The current study demonstrated for the first time that MR C57BL/6J mice on HFD are protected from developing obesity despite having increased energy intake and food absorption. These MR mice on HFD also remain glucose tolerant and insulin sensitive, effects possibly associated with increased adiponectin and hepatic FGF21 gene and protein expression. In addition, growth in MR mice on HFD was restricted without affecting motor coordination but there was a concomitant decrease in bone mass.
MR mice on HFD showed decreased body weight gain despite exhibiting hyperphagia as previously reported for rats and mice fed low fat diets 
. Although hyperphagia could be attributed to the increased glutamic acid in the diets to compensate for the reduced methionine concentrations, it has been shown that serum glutamate was not increased in MR rats 
. Moreover, the MR effects in rats could be reversed by cysteine supplementation which verified that the observed effects are specific to sulfur-containing amino acids 
Absorption efficiency of the diet was also improved in the MR mice on HFD compared to CF counterparts. This effect of MR on mice fed HFD could be explained by stimulation of β-adrenergic receptor causing increased energy expenditure and fat oxidation, as previously reported 
The effect of MR on the body composition of C57BL/6J mice corresponded with the observed increase of plasma adiponectin and the decrease of IGF-1, insulin and leptin levels 
. The MR mice on HFD in this study showed increased adiponectin levels by 68% which has also been reported to potentiate insulin sensitizing effects through the activation of PPARγ signaling 
. Plasma leptin levels are associated with adipose tissue mass 
, and this study showed that the percent of perigonadal fat in MR mice on HFD was 64% smaller compared to its CF counterparts, which could explain the 95% reduction of plasma leptin levels (). Interestingly, MR mice on HFD also had a 16-fold increase in plasma levels of FGF21 compared to their CF counterparts. FGF21 is a recently discovered hormone with potent effects on glucose homeostasis 
. Taken together, the hormone profile in MR mice on HFD is characteristic of insulin sensitive animals.
Improvement of glucose homeostasis and delayed onset of type 2 diabetes was followed by monitoring of glucose and insulin levels during the course of the study. MR mice on HFD did not present fasting hyperglycemia and hyperinsulinemia and showed an improved HOMA-IR index. This data agrees with reports on aged MR mice that showed lower glucose and insulin levels compared to aged CF mice 
. MR mice on HFD also exhibited improved glucose tolerance and peripheral insulin sensitivity without affecting hepatic glucose production. The similar expression levels of hepatic gluconeogenic genes, G6pase
, could partly explain similar hepatic glucose production in both groups. These data are also in agreement with reports on aged MR rats that had improved insulin sensitivity compared to aged CF rats 
. It is important to mention that despite pyroglutamic acid feeding to diabetic rats and mice alleviates the symptoms of type 2 diabetes 
and that the nitrogen balance of our diets was maintained by increasing levels of glutamic acid, the observed MR effects in this study are due to reduced levels of sulfur amino acids since these effects are reversed by cysteine supplementation of the MR diet in rats 
Insulin resistance is characterized by the accumulation of intrahepatic lipids due to the imbalances in the input, oxidation, synthesis and output of free-fatty acids by the hepatocytes 
. Our studies showed that the MR mice on HFD had decreased accumulation of hepatic lipids as well as a hormone profile that promotes insulin sensitivity. In addition, the hepatic gene expression data agreed with reports showing the upregulation of Pparγ
in MR rats 
and its target genes: Cd36
. In fact, rosiglitazone, a Pparγ
agonist, was reported to ameliorate hepatic steatosis and improve insulin sensitivity 
. However, this is in contrast with studies showing hepatic overexpression of Pparγ
in mouse models lead to adipogenic lipogenesis 
and that Pparγ
are upregulated during hepatic steatosis 
. Nevertheless, decreased in hepatic Scd1
gene expression in this model coincided with observations in MR rats 
suggesting increased β-oxidation 
. These data, therefore, suggest that MR mice on HFD are protected from developing hepatic steatosis which could attenuate type 2 diabetes.
Another plausible explanation for the protection against insulin resistance by MR on mice fed HFD could be on the effects of FGF21. MR mice on HFD showed a 20-fold upregulation of hepatic Fgf21
gene expression which corresponded with a 16-fold increase of plasma FGF21. Therapeutic administration of FGF21 in diabetic rodent models and overexpression of FGF21 in mice were previously shown to improve glucose homeostasis 
. In contrast, Fisher et al.
reported that obese mice have increased hepatic mRNA and circulating plasma FGF21 levels compared to lean mice, but this was attributed to FGF21 resistance and reduced glucose clearance rate 
. Xu et al.
however showed that injecting recombinant FGF21 into obese mice reversed hepatic steatosis, increased energy expenditure and improved insulin sensitivity 
. These observations were partly explained by Dutchak et al.
who reported that white adipose tissue of FGF21-deficient mice had a decrease in PPARγ activity 
. In the latter studies, it was proposed that FGF21 enhanced PPARγ activity in white adipose tissue by suppressing PPARγ sumoylation, which leads to improved insulin sensitivity 
. It is also likely that, in mice fed HFD, FGF21 could be exerting an important role in the liver where MR led to the transcriptional upregulation of Pparγ
gene expression which correlated with improved glucose homeostasis and decreased hepatic steatosis. Thus, the robust effects of MR on FGF21 could possibly explain, at least in part, the protection against type 2 diabetes in HFD mice.
MR mice on HFD presented stunted growth and reduced bone density. Although we did not observe any adverse effect on the motor function in the MR mice on HFD, the plasma biomarker for type 1 collagen degradation, CTX-1, was increased compared to the CF mice on HFD. Serum CTX-1 has been reported to be a specific marker for bone resorption 
. The growth restriction observed in the MR mice could be further explained by the effect of MR on FGF21 
and its subsequent effects on the growth hormone 
. De Sousa-Coelho et al.
reported that amino acid deprivation in mouse liver and HepG2 cells induced FGF21, a target of activating transcription factor 4 (ATF4) 
. In addition, Inagaki et al
. showed that FGF21 transgenic animals had decreased levels of plasma IGF-1 and were smaller than the wild-type counterparts 
. These investigators also showed that the effects of FGF21 were mediated by a decrease in phosphorylated Stat5 and an increase in Jak2 phosphorylation 
. Although hepatic Stat5a
gene expression was not affected in our studies, Jak2
gene expression was increased in MR mice on HFD. Therefore, our data could also suggest that FGF21 interrupts growth hormone signaling downstream of Jak2. Furthermore, Wei et al.
showed that, compared to their wild-type littermates, transgenic mice overexpressing FGF21 had lower BMD, which was associated with the upregulation of Pparγ2
in osteoblasts and increased levels of urinary and serum CTX-1 
. Rosiglitazone, a PPARγ agonist, increased bone loss in the wild-type mice but had no additive effect on FGF21 knockout mice suggesting that FGF21 could be causing decreased bone mass via PPARγ signaling 
. Taken together, decreased bone mass in MR mice on HFD could be explained, at least in part, by enhanced FGF21 activity. It is, however, important to mention that the mice used in our experiments were 8 weeks old at the beginning of the studies and had not reached their peak growth. Therefore, studies using aged mice that have reached maximum growth as well as young mice with fast growth rates are necessary to elucidate the effects of MR on the bone remodeling.
In conclusion, studies using MR mice on HFD confirmed that MR protects rodents from developing obesity, insulin resistance and type 2 diabetes, conditions that are observed during aging. The MR effects may be associated with the reduction of hepatic lipid accumulation and favorable hormonal changes associated with insulin sensitivity. MR significantly upregulated the expression of FGF21 at the gene and protein levels and its enhanced activity is proposed to be involved in the bone remodeling changes observed in MR mice on HFD. Overall, these results reveal beneficial and unfavorable effects of MR.