- Click here for explanation of classification scheme.

The purpose of this series of 3 studies was the following:

• to test (using primary cultures of human adipocytes) the hypothesis that calcitrophic hormones may act on adipocytes to increase Ca²⁺ and a corresponding marked inhibition of lipolysis;
• to test (in transgenic mice expressing the agouti gene) the hypothesis that dietary calcium could reduce adipocyte mass by supporessing 1,25-(OH)₂-D;
• to test the concept of adiposity epidemiologically using the NHANES III data set.  

In Vitro (Primary Cultures of Human Adipocytes) Study: Study used human subcutaneous adipose tissue obtained from patients with no known history of metabolic disorders undergoing abdominal surgery

Mice Study: Male transgenic mice expressing agouti specifically in adipocytes under the control of the aP2 promoter (these mice exhibit a normal pattern of leptin expression and activity similar to that found in humands and exhibit a human pattern (adipocte-specific) of agouti expression.  These mice have been found to be useful modesl for diet-induced obesity).

Human Study: Adults completing all 3 phases of the NHANES III cross-sectionial survey conducted between 1988 and 1994: (1) interview; (2) physical examination; and (3) laboratory examination.

 

In Vitro (Primary Cultures of Human Adipocytes) Study: Study used human subcutaneous adipose tissue obtained from patients with known history of metabolic disorders

Mice Study: NA

Human Study: Adult respondents were excluded from this analysis if they couldn't provide complete, usable body composition/anthropometic data (e.g., amputees and individuals with casts), used insulin, or were pregnant, recently pregnant, or currently breastfeeding  

 

 

 

Recruitment Not indicated for NHANES III analysis

Design

1. In Vitro (Primary Cultures of Human Adipocytes) Study: Human subcutaneous adipose tissue was isolated by washing, mincing, collagenase digestion, and filtration and cultured in Dulbecco's modified Eagle's medium supplemented with 1% fetal bovine serum, penicillin, streptomycin, and gentamicin. Cells were cultured in suspension and maintained in a thin layer at the top of the culture media which was changed daily. Cells were studies approximately 72 hours after isolation and were serum-starved prior to study. Effects of both 1,25-(OH)₂-D and parathyroid hormone (PTH) on intracellular Ca²⁺ on adipocyte lipolysis were measured. 
2. Mice Study: Mice were placed at 6 weeks of age on a modified AIN 93-G diet with suboptimal calcium (0.4%), sucrose (sole CHO source), and fat (increased to 25% of energy with lard). Mice were then randomized into 4 groups: (a) basal group continued above diet with no modification; (b) a high calcium group ate basal diet supplemented with CaCO₃ to increase dietary calcium by threefold to 1.2%; (c) a medium dairy diet, in which 25/% of protein was replaced by non-fat dry milk and dietary calcium was increased to 1.2%; and (d) a high dairy group in which 50% of protein was replaced by non-fat dry milk, which increased calcium to 2.4%. Food intake and spillage were measured on a daily basis, and animals were weighed on a weekly basis. At end of the 6 week feeding period, mice were killed by exsanguination under isoflurane anesthesia, and blood wasa collected via cardiac puncture for glucose and insulin measurements. Fat pads (epididymal, perirenal, abdominal, and subscapular) were dissected, weighed immediately, frozen in liquid nitrogen, and stores at -80 degrees C. Fatty acid synthase activite and mRNA levels were measured in abdominal fat.   

Human Study: After controlling for various variables (energy intake, activity level, age, race, and ethnicity), the odds ratios (OR) of being in the highest to lowest quartiles of calcium intake were determined.     

Statistical Analysis

Mice Study: Data were evaluated for statistical significance using one-way ANOVA or t test, depending on the number of comparisons to be made. All data sets with multiple comparisons were analyzed using ANOVA followed by separation of significantly different group means via test the least significant difference using SPSS-PC.

Human Study: OR for % body fat and corresponding 95% confidence intervals were estimated using multiple logistic regression analysis with a robust variance estimation method using SUDAAN. Point estimates for all parameters were weighted to reflect population distribution of each and variances were calculated using SUDAAN. Analyses were conducted separately for men and women, and all odds rations were adjusted for age (age was included in model as a continuous variable). Other covariates in the model were calorie intake, race/ethnicity, and activity level.  

Timing of Measurements

In Vitro (Primary Cultures of Human Adipocytes) Study: Cells were studied approximately 72 hours after isolation.

Mice Study: Mice were killed and measurements taken after mice had been on the various diets for a 6-week period.   

 Dependent Variables

In Vitro (Primary Cultures of Human Adipocytes) Study:

1. Lipolysis-after adipocytes were incubated for 4 hours in presence or absence of forskolin, gylcerol release was measured.
2. Intracellular Ca^2+-determined fluorometrically. Cells were wshed with HEPES-buffered salt solution loaded with Fura-2-acetoxymethyl ester for 45 inutes at 37 degrees C. in the dark. Cells were rinsed 3 times, resuspended, and then intracellular Ca²⁺ was measured using dual excitation fluorometry.   

Mice Study:

1. Weight change
2. Core temperature-measured via a thermo-couple, with all temperature measurements made between 8am and 9am
3. Fatty acid synthase activity-immediately post-death, adipose tissue was isolated and fatty acid synthase activity was measured in cytosolic extracts by measuring oxidation rate of NAHPH. Enzyme activity was protein corrected using Coomassie blue dye.
4. mRNA levels-Total RNA was exbracted using cesium choloride density gradient, electrophoresed, subjected to Northern blot analysis, and then hybridized with radiolabled rat cDNA probe for fatty acid synthase using standard methods. Autoradiographs were quantified densitometricaly. All blots were stripped and reprobed with beta-actin as a loading control.  
5. Fasting Insulin and Glucose 

Human Study:

Body Fat Quartile-body composition was measured using anthropometric and bioelectical impedence data colelcted during physical examination of NHANES III study, with % body fat calculated using regression equations derived by Segal.

Independent Variables

In Vitro (Primary Cultures of Human Adipocytes) Study: 

1. Parathyroid Hormone (PTH) Administration
2. 1,25-(OH)₂-D Administration

Mice Study:

Diet (basal diet: low calcium/high fat/high sucrose, basal diet with 25% of protein replaced by non-fat dry milk, basal diet with 50% of protein replaced by non-fat dry milk, or basal diet supplemented to 1.2% calcium with CaCO₃

Human Study:

1. Calcium Intake (measured in mg/day)
2. Dairy Consumption (measured in servings/month) 

Control Variables

Human Study: age, caloric intake, race/ethnicity, and activity level

 

Initial N: Unknown for mice study and human study 

Attrition (final N):

Mice study: 10 per diet group/assignment (total N=40)

Human study: 380 women and 7114 men

Age:

Mice study: mice were started on diets at 6 weeks of age and stayed on assigned diet for a 6 week feeding period  

Human Study: Women were an average of 28.7+0.4 years, men were an average of 43.5+0.44 years

Ethnicity:

No specific information was provided, except that HNANES III data set utilized a large cross-sectional survey conducted between 1988-1994 that followed a complex, 4-stage probabiliyt sampling scheme designed to represent the entire U.S. civilian noninstitutionalized population over age of 2 months.   

Anthropometrics and Other relevant demographics:

In Human Study: 

Mean Body Mass Index- Women: 25.7+0.4   Men: 26.6+0.11

Body Fat (%)- Women: 32.7+0.6  Men: 25+0.2

Calcium Intake (mg/day)-  Women: 720+52   Men: 965+15

Dairly Product Consumption (monthly frequency)-  Women: 54.7+3    Men: 51.4+1

Energy Intake (kcal/day)-  Women: 1896+68   Men: 2656+28

Dietary Fat (g/day)- Women: 74+4  Men: 102+2  

 

In Vitro (Primary Cultures of Human Adipocytes) Study:

Both 1,25-(OH)₂-D and PTH stimulate significant, sustained increases in intracellular Ca²⁺ in primary cultures of human adipocytes (P<0.001).  1,25-(OH)₂-D additionaly resulted in marked (83%) inhibition of forskolin-stimulated lipolysis (P<0.001) in human adipocytes, while PTH treatment exerted little effect on lipolysis. 

Mice Study:

1. Mice consuming a basal diet exhibited weight gain of 24%, which was reduced by 26 and 29% by the high calcium and medium dairy diets, respectively (P<0.04) and further reduced by 39% by the high dairy diet (P<0.04). These differences occurred even though there was no difference in food intake.   
2. Core temperature measurements (an indirect metabolic index) were in line with weight gain results, indicating increases in core temperature in all 3 high calcium diets (P<0.03). This increase, along with the lack of difference in food intake, indicate a shift in efficiency of energy metabolism from energy storage to thermogenesis.
3. Studies of fatty acid synthase also reflected this shift in energy storage. The basal diet casued a 2.6-fold increase in fatty acid synthase activity, with the effect markedly attenuated by all 3 high calcium diets (P<0.002). The diets resulted in corresponding decreases in adipocyte fatty acid synthase mRNA, with a 27% reduction on the high calcium diet and a 51% reduction on the medium and high dairy diets (P<0.01). 
4. The basal diet resulted in a marked (67%) suppression of lypolysis (P<0.0001). In contrast, lipolysis was stimuluated 3.4- to 5.2-fold by the high calcium diets (P<0.015), withi greater effects from the high dairy diets than from high calcium diets. 
5. Evaluation of fat pad mass after 6 weeks of dietary treatment further supported these findings. All 3 high calcium diets caused a 36% decrease in mass of epidymal, abdominal, perirenal, and subscapuular adipose tissue compartments (P<0.001). Epidymal and subscapular fat pad mass was decreaed by about 50% by all 3 diets, while the abdominal fat pads exhibited greater reductions on the medium and high dairy diets than on high calcium diet (P<0.001). 
6. Plasma glucose and insulin were increased on the basal (low calcium) diet, with an incrase in fasting glucose from 98+10 to 130+11 mg/dl (P<0.02) and a corresponding degree of compensatory hyperinsulimemia. However, these increases were attenuated by the high calcium and medium dairy diets and prevented by the high dairy diet.   

  

Human Study:

After controlling for age, actvity level, energy intake, race, and ethnicity, the OR of being in the highest quartile of body fat was markedly decreased from 1.00 for the first quartile of calcium intake (reflecting lowest level of calcium intake) to 0.75, 0.40, and 0.16 for the 2nd, 3rd, and 4th quartiles, respectively (multiple R² =0.20; P=0.0009) in adult women. In a similar manner, the regression model for men indicated an inverse relationship between calcium and dairy intakes and body fat (multiple R² =0.40; P=0.0006), though a compariable dose-responsive decrease in relative risk by quartile of calcium intake wasn't evident from the model.

 

 

Results of the in vitro, mice, and human studies documented in this report indicate that:

1. Dietary calcium appears to modulate effeciency of energy utilization, with low calcium diets favoring increased efficiency of energy storage and higher calcium diets decreasing energy efficienty and instead favoring the occurrence of increased thermogenesis.
2. Increasing dietary calcium suppresses adipocyte intracellular Ca²⁺ which as a result modulates energy metabolism and attenuates obesity risk.

University/Hospital:	University of Tennessee Knoxville
Not-for-profit	0

1. Little information was provided about how dietary data (in human study) was obtained.
2. Mice study indicated that the high dairy diet resulted in greater attenuation of weight gain than did the medium dairy or supplemental calcium diets.    
3. At end of article, it was noted that funding for this research was provided by the National Dairy Council.