Protein Leverage: Theoretical Foundations and Ten Points of Clarification

Much attention has been focused on fats and carbohydrates as the nutritional causes of energy overconsumption and obesity. In 2003, a model of intake regulation was proposed in which the third macronutrient, protein, is not only involved but is a primary driver of calorie intake via its interactions with carbohydrates and fats. This model, called protein leverage, posits that the strong regulation of protein intake causes the overconsumption of fats and carbohydrates (hence total energy) on diets with a low proportion of energy from protein and their underconsumption on diets with a high proportion of protein. Protein leverage has since been demonstrated in a range of animal studies and in several studies of human macronutrient regulation, and its potential role in contributing to the obesity epidemic is increasingly attracting discussion. Over recent years, however, several misconceptions about protein leverage have arisen. Our aim in this paper is to briefly outline some key aspects of the underlying theory and clarify 10 points of misunderstanding that have the potential to divert attention from the substantive issues.     

The influence of ambient temperature and the energy and protein content of food on nitrogenous excretion in the Egyptian fruit bat (Rousettus aegyptiacus)

The diets of frugivorous and nectarivorous vertebrates contain much water and generally have high energy but low protein contents. Therefore, we tested the prediction that to save energy under conditions of high energy demands and high water intake, frugivorous Egyptian fruit bats (Rousettus aegyptiacus) will increase both the absolute quantity and the proportion of ammonia in their urine. We also examined whether such changes occur when protein intake is low and water intake is high. We did three feeding trials. In trials 1 and 2, bats were fed one of four liquid diets containing constant soy protein concentrations but varying in sucrose concentration and were kept at ambient temperatures (T(a)) of 30 degrees Celsius and 12 degrees Celsius, respectively. In trial 3, bats were kept at Ta=12 degrees Celsius and fed one of four liquid diets with equal sucrose concentrations but varying protein concentrations. In trial 1, food intake at a sucrose concentration of 256 mmol/kg H(2)O was initially high but decreased to a constant rate with further increases in sucrose concentration, while in trial 2, food intake decreased exponentially with increasing sucrose concentration. As predicted, at 12 degrees Celsius with varying sucrose concentration, both the absolute quantity and the fraction of ammonia in the bats' urine increased significantly with food intake (P<0.02), while the absolute quantity of urea and the fraction of urea nitrogen excreted decreased significantly with food intake (P<0.03). Varying sucrose concentration had no significant effect on nitrogen excretion at Ta=30 degrees Celsius. Varying protein concentration had no significant effect on nitrogen excretion at Ta=12 degrees Celsius. We suggest that Egyptian fruit bats can increase ammonia excretion in response to increased energetic demands, and we calculate that they can save energy equal to approximately 2% of their daily metabolic rate by doing so.     

Testing protein leverage in lean humans: a randomised controlled experimental study

A significant contributor to the rising rates of human obesity is an increase in energy intake. The ‘protein leverage hypothesis’ proposes that a dominant appetite for protein in conjunction with a decline in the ratio of protein to fat and carbohydrate in the diet drives excess energy intake and could therefore promote the development of obesity. Our aim was to test the ‘protein leverage hypothesis’ in lean humans by disguising the macronutrient composition of foods offered to subjects under ad libitum feeding conditions. Energy intakes and hunger ratings were measured for 22 lean subjects studied over three 4-day periods of in-house dietary manipulation. Subjects were restricted to fixed menus in random order comprising 28 foods designed to be similar in palatability, availability, variety and sensory quality and providing 10%, 15% or 25% energy as protein. Nutrient and energy intake was calculated as the product of the amount of each food eaten and its composition. Lowering the percent protein of the diet from 15% to 10% resulted in higher (+12±4.5%, p = 0.02) total energy intake, predominantly from savoury-flavoured foods available between meals. This increased energy intake was not sufficient to maintain protein intake constant, indicating that protein leverage is incomplete. Urinary urea on the 10% and 15% protein diets did not differ statistically, nor did they differ from habitual values prior to the study. In contrast, increasing protein from 15% to 25% did not alter energy intake. On the fourth day of the trial, however, there was a greater increase in the hunger score between 1–2 h after the 10% protein breakfast versus the 25% protein breakfast (1.6±0.4 vs 25%: 0.5±0.3, p = 0.005). In our study population a change in the nutritional environment that dilutes dietary protein with carbohydrate and fat promotes overconsumption, enhancing the risk for potential weight gain.     

Quantitative analysis of the energy requirements for development of obesity

OBJECTIVE: Obesity is typically developed over long time and reflected in an energy imbalance, which is too small to be measured and controlled. Our objective is to formulate a mathematical model for the relation between the change in body mass and the values of the energy intake and the energy expenditure, controlled by the physical activity factor PAF. DATA AND THEORY: The uncontrolled components of energy expenditure increases as result of body mass increase: expenditure of a larger mass and expenditure to convert matter in intake into tissue. Both contributions depend on the fraction of fat in the added tissue. Based on data from the literature, the fraction of fat in added tissue and the energy required to convert energy into tissue are estimated and included in the model. RESULTS: Application of the theory shows that an increase in body mass of 1 kg/year corresponds to an energy imbalance of 71 kJ/d for men. Of this imbalance, 82% are stored as new tissue, while 18% are used for energy conversion. If a man in steady state changes energy intake by 0.1 MJ/d, keeping the physical activity factor constant, then the corresponding increase in steady-state body mass is 1.77 kg/PAF, and it will take 320/PAF days before half the change of body mass has taken place. A typical value for PAF is 1.8. CONCLUSION: Energy-based theoretical relations between the various factors involved in energy balance help identifying and quantifying the components of the energy balance and understanding their relations during development of obesity. The inclusion of increased energy expenditure to convert food energy to tissue changes previous estimates of the energy imbalance by about 20 percent.     

The responses of rats to various combinations of energy and protein. II. Diets made from natural ingredients

Natural diets with metabolizable energy levels of 8.5, 10.0, 11.5 or 13.0 MJ/kg and protein:energy ratios of 1:1, 1.33:1, 1.67:1 or 2:1 %:MJ/kg were fed ad libitum for 28 days to male and female weanling rats. Records of food intake and bodyweight were maintained weekly, and at post mortem examination body length, abdominal fat, liver and kidney weights were measured. Food intake was reduced when dietary energy level increased but this reduction was not sufficient to prevent energy intake increasing, especially in males. Female rats showed only small increases in energy intakes as dietary energy levels rose. The increase in energy intake at higher dietary energy levels increased food conversion efficiency, weight gain and abdominal fat deposition. The responses of male rats were greater than females. Protein intake had a smaller and less consistent effect than energy intake. Increased protein:energy ratio resulted in higher absolute and relative liver and kidney weights and greater body length. This reflected the increase of bodyweight gain at higher protein:energy ratios.     

Modulatory role of food, feeding regime and physical exercise on body weight and insulin resistance

Energy intake and expenditure is a highly conserved and well-controlled system with a bias toward energy intake. In times of abundant food supply, individuals tend to overeat and in consequence to increase body weight, sometimes to the point of clinical obesity. Obesity is a disease that is not only characterized by enormous body weight but also by rising morbidity for diabetes type II and cardiovascular complications. To better understand the critical factors contributing to obesity we performed the present study in which the effects of energy expenditure and energy intake were examined with respect to body weight, localization of fat and insulin resistance in normal Wistar rats. It was found that a diet rich in fat and carbohydrates similar to "fast food" (cafeteria diet) has pronounced implication in the development of obesity, leading to significant body weight gain, fat deposition and also insulin resistance. Furthermore, an irregularly presented cafeteria diet (yoyo diet) has similar effects on body weight and fat deposition. However, these rats were not resistant to insulin, but showed an increased insulin secretion in response to glucose. When rats were fed with a specified high fat/carbohydrate diet (10% fat, 56.7% carbohydrate) ad lib or at the beginning of their activity phase they were able to detect the energy content of the food and compensate this by a lower intake. They, however, failed to compensate when food was given in the resting phase and gained more body weight as controls. Exercise, even of short duration, was able to keep rats on lower body weight and reduced fat deposition. Thus, inappropriate food intake with different levels of energy content is able to induce obesity in normal rats with additional metabolic changes that can be also observed in humans.     

Protein-leverage in mice: the geometry of macronutrient balancing and consequences for fat deposition

Objective: The Protein‐Leverage Hypothesis proposes that humans regulate their intake of macronutrients and that protein intake is prioritized over fat and carbohydrate intake, causing excess energy ingestion when diets contain low %protein. Here we test in a model animal, the mouse: (i) the extent to which intakes of protein and carbohydrate are regulated; (ii) if protein intake has priority over carbohydrates so that unbalanced foods low in %protein leads to increased energy intake; and (iii) how such variations in energy intake are converted into growth and storage. Methods and Procedures: We fed mice one of five isocaloric foods having different protein to carbohydrate composition, or a combination of two of these foods (N = 15). Nutrient intake and corresponding growth in lean body mass and lipid mass were measured. Data were analyzed using a geometric approach for analyzing intake of multiple nutrients. Results: (i) Mice fed different combinations of complementary foods regulated their intake of protein and carbohydrate toward a relatively well‐defined intake target. (ii) When mice were offered diets with fixed protein to carbohydrate ratio, they regulated the intake of protein more strongly than carbohydrate. This protein‐leverage resulted in higher energy consumption when diets had lower %protein and led to increased lipid storage in mice fed the diet containing the lowest %protein. Discussion: Although the protein‐leverage in mice was less than what has been proposed for humans, energy intakes were clearly higher on diets containing low %protein. This result indicates that tight protein regulation can be responsible for excess energy ingestion and higher fat deposition when the diet contains low %protein.     

Adiponectin stimulates AMP-activated protein kinase in the hypothalamus and increases food intake

Adiponectin has been shown to stimulate fatty acid oxidation and enhance insulin sensitivity through the activation of AMP-activated protein kinase (AMPK) in the peripheral tissues. The effects of adiponectin in the central nervous system, however, are still poorly understood. Here, we show that adiponectin enhances AMPK activity in the arcuate hypothalamus (ARH) via its receptor AdipoR1 to stimulate food intake; this stimulation of food intake by adiponectin was attenuated by dominant-negative AMPK expression in the ARH. Moreover, adiponectin also decreased energy expenditure. Adiponectin-deficient mice showed decreased AMPK phosphorylation in the ARH, decreased food intake, and increased energy expenditure, exhibiting resistance to high-fat-diet-induced obesity. Serum and cerebrospinal fluid levels of adiponectin and expression of AdipoR1 in the ARH were increased during fasting and decreased after refeeding. We conclude that adiponectin stimulates food intake and decreases energy expenditure during fasting through its effects in the central nervous system.     

AMP-activated protein kinase plays a role in the control of food intake

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that acts as an intracellular energy sensor maintaining the energy balance within the cell. The finding that leptin and adiponectin activate AMPK to alter metabolic pathways in muscle and liver provides direct evidence for this role in peripheral tissues. The hypothalamus is a key regulator of food intake and energy balance, coordinating body adiposity and nutritional state in response to peripheral hormones, such as leptin, peptide YY-(3-36), and ghrelin. To date the hormonal regulation of AMPK in the hypothalamus, or its potential role in the control of food intake, have not been reported. Here we demonstrate that counter-regulatory hormones involved in appetite control regulate AMPK activity and that pharmacological activation of AMPK in the hypothalamus increases food intake. In vivo administration of leptin, which leads to a reduction in food intake, decreases hypothalamic AMPK activity. By contrast, injection of ghrelin in vivo, which increases food intake, stimulates AMPK activity in the hypothalamus. Consistent with the effect of ghrelin, injection of 5-amino-4-imidazole carboxamide riboside, a pharmacological activator of AMPK, into either the third cerebral ventricle or directly into the paraventricular nucleus of the hypothalamus significantly increased food intake. These results suggest that AMPK is regulated in the hypothalamus by hormones which regulate food intake. Furthermore, direct pharmacological activation of AMPK in the hypothalamus is sufficient to increase food intake. These findings demonstrate that AMPK plays a role in the regulation of feeding and identify AMPK as a novel target for anti-obesity drugs.     

Obesity: the role of hypothalamic AMP-activated protein kinase in body weight regulation

Obesity is rapidly increasing and is of great public health concern worldwide. Although there have been remarkable developments in obesity research over the past 10 years, the molecular mechanism of obesity is still not completely understood. Body weight results from the balance between food intake and energy expenditure. Recent studies have found that hypothalamic AMP-activated protein kinase plays a key role in regulating these processes. Leptin, insulin, glucose and alpha-lipoic acid have been shown to reduce food intake by lowering hypothalamic AMP-activated protein kinase activity, whereas ghrelin and glucose depletion increase food intake by increasing hypothalamic AMP-activated protein kinase activity. In addition, this enzyme plays a role in the central regulation of energy expenditure. These findings indicate that hypothalamic AMP-activated protein kinase is an important signal molecule, which integrates nutritional and hormonal signals and modulates feeding behavior and energy expenditure.     

Increased hypothalamic melanin concentrating hormone gene expression during energy restriction involves a melanocortin-independent, estrogen-sensitive mechanism

Increased expression of melanin concentrating hormone (MCH), an orexigenic neuropeptide produced by neurons in the lateral hypothalamic area (LHA), is implicated in the effect of energy restriction to increase food intake. Since melanocortins inhibit Mch gene expression, this effect of energy restriction to increase Mch signaling may involve reduced hypothalamic melanocortin signaling. Consistent with this hypothesis, we detected increased hypothalamic Mch mRNA levels in agouti (Ay) mice (by 102%; P < 0.05), a model of genetic obesity resulting from impaired melanocortin signaling, compared to wild-type controls. If reduced melanocortin signaling mediates the effect of energy restriction, hypothalamic Mch gene expression in Ay mice should not be increased further by energy restriction, since melanocortin signaling is impaired in these animals regardless of nutritional state. We therefore investigated the effects of energy restriction on hypothalamic Mch gene expression in both Ay mice and in wild-type mice with diet-induced obesity (DIO). Responses in these mice were compared to those induced by administration of 17beta-estradiol (E2) at a dose previously shown to reduce food intake and Mch expression in rats. In both Ay and DIO mice, energy restriction increased hypothalamic Mch mRNA levels (P < 0.05 for each) via a mechanism that was fully blocked by E2. However, E2 did not lower levels of Mch mRNA below basal values in Ay mice, whereas it did so in DIO mice. Thus, the effect of energy restriction to increase hypothalamic Mch gene expression involves an E2-sensitive mechanism that is not altered by impaired melanocortin signaling. By comparison, impaired melanocortin signaling increases hypothalamic Mch gene expression via a mechanism that is insensitive to E2. These findings suggest that while both energy restriction and reduced melanocortin signaling stimulate hypothalamic Mch gene expression, they do so via distinct mechanisms.     

Decreased energy levels can cause and sustain obesity

Obesity has reached epidemic proportions and has become one of the major health problems in developed countries. Current theories consider obesity a result of overeating and sedentary life style and most efforts to treat or prevent weight gain concentrate on exercise and food intake. This approach does not improve the situation as may be seen from the steep increase in the prevalence of obesity. This encouraged us to reanalyse existing information and look for biochemical basis of obesity. Our approach was to ignore current theories and concentrate on experimental data which are described in scientific journals and are available from several databases. We developed and applied a Knowledge Discovery in Databases procedure to analyse metabolic data. We began with the contradictory information: in obesity, more calories are consumed than used up, suggesting that obese people should have excess energy. On the other side, obese people experience fatigue and decreased physical endurance that indicates diminished energy supply in the body. The result of our work is a chain of metabolic events leading to obesity. The crucial event is the inhibition of the TCA cycle at the step of aconitase. It disturbs energy metabolism and results in ATP deficiency with simultaneous fat accumulation. Further steps in obesity development are the consequences of diminished energy supply: inhibition of beta-oxidation, leptin resistance, increase in appetite and food intake and a decrease in physical activity. Thus, our theory shows that obesity does not have to be caused by overeating and sedentary life-style but may be the result of the "obese" change in metabolism which is forcing people to overeat and save energy to sustain metabolic functions of cells. This "obese" change is caused by environmental factors that activate chronic low-grade inflammatory process in the body linking obesity with the environment of developed countries.     

The responses of rats to various combinations of energy and protein. I. Diets made from purified ingredients

Male and female weanling rats were fed ad libitum for 28 days on purified diets with metabolizable energy levels of 8.0, 9.5, 11.0 or 12.5 MJ/kg and protein:energy ratios of 1:1, 1.33:1, 1.67:1 or 2:1 %:MJ/kg at each energy level. Major nutrients were balanced in proportion to energy and protein. The following parameters were measured: food intake, bodyweight, body length, abdominal fat, liver and kidney weights. Increasing dietary energy level reduced food intake but the reduction was not sufficient to prevent an increase in energy intake. This was reflected by increases in bodyweight, body length, abdominal fat, and relative liver and kidney weights, especially in male rats. Higher energy intake increased weight gain and food conversion efficiency to a greater extent than higher protein intake. The response to protein intake at different energy levels was not consistent. There was no common protein:energy ratio for overall good performance. It is concluded that rat growth and other features can be controlled by the alteration of dietary energy and protein levels.     

Do Obese Eat Faster Than Lean Subjects? Food Intake Studies in Pima Indian Men

Food intake rate has previously been derived from observation of eating behavior in laboratory settings or in public eating establishments. Although it has been suggested that obese individuals eat faster than lean individuals, observations of such an “obese eating style” have yielded mixed results. In the present study, the relationship between ad-libitum food intake rate and obesity was evaluated over 4 days on a metabolic ward in 28 healthy Pima Indian men (Mean ± SD; 29 ± 7 y, 100.4 ± 27.1 kg, 33 ± 10% body fat) using an automated food selection system containing a large variety of foods . Total energy intake averaged 18829 ± 3299 kJ/d consisting of 47 ± 4,40 ± 3, and 13 ± 1 percent of carbohydrate, fat and protein, respectively. The average meal duration was 25 ± 7 min. Food intake rate was 68 ± 21 g/min while carbohydrate, fat and protein intake rates were 23 ± 6, 9 ± 3 and 6 ± 2 g/min, respectively. Food intake rate correlated negatively with %body fat (1=0.61, P<0.01). Similar relationships were found between the intake rates of carbohydrate, fat and protein and body fatness. Only prospective studies will indicate whether a slow food intake rate may contribute to the etiology of obesity by possibly reducing satiety .     

Increased adiposity in DNA binding-dependent androgen receptor knockout male mice associated with decreased voluntary activity and not insulin resistance

In men, as testosterone levels decrease, fat mass increases and muscle mass decreases. Increased fat mass in men, in particular central obesity, is a major risk factor for type 2 diabetes, cardiovascular disease, and all-cause mortality. Testosterone treatment has been shown to decrease fat mass and increase fat-free mass. We hypothesize that androgens act directly via the DNA binding-dependent actions of the androgen receptor (AR) to regulate genes controlling fat mass and metabolism. The aim of this study was to determine the effect of a global DNA binding-dependent (DBD) AR knockout (DBD-ARKO) on the metabolic phenotype in male mice by measuring body mass, fat mass, food intake, voluntary physical activity, resting energy expenditure, substrate oxidation rates, serum glucose, insulin, lipid, and hormone levels, and metabolic gene expression levels and second messenger protein levels. DBD-ARKO males have increased adiposity despite a decreased total body mass compared with wild-type (WT) males. DBD-ARKO males showed reduced voluntary activity, decreased food intake, increased serum leptin and adiponectin levels, an altered lipid metabolism gene profile, and increased phosphorylated CREB levels compared with WT males. This study demonstrates that androgens acting via the DNA binding-dependent actions of the AR regulate fat mass and metabolism in males and that the increased adiposity in DBD-ARKO male mice is associated with decreased voluntary activity, hyperleptinemia and hyperadiponectinemia and not with insulin resistance, increased food intake, or decreased resting energy expenditure.     

Difructose anhydride III decreases body fat as a low-energy substitute or by decreasing energy intake in non-ovariectomized and ovariectomized female rats

We evaluated the effects of difructose anhydride III (DFAIII) on body weights of ovariectomized rats, which are a good model for obesity by estrogen deficiency-induced overeating. Female rats (10 weeks old) were subjected to ovariectomy or sham operation and then fed with or without a diet containing 3% or 6% DFAIII for 33 days or pair-fed control diet during the same period. Rats fed DFAIII showed significantly decreased food intake, energy intake, body weight gain, body energy accumulation, and fat tissue weight than control group, regardless of ovariectomy. DFAIII may decrease body fat dependent of reduced food/energy intake. Compared with the respective pair feeding groups, rats fed DFAIII showed significantly decreased body energy and fat tissue weight, regardless of ovariectomy, suggesting its potential as a low-energy substitute for high-energy sweeteners. The low energy of DFAIII may contribute to decreased body fat, which may not be dependent on obesity.     

Cytokines of the LIF/CNTF family and metabolism

The incidence of obesity is increasing worldwide. Obesity is accompanied by a chronic inflammatory state that increases the risk of metabolic diseases such as insulin-resistance and type 2 diabetes. Over the past two decades, interest in immunomodulatory cytokines as potential mediators and/or targets for treatment or prevention of obesity and metabolic syndrome has increased. In this review, we summarize studies that revealed the effects of LIF family cytokines on adipose tissue, energy expenditure and food intake, highlighting the importance of gp130/LIFRβ signaling in obesity and obesity-related metabolic diseases.     

Role of Hypothalamic Reactive Astrocytes in Diet-Induced Obesity

Hypothalamus is a brain region that controls food intake and energy expenditure while sensing signals that convey information about energy status. Within the hypothalamus, molecularly and functionally distinct neurons work in concert under physiological conditions. However, under pathological conditions such as in diet-induced obesity (DIO) model, these neurons show dysfunctional firing patterns and distorted regulation by neurotransmitters and neurohormones. Concurrently, resident glial cells including astrocytes dramatically transform into reactive states. In particular, it has been reported that reactive astrogliosis is observed in the hypothalamus, along with various neuroinflammatory signals. However, how the reactive astrocytes control and modulate DIO by influencing neighboring neurons is not well understood. Recently, new lines of evidence have emerged indicating that these reactive astrocytes directly contribute to the pathology of obesity by synthesizing and tonically releasing the major inhibitory transmitter GABA. The released GABA strongly inhibits the neighboring neurons that control energy expenditure. These surprising findings shed light on the interplay between reactive astrocytes and neighboring neurons in the hypothalamus. This review summarizes recent discoveries related to the functions of hypothalamic reactive astrocytes in obesity and raises new potential therapeutic targets against obesity.     

Beyond skin color: emerging roles of melanin-concentrating hormone in energy homeostasis and other physiological functions

Melanin-concentrating hormone (MCH) is a cyclic peptide that mediates its effects by the activation of two G-protein-coupled seven transmembrane receptors (MCHR1 and MCHR2) in humans. In contrast to its primary role in regulating skin color in fish, MCH has evolved in mammals to regulate dynamic physiological functions, from food intake and energy expenditure to behavior and emotion. Chronic infusion or transgenic expression of MCH stimulates feeding and increases adipocity, whereas targeted deletion of MCH or its receptor (MCHR1) leads to resistance to diet-induced obesity with increased energy expenditure and thermogenesis. The involvement of MCH in energy homeostasis and in brain activity has also been validated in mice treated with non-peptide antagonists, suggesting that blockade of MCHR1 could provide a viable approach for treatment of obesity and certain neurological disorders. This review focuses on emerging roles of MCH in regulating central and peripheral mechanisms.