The impact of low-fat and full-fat dairy foods on symptoms of gastroesophageal reflux disease: an exploratory analysis based on a randomized controlled trial.

PURPOSE: Gastroesophageal reflux disease (GERD) is a widely prevalent condition. High consumption of dairy foods and dietary fat are associated with worse GERD symptoms. However, existing data are inconsistent and mostly based on observational studies. The purpose of this exploratory analysis of a randomized controlled trial was to investigate the impact of low-fat and full-fat dairy food consumption on GERD symptoms. METHODS: Seventy-two participants with metabolic syndrome completed a 4-week wash-in diet during which dairy intake was limited to three servings of nonfat milk per week. Participants were then randomized to either continue the limited dairy diet or switch to a diet containing 3.3 servings per day of either low-fat or full-fat milk, yogurt and cheese for 12 weeks. Here, we report intervention effects on the frequency of acid reflux, and the frequency and severity of heartburn, exploratory endpoints assessed by a questionnaire administered before and after the 12-week intervention. RESULTS: In the per-protocol analysis (n = 63), there was no differential intervention effect on a cumulative heartburn score (p = 0.443 for the time by diet interaction in the overall repeated measures analysis of variance). Similarly, the intervention groups did not differentially affect the odds of experiencing acid regurgitation (p = 0.651). The intent-to-treat analyses (n = 72) yielded similar results. CONCLUSION: Our exploratory analyses suggest that, in men and women with the metabolic syndrome, increasing the consumption of either low-fat or full-fat dairy foods to at least three servings per day does not affect common symptoms of GERD, heartburn and acid regurgitation compared to a diet limited in dairy. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02663544, registered on January 26, 2016.

Assessing the validity of plasma phospholipid fatty acids as biomarkers of dairy fat intake using data from a randomized controlled intervention trial.

BACKGROUND: Plasma phospholipid pentadecanoic acid (C15:0), heptadecanoic acid (C17:0), and trans-palmitoleic acid (trans-C16:1n-7) are correlates of dairy fat intake. However, their relative concentrations may be influenced by other endogenous factors, such as liver fat content, and their validity as biomarkers of dairy fat intake has yet to be established. OBJECTIVES: We investigated whether liver fat content modifies relations between concentrations of C15:0, C17:0, and trans-C16:1n-7 (alone and in combination with iso-C17:0) and known dairy fat intake in the context of a randomized controlled intervention study. We further examined the proportion of dairy fat intake explained by these fatty acids on their own and when considering liver fat content. METHODS: We used data from a 12-wk intervention trial in which participants (n = 62) consumed diets limited in dairy (0.3 g/d of dairy fat), rich in low-fat dairy (8.7 g/d of dairy fat), or rich in full-fat dairy (28.5 g/d of dairy fat). We used linear regression models to examine relations between relative fatty acid concentrations and grams per day of dairy fat intake, liver fat percentage, and their interaction. RESULTS: Only trans-C16:1n-7 in isolation (ß: 0.0004 ± 0.0002, P = 0.03) and combined with iso-C17:0 (ß: 0.002 ± 0.0005, P < 0.0001) were consistently positively associated with dairy fat intake regardless of liver fat content. Trans-C16:1n-7 combined with iso-C17:0 also explained the greatest proportion of variation (35.4%) in dairy fat intake. C15:0 and C17:0 were not associated with dairy fat intake after adjusting for liver fat and were predicted to be higher in relation to increased dairy fat intake only among individuals with elevated liver fat. CONCLUSIONS: The potential for liver fat to affect relative plasma phospholipid concentrations of C15:0 and C17:0 raises questions about their validity as biomarkers of dairy fat intake. Of the fatty acid measures tested, trans-C16:1n-7 combined with iso-C17:0, especially with adjustment of liver fat, age, and sex, may provide the most robust estimate of dairy fat consumption.

Impact of low-fat and full-fat dairy foods on fasting lipid profile and blood pressure: exploratory endpoints of a randomized controlled trial.

BACKGROUND: Dietary guidelines traditionally recommend low-fat dairy because dairy's high saturated fat content is thought to promote cardiovascular disease (CVD). However, emerging evidence indicates that dairy fat may not negatively impact CVD risk factors when consumed in foods with a complex matrix. OBJECTIVE: The aim was to compare the effects of diets limited in dairy or rich in either low-fat or full-fat dairy on CVD risk factors. METHODS: In this randomized controlled trial, 72 participants with metabolic syndrome completed a 4-wk run-in period, limiting their dairy intake to ≤3 servings/wk of nonfat milk. Participants were then randomly assigned to 1 of 3 diets, either continuing the limited-dairy diet or switching to a diet containing 3.3 servings/d of either low-fat or full-fat milk, yogurt, and cheese for 12 wk. Exploratory outcome measures included changes in the fasting lipid profile and blood pressure. RESULTS: In the per-protocol analysis (n = 66), there was no intervention effect on fasting serum total, LDL, and HDL cholesterol; triglycerides; free fatty acids; or cholesterol content in 38 isolated plasma lipoprotein fractions (P > 0.1 for all variables in repeated-measures ANOVA). There was also no intervention effect on diastolic blood pressure, but a significant intervention effect for systolic blood pressure (P = 0.048), with a trend for a decrease in the low-fat dairy diet (-1.6 ± 8.6 mm Hg) compared with the limited-dairy diet (+2.5 ± 8.2 mm Hg) in post hoc testing. Intent-to-treat results were consistent for all endpoints, with the exception that systolic blood pressure became nonsignificant (P = 0.08). CONCLUSIONS: In men and women with metabolic syndrome, a diet rich in full-fat dairy had no effects on fasting lipid profile or blood pressure compared with diets limited in dairy or rich in low-fat dairy. Therefore, dairy fat, when consumed as part of complex whole foods, does not adversely impact these classic CVD risk factors. This trial was registered at clinicaltrials.gov as NCT02663544.

The impact of diets rich in low-fat or full-fat dairy on glucose tolerance and its determinants: a randomized controlled trial.

BACKGROUND: Dairy foods, particularly yogurt, and plasma biomarkers of dairy fat intake are consistently inversely associated with incident type 2 diabetes. Yet, few trials assessing the impact of dairy on glucose homeostasis include fermented or full-fat dairy foods. OBJECTIVES: We aimed to compare the effects of diets rich in low-fat or full-fat milk, yogurt, and cheese on glucose tolerance and its determinants, with those of a limited dairy diet. METHODS: In this parallel-design randomized controlled trial, 72 participants with metabolic syndrome completed a 4-wk wash-in period, limiting dairy intake to ≤3 servings/wk of nonfat milk. Participants were then randomly assigned to either continue the limited dairy diet, or switch to a diet containing 3.3 servings/d of either low-fat or full-fat dairy for 12 wk. Outcome measures included glucose tolerance (area under the curve glucose during an oral-glucose-tolerance test), insulin sensitivity, pancreatic ß-cell function, systemic inflammation, liver-fat content, and body weight and composition. RESULTS: In the per-protocol analysis (n = 67), we observed no intervention effect on glucose tolerance (P = 0.340). Both the low-fat and full-fat dairy diets decreased the Matsuda insulin sensitivity index (ISI) (means ± SDs -0.47 ± 1.07 and -0.25 ± 0.91, respectively) and as compared with the limited dairy group (0.00 ± 0.92) (P = 0.012 overall). Body weight also changed differentially (P = 0.006 overall), increasing on full-fat dairy (+1.0 kg; -0.2, 1.8 kg) compared with the limited dairy diet (-0.4 kg; -2.5, 0.7 kg), whereas the low-fat dairy diet (+0.3 kg; -1.1, 1.9 kg) was not significantly different from the other interventions. Intervention effects on the Matsuda ISI remained after adjusting for changes in adiposity. No intervention effects were detected for liver fat content or systemic inflammation. Findings in intent-to-treat analyses (n = 72) were consistent. CONCLUSIONS: Contrary to our hypothesis, neither dairy diet improved glucose tolerance in individuals with metabolic syndrome. Both dairy diets decreased insulin sensitivity through mechanisms largely unrelated to changes in key determinants of insulin sensitivity.This trial was registered at clinicaltrials.gov as NCT02663544.

Vitamin D in human serum and adipose tissue after supplementation.

BACKGROUND: Serum 25-hydroxyvitamin D [25(OH)D] concentration is an indicator of vitamin D exposure, but it is also influenced by clinical characteristics that affect 25(OH)D production and clearance. Vitamin D is the precursor to 25(OH)D but is analytically challenging to measure in biological specimens. OBJECTIVES: We aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification of vitamins D3 and D2 in serum and to explore the potential of circulating vitamin D as a biomarker of exposure in supplementation trials. METHODS: The method was validated using guideline C62-A from the Clinical and Laboratory Standards Institute and was applied in 2 pilot clinical trials of oral vitamin D3 supplementation. Pilot study 1 included 22 adults randomly assigned to placebo or 2000 IU/d. Blood was collected at baseline, 1, 3, 6, and 12 mo. Pilot study 2 included 15 adults randomly assigned to 2000 or 4000 IU/d. Blood and subcutaneous (SUBQ) adipose tissue were collected at baseline and 3 mo. RESULTS: In study 1, mean change (baseline to 3 mo) in serum vitamin D3 was -0.1 ng/mL in the placebo group and 6.8 ng/mL in the 2000 IU/d group (absolute difference: 6.9; 95% CI: 4.5, 9.3 ng/mL). In study 2, mean change (baseline to 3 mo) in serum vitamin D3 was 10.4 ng/mL in the 2000 IU/d group and 22.2 ng/mL in the 4000 IU/d group (fold difference: 2.15; 95% CI: 1.40, 3.37). Serum and adipose tissue vitamin D3 concentrations were correlated, and the dose-response of vitamin D3 in adipose mirrored that in serum. CONCLUSIONS: We validated a sensitive, robust, and high-throughput LC-MS/MS method to quantify vitamins D3 and D2 in serum. Serum and SUBQ adipose tissue vitamin D3 concentrations increased proportionally to dose with 3 mo of daily supplementation.These trials were registered at clinicaltrials.gov as NCT00552409 (pilot study 1) and NCT01477034 (pilot study 2).

Consuming glucose-sweetened, not fructose-sweetened, beverages increases fasting insulin in healthy humans.

Fructose-, compared to glucose-, sweetened beverages increase liver triglyceride content in the short-term, prior to weight gain. In secondary analyses of a randomized cross-over design study during which 24 healthy adults consumed 25% of their estimated energy requirement in the form of glucose-, fructose-, and high-fructose corn syrup-sweetened beverages in addition to an identical ad libitum diet for three periods of 8 days each, we investigated the hypothesis that fructose in sweetened beverages also triggers insulin resistance in the short term. Total energy intake, body weight, and fasting glucose did not differ among diet phases. However, there was a significant trend for higher fasting insulin (p = 0.042 for trend) and, among normal-weight participants, homeostasis model assessment index of insulin resistance (p = 0.034 for diet × adiposity interaction) according to the glucose content of the beverages. In conclusion, in contrast to our hypothesis, insulin resistance was increased with higher glucose vs. fructose content of the beverages in this short-term trial.

Intraindividual Variation in Markers of Intestinal Permeability and Adipose Tissue Inflammation in Healthy Normal-Weight to Obese Adults.

BACKGROUND: Intestinal permeability and adipose tissue inflammation are considered mechanistic links in the relationship between diet, obesity, and chronic disease. However, methods to measure both are not well standardized, and the reliability of commonly used measures is not known. METHODS: We calculated the intraclass correlation coefficient (ICC) for several common measures of intestinal permeability and adipose tissue inflammation from a randomized clinical trial of cross-over design in which normal-weight (n = 12) or overweight/obese (n = 12) individuals each completed three 8-day dietary intervention periods. RESULTS: For biomarkers of intestinal permeability, plasma zonulin, and lipopolysaccharide-binding protein, ICCs were "excellent" (i.e., >0.9). The direct measure of intestinal permeability, the lactulose/mannitol test, exhibited "fair" reliability (ICC = 0.53). A wider range of ICCs (0.6-0.9), suggesting "good" to "excellent" reliability, were obtained for measures of adipose tissue expression of genes encoding major mediators of inflammation. Similarly, individual immune cell populations isolated from adipose tissue, expressed as a percentage of all CD45+ cells, also had "good" to "excellent" ICCs. However, when these populations were expressed as number of cells per gram of tissue, ICC values were "fair," falling below 0.6. CONCLUSIONS: Due to the repeated measures design, our study offered a unique opportunity to assess reliability of commonly used biomarkers of intestinal permeability and adipose tissue inflammation. Our findings suggest that these measures were generally highly reliable in the short-term. IMPACT: Along with other factors, particularly validity, the demonstrated reliabilities can help inform the choice of endpoints in studies of intestinal permeability and adipose tissue inflammation.

Contribution of Adipose Tissue Inflammation to the Development of Type 2 Diabetes Mellitus.

The objective of this comprehensive review is to summarize and discuss the available evidence of how adipose tissue inflammation affects insulin sensitivity and glucose tolerance. Low-grade, chronic adipose tissue inflammation is characterized by infiltration of macrophages and other immune cell populations into adipose tissue, and a shift toward more proinflammatory subtypes of leukocytes. The infiltration of proinflammatory cells in adipose tissue is associated with an increased production of key chemokines such as C-C motif chemokine ligand 2, proinflammatory cytokines including tumor necrosis factor α and interleukins 1ß and 6 as well as reduced expression of the key insulin-sensitizing adipokine, adiponectin. In both rodent models and humans, adipose tissue inflammation is consistently associated with excess fat mass and insulin resistance. In humans, associations with insulin resistance are stronger and more consistent for inflammation in visceral as opposed to subcutaneous fat. Further, genetic alterations in mouse models of obesity that reduce adipose tissue inflammation are-almost without exception-associated with improved insulin sensitivity. However, a dissociation between adipose tissue inflammation and insulin resistance can be observed in very few rodent models of obesity as well as in humans following bariatric surgery- or low-calorie-diet-induced weight loss, illustrating that the etiology of insulin resistance is multifactorial. Taken together, adipose tissue inflammation is a key factor in the development of insulin resistance and type 2 diabetes in obesity, along with other factors that likely include inflammation and fat accumulation in other metabolically active tissues. © 2019 American Physiological Society. Compr Physiol 9:1-58, 2019.

The short-term and long-term effects of bariatric/metabolic surgery on subcutaneous adipose tissue inflammation in humans.

CONTEXT: The mechanisms mediating the short- and long-term improvements in glucose homeostasis following bariatric/metabolic surgery remain incompletely understood. OBJECTIVE: To investigate whether a reduction in adipose tissue inflammation plays a role in the metabolic improvements seen after bariatric/metabolic surgery, both in the short-term and longer-term. DESIGN: Fasting blood and subcutaneous abdominal adipose tissue were obtained before (n=14), at one month (n=9), and 6-12months (n=14) after bariatric/metabolic surgery from individuals with obesity who were not on insulin or anti-diabetes medication. Adipose tissue inflammation was assessed by a combination of whole-tissue gene expression and flow cytometry-based quantification of tissue leukocytes. RESULTS: One month after surgery, body weight was reduced by 13.5±4.4kg (p<0.001), with improvements in glucose tolerance reflected by a decrease in area-under-the-curve (AUC) glucose in 3-h oral glucose tolerance tests (-105±98mmol/L * min; p=0.009) and enhanced pancreatic ß-cell function (insulinogenic index: +0.8±0.9pmol/mmol; p=0.032), but no change in estimated insulin sensitivity (Matsuda insulin sensitivity index [ISI]; p=0.720). Furthermore, although biomarkers of systemic inflammation and pro-inflammatory gene expression in adipose tissue remained unchanged, the number of neutrophils increased in adipose tissue 15-20 fold (p<0.001), with less substantial increases in other leukocyte populations. By the 6-12month follow-up visit, body weight was reduced by 34.8±10.8kg (p<0.001) relative to baseline, and glucose tolerance was further improved (AUC glucose -276±229; p<0.001) along with estimated insulin sensitivity (Matsuda ISI: +4.6±3.2; p<0.001). In addition, improvements in systemic inflammation were reflected by reductions in circulating C-reactive protein (CRP; -2.0±5.3mg/dL; p=0.002), and increased serum adiponectin (+1358±1406pg/mL; p=0.003). However, leukocyte infiltration of adipose tissue remained elevated relative to baseline, with pro-inflammatory cytokine mRNA expression unchanged, while adiponectin mRNA expression trended downward (p=0.069). CONCLUSION: Both the short- and longer-term metabolic improvements following bariatric/metabolic surgery occur without significant reductions in measures of adipose tissue inflammation, as assessed by measuring the expression of genes encoding key mediators of inflammation and by flow cytometric immunophenotyping and quantification of adipose tissue leukocytes.

No differential effect of beverages sweetened with fructose, high-fructose corn syrup, or glucose on systemic or adipose tissue inflammation in normal-weight to obese adults: a randomized controlled trial.

BACKGROUND: Sugar-sweetened beverage (SSB) consumption and low-grade chronic inflammation are both independently associated with type 2 diabetes and cardiovascular disease. Fructose, a major component of SSBs, may acutely trigger inflammation, which may be one link between SSB consumption and cardiometabolic disease. OBJECTIVE: We sought to determine whether beverages sweetened with fructose, high-fructose corn syrup (HFCS), and glucose differentially influence systemic inflammation [fasting plasma C-reactive protein and interleukin-6 (IL-6) as primary endpoints] acutely and before major changes in body weight. Secondary endpoints included adipose tissue inflammation, intestinal permeability, and plasma fetuin-A as potential mechanistic links between fructose intake and low-grade inflammation. DESIGN: We conducted a randomized, controlled, double-blind, crossover design dietary intervention (the Diet and Systemic Inflammation Study) in 24 normal-weight to obese adults without fructose malabsorption. Participants drank 4 servings/d of fructose-, glucose-, or HFCS-sweetened beverages accounting for 25% of estimated calorie requirements while consuming a standardized diet ad libitum for three 8-d periods. RESULTS: Subjects consumed 116% of their estimated calorie requirement while drinking the beverages with no difference in total energy intake or body weight between groups as reported previously. Fasting plasma concentrations of C-reactive protein and IL-6 did not differ significantly at the end of the 3 diet periods. We did not detect a consistent differential effect of the diets on measures of adipose tissue inflammation except for adiponectin gene expression in adipose tissue (P = 0.005), which was lowest after the glucose phase. We also did not detect consistent evidence of a differential impact of these sugars on measures of intestinal permeability (lactulose:mannitol test, plasma zonulin, and plasma lipopolysaccharide-binding protein). CONCLUSION: Excessive amounts of fructose, HFCS, and glucose from SSBs consumed over 8 d did not differentially affect low-grade chronic systemic inflammation in normal-weight to obese adults. This trial was registered at clinicaltrials.gov as NCT01424306.

Improvements in glycemic control after gastric bypass occur despite persistent adipose tissue inflammation.

OBJECTIVE: Type 2 diabetes commonly goes into remission following Roux-en-Y gastric bypass (RYGB). As the mechanisms remain incompletely understood, a reduction in adipose tissue inflammation may contribute to these metabolic improvements. Therefore, whether RYGB reduces adipose tissue inflammation compared with equivalent weight loss from an intensive lifestyle intervention was investigated. METHODS: Sixteen people with obesity and type 2 diabetes were randomized to RYGB or lifestyle intervention. Fasting blood and subcutaneous abdominal adipose tissue were obtained before and after the loss of ∼7% of baseline weight. Adipose tissue inflammation was assessed by whole-tissue gene expression and flow cytometry-based quantification of tissue leukocytes. RESULTS: At 7% weight loss, insulin and metformin use were reduced among the RYGB but not the Lifestyle cohort, while fasting glucose and insulin declined in both. Adipose tissue inflammation increased modestly after RYGB and to a similar extent following nonsurgical weight loss. In both groups, the number of neutrophils increased severalfold (P < 0.001), mRNA levels of the proinflammatory cytokine interleukin-1ß increased (P = 0.037), and mRNA expression of the anti-inflammatory and insulin-sensitizing adipokine adiponectin decreased (P = 0.010). CONCLUSIONS: A reduction in adipose tissue inflammation is not one of the acute weight loss-independent mechanisms through which RYGB exerts its antidiabetes effects.

No difference in ad libitum energy intake in healthy men and women consuming beverages sweetened with fructose, glucose, or high-fructose corn syrup: a randomized trial.

BACKGROUND: Increased energy intake is consistently observed in individuals consuming sugar-sweetened beverages (SSBs), likely mainly because of an inadequate satiety response to liquid calories. However, SSBs have a high content of fructose, the consumption of which acutely fails to trigger responses in key signals involved in energy homeostasis. It is unclear whether the fructose content of SSBs contributes to the increased energy intake in individuals drinking SSBs. OBJECTIVE: We investigated whether the relative amounts of fructose and glucose in SSBs modifies ad libitum energy intake over 8 d in healthy adults without fructose malabsorption. DESIGN: We conducted 2 randomized, controlled, double-blind crossover studies to compare the effects of consuming 4 servings/d of a fructose-, glucose-, or aspartame-sweetened beverage (study A; n = 9) or a fructose-, glucose-, or high-fructose corn syrup (HFCS)-sweetened beverage (study B; n = 24) for 8 d on overall energy intake. SSBs were provided at 25% of estimated energy requirement, or an equivalent volume of the aspartame-sweetened beverage, and consumption was mandatory. All solid foods were provided at 125% of estimated energy requirements and were consumed ad libitum. RESULTS: In study A, ad libitum energy intake was 120% ± 10%, 117% ± 12%, and 102% ± 15% of estimated energy requirements when subjects consumed the fructose-, glucose-, and aspartame-sweetened beverages. Energy intake was significantly higher in the fructose and glucose phases than in the aspartame phase (P < 0.003 for each), with no difference between the fructose and glucose phases (P = 0.462). In study B, total energy intake during the fructose, HFCS, and glucose phases was 116% ± 14%, 116% ± 16%, and 116% ± 16% of the subject's estimated total energy requirements (P = 0.880). CONCLUSIONS: In healthy adults, total 8-d ad libitum energy intake was increased in individuals consuming SSBs compared with aspartame-sweetened beverages. The energy overconsumption observed in individuals consuming SSBs occurred independently of the relative amounts of fructose and glucose in the beverages. These trials were registered at clinicaltrials.gov as NCT00475475 and NCT01424306.

Macrophage metalloelastase (MMP12) regulates adipose tissue expansion, insulin sensitivity, and expression of inducible nitric oxide synthase.

Macrophage metalloelastase, a matrix metallopeptidase (MMP12) predominantly expressed by mature tissue macrophages, is implicated in pathological processes. However, physiological functions for MMP12 have not been described. Because mRNA levels for the enzyme increase markedly in adipose tissue of obese mice, we investigated the role of MMP12 in adipose tissue expansion and insulin resistance. In humans, MMP12 expression correlated positively and significantly with insulin resistance, TNF-α expression, and the number of CD14(+)CD206(+) macrophages in adipose tissue. MMP12 was the most abundant matrix metallopeptidase detected by proteomic analysis of conditioned medium of M2 macrophages and dendritic cells. In contrast, it was detected only at low levels in bone marrow derived macrophages and M1 macrophages. When mice received a high-fat diet, adipose tissue mass increased and CD11b(+)F4/80(+)CD11c(-) macrophages accumulated to a greater extent in MMP12-deficient (Mmp12(-/-)) mice than in wild-type mice (Mmp12(+/+)). Despite being markedly more obese, fat-fed Mmp12(-/-) mice were more insulin sensitive than fat-fed Mmp12(+/+) mice. Expression of inducible nitric oxide synthase (Nos2) by Mmp12(-/-) macrophages was significantly impaired both in vivo and in vitro, suggesting that MMP12 might mediate nitric oxide production during inflammation. We propose that MMP12 acts as a double-edged sword by promoting insulin resistance while combatting adipose tissue expansion.

n3 PUFAs do not affect adipose tissue inflammation in overweight to moderately obese men and women.

Recent studies have indicated that omega-3 (n3) polyunsaturated fatty acids (PUFAs) decrease adipose tissue inflammation in rodents and in morbidly obese humans. We investigated whether a diet rich in n3 PUFAs from both marine and plant sources reduces adipose tissue and systemic inflammation in overweight to moderately obese adults. We conducted a randomized, single-blind, parallel-design, placebo-controlled feeding trial. Healthy men and women with a body mass index between 28 and 33 kg/m(2) consumed a diet rich in n3 PUFAs (3.5% of energy intake; n = 11) from plant and marine sources or a control diet (0.5% of energy intake from n3 PUFAs; n = 13). These diets were consumed for 14 wk (ad libitum for 12 wk). All foods were provided for the entire study period. Subcutaneous abdominal adipose tissue and fasting plasma were collected after the first 2 wk with the control diet and again at the end of the 14-wk dietary period. The primary outcome of this ex post analysis was the adipose tissue gene expression of 13 key mediators of inflammation. Adipose tissue gene expression of inflammatory mediators did not differ between the 2 groups, after adjustment for weight change. Furthermore, none of the 5 plasma markers of systemic inflammation differed significantly as an effect of diet treatment. We conclude that a relatively high dose of n3 PUFAs from plant and marine sources did not significantly lower adipose tissue or systemic inflammation in overweight to moderately obese healthy men and women over 14 wk.

Characterizing and quantifying leukocyte populations in human adipose tissue: impact of enzymatic tissue processing.

Adipose tissue inflammation is a major mechanistic link between obesity and chronic disease. To isolate and characterize specific leukocyte populations, e.g. by flow cytometry, tissue needs to be processed to digest the extracellular matrix. We have systematically compared the impact of different commonly used collagenase preparations, digestion times, and normalization strategies on the reproducibility of flow cytometric phenotyping of adipose tissue leukocyte populations. Subcutaneous adipose tissue was obtained from 11 anonymous donors undergoing elective procedures at a plastic surgery clinic in Seattle, WA. We found that collagenase alone consistently produced better cell yields (p=0.007) than when combined with additional proteases such as the commercially available liberases. Moreover, liberase significantly degraded the cell surface expression of CD4 (p<0.001) on T cells and to a lesser extent CD16 (p=0.058) on neutrophils. Extension of the digestion interval from 30 to 120 min did not significantly impact cell viability (p=0.319) or yield (p=0.247). Normalization by either 'live-gate' or percentage of CD45(pos) leukocytes exhibited the lowest coefficient of variation for tissue digests between 60 and 75 min, compared to normalization per gram of tissue, which consistently exhibited the greatest variability. Our data suggest that digestion of adipose tissue using pure collagenase for 60-75 min provides the best cell yield and viability, with minimal degradation of cell surface markers used to identify immune cell subpopulations, and best reproducibility independent of the normalization strategy.

Binding of activating transcription factor 6 to the A5/Core of the rat insulin II gene promoter does not mediate its transcriptional repression.

Pancreatic ß-cells have a well-developed endoplasmic reticulum due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. It has been previously reported that overexpression of activating transcription factor 6 (ATF6) reduces insulin gene expression in part via upregulation of small heterodimer partner. In this study, we investigated whether ATF6 directly binds to the insulin gene promoter, and whether its direct binding represses insulin gene promoter activity. A bioinformatics analysis identified a putative ATF6 binding site in the A5/Core region of the rat insulin II gene promoter. Direct binding of ATF6 was confirmed using several approaches. Electrophoretic mobility shift assays in nuclear extracts from MCF7 cells, isolated rat islets and insulin-secreting HIT-T15 cells showed ATF6 binding to the native A5/Core of the rat insulin II gene promoter. Antibody-mediated supershift analyses revealed the presence of both ATF6 isoforms, ATF6α and ATF6ß, in the complex. Chromatin immunoprecipitation assays confirmed the binding of ATF6α and ATF6ß to a region encompassing the A5/Core of the rat insulin II gene promoter in isolated rat islets. Overexpression of the active (cleaved) fragment of ATF6α, but not ATF6ß, inhibited the activity of an insulin promoter-reporter by 50%. However, the inhibitory effect of ATF6α was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity.

Lack of preservation of insulin gene expression by a glucagon-like peptide 1 agonist or a dipeptidyl peptidase 4 inhibitor in an in vivo model of glucolipotoxicity.

UNLABELLED: Prolonged exposure of pancreatic beta-cells to elevated levels of glucose and fatty acids adversely affects insulin secretion and gene expression. AIM: To examine whether the GLP-1 agonist exenatide or the inhibitor of the GLP-1-degrading enzyme dipeptidyl peptidase 4 (DPP-4) sitagliptin rescue insulin gene expression in rats infused for 72h with glucose+Intralipid, independently from their glucose-lowering action. METHODS: Wistar rats were infused alternatively with glucose or Intralipid for cycles of 4h each for a total of 72h. The animals received exenatide (5microg/kg/day IV) or sitagliptin (5mg/kg/day IV) continuously starting 4 days prior to and continuing throughout the 3-day infusion period. RESULTS: Plasma glucose, fatty acids, insulin and C-peptide levels were unaffected by exenatide or sitagliptin treatment during the infusion period. Insulin mRNA levels increased in response to the glucose infusion, but this increase was abolished in islets from rats receiving glucose+Intralipid. Neither exenatide nor sitagliptin administration rescued insulin mRNA in glucose+Intralipid infused rats. CONCLUSIONS: Neither a GLP-1 agonist nor a DPP-4 inhibitor, at doses that do not alter blood glucose levels, prevented the inhibition of insulin gene expression in this in vivo model of glucolipotoxicity.

Involvement of Per-Arnt-Sim Kinase and extracellular-regulated kinases-1/2 in palmitate inhibition of insulin gene expression in pancreatic beta-cells.

OBJECTIVE: Prolonged exposure of pancreatic beta-cells to simultaneously elevated levels of fatty acids and glucose (glucolipotoxicity) impairs insulin gene transcription. However, the intracellular signaling pathways mediating these effects are mostly unknown. This study aimed to ascertain the role of extracellular-regulated kinases (ERKs)1/2, protein kinase B (PKB), and Per-Arnt-Sim kinase (PASK) in palmitate inhibition of insulin gene expression in pancreatic beta-cells. RESEARCH DESIGN AND METHODS: MIN6 cells and isolated rat islets were cultured in the presence of elevated glucose, with or without palmitate or ceramide. ERK1/2 phosphorylation, PKB phosphorylation, and PASK expression were examined by immunoblotting and real-time PCR. The role of these kinases in insulin gene expression was assessed using pharmacological and molecular approaches. RESULTS: Exposure of MIN6 cells and islets to elevated glucose induced ERK1/2 and PKB phosphorylation, which was further enhanced by palmitate. Inhibition of ERK1/2, but not of PKB, partially prevented the inhibition of insulin gene expression in the presence of palmitate or ceramide. Glucose-induced expression of PASK mRNA and protein levels was reduced in the presence of palmitate. Overexpression of wild-type PASK increased insulin and pancreatic duodenal homeobox-1 gene expression in MIN6 cells and rat islets incubated with glucose and palmitate, whereas overexpression of a kinase-dead PASK mutant in rat islets decreased expression of insulin and pancreatic duodenal homeobox-1 and increased C/EBPbeta expression. CONCLUSIONS: Both the PASK and ERK1/2 signaling pathways mediate palmitate inhibition of insulin gene expression. These findings identify PASK as a novel mediator of glucolipotoxicity on the insulin gene in pancreatic beta-cells.

The stability and transactivation potential of the mammalian MafA transcription factor are regulated by serine 65 phosphorylation.

The level of the MafA transcription factor is regulated by a variety of effectors of beta cell function, including glucose, fatty acids, and insulin. Here, we show that phosphorylation at Ser(65) of mammalian MafA influences both protein stability and transactivation potential. Replacement of Ser(65) with Glu to mimic phosphorylation produced a protein that was as unstable as the wild type, whereas Asp or Ala mutation blocked degradation. Analysis of MafA chimeric and deletion constructs suggests that protein phosphorylation at Ser(65) alone represents the initial degradation signal, with ubiquitinylation occurring within the C terminus (amino acids 234-359). Although only wild type MafA and S65E were polyubiquitinylated, both S65D and S65E potently stimulated transactivation compared with S65A. Phosphorylation at Ser(14) also enhanced activation, although it had no impact on protein turnover. The mobility of MafA S65A was profoundly affected upon SDS-PAGE, with the S65E and S65D mutants influenced less due to their ability to serve as substrates for glycogen synthase kinase 3, which acts at neighboring N-terminal residues after Ser(65) phosphorylation. Our observations not only illustrate the sensitivity of the cellular transcriptional and degradation machinery to phosphomimetic mutants at Ser(65), but also demonstrate the singular importance of phosphorylation at this amino acid in regulating MafA activity.

Cyclical and alternating infusions of glucose and intralipid in rats inhibit insulin gene expression and Pdx-1 binding in islets.

OBJECTIVE: Prolonged exposure of isolated islets of Langerhans to elevated levels of fatty acids, in the presence of high glucose, impairs insulin gene expression via a transcriptional mechanism involving nuclear exclusion of pancreas-duodenum homeobox-1 (Pdx-1) and loss of MafA expression. Whether such a phenomenon also occurs in vivo is unknown. Our objective was therefore to ascertain whether chronic nutrient oversupply inhibits insulin gene expression in vivo. RESEARCH DESIGN AND METHODS: Wistar rats received alternating 4-h infusions of glucose and Intralipid for a total of 72 h. Control groups received alternating infusions of glucose and saline, saline and Intralipid, or saline only. Insulin and C-peptide secretion were measured under hyperglycemic clamps. Insulin secretion and gene expression were assessed in isolated islets, and beta-cell mass was quantified by morphometric analysis. RESULTS: Neither C-peptide secretion nor insulin sensitivity was different among infusion regimens. Insulin content and insulin mRNA levels were lower in islets isolated from rats infused with glucose plus Intralipid. This was associated with reduced Pdx-1 binding to the endogenous insulin promoter, and an increased proportion of Pdx-1 localized in the cytoplasm versus the nucleus. In contrast, MafA mRNA and protein levels and beta-cell mass and proliferation were unchanged. CONCLUSIONS: Cyclical and alternating infusions of glucose and Intralipid in normal rats inhibit insulin gene expression without affecting insulin secretion or beta-cell mass. We conclude that fatty acid inhibition of insulin gene expression, in the presence of high glucose, is an early functional defect that may contribute to beta-cell failure in type 2 diabetes.