Progress 09/01/09 to 08/31/13
Outputs
Target Audience: Nutritionists and nutrition scientists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Provided an opportunity for graduate students in nutrition to gain experience with designing and executing a nutrition intervention trial. This included working with the IRB, developing a study design, volunteer recruitment, data analyses and preparation of a scientific manuscript. How have the results been disseminated to communities of interest? Results from this study have been published in Endocrinology as noted in our earlier progress reports. Our most recent data has been presented in abstract form at two different scientific venues. The effect of selective amino acid supplementation on calcium absorption during a low protein diet. Jessica D Bihuniak, Rebecca R Sullivan, Christine A Simpson, Donna M Caseria, Kimberly O O’Brien, Jane E Kerstetter, Karl L Insogna Experimental Biology Annual Meeting, April 2013 The effect of selective amino acid supplementation on calcium absorption during a low protein diet. Jessica D Bihuniak, Rebecca R Sullivan, Christine A Simpson, Donna M Caseria, Kimberly O O’Brien, Jane E Kerstetter, Karl L Insogna University of Connecticut College of Agriculture and Natural Resources Graduate Student Research Forum, April 2013 What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Aim 1. As of December 2012, 14 subjects successfully completed the study protocol. Sample and data analyses were completed during the winter and spring of 2013. There was a decline in urinary calcium during all three experimental diets, however, this change only reached statistical significance during the control diet (P = 0.008). By day 5, urinary calcium was significantly greater with the dibasic amino acid supplementation (3.75 ± 0.49 mmol/d) than the control diet (2.86 ± 0.32 mmol/d, P = .039). There was no significant difference in calcium absorption between the diet supplemented with aromatic amino acids and the control diet (22.9±2.0% vs. 22.3±1.4% respectively, P = 0.643). However, there was a non-significant trend toward increased calcium absorption with dibasic amino acids supplementation as compared to the control diet (25.2±1.4% vs. 22.3±1.4%, P = 0.094), representing a mean difference of 11.51%. Because dietary calcium was fixed at 20 mmol/d, the intestinal absorption on the low protein diet supplemented with dibasic amino acids was 0.59 ± 0.33 mmol/d higher than on the control diet. The modest increase in intestinal calcium absorption mirrored the change in urinary calcium observed during the two diets. Aim 2. The majority of this aim was completed during the second year of the grant (9/1/10-8/31/11). The studies in Caco-2 cells are ongoing. The remainder of these studies related to this specific aim have been published (Gaffney-Stomberg, Endocrinology 151:1071, 2010). Aim 3. This aim was completed during the second year of the grant and the results have been published (Gaffney-Stomberg, Endocrinology 151:1071, 2010). Results for aim 2 and 3 were provided in our 9/1/2009 – 8/31/2010 and 9/1/2010 - 8/31/2011 reports.
Publications
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: The Aims of this project are to: 1. Determine which functional group of amino acids is most responsible for the effects of dietary protein on intestinal calcium absorption. This study seeks to recapitulate the effect of increasing dietary protein on calcium absorption by adding selected amino acids to a low protein diet such that their total dietary content is equal to that of our high-normal protein diet. We will determine which group of amino acids most improves calcium absorption.2. Determine if dietary protein regulates calcium transport by transcellular and/or paracellular routes. Isolated enterocyte brush-border membrane vesicles from rats habituated to either low or high protein diets will be used to quantify rates of calcium uptake as an index of transcellular calcium transport. Caco2 cell monolayers will be used to determine if dietary protein augments paracellular calcium transport. 3. Determine if amino acids/peptides augment expression of the intestinal calcium transporter, TRPV6 (Transient Receptor Potential Channel 6). TRPV6 is the principal identified calcium transporter in the duodenum. Although TRPV5 is also expressed in the gut, its role in rat intestinal calcium transport is thought to be minor. We will use quantitative PCR and western analyses to determine if the levels of TRPV6 transcript and/or TRPV6 protein expression are altered in enterocytes by changing dietary protein intake in rats. We will also determine if peptide mixtures directly affect TRPV6 expression in vitro in CaCo2 cells. Progress towards these specific aims: Aim 1. To date, 12 subjects have successfully completed the study protocol. The last 2 study subjects are currently enrolled and will be finishing in December 2012. Aim 2. This aim was completed during the second year of the grant (9/1/10-8/31/11) and we are working towards a publication. Aim 3. This aim was completed during the second year of the grant and the results have been published (Gaffney-Stomberg, Endocrinology 151:1071, 2010). Results for aim 2 and 3 were provided in our 9/1/2009 - 8/31/2010 and 9/1/2010 - 8/31/2011 reports. PARTICIPANTS: Jessica Binhuniak, M.S., R.D. worked on this project as a doctoral candidate in the School of Nutritional Sciences at the University of Connecticut. TARGET AUDIENCES: The target audience for this project is the Nutritional Science community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Dr. Insogna spoke at the Endocrine Symposia "Endo 2010" Clinical Symposia June 19-22, 2010 program titled: Give Me a Break! Skeletal Complications of Medications. Dr. Insognas presentation was titled "Proton pump inhibitors and fracture risk: de nada or more aggita"
Publications
- No publications reported this period
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: The Aims of this project are to: 1. Determine which functional group of amino acids is most responsible for the effects of dietary protein on intestinal calcium absorption. This study seeks to recapitulate the effect of increasing dietary protein on calcium absorption by adding selected amino acids to a low protein diet such that their total dietary content is equal to that of our high-normal protein diet. We will determine which group of amino acids most improves calcium absorption.2. Determine if dietary protein regulates calcium transport by transcellular and/or paracellular routes. Isolated enterocyte brush-border membrane vesicles from rats habituated to either low or high protein diets will be used to quantify rates of calcium uptake as an index of transcellular calcium transport. Caco2 cell monolayers will be used to determine if dietary protein augments paracellular calcium transport. 3. Determine if amino acids/peptides augment expression of the intestinal calcium transporter, TRPV6 (Transient Receptor Potential Channel 6). TRPV6 is the principal identified calcium transporter in the duodenum. Although TRPV5 is also expressed in the gut, its role in rat intestinal calcium transport is thought to be minor. We will use quantitative PCR and western analyses to determine if the levels of TRPV6 transcript and/or TRPV6 protein expression are altered in enterocytes by changing dietary protein intake in rats. We will also determine if peptide mixtures directly affect TRPV6 expression in vitro in CaCo2 cells. Progress towards these specific aims: Aim 1. To date, we have completed absorption studies in 7 subjects each of whom has completed one of the three interventions. We are actively recruiting and screening subjects. Aim 2. As noted in last years progress report, we completed our brush-border membrane vesicles studies and those findings have been published. We have now completed in vitro experiments using Caco-2 Bbe cells to explore the impact of dietary protein on paracelluar calcium transport and have summarized our findings in the "Outcomes Section" of the report. Aim 3. We have successfully isolated RNA from the duodenum of rats habituated to the 5%, 20% or 40% (n=6-9 per group) casein protein diet for one week and from Caco-2 Bbe cells exposed to 2X amino acids (twice the amount of amino acids found in normal growth media) for 6 hours. We used quantitative PCR to assess changes in the level of expression of intestinal transporter TRPV6. In addition, we performed a whole genome microarray screen using RNA isolated from enterocytes isolated from the duodenum of rats habituated to the 5%, and 40% diets to explore the possibility that novel calcium transporters are regulated by dietary protein. We have summarized our findings for Aim 3 in the "Outcomes Section" of the report. PARTICIPANTS: Jessica Bihuniak, M.S., R.D worked on this project as a Ph.D student in the Nutritional Sciences Department at the University of Connecticut. TARGET AUDIENCES: This research is primarily for the nutritional science community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results from experiments addressing Aim 2. We explored whether exposing Caco-2 cell monolayers to amino acids in the presence of a calcium gradient results in increased unidirectional calcium flux which would be indicative of increased paracellular Ca transport. Since a calcium gradient would be expected in vivo after consumption of a calcium-containing meal, these experimental conditions more closely model the in vivo environment. When exposed to 100 mM extracellular calcium, the transcellular calcium transporters are saturated and the paracellular pathway of calcium absorption predominates. Under these conditions, we found that unidirectional calcium flux was increased ~34% in Caco-2 cells exposed to 2X amino acids compared to control (p<0.05). Raffinose, a non-digestible oligsaccharide which has been reported to decrease transepithelial resistance (TER) and increase paracellular calcium flux across Caco-2 cells increased calcium flux by ~50% versus control (p<0.05). Consistent with a previous report, raffinose resulted in increased calcium flux by decreasing TER across the Caco-2 cell monolayer. Interestingly, amino acids resulted in increased paracellular calcium flux without a global change in tissue resistance. Results from experiments addressing Aim 3. By qPCR we found no change in the level of expression of TRPV6 and TRPV5 is enterocytes from animals eating a 40% protein diet as compared to animals on a 5% diet (Gaffney-Stomberg, Endocrinology 151:1071, 2010). We were not able to analyze TRPV6 or TRPV5 expression by Western blotting because we could not find a suitable antibody for either. However, the microarray screen identified one gene known to be involved in paracellular calcium flux, claudin-2. Claudin-2 was upregulated by 2.9-fold in the duodenal mucosa harvested from rats in the high protein group (40% protein diet). QPCR analysis of the same RNA samples used in the microarray screen confirmed that claudin-2 was upregulated (2.5 fold) in the high protein group compared to the low protein group (5% protein diet). When we evaluated claudin-2 expression by qPCR using RNA isolated from duodenum of rats consuming 5%, 20%, and 40% protein diets we found a dose-dependent increase in claudin-2 expression as dietary protein was increased. Additionally, incubating Caco-2 Bbe cells with 2X amino acids resulted in a 48% increase in claudin-2 expression. The observation that TRPV6 expression was not increased in Caco-2 cells incubated with amino acids suggests a specific effect of amino acids on claudin-2 expression rather than a global increase in gene expression due to changes in metabolic substrates.
Publications
- Wright MJ, Sullivan RR, Gaffney-Stomberg E, Caseria DM, OBrien KO, Proctor DD, Simpson CA, Kerstetter JE, Insogna KL. Inhibiting gastric acid production does not affect intestinal calcium absorption in young, healthy individuals: a randomized, crossover, controlled clinical trial. J Bone Miner Res. 2010 Oct; 25(10):2205-11.
- Kerstetter JE, Kenny AM, Insogna KL. Dietary protein and skeletal health: a review of recent human research. Curr Opin Lipidol. 2011 Feb;22(1):16-20.PMID: 21102327.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: The Aims of this project are to: 1. Determine which functional group of amino acids is most responsible for the effects of dietary protein on intestinal calcium absorption. This study seeks to recapitulate the effect of increasing dietary protein on calcium absorption by adding selected amino acids to a low protein diet such that their total dietary content is equal to that of our high-normal protein diet. We will determine which group of amino acids most improves calcium absorption.2. Determine if dietary protein regulates calcium transport by transcellular and/or paracellular routes. Isolated enterocyte brush-border membrane vesicles from rats habituated to either low or high protein diets will be used to quantify rates of calcium uptake as an index of transcellular calcium transport. Caco2 cell monolayers will be used to determine if dietary protein augments paracellular calcium transport. 3. Determine if amino acids/peptides augment expression of the intestinal calcium transporter, TRPV6 (Transient Receptor Potential Channel 6). TRPV6 is the principal identified calcium transporter in the duodenum. Although TRPV5 is also expressed in the gut, its role in rat intestinal calcium transport is thought to be minor. We will use quantitative PCR and western analyses to determine if the levels of TRPV6 transcript and/or TRPV6 protein expression are altered in enterocytes by changing dietary protein intake in rats. We will also determine if peptide mixtures directly affect TRPV6 expression in vitro in CaCo2 cells. Progress towards these specific aims: Aim 1. We have amended our protocol to reflect the recommendations made by our biostatistician, James Dziura, PhD, MPH. Dr. Dziura felt that we would be underpowered if we undertook 5 interventions, would have an excessive attrition rate and unduly prolong the study. Furthermore, Dr. Dziura suggested reducing the study to 3 interventions. Based on available data we have removed the branch-chain amino acids because they are least likely to have an effect on calcium absorption. We have already established that a high protein diet (2.1 g/kg/d) increases calcium absorption in this population, which is why we have also chosen to eliminate the high protein group. We have obtained IRB approval and are in the process of recruiting and screening subjects. Aim 2. We have completed our brush-border membrane vesicles studies and have summarized our findings in the Outcomes section of the report. We are in the process of growing Caco-2 Bbe cells for paracellular transport studies. Aim3. As stated in our grant, we will begin conducting experiments to address aim 3 during the second year of funding. PARTICIPANTS: Jessica Bihuniak Masters candidate in the Department of Nutritional Sciences at the University of Connecticut Erin Gaffney-Stomberg doctoral candidate in the Department of Nutritional Sciences at the University of Connecticut TARGET AUDIENCES: The nutritional science community is our target audience. PROJECT MODIFICATIONS: See modifications in the clinical study reported under Aim 1 in the section Outputs.
Impacts
Results from Aim 2. To explore the mechanisms underlying dietary proteins effect on intestinal Ca absorption, female Sprague-Dawley rats were fed a control (20%), low (5%), or high (40%) protein diet for 7 days and Ca balance was measured during days 4-7. On day 7, duodenal mucosa was harvested and brush border membrane vesicles (BBMV) prepared to evaluate Ca uptake. By day 7, UCa was more than two-fold higher in the 40% protein group compared to control (4.2 mg/d vs. 1.7 mg/d, p <0.05). Rats consuming the 40% protein diet both absorbed and retained more Ca compared to the 5% protein group (absorption: 48.5% vs. 34.1% and retention: 45.8% vs. 33.7% respectively, p <0.01). Initial rates of Ca uptake were increased in BBMV prepared from rats consuming the high protein diet. Vmax was higher in the BBMV prepared from the high protein group compared to those from the low protein group (90 vs. 36 nmol Ca/ mg protein/ min, p <0.001; 95% CI: 46-2486 and 14-55, respectively). Km was unchanged (2.2 mM vs. 1.8 mM, respectively; p = 0.19). We conclude that in rats as in humans, acute increases in protein intake result in hypercalciuria due to augmented intestinal Ca absorption. BBMV Ca uptake studies suggest that higher protein intake improves Ca absorption, at least in part, by increasing transcellular Ca uptake. The increase in Vmax with no change in Km suggests that either more transporters are present on the apical membrane of enterocytes isolated from animals in the high protein diet or that the activity of the transporters is increased or both.
Publications
- Kerstetter J, Gaffney-Stomberg E, Simpson C, Insogna K, Sun B-H, Cucchi C. High Protein Intake Increases Duodenal Transcellular Calcium Absorption in Rats. J Bone Min Res 24 (Suppl 1), 2009
- Cucchi C, Gaffney-Stomberg E, Sun B-H, Kerstetter J, Insogna K. A high protein diet induces intestinal iron transporter expression and improves iron absorption in rats. FASEB J. 2010 24:229.1
- Sullivan R, Rosewater I, Caseria D, OBrien K, Kerstetter J, Insogna K. Six weeks of a low protein diet impairs calcium homeostasis. FASEB J. 2010 24:lb356
- Gaffney-Stomberg E-G, Sun B-h, Cucchi C, Simpson C, Gundberg C, Kerstetter J, Insogna K. The Effect of Dietary Protein on Intestinal Calcium Absorption in Rats Endocrinology 151:1071, 2010
- Surdykowski A, Kenny A, Insogna K, Kerstetter J. Optimizing bone health in older adults: the importance of dietary protein. Aging Health 6 345-357, 2010. PMID 20657805. PMC 2840679.
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