AGE

, Volume 30, Issue 4, pp 201–208

Pilot study for an age- and gender-based nutrient signaling system for weight control

Authors

    • Biochemistry ProgramBeloit College
Article

DOI: 10.1007/s11357-008-9049-0

Cite this article as:
Ordman, A.B. AGE (2008) 30: 201. doi:10.1007/s11357-008-9049-0

Abstract

Nutrient signaling has recently shown how nutraceuticals regulate specific functions of the brain and adipose tissue. In this pilot study to find an effective nutrient signaling system to cause weight loss, a double-blind placebo-controlled trial using leucine, olive oil, and fish oil was conducted on volunteers to signal metabolic and appetite effects to regulate body weight, while controls took only fiber. Men and women aged 18–26 and 39–62 years were given different dosages that they took orally twice daily for 14 days while recording body weight, followed by 2 weeks to check rebound. Most young men and women lost weight on low dose leucine and olive oil. Mature men required higher doses. Mature women’s weight was affected least, though results are consistent with a hypothesis that sufficient leucine and docosahexaenoic acid would be effective. Determining how age affects signaling pathways by nutrients will be important to reduce risk of chronic disease associated with age and obesity. This pilot study has led to hypotheses of practical strategies.

Keywords

LeucineFish oilOleic acidBranched chain amino acidsDocosahexaenoic acidDHAOlive oilmTORObesityWeight loss

Abbreviations

BCAA

Branched chain amino acids

Cntl

Control

DHA

Docosahexaenoic acid

EPA

Eicosapentaenoic acid

Fsh

Fish oil

Hi

High dose (12 g/day in divided doses)

Lo

Low dose (6 g/day in divided doses)

Leu

Leucine

M

Men

mTOR

Mammalian Target of Rapamycin

O

Older

Olv

Olive oil

W

Women

Y

Younger

Introduction

Obesity is a major factor increasing the risk of chronic disease with age (WHO 2004; Woo et al. 2001). The rate of obesity has increased significantly worldwide (Bray 2004). In well-controlled longterm studies, people on many popular diet plans fail to lose weight (Zivkovic et al. 2007).

In the past decade, biological signaling pathways have been recognized to control many metabolic processes (Bhalla and Iyengar 1999). Studies began to recognize that beyond providing nutrition, certain molecules in food activate signaling pathways relevant to weight control. In 2006, three studies indicated how nutrients may signal metabolic controls related to body weight.

The first study was done with leucine. Other studies (She et al. 2007; Eley et al. 2007) have shown that branched chain amino acids (BCAA) produce anabolic responses to build muscle. But BCAA also stimulate the nutrient signaling pathway known as the mammalian Target of Rapamycin (mTOR) pathway (Sabatini et al. 1994). The protein regulated by the mTOR pathway is a serine-threonine kinase that regulates cell-cycle progression and growth by sensing changes in energy status. Cota et al. (2006) described the complex effects of mTOR signaling and then demonstrated in rats that leucine produces two signals in the brain in specific regions of the hypothalamus and the arcuate nucleus. They also demonstrated that those two signals decreased both food intake and body weight.

The second study was done with oleic acid. A signaling system proposed by Hsu and Huang (2006) could regulate metabolism in adipose tissue in rats. They proposed two signals that would occur in fat cells in response to oleic acid. Fatty acid catabolic genes would be upregulated while lipid storage genes would be downregulated.

The third signaling system was found by Kim et al. (2006) using docosahexaenoic acid (DHA). This fatty acid commonly associated with fish oil was shown to produce two important signals, one inhibiting adipocyte differentiation and another causing apoptosis in preadipocytes.

The goal of this pilot study was to begin exploring which different combinations and dosages of branched chain amino acids and fatty acids (Table 1) might affect weight loss in humans as a function of age and gender. Given the often contradictory reports from obesity studies, specific hypotheses were set forth based on familiarity with nutritional literature and the above citations. Although the hypotheses may be controversial, clearly stated hypotheses invite direct experimental exploration.
Table 1

Study considerations. The following variables were evaluated in this study, using abbreviations shown

Category

Symbol

Details 

Dosage

Lo

Low

6 g/day

Hi

High

12 g/day

Branched chain amino acid

Leu

Leucine

 

Fatty acid

Olv

Olive oil

 

Fsh

Fish oil

 

Control

Cntl

Cellulose

 

Gender

M

Men

 

W

Women

 

Age

O

Older

39–62 years

Y

Younger

18–24 years

Hypotheses

  1. 1.

    Eating the combination of leucine and a fatty acid will cause weight loss, because the signals will work synergistically.

     
  2. 2.

    Eating twice the dosage of leucine and a fatty acid will make weight loss more likely because stronger signals will be delivered. The initial doses selected for this study were extrapolated from the rat studies.

     
  3. 3.

    Fish oil is more likely to cause weight loss than olive oil, because DHA is essential in the diet while oleic acid is not.

     
  4. 4.

    It will be harder to cause weight loss in older (O) than younger (Y) people because signaling pathways may be less responsive.

     
  5. 5.

    It will be harder to cause weight loss in women (W) than in men (M) because women have evolved to survive to care for offspring.

     

These hypotheses resulted in the design of this study. I extrapolated combinations and dosages of nutrients from in vitro and rodent studies. The purpose of the study was to identify specific protocols that might have an effect in a trial large enough to achieve statistical significance. This pilot study provided an efficient way to determine protocols most likely to succeed in producing significant weight loss.

Materials and methods

Study population

The Beloit College Institutional Review Board approved the trial. Of 68 healthy volunteers who provided informed consent after the procedure had been fully explained, 8 withdrew without reason and 1 developed an illness unrelated to the study. Characteristics of the 59 who actively participated in the study are shown in Table 2.
Table 2

Characteristics of participants

No.

Age (years)

Weight (kg)

Dosages (g/day)

Group

Max

Min

Min

Max

Leu

Olv

Fsh

Cntl

Women over 38 years

7

58

44

58.5

114.3

12

12

0

0

5

52

40

68.0

99.3

12

0

12

0

5

59

39

56.7

125.6

6

6

0

0

4

58

47

70.3

100.7

0

0

0

12

Women 18–26 years

10

23

18

54.9

85.7

12

12

0

0

1

21

n/a

63.0

n/a

12

6

6

0

2

23

22

62.6

66.2

12

0

12

0

3

20

18

71.2

78.5

6

6

0

0

2

24

19

69.4

77.1

0

0

0

12

Men over 38 years

2

53

45

127.0

128.8

12

6

6

0

2

62

58

77.1

106.6

6

6

0

0

4

62

41

95.3

117.5

0

0

0

12

Men 18–26 years

4

22

19

96.2

138.8

12

6

6

0

4

23

18

62.6

97.5

6

6

0

0

4

26

24

73.9

92.1

0

0

0

12

Fifteen groups took different dosages of leucine, olive oil, fish oil, and cellulose when waking in the morning and again 0–3 h before the evening meal

Regimen and compliance

All signaling molecules were provided in 1-g capsules. Half of the daily dosage was taken upon waking in the morning, 0 to 3 h before breakfast. People then weighed themselves before eating. The second half of the pills was taken 0–3 h before the evening meal. Experimental groups swallowed high (12 g/day) or low (6 g/day) dosages of the BCAA leucine. They also took 6–12 g/day of either fish oil containing 0.12 g DHA and 0.18 g EPA/g fish oil, and/or olive oil containing 0.85 g oleic acid/g olive oil. Controls took cellulose tablets. Participants did not know which tablets they were taking. They took tablets for 14 days. They were encouraged to maintain normal behavior, not modifying eating or exercise habits during the trial. After 2 weeks of taking tablets, they recorded their weights 1 and 2 weeks later so that rebound effects could be evaluated.

Participants recorded the times they took the tablets and recorded their weight each day. They were also asked to maintain a diary of possibly relevant events or feelings. All participants had email addresses and received daily messages about the importance of the accuracy of their record keeping. Because of health considerations (Partnership For Healthy Weight Management 1999) in the IRB proposal, people were instructed not to take tablets on days in which their weight had dropped more than 0.91 kg (2 lb)/week.

Endpoints

Weight loss was measured as the difference between weight the first morning and the last morning after taking tablets. Rebound was taken as the difference between the last weighing after taking tablets and the weight 2 weeks later. To account for behavioral effects, participants were aware that this was a weight loss study where eating and exercise patterns were not to be modified, but did not know about the study design relating to age, gender, experimental, and control groups, or what the various tablets contained.

Statistics

Given safety considerations on weight loss and availability of resources, this pilot study was designed realizing there are more variables than were likely to be analyzed satisfactorily given the sample sizes in each cell. Application of analysis of variance (ANOVA) using the statistical software JMP did not suggest any differences among treatments significant at the 5% level. Statistical significance would have been obtained if every subject taking both leucine and a fatty acid had lost the maximum weight permitted (0.91 kg) and every control had lost no weight. The truncation of weight loss at 0.91 kg/week, reached by some treatment subjects, results in underestimation of average effect of the treatments.

Results

Public advertising through Beloit College networks was used to recruit volunteers. Table 3 shows the responses received that led to 59 weight records analyzed for this trial. During instruction of volunteers including informed consent, many withdrew once they understood clearly what a blind controlled study involved. The 59 people finishing the trial remembered to record 97.5% of 826 daily weights. Complete rebound data was not provided by 12 of 59 who turned in 14-day weighing records.
Table 3

Participant dairies

Record Type

Number

Signed informed consents

72

Assigned to receive tablets

68

Participants providing records

60

14-day weight record

59

2-week rebound record

48

Diary comments received

31

Comment not tablet related

13

Public advertising invited college-related audiences to consider participation; responses after public announcements of the trial are shown

Design of this pilot study has more effects than can be analyzed satisfactorily with available sample sizes. An analysis with “best possible” outcome did show that significance would have been possible with maximum weight loss difference from one group to another. The short duration does not allow greater weight loss that would be possible with a longer trial. Analysis of the values from weight records submitted by the participants is consistent with their accurately recording their weights.

Participants were provided daily diaries to record any comments in response to their experience. Table 4 shows the nature of those comments. All participants were encouraged to withdraw if their response to the tablets caused health concerns. No one gave any response on withdrawing from the study except the person who reported being diagnosed with mononucleosis on the second day of taking tablets. Females over 38 provided the highest level of comments, but none withdrew.
Table 4

Possible side effects

Sex

Age (years)

Dosage

No. in group

No. diaries received

Comments

F

Over 38

Control

4

1

Trouble sleeping

F

Over 38

L1O1

5

1

Decreased appetite

F

Over 38

L2F2

5

2

Reduced appetite 1 day; feel bloated

F

Over 38

L2O2

7

4

Excess elimination; painful gas and bloating; swollen hands/joints  day; flu last 4 days of rebound

M

Over 38

Control

4

1

No appetite 1 day

M

Over 38

L1O1

2

2

More energy; joints hurt 1 day

M

Over 38

L2O1F1

2

1

Constipated

F

18–26

Control

2

1

Nauseous 1 day

F

18–26

L2O2

9

2

Hungrier; feel great, more energy

M

18–26

Control

4

2

Hungrier; less tired

M

18–26

L2O1F1

4

1

Eating healthier foods

Of 59 people taking tablets for 14 days, 18 made comments that may be related. No comment came from more than 1 person in a group

This study was designed based on available resources to explore the five hypotheses presented in the introduction. The 59 participants were divided into 15 groups based on age, gender, and dosages. Table 5 summarizes weight change from first to last day of taking tablets, and weight regain (rebound) during the next two weeks. Values with negative signs indicate weight loss.
Table 5

Weight change and rebound

No. in group

Sex

Dosagesa

Lgst 2 week loss

Avg 2 week loss

Smlst 2 week loss

% Loss

% N.C.

% Gain

Lgst 2 week rebnd

Avg 2 week rebnd

Smlst 2 week rebnd

% Loss

% N.C.

% Gain

Leu

Olv

Fsh

Cntl

Young women

1

F

2

1

1

0

−0.9

−0.9

n.d.

100%

  

0.9

0.9

n.d.

100

  

3

F

1

1

0

0

−1.8

−1.0

0.0

67%

33%

 

−0.8

−0.4

0.0

 

67%

33%

10

F

2

2

0

0

−3.2

−0.6

1.4

60%

10%

30%

−1.8

−0.4

0.5

20%

40%

40%

2

F

2

0

2

0

−0.5

−0.2

0.0

50%

50%

 

n.d.

−0.2

n.d.

 

100

 

2

F

0

0

0

2

−0.5

−0.2

0.0

50%

50%

 

−0.2

−0.2

n.d.

 

50%

50%

Women over 38

5

F

2

0

2

0

−1.1

0.1

1.6

60%

 

40%

−1.4

−0.2

0.2

20%

40%

40%

7

F

2

2

0

0

−1.8

−0.4

0.5

43%

14%

43%

−1.8

−0.5

0.7

57%

14%

28%

5

F

1

1

0

0

−1.3

−0.2

0.9

40%

20%

40%

−1.8

−0.4

0.6

60%

20%

20%

4

F

0

0

0

2

−0.9

0.0

1.4

50%

25%

25%

0.0

0.2

0.9

 

75%

25%

Young men

4

M

2

1

1

0

−3.2

−0.9

1.3

75%

 

25%

−2.7

−0.8

0.5

25%

50%

25%

4

M

1

1

0

0

−1.6

−0.2

0.9

25%

50%

25%

0.0

0.0

n.d.

  

100%

4

M

0

0

0

2

−1.8

−1.0

−0.5

100%

  

−0.9

0.2

1.8

25%

25%

50%

Men over 38

2

M

2

1

1

0

−3.6

−2.0

−0.5

100%

  

−0.9

−0.5

0.0

 

50%

50%

2

M

1

1

0

0

−0.7

−0.7

−0.7

100%

  

−3.6

−1.9

−0.1

  

100%

4

M

0

0

0

2

−2.3

−0.3

0.9

25%

50%

25%

0.9

1.7

2.5

50%

50%

 

a1 = Lo Dose, 2 = Hi Dose

Lgst Largest, Avg average, Smlst smallest, Leu leucine, Olv olive oil, Fsh fish oil, Cntl cellulose, n.d. no data received, N.C. no change, rebnd rebound

The maximum, minimum and average individual weight changes are shown for each group after 2 weeks taking tablets, and rebound 2 weeks after the last tablets were taken. Total daily dosage is shown. Weight loss (kg) is shown as a negative number

Discussion

Although knowledge related to nutrition and aging is changing rapidly, “the complex interrelationship between energy intake, energy expenditure and specific physical activity requirements for current humans remains very similar to that originally selected for Stone Age men and women who lived by gathering and hunting” (Cordain et al. 1998). The results of this study may provide further insight into how to reveal those relationships and what they may be. The design of this study also demonstrates the value of double-blind design in weight loss studies where placebo effects can be substantial. The study tested the five hypotheses in the Introduction.

The average weight loss results were consistent with hypotheses 1 and 2, although there were individuals who did not lose weight. Eating the combination of leucine and a fatty acid did result in weight loss, and eating twice the dosage of leucine and a fatty acid resulted in more weight loss, as shown in Fig. 1.
https://static-content.springer.com/image/art%3A10.1007%2Fs11357-008-9049-0/MediaObjects/11357_2008_9049_Fig1_HTML.gif
Fig. 1

Influence of leucine dose on weight loss. Weight loss by all participants was averaged based on dosage of leucine taken

Resources for the study allowed initial exploration of hypothesis 3. Fish oil tablets contained only 12% DHA, while the olive oil contained 85% oleic acid. A comparison of those taking 6 g olive oil (5.1 g oleic) and 6 g fish oil (0.72 g DHA) versus 12 g olive oil is shown in Fig. 2. These results reflect that the combination of the two fatty acids may have a greater effect than either alone. Based on these results and reports mentioned in the Introduction, the signaling systems of DHA and oleic acid indicated in Table 6 may be different and interact synergistically.
https://static-content.springer.com/image/art%3A10.1007%2Fs11357-008-9049-0/MediaObjects/11357_2008_9049_Fig2_HTML.gif
Fig. 2

Influence of fish and olive oil on weight loss. All dosages included 12 g leucine/day. Endpoint S1 is weight loss, S2 is rebound; olv olive oil, fsh fish oil, Total daily divided doses are: Hi = 12 g/day, Lo = 6 g/day

Table 6

Possible weight signals by nutrients

Nutrient example

Nutrient class

Signal 1

Signal 2

Reference

Leucine

Branched chain amino acid

To the brain to decrease appetite

To the brain to increase metabolic rate

Sabatini et al. 1994

Oleic Acid

Polyunsaturated fatty acids

To adipose tissue to upregulate fat catabolism

To adipose tissue to downregulate lipid storage gene expression

Hsu and Huang 2006

DHA

Omega-3 fatty acids

To adipocytes to inhibit differentiation

To pre-adipocytes to stimulate apoptosis

Kim et al. 2006

Hypotheses 4 and 5 are consistent with our results, as mature women lost the least weight for every dosage for which they were tested. Women may have evolved to retain body weight after maturity even under conditions of scarcity (Cordain et al. 2005). A systematic review of weight loss studies did not find gender to be a significant factor in the type of fat that was lost during dieting (Chaston and Dixon 2008), while a study of weight loss after bariatric surgery found more weight loss in females (Melton et al. 2008), so hypothesis 5 is particularly controversial.

What conclusions can be drawn? Many individuals who consumed leucine, oleic acid, and DHA lost weight. The loss was not statistically significant in this pilot study. The rate of obesity internationally has increased substantially in recent decades (Bray 2004). The rise in obesity is a major risk factor for heart disease, cancer, diabetes, and other chronic diseases associated with aging (Woo et al. 2001). I have been unable to locate any other double-blind study of a weight loss nutraceutical, despite the wide range of diet products that are commercially available.

This study has resulted in revised hypotheses for nutrient signals likely to contribute to weight loss. For men and young women, 12 g leucine with 6 g olive oil and 6 g fish oil was most effective. For mature women, 12 g leucine and 12 g olive oil were most effective, but individual results were variable, as shown in Table 7. Because DHA and oleic acid appear to have different signal pathways, different combinations and dosages of the two different fatty acids may be effective.
Table 7

Results of mature women on 12 g leucine and 12 g olive oil

Year of birth

Initial weight (kg)

2 week loss (kg)

2 week rebd (kg)

1966

90.7

−1.8

0.5

1956

70.3

−0.9

−1.4

1964

58.5

−0.9

0.7

1948

114.3

0.0

0.0

1953

94.8

0.2

−0.7

1962

59.4

0.2

−0.5

1963

94.8

0.5

−1.8

Although there was an average loss of weight during tablets and rebound, the leuHiolvHi signals were not sufficient to cause weight loss in all mature women

Based on the possible efficacy and demonstrated safety shown in this study, a new study of longer duration with appropriate dosages for young and mature male and female groups is planned.

In conclusion, this study demonstrates that leucine, olive oil, and fish oil may cause weight loss through signaling mechanisms to the brain and adipose tissue. Polyunsaturated fatty acids like oleic acid and omega-3 fatty acids like DHA may have different signaling pathways that can act synergistically.

Acknowledgements

The supplements used in this study were provided by MDR. Professor Paul Campbell, Doreen Dalman and Jhaunell Reid of Beloit College assisted respectively in the ANOVA analysis and writing of the manuscipt, the blinding of the study, and the data input and proofreading.

Copyright information

© American Aging Association, Media, PA, USA 2008