Bassil, Maya S.a; Gougeon, Réjeanneb
Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance. This is classically defined as blunted insulin-induced suppression of hepatic glucose production and uptake into peripheral tissues, including muscle, and of lipolysis in adipose tissue. As insulin also modulates protein metabolism by stimulating synthesis and suppressing breakdown, derangement in muscle protein anabolism in T2DM is expected, albeit not found using some experimental protocols. In this review we explore the potential clinical manifestations of altered muscle protein anabolism in T2DM and discuss recent studies of whole body and muscle protein anabolism in response to insulin, amino acids, and exercise.
MUSCLE MASS AND STRENGTH
Clinically, obese people with T2DM are not perceived to have muscle loss, a reason for protein metabolism abnormalities not having been considered until recently as significant. Despite usually having greater muscle mass than lean controls due to larger body size and obesity [1▪], T2DM appears to cause poorer muscle performance, a factor contributing to increased disabilities in older age. Lower leg and arm muscle strength have been reported in a cross-section of older men with T2DM compared with healthy controls . Similarly, older patients with T2DM had a 30% greater decline over 3 years in leg muscle strength and quality than healthy age-matched controls . Impairments in muscle strength in T2DM have been strongly correlated with intramuscular fat storage which is twice that found in controls . However, intramyocellular triglyceride content is not an independent biomarker of insulin resistance, as its increase in chronic exercise and obesity has opposing effects on insulin sensitivity .
T2DM also aggravates the loss of muscle mass even to the point of sarcopenia associated with aging. Older adults with T2DM followed for 6 years had accelerated loss of appendicular muscle mass compared with controls, interestingly, with more pronounced loss in newly diagnosed diabetes . In agreement with this, the Korean Sarcopenic Obesity Study showed that sarcopenia was more prevalent among older people with T2DM (15.7 versus 6.9%) . The Third National Health and Nutrition Survey revealed an inverse relationship between skeletal muscle index (ratio of skeletal muscle mass to total body weight), and insulin resistance [using the Homeostatic Model Assessment (HOMA-IR)], glycated haemoglobin (A1C), and prevalence of prediabetes and diabetes [8▪▪]. Consistent with this, muscle of patients with T2DM has increased expression of myostatin mRNA, a peptide hormone known to negatively regulate skeletal muscle mass [9▪].
RESPONSE TO INSULIN
Insulin stimulates protein anabolism in muscle fibers from healthy individuals by increasing protein synthesis and suppressing breakdown . Thus, insulin resistance in T2DM should impair protein metabolism concurrent with that proven to occur for that of glucose and lipids. Under in-vivo physiological conditions, insulin increases postprandially, but meal studies cannot distinguish its effect on protein anabolism from that of absorbed nutrients. To isolate the effect of insulin, the hyperinsulinemic, euglycemic clamp has been used, with serum concentrations maintained at typical postprandial concentrations (500–600 pmol/l) and glucose is infused to maintain glycemia at 5.5 mmol/l. Amino acid tracers (13C or 14C leucine, L-ring 2H5-phenylalanine) are infused to assess whole body and/or muscle protein kinetics. Ideally, a mixture of amino acids is also infused to maintain basal fasting plasma concentrations (isoaminoacidemia). This prevents plasma amino acids from falling below fasting levels due to suppression of protein breakdown by insulin, and thus creating a nonphysiological limitation of amino acid availability for synthesis . Using hyperinsulinemic, euglycemic, isoaminoacidemic clamps, we found insulin resistance of whole body net protein anabolism, mostly due to blunted stimulation of synthesis, in overweight and obese men with poorly controlled T2DM compared with weight matched nondiabetic men (Fig. 1) . Women studied in the same manner did not show this added diabetes effect possibly because the obese women have more insulin resistance of protein anabolism versus lean patients . Similar results were found in obese, hyperglycemic men with T2DM with glycemia clamped at 8 mmol/l to simulate the more typical postabsorptive milieu of T2DM, instead of being lowered to 5.5 mmol/l to begin the clamp [1▪]. This hyperglycemia was associated with a more accelerated turnover rate (higher protein synthesis and breakdown) than during euglycemia. These whole body protein data suggest that most of the impairments exist in muscles, as they account for most of the insulin-induced protein anabolism , and thus wherein most insulin resistance takes place.
Our findings contrast with those of others reporting normal whole body  and muscle  protein anabolism in T2DM in response to insulin. This discrepancy might be due to the lack of controlling for sex, body composition, previous protein and energy intake differences, or especially the absence of concurrent amino acid infusion to maintain fasting levels during these clamps. Inconsistent findings might also stem from differences in prior glucose control as hyperglycemia was normalized by insulin infusion for 11 days  or overnight  before assessing insulin-induced protein anabolism in some protocols, but not others [1▪,12]. It remains to be determined with well designed clamp studies that control for these confounding variables, whether good prior glycemic control improves muscle insulin resistance of protein metabolism.
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RESPONSES TO AMINO ACIDS
Amino acids not only stimulate muscle protein anabolism by serving as substrates for protein synthesis but also as nutritional signals in the mRNA translation initiation pathway leading to protein synthesis. Among amino acids, the essential branched-chain, leucine, is the most potent by activating the mammalian target of rapamycin-complex 1 (mTORC1), which then triggers signal transduction events that promote protein synthesis in skeletal muscle . The exact mechanisms of leucine action are still not clear but appear to involve Rag proteins, a family of guanosine triphosphatases that interact with mTORC1. This promotes its intracellular perinuclear localization, which favors its activation by Rheb . Furthermore, leucine may also contribute to anabolism by suppressing muscle protein breakdown via inhibition of the ubiquitin–proteasome pathway  and possibly autophagy, through mTORC1 activation or independently .
Amino acids stimulate muscle protein anabolism in an independent and synergistic fashion with insulin . This is why under normal physiological conditions, the greatest protein anabolism occurs in the fed state, during which concentrations of insulin and amino acids are elevated. Thus, one might anticipate that insulin resistance of protein anabolism in T2DM would be predicted to be maximal in this state. However, postprandial muscle protein synthesis was found to be normal in hyperglycemic T2DM men after the consumption of carbohydrate and protein hydrolysate taken in repeated boluses . We have also recently reported ‘normalized’ whole body protein anabolism in T2DM during clamp simulating fed state concentrations of insulin and amino acids [1▪]. Obese men with T2DM had a whole-body protein anabolic response comparable to that found in lean men during the hyperinsulinemic, hyperglycemic, hyperaminoacidemic clamp. This was supported by signaling data from muscle biopsies collected during the clamps [23▪▪], in which the magnitude of increase in phosphorylation of mTOR and its downstream substrate, ribosomal protein S6 (rpS6) in response to hyperinsulinemia and hyperaminocidemia (Fig. 2a and b) was comparable to that of lean men studied under similar conditions . Thus, we concluded that postprandial hyperaminoacidemia can overcome insulin resistance of protein anabolism in T2DM.
One possible explanation for these findings is the ample amino acid or protein administered, which if extrapolated over one day exceeds the recommended daily adult protein intake. Although the total amount of amino acid infused during the clamp study (30 g) [1▪] corresponds to a typical adult meal, the total amount of protein consumed during the study by Manders et al. was very high (134 g protein hydrolysate taken in 12 repeated boluses over 6 h). Thus, it remains to be determined at what levels and durations of hyperaminoacidemia muscle protein anabolism become normalized in T2DM, as there could be a shift in dose-response to the right, requiring larger amounts of exogenous protein than recommended requirements. This could explain the clinical signs of abnormal muscle protein anabolism in T2DM that start to become apparent in old age concurrent with a decrease in protein intake  and specifically in serum branched-chain amino acids (BCAA).
THE DILEMMA OF AMINO ACIDS/PROTEIN IMPROVING VERSUS IMPAIRING METABOLIC CONTROL
Increasing dietary protein/amino acid intake has been recommended as part of the dietary management to improve insulin sensitivity and blood glucose control in T2DM . High protein, hypoenergetic diets can cause weight loss with maintenance of lean body mass. Furthermore, certain individual amino acids including leucine augment insulin secretion, despite the blunted glucose insulinotropic response in T2DM . This is likely to be a factor in low carbohydrate, high protein diets improving glycemic control in untreated T2DM . Increasing protein and amino acid intake, leucine in particular, has been suggested as a nutritional strategy to improve muscle protein anabolism in T2DM, especially in the elderly with decreased muscle mass [28,29]. This is thought to increase muscle protein anabolism both directly by activating protein synthesis and by stimulating insulin secretion. Amino acid composition and digestibility of specific protein sources may also play roles. Muscle protein synthesis was stimulated significantly more in response to 20 g whey versus casein in prediabetic older men [30▪]. This was partly attributed to whey's higher leucine content. A high protein (30% of energy mostly from animal food sources), low carbohydrate (30%), weight maintaining diet for 5 weeks in men with untreated T2DM was associated with improved nitrogen balance with no change in body composition [31▪]. Notable is that integrated 24 h plasma concentrations of leucine and the other BCAA, tyrosine, and phenylalanine were increased up to 12-fold for leucine. In contrast, long-term leucine supplementation (7.5 g per day) did not affect muscle mass in older men with T2DM. However, these patients did not have compromised muscle mass at baseline, and their diet composition was not controlled, though protein intake was ‘adequate’ (∼1.0 g per day) [32▪▪]. It remains to be established whether persons with T2DM with reduced protein intake and/or muscle mass would benefit from leucine supplementation, as was recently demonstrated in the healthy elderly consuming protein at the RDA level (0.8 g/kg body weight per day) .
In contrast, several lines of evidence have been published that have been interpreted as protein, especially leucine, worsening glucoregulation. A plethora of recent metabolomic studies have all come up with serum BCAA as a signal of diabetes risk, including prediction of later T2DM . Epidemiological studies have revealed a strong positive association between increased intake of animal protein rich in BCAA and the risk of insulin resistance and T2DM . Furthermore, raising serum amino acids to postprandial concentrations blunts glucose uptake in healthy humans [21,36]. At the molecular level, this was attributed to leucine-induced overactivation of ribosomal protein (rp) S6 kinase 1 (S6K1), a substrate of the mTORC1 signaling pathway , which phosphorylates the insulin receptor substrate (IRS)-1 at specific serine residues . This, in turn, inhibits IRS-1 and deactivates Akt signaling resulting in attenuated translocation of GLUT4 to the plasma membrane, and hence, reduced glucose uptake.
These mechanisms were extrapolated to insulin resistant states to postulate that increased protein intake in T2DM could further contribute to peripheral insulin resistance of glucose metabolism . However, we have recently demonstrated in hyperglycemic men with T2DM during hyperisulinemic clamps that whole-body glucose uptake was not further impaired by hyperaminoacidemia. This was supported by unchanged IRS-1 serine phosphorylation during hyperaminoacidemia despite increased mTORC1 phosphorylation (Fig. 2c and d) [23▪▪]. We suggest that in such hyperglycemic T2DM, insulin-stimulated glucose uptake is already highly attenuated, as is hyperphosphorylation of IRS-1 serine residues, such that hyperaminoacidemia does not aggravate it. Another key metabolic abnormality of T2DM that is thought to be worsened by excess leucine intake is pancreatic β cell apoptosis. Leucine-induced mTORC1 phosphorylation in cultured β cells, concurrent with high levels of glucose, insulin, and insulin-like growth factor (IGF)-1 in T2DM might ‘overstimulate’ β cell proliferation and precipitate β cell senescence . However, there exists no evidence to support this hypothesis in humans. In summary, inconsistent findings arise among those with unambiguous clinical benefits favoring higher protein/amino acid intakes with negative associations derived from epidemiologic/metabolomic and some more fundamental studies. Reconciliation of these findings requires highly targeted whole-body and organ/tissue studies at a mechanistic level, in persons with T2DM.
RESPONSES TO EXERCISE
Resistance exercise generates anabolic signals that stimulate muscle protein synthesis and is also known to improve insulin sensitivity independently of any effect it might have on weight loss. Physical activity has been recommended to prevent age-related loss of muscle mass. In a recent report, Wall et al.[40▪] demonstrated that electrical stimulation of leg muscle contraction was sufficient to increase muscle protein synthesis in older T2DM men during a 4 h recovery period. Exercise prior to ingestion of a protein bolus (20 g) led to higher muscle protein synthesis versus no exercise in prediabetic aged men , at a magnitude that was comparable to that observed in young men. Longer term exercise regimens over 3–4 months have also been proven to increase muscle mass  and strength [43▪,44▪] in T2DM and improvements were comparable with those of healthy controls [44▪].
Age-related loss of muscle mass and function is accelerated with T2DM that might be due to insulin resistance of protein anabolism with concurrent insufficient protein intake and physical activity. Indeed, in T2DM, muscle protein anabolism is normal in response to ample supply of BCAA, especially leucine, or to exercise. More research is required on the human muscle cellular and molecular mechanisms that reveal strategies for intervention to concurrently correct glucose and protein metabolic abnormalities. The dilemma is to identify the right balance, that is, optimal protein intake combined with exercise prescription to protect from muscle loss, while not aggravating good glycemic control.
We thank Dr Errol B. Marliss, Dr Stéphanie Chevalier, and Dr Terry P. Combs for their valuable edits and additions based on the critical reading of the manuscript.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 116–117).
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