As indicated previously, a LoBAG diet theoretically could result in a positive protein balance and an increase in FFM for several reasons. First, the postprandial insulin response to the LoBAG30 diet is vigorous  and insulin is reported to decrease whole body protein degradation and possibly stimulate protein synthesis [9, 25, 26], although this effect is blunted in men with type 2 diabetes .
Second, total IGF-I is increased by ~ 30% and free IGF-I is likely to be increased further because of a marked decrease in IGFBP-1 associated with meal ingestion . That the total IGF-I was increased but IGFBP-3 was not  also suggests that the additional IGF-I was either bound to other IGF-I binding proteins  and/or the free IGF-I was markedly elevated when the subjects were ingesting the LoBAG diet.
The effect of IGF-I on protein anabolism has been reported to occur only at high and perhaps unphysiological concentrations, and was attributed to its insulinmimetic activity . However, others have clearly reported that IGF-I stimulates skeletal muscle protein synthesis at physiological levels, an effect independent of its insulin-like effect .
In addition, IGF-I is reported to not only stimulate protein synthesis and to accelerate amino acid clearance [9, 29–32] but possibly to inhibit degradation as well . Whether the in vivo metabolic effects of IGF-I are independent of or depend on it being bound to carrier proteins remains an unresolved issue .
Third, an increase in circulating total amino acid concentration and specifically BCAAs stimulate protein synthesis [9, 27]. A maximal stimulation was reported to occur with an increase in total amino acids of 2–3 fold . In the present study, the 24 hour integrated net area in the 3 controls was increased 2-4 fold, and ~13 fold with a LoBAG30 diet, (>4 times that in the controls). There also was a large increase in BCAAs, particularly leucine (12 fold) which, in the presence of a high insulin concentration, should strongly stimulate net protein accumulation [25, 26, 35], and it was sustained for a prolonged period of time.
In contrast, an increased cortisol and glucagon concentration could oppose the anabolic stimulation by insulin, IGF-I, total and BCAAs. The 24-hour cortisol profile remained unchanged (Figure 2). However, the 24-hour urinary free cortisol was increased (Table 3). The latter suggests that the free, presumably active form of circulating cortisol, could have been increased. We previously observed an increase in urinary free cortisol in subjects ingesting a LoBAG20 diet but this was not statistically significant. We did not do a 24-hr serum cortisol profile in that study .
We were somewhat surprised that the serum total cortisol was not increased because several years ago we demonstrated a post-meal increase in cortisol and adrenocorticotropin (ACTH) when young, normal subjects ingested a diet containing 4 g protein/kg body weight over a 12-hour period in the form of 3 identical meals . A small but significant increase also occurred when the protein content was 1 g/kg body weight. That an increase did not occur in the present study suggests that the protein content was not sufficient for an increase to be observed, or more likely, the subjects being older, more obese and with type 2 diabetes, do not respond to an increase in dietary protein as well as do young people without diabetes. It also is possible that a metabolic adaptation to the increased protein content occurred over 5 weeks. The previous study was only a single day study. Nevertheless, a subtle increase may have been present in the current study as indicated by the increase in urinary free cortisol.
That the fasting glucagon concentration was unchanged in the present study was expected. That the 24 hr integrated net and total glucagon area responses were further elevated (5 fold and 20%, respectively) as a consequence of ingestion of the LoBAG30 diet was somewhat unexpected (Figure 3). Ingestion of a LoBAG30 diet in a previous study resulted in only a modest and not significant further elevation in glucagon when compared to the control diet (15% protein) . However, a clearly elevated net area response (2.6 fold) was present when the carbohydrate content was reduced to 20% in a LoBAG20 diet . We previously reported that high carbohydrate diets modestly decrease the circulating glucagon, in normal young subjects, whereas high protein or high fat diets greatly increased it in single day studies. However, with the typical ratios found in the American diet (45–55% CHO, 10-15% Pro) there generally is little change in glucagon concentrations after meals [8, 37].
An elevated glucagon concentration has been reported to increase amino acid clearance and stimulate urea synthesis, whereas a raised glucose concentration inhibits it . Thus, the metabolic disposal of the increased absorbed amino acids derived from ingesting the LoBAG diet could have been accelerated by the increased glucagon concentration. The total amino acid concentration itself also has been reported to regulate the urea synthesis rate in a concentration-dependent fashion (reviewed in ).
Glucagon given as an IV bolus, also has been reported to stimulate a transient rise in IGFBP-1 . However, even though the 24 hour net glucagon concentration increased 5 fold in the present study (Figure 3) the IGFBP-1 decrease was similar to that when the subjects ingested the control diet .
As we have reported previously, a LoBAG30 diet strongly stimulates an increase in insulin [1–3]. The insulin concentration is further increased relative to the glucose concentration. Thus, the calculated insulin resistance also is increased. We consider this resistance to be physiologic and to be due to the increased protein as well as fat content. In any regard, the increase in insulin stimulated by protein ingestion but without a decrease in glucose into the hypoglycemic range is important since it allows the insulin to inhibit proteolysis, and thus facilitate a net increase in protein synthesis stimulated by the increased BCAAs, particularly leucine. In addition, a modest increase in cortisol could induce a mild insulin-resistant state and thus, limit the effect of insulin on glucose metabolism. The ingested protein-stimulated increase in glucagon facilitates deamination of amino acids and urea formation i.e. it facilitates the disposition of the remaining amino acids . The net effect is to rapidly remove from the circulation those absorbed amino acids not removed by the gut cells. The deaminated amino acids are largely converted to glucose through gluconeogenesis and could replace endogenous gluconeogenic substrates [40, 41]. The result of the entire integrated process is the amino acids are disposed of relatively rapidly without a significant change in plasma glucose [42–44]. Overall, it facilitates body protein homeostasis or in the case of a LoBAG diet, a positive nitrogen balance and an eventual possible modest increase in protein mass.
Of considerable interest, even though the increase in total serum amino acid net area response was approximately 4 fold greater when the subjects ingested a LoBAG30 diet (Figure 2), the total amino acid concentration had returned to the fasting baseline by the following morning indicating complete disposal, either through metabolism (deamination) and/or from incorporation into protein.
Amino acids also can be temporarily stored in skeletal muscle , but this is not likely to have been of importance. It also has been reported that during the day not only are the diet-derived amino acids oxidized but additional new protein is synthesized due presumably to the increase in insulin, BCAAs, etc. and to which an increase in free IGF-I, as indicated here, could contribute. During the night net proteolysis results in a stable protein mass . This may be the case even when the protein content is increased as in the current study.
Data obtained in this and previous studies indicated that the higher content of protein (30%) in a LoBAG diet resulted in a relatively large calculated positive protein balance, whereas the control diet, (55% carbohydrate, 15% protein, 30% fat), resulted in a calculated neutral balance . However, in the present study, the dietary nitrogen was directly quantified, and not just calculated from food tables. In addition to the urinary nitrogen used in our calculation of protein balance, the fecal nitrogen also was quantified. These directly measured results indicate that the net protein balance, as estimated from nitrogen balance, was negative with the control diet, but was positive with a LoBAG30 diet.
A more accelerated loss of appendicular lean body mass and an increased risk of sarcopenia with aging has been reported in people with type 2 diabetes [47, 48]. That is, nitrogen balance is negative and greater than in those without diabetes. However, this change in balance would be subtle and far less than anticipated with the negative nitrogen balance observed here.
The current data do not indicate an overall increase in fat-free mass and presumably protein mass (Table 5). The CT data also did not indicate an increase in muscle mass or a reduction in thigh and abdominal fat. However, the urinary creatinine data suggest an increase in muscle mass may have occurred , although not sufficient for it to be detected over this time frame. Thus, a larger and longer duration study is needed to determine if the demonstrated positive nitrogen balance results in a demonstrable increase in muscle mass and function.
Regarding the time duration required to observe a change in lean mass or muscle mass, a decrease in mid thigh muscle was determined by CT in 10 healthy men and women (age 55–77 years) after 14 weeks on a low protein (0.8 g/kg/day) diet . Also in healthy male subjects, Westerterp-Plantera and associates have reported a positive protein balance and a negative fat balance in a 4 day comparison of a 30% vs. 10% protein diet . The same group reported an increase in fat free mass of 0.73 Kg (1.6 pounds) independent of a change in body weight over a 3 month increased dietary protein intervention. It also resulted in a negative fat balance . In another study, an increase in muscle mass was observed within 4 weeks when testosterone was administered during exercise training .