Our results demonstrate a reduced fat mass accumulation with intake of diets containing whey, high calcium and high vitamin D. The current study demonstrates for the first time that the impact of these components in reducing fat mass accumulation is effective with either a high fat or high sucrose diet, and that the alterations in substrate oxidation in the liver differ with composition of base diet. Further, to our knowledge, this is the first study to demonstrate that high whey, calcium and vitamin D intakes increases insulin receptor expression in muscle coincident with an increase in lean mass regardless of base diet.
Our results showing reduced fat mass accumulation with whey, high calcium and high vitamin D diets compared to no whey, suboptimal dietary calcium and vitamin D is consistent with a variety of epidemiological trials in humans [2
], and a follow-up of an intervention study [4
]. In addition, the results of several calcium or dairy product intervention trials supports that calcium or dairy products enhance weight and fat mass loss in humans and animals [21
]. However, not all weight loss trials support this conclusion [23
]. In addition, several animal studies also support that dairy product intake reduces fat mass accumulation [6
]. Therefore, the results of the current study confirm that the combination of several components of dairy products (whey, calcium and vitamin D) reduce fat mass accumulation in rodents.
HD diets reduced fat mass and increased lean mass regardless of whether the base diet contained high fat or high sucrose, suggesting that the primary mechanisms mediating these changes may be similar with both dietary backgrounds. The increased lean mass with high whey, calcium and vitamin D intake, with no differences in body weight, is the only consistent difference of the factors assessed across dietary backgrounds noted in this study that may influence energy utilization in tissues. Although the process of fat and lean mass accumulation may have similar energy costs, lean mass per kilogram is more metabolically active and requires greater energy utilization than fat mass. Thus, the increase in lean body mass may shift energy away from fat stores. The increase in insulin receptor expression in muscle is consistent with the increase in lean mass noted in this study. Insulin acts through the insulin receptor, a member of the receptor tyrosine kinase family and results from multiple studies suggest that insulin stimulates muscle cell hypertrophy [25
]. Further, vitamin D status is associated with reduced risk of diabetes or higher insulin sensitivity [27
], calcium intake is associated with reduced risk for metabolic syndrome [28
], and whey protein may improve insulin sensitivity [29
]. In addition, a vitamin D response element sequence has been found in the insulin receptor gene promoter [30
]. Consistent with this, 1,25(OH)2
D increased the expression and transcription of the insulin receptor in pro-monocytic lymphoma cells [31
]. The results of the current study are consistent with these data and suggest that dietary vitamin D, calcium and/or whey may enhance insulin action in muscle fibers through increased insulin receptor levels, potentially leading to increased lean mass. Therefore, it is hypothesized that dairy product components may improve lean mass, or contribute to maintenance of lean mass, without altering accumulation of body weight and thus it is critical to consider distribution of weight in the action of dairy products.
Other factors measured in this study that may impact muscle mass include myostatin and VEGF. Myostatin is a critical negative regulator of skeletal muscle cell hypertrophy, thus myostatin null mice (Mystn −/−) [32
] have extraordinary amount of muscle primarily due to an abnormal increase in muscle fiber number, even though subtle increases in muscle fiber size have been noted. VEGF is important for the maintenance of the skeletal muscle capillaries [33
]. Further, inhibition of VEGF production reduces skeletal muscle vascularization [35
] and inhibition of VEGF during chronic electrical stimulation or exercise training inhibits skeletal muscle angiogenesis [33
]. In the current study, there was no difference in either myostatin or VEGF mRNA expression between the LD and HD diets, suggesting that these factors may not play a role in the increase in lean mass with the HD diet.
The results of several studies suggest that a high calcium diet is associated with increased fat oxidation [10
]. However, not all studies have shown an impact of higher calcium intake on fat oxidation [14
]. It is intriguing that in the current study, high whey, calcium and vitamin D intake modulates substrate utilization with a lower glucose oxidative capability with a high fat background, but not during high sucrose intake. Although tissue specific substrate oxidation may not reflect overall body oxidation, these results may contribute to understanding the discrepancy in the published results, as the macronutrient background is not considered in the studies to date. In addition, PGC-1α expression was increased in the muscle in the HD compared to LD fed groups with a high fat diet. The transcriptional co-activator, PGC-1α, is a key regulator of mitochondrial biogenesis. However, glucose oxidation and PGC-1α were not altered in the liver of animals consuming the high sucrose diets and there were no differences in palmitate oxidation in either tissue between LD and HD containing diets. Cumulatively, the results of the current study suggest that the alterations in substrate oxidation mediated by higher whey, calcium and vitamin D intake noted during consumption of a high fat, but not a high sucrose background, are unlikely to contribute to changes in body composition because a lower body fat and higher lean mass accumulation was achieved with both dietary backgrounds.
In the current study, the increase in lean mass may play an important role in the coincident reduction in fat mass by shifting energy utilization. In several studies, dairy products enhance weight loss greater than calcium supplementation alone [21
], suggesting another factor in addition to calcium in dairy products may impact body composition. Several factors in dairy products are proposed to contribute to energy balance or body composition, including protein or amino acid composition, calcium, or vitamin D. One possibility is the branched chain amino acid composition in milk, particularly leucine, is proposed to contribute to increased muscle mass [37
]. In the current study the protein source was casein for all experimental diets, therefore the difference in amino acid composition between the diets was minimal. On the other hand, the HD diets contained whey proteins which are proposed to improve muscle mass, particularly under conditions of resistance training [38
]. It is possible that the whey component of the high dairy product intake contributes to the increase in muscle mass. Alternatively, improved vitamin D status may also contribute to lean mass [39
]. Support for this hypothesis is provided by studies in vitamin D receptor knock out mouse in which muscle fiber size is reduced [40
]. Therefore, future studies are warranted to investigate which component, or combination of components, of the HD diet compared to the LD diet promoted an increase in lean mass and increases insulin receptor expression in muscle.
The strengths of the study are the comprehensive metabolic assessments, and body composition measurements. The limitations of the study are that the study focuses on metabolism in a rodent model, the use of growing animals and that the study applies to accumulation of body weight and may not apply to energy restricted regimens.
In summary, these results support that high whey, calcium and vitamin D intake prevents excess accumulation of fat mass with both high sucrose or high fat diets. Consistent results across macronutrient backgrounds suggest increased lean mass occurs with high whey, calcium and vitamin D intake compared to low intakes of these components. Finally, higher whey, calcium and vitamin D intake promotes increased expression of the insulin receptor in muscle in this rodent model, independent of dietary background. The specific component or components in the diets and the role of the insulin receptor in mediating the increase in lean mass warrants further investigation.