libitum energy intakes of animals consuming the 20% casein diet), total diet restriction (dietary intake was 60% of the ad libitum intake of 20% casein diet group), aflatoxin administration, and whole body irradiation. Cellular proliferation was measured in sections of jejunum, ileum, proximal colon, and distal colon with the [3H]thymidine technique. Restricting energy or total diet intakes by 40% from ad libitum levels reduced proliferation in epithelial cells throughout the intestine. In comparison to the 5% casein diet, the 20% casein diet resulted in modestly lower cellular proliferation in all intestinal segments. Radiation induced a decrease in cellular proliferation in the jejunum and ileum; this decrease was prevented by a 20% casein diet. Pretreatment with aflatoxin B1 decreased intestinal cell proliferation throughout the intestine, and this decrease was not influenced by the protein content of the diet.

Sodium Butyrate Inhibits Cell Growth and Stimulates p21WAF1/CIP1 Protein in Human Colonic Adenocarcinoma Cells Independently of p53 Status

Butyric acid, one of the short-chain fatty acids produced by microbial fermentation in the colon, exhibits antiproliferative activities in various cancer cell lines. The initial objective of the study was to assess whether the effect of sodium butyrate (NaB) on cell growth differed by p53 status of the cells. Four human colorectal adenocarcinoma cell lines were used: HT29 (p53 point mutation), Caco2 (p53 truncation), LS513 (p53 wild type), and Lovo (p53 wild type). NaB significantly inhibited cell growth in all four cell lines. NaB arrested HT29 and LS513 cells in G0/G1 and Caco2 and Lovo in G2-phase. A second objective was to determine whether NaB similarly affected the cyclin-dependent kinase inhibitor, p21WAF1/CIP1. In all cell lines, p21 mRNA levels were immediately elevated after NaBexposure, and p21 protein levels were increased within 6 h. NaB increased p21 promoter activity in both Caco2 and Lovo, suggesting p53 independence. NaB did not influence p21 mRNA stability. Although three DNase I hypersensitivity sites were identified in the region of the p21 gene, induction of p21mRNAbyNaBwas not accompanied by relaxation of the chromatin in the region of the p21 gene.

Baseline Correlates of Insulin Resistance in Inner City High‐BMI African‐American Children

To characterize the influence of diet‐, physical activity–, and self‐esteem‐related factors on insulin resistance in 8–10‐year‐old African‐American (AA) children with BMI greater than the 85th percentile who were screened to participate in a community‐based type 2 diabetes mellitus (T2DM) prevention trial. In 165 subjects, fasting glucose‐ and insulin‐derived values for homeostasis model assessment of insulin resistance (HOMA‐IR) assessed insulin resistance. Body fatness was calculated following bioelectrical impedance analysis, and fitness was measured using laps from a 20‐m shuttle run. Child questionnaires assessed physical activity, dietary habits, and self‐esteem. Pubertal staging was assessed using serum levels of sex hormones. Parent questionnaires assessed family demographics, family health, and family food and physical activity habits. Girls had significantly higher percent body fat but similar anthropometric measures compared with boys, whereas boys spent more time in high‐intensity activities than girls. Scores for self‐perceived behavior were higher for girls than for boys; and girls desired a more slender body. Girls had significantly higher insulin resistance (HOMA‐IR), compared with boys (P < 0.01). Adjusting for age, sex, pubertal stage, socioeconomic index (SE index), and family history of diabetes, multivariate regression analysis showed that children with higher waist circumference (WC) (P < 0.001) and lower Harters scholastic competence (SC) scale (P = 0.044) had higher insulin resistance. WC and selected self‐esteem parameters predicted insulin resistance in high‐BMI AA children. The risk of T2DM may be reduced in these children by targeting these factors.

Taking Action Together: A YMCA-based protocol to prevent Type-2 Diabetes in high-BMI inner-city African American children

BackgroundAssociated with a tripling in obesity since 1970, type 2 diabetes mellitus (T2DM) in children has risen 9-10 fold. There is a critical need of protocols for trials to prevent T2DM in children.Methods/DesignThis protocol includes the theory, development, evaluation components and lessons learned from a novel YMCA-based T2DM prevention intervention designed specifically for high-BMI African American children from disadvantaged, inner-city neighborhoods of Oakland, California. The intervention was developed on the basis of: review of epidemiological and intervention studies of pediatric T2DM; a conceptual theory (social cognitive); a comprehensive examination of health promotion curricula designed for children; consultation with research, clinical experts and practitioners and; input from community partners. The intervention, Taking Action Together, included culturally sensitive and age-appropriate programming on: healthy eating; increasing physical activity and, improving self esteem.DiscussionEvaluations completed to date suggest that Taking Action Together may be an effective intervention, and results warrant an expanded evaluation effort. This protocol could be used in other community settings to reduce the risk of children developing T2DM and related health consequences.Trial registrationClinicalTrials.gov NCT01039116.

Prevention of type 2 diabetes in youth: etiology, promising interventions and recommendations.

Ritchie LD, Ganapathy S, Woodward-Lopez G, Gerstein DE, Fleming SE. Prevention of type 2 diabetes in youth: Etiology, promising interventions and recommendations. Pediatric Diabetes 2003: 4: 174—209. # Blackwell Munksgaard, 2003 Lorrene D. Ritchie, Sujatha Ganapathy, Gail Woodward-Lopez, Dana E. Gerstein and Sharon E. Fleming Center for Weight and Health, College of Natural Resources, University of California; and Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA

Carbohydrate intake and cardiometabolic risk factors in high BMI African American children.

The aim of this study was to evaluate the relationship between intakes of subgroups of energy-providing carbohydrate, and markers of cardiometabolic risk factors in high BMI African American (AA) children.A cross sectional analysis was performed on data from a sample of 9-11 year old children (n = 95) with BMI greater than the 85th percentile. Fasting hematological and biochemical values for selected markers of cardiometabolic risk factors were related to intakes of carbohydrates and sugars.After adjusting for gender, pubertal stage and waist circumference, multivariate regression analysis showed that higher intakes of carbohydrate (with fat and protein held constant) were associated with higher plasma concentrations of triglycerides (TG), VLDL-C, IDL-C, and worse insulin resistance (homeostasis model assessment of insulin resistance, HOMA-IR). After dividing carbohydrate into non-sugar versus sugar fractions, sugars were significantly related to higher TG, VLDL-C, IDL-C, lower adipocyte fatty acid insulin sensitivity (ISI-FFA), and was closely associated with increased HOMA-IR. Similar trends were observed for sugars classified as added sugars, and for sugars included in beverages. Further dividing sugar according to the food group from which it was consumed showed that consuming more sugar from the candy/soda food group was highly significantly associated with increased TG, VLDL-C, IDL-C and closely associated with increased HOMA-IR. Sugars consumed in all fruit-containing foods were significantly associated with lower ISI-FFA. Sugars consumed as fruit beverages was significantly associated with VLDL-C, IDL-C and ISI-FFA whereas sugars consumed as fresh, dried and preserved fruits did not show significant associations with these markers.Sugars consumed from in all dairy foods were significantly associated with higher TG, VLDL-C and IDL-C, and with significantly lower HDL-C and ISI-FFA. These effects were associated with sugars consumed in sweetened dairy products, but not with sugars consumed in unsweetened dairy products. This analysis suggests that increases in carbohydrate energy, especially in the form of sugar, may be detrimental to cardiometabolic health in high BMI children.

Lp(a)-cholesterol is associated with HDL-cholesterol in overweight and obese African American children and is not an independent risk factor for CVD

BackgroundThe role of Lipoprotein (a) cholesterol {Lp(a)-C}as an additional and/or independent risk factor for cardiovascular disease (CVD) is not clear. We evaluated the associations between Lp(a)-C and other CVD risk factors including plasma lipoprotein concentrations and body fatness in overweight and obese African American children.MethodsA cross-sectional analysis was carried out using data from a sample of 121 African American children aged 9-11 years with Body Mass Index (BMI)s greater than the 85th percentile. Body height, weight and waist circumference (WC) were measured. Fasting plasma concentrations of Lp(a)-C, Total cholesterol (TC), High density lipoprotein cholesterol (HDL-C), Very low density lipoprotein cholesterol (VLDL-C), Intermediate density lipoprotein cholesterol (IDL-C), Low density lipoprotein cholesterol (LDL-C), and Triacylglycerides (TAG) were analyzed using the vertical auto profile (VAP) cholesterol method.ResultsAfter adjusting for child age, gender, and pubertal status, Lp(a)-C was positively associated with both HDL-C and TC, and negatively associated with VLDL-C and TAG. Including BMIz and WC as additional covariates did not alter the direction of the relationships between Lp(a)-C and the other lipoproteins. Finally, after adjusting for the other plasma lipoproteins, Lp(a)-C remained strongly associated with HDL-C, whereas the associations of Lp(a)-C with the other lipoproteins were not significant when HDL-C was simultaneously included in the regression models.ConclusionsLp(a)-C was positively associated with HDL-C and this association is not influenced by other lipoprotein subclasses or by the degree of obesity. We conclude that Lp(a) cholesterol is not an independent risk factor for CVD in African American children.

Insulin Resistance is Improved in Overweight African American Boys but Not in Girls Following a One-Year Multidisciplinary Community Intervention Program

AIM To assess potential for effectiveness, in a non-randomized pilot study, of a community-based lifestyle intervention program to reduce the risk for type 2 diabetes mellitus in overweight African American (AA) children. RESEARCH DESIGN Sample of 165 9-11 year-old AA children with body mass index (BMI) >85th percentile were recruited from local recreational sites, schools and churches. Participants self-selected to attend one of two study sites, blinded to the specifics of the intervention administered at each site. The intervention group received a programmatically focused 2-week summer camp with once-a-week community-based exercise, nutrition, and behavioral modification sessions, and their families were invited to monthly nutrition educational sessions. Control group participants received a 2-week conventional YMCA summer camp and their families received nutrition and physical activity education material through the mail. Baseline assessment and 1-year follow-up were conducted in collaboration with the YMCA of the East Bay and Childrens Hospital Oakland, CA, with 109 participants (66%) having pre/post data. RESULTS After one-year of intervention, treatment boys showed a drop in homeostasis model assessment of insulin-resistance (HOMA-IR) (-0.58 vs +0.17; p = 0.003), fasting glucose (Gf, mg/dL) (mean change: -2.9 vs +0.4; p = 0.126) and fasting insulin (If, microU/mL) (-2.2 vs +0.7; p = 0.009) compared to control boys, after accounting for baseline differences and pubertal stage of the child. Treatment girls had similar changes to the control girls in HOMA-IR (-0.02 vs -0.17; p = 0.66), Gr (-0.3 vs +1.4; p = 0.29) and If (+0.03 vs +0.17; p = 0.57). CONCLUSION After one year, this community-based intervention program effectively improved insulin resistance and thus reduced risk for type 2 diabetes mellitus in overweight AA boys but did not change the risk in girls compared to control children.