Effect of l-arginine on energy metabolism, skeletal muscle and brown adipose tissue in South Asian and Europid prediabetic men: a randomised double-blinded crossover study

The South Asian population, comprising 20% of the total world population, is particularly vulnerable to developing obesity and type 2 diabetes mellitus. Currently, South Asians living in the Netherlands have a sixfold higher risk of developing type 2 diabetes compared with native Dutch Europids and are at higher risk of developing diabetes-related complications, including cardiovascular disease [1‐4]. An important contributor is their highly common disadvantageous metabolic phenotype, consisting of central obesity, insulin resistance and dyslipidaemia [5]. This disadvantageous phenotype likely results from extrinsic factors such as migration followed by adaptation of a western lifestyle and less physical activity as well as intrinsic factors such as a disturbed energy metabolism (e.g. a reduced [fat] oxidative capacity) [6]. The current epidemic of type 2 diabetes results in high morbidity and mortality and novel treatment options to combat ‘diabesity’ are urgently needed. Since reduction of energy intake has been proven to be often ineffective in the long term due to poor compliance [7], shifting energy balance towards higher energy expenditure is an attractive therapeutic strategy.

Skeletal muscle mitochondrial function plays a pronounced role in whole-body energy expenditure and is closely associated with insulin sensitivity [8]. Poor skeletal muscle oxidative capacity may play a crucial role in the development of obesity-induced peripheral insulin resistance (and subsequently type 2 diabetes), so enhancing skeletal muscle mitochondrial function may improve whole-body glucose metabolism. Skeletal muscle and possibly also brown adipose tissue (BAT) are known to be involved in human energy metabolism. Due to the presence of uncoupling protein-1 (UCP-1) in the inner membrane of its mitochondria, BAT is able to combust relatively large amounts of fatty acids and glucose, resulting in dissipation of energy as heat rather than the production of ATP. Interestingly, a reduction in BAT volume and activity has been associated with both adiposity [9] and diabetic status [10] and we recently showed reduced BAT volume in young South Asian men compared with young Europid men [11]. Thus, enhancing BAT volume and activity could be a potential therapeutic approach to ameliorate type 2 diabetes risk, especially in South Asians. So far, significant BAT recruitment in humans has only been shown by means of prolonged intermittent cold exposure (i.e. cold acclimation) [12‐14] or massive weight loss [15]. Other means to recruit BAT are urgently awaited.

Animal studies have suggested that nitric oxide (NO) plays a pivotal role in regulating mitochondrial biogenesis in skeletal muscle and BAT [16]; mice that lack the endothelial enzyme NO synthase (NOS), which catalyses the conversion of l-arginine to NO, display lower BAT volume, fewer mitochondria in BAT and muscle, lower energy expenditure and aggravated insulin resistance [17]. In addition, enhancing NO bioavailability by supplementation of its precursor l-arginine improves glucose metabolism and reduces fat mass in both animals and humans [18, 19], possibly due to effects on skeletal muscle respiration and BAT activity leading to enhanced energy expenditure [20‐22]. Interestingly, South Asian individuals exhibit lower flow-mediated vasodilatation compared with Europids [23], pointing towards lower NO bioavailability. Thus, increasing NO bioavailability might be a promising approach to enhance skeletal muscle mitochondrial function as well as BAT activity, thereby exerting positive effects on whole-body energy and substrate metabolism.

The primary aim of the current study was to investigate the effects of 6 weeks of l-arginine supplementation on energy metabolism by BAT in overweight prediabetic Dutch South Asian and Dutch Europid men. In addition, we assessed the effects of l-arginine on skeletal muscle metabolism as well as glucose metabolism.

For more details of methods, see electronic supplementary materials (ESM) Methods.

In the current study, we show that l-arginine, the precursor of NO, does not affect energy expenditure, cold-induced BAT activity or skeletal muscle mitochondrial respiration in Europid and South Asian men with prediabetes. l-arginine improves glucose tolerance only in Europid men, possibly by improving peripheral insulin sensitivity.

Contrary to our hypothesis, l-arginine did not enhance either BAT volume or BAT activity as measured via [¹⁸F]FDG uptake. Preclinical studies in which the conversion of l-arginine to NO was diminished showed that this resulted in lower BAT volume as well as fewer mitochondria in BAT and muscle [16, 17]. Furthermore, a study in sheep showed that dietary supplementation with arginine enhanced BAT volume by 60% [36]. Although the current study suggests that l-arginine does not affect BAT, at least in humans, it cannot be excluded that l-arginine affects BAT oxidation since we used a tracer that only measures glucose uptake rather than oxidation. Therefore, future studies using alternative tracers, such as [¹¹C]acetate [37] will be needed to assess the effect of l-arginine on oxidative metabolism in BAT.

Furthermore, we previously showed that healthy lean South Asian men exhibit reduced BMR, even after correction for fat-free mass, and reduced BAT volume compared with Europid men [11]. However, in the current study, the difference in BMR disappeared after correction for fat-free mass. Curiously, NST was significantly higher in South Asians, while both BAT activity and detectable BAT volume were equal when comparing South Asian and Europid men. This is in line with findings of a previous study by Admiraal et al [38] but contradicts our previous notion that BAT volume and activity are lower in South Asian men, at least as measured via [¹⁸F]FDG uptake.

Furthermore, in our study, we did not find that l-arginine had any effect on body composition, including fat mass, as had previously been reported after l-arginine supplementation in obese individuals for 21 days [18] and 12 weeks [39]. A plausible explanation is that our treatment period of 6 weeks might have been too short to induce a reduction in fat mass. The reduction in fat mass observed after 21 days in the study of Lucotti et al [18] might have been caused by the interaction between l-arginine supplementation and the hypoenergetic diet and exercise intervention.

Our finding that l-arginine treatment improves glucose metabolism in overweight and obese prediabetic Europid men is in line with previous studies. Lucotti et al [18] showed that in obese individuals with type 2 diabetes, 21 days of l-arginine supplementation in a dose comparable with ours (8.3 g/day), on top of a hypoenergetic diet and exercise intervention, lowered fasting and postprandial glucose levels compared with placebo treatment. Moreover, l-arginine further reduced insulin levels, suggesting improved insulin sensitivity. Indeed, supplementation with l-arginine for 18 months improved insulin sensitivity [35]. In our study, the lower glucose excursion during OGTT upon l-arginine treatment in Europids, combined with the lower insulin excursion and tendency towards increased Matsuda index, points to improved insulin sensitivity. By quantifying [¹⁸F]FDG uptake, we could not identify the metabolic organ that was most responsible for this beneficial metabolic effect. Still, several other mechanisms are plausible when seeking to explain the improved glucose metabolism. For instance, due to NO-induced vasodilatation, l-arginine can increase blood flow and thereby glucose supply to metabolic tissues [40]. Indeed, in individuals with type 2 diabetes, a 3 month intervention with l-arginine decreased vascular resistance and increased blood flow, further supporting this hypothesis [41].

An interesting result in the current study is the lack of effect of l-arginine on glucose metabolism in men of South Asian ethnicity. Since, to our knowledge, this is the first study in which individuals of South Asian descent have been treated with l-arginine, we can only speculate on possible underlying mechanisms. A recent study has shown that endothelial cells isolated from South Asian men display lower expression levels of endothelial NOS, one of the enzymes that converts l-arginine into NO [42], compared with cells isolated from a matched control group of European origin [43]. A reduced functionality of endothelial NOS in South Asians may result in lower NO formation and thus less-beneficial NO-mediated effects on organ perfusion and glucose metabolism. Thus, in South Asians, other routes to enhance NO availability should be explored.

We also observed a lower skeletal muscle oxidative capacity in South Asian men in the current study. Skeletal muscle mitochondrial oxidative capacity has been repeatedly linked to metabolic dysfunction, such as insulin resistance, although the causal relationships are less clear [44]. Recently, it has been reported that lean African-American women, who showed lower peripheral insulin sensitivity compared with matched white women, also showed lower skeletal muscle mitochondrial respiration [45], indicating a role for ethnicity in these impairments. Thus, ethnically inherited defects in mitochondrial capacity may render South Asians more prone to the development of disturbances in skeletal muscle energy metabolism and insulin resistance.

This study is not without limitations. At baseline, South Asian and Europid men were not fully comparable with respect to several metabolic variables. Participants were equal with respect to BMI but South Asians generally have a different body composition with more fat mass and less lean mass compared with Europids [11]. In the current study, however, fat mass did not significantly differ between ethnicities. Furthermore, at baseline, Europid men were more insulin sensitive, as was evident from a lower Matsuda index after placebo treatment. Europid men also showed signs of greater beta cell failure compared with South Asians, as apparent from the OGTT data. It can also be questioned whether beta cell mas was equal between both ethnicities at baseline. One of the effects of l-arginine is stimulation of insulin release by beta cells and this could have influenced the response to l-arginine in both ethnicities. However, if that was the case, one would have expected a lower rather than a higher response in the Europid group. Furthermore, BMR was lower in South Asian men, although this difference disappeared after correction for free-fat mass. L-Arginine may also have influenced appetite. Although participants consumed a standardised meal the evening before the study, we cannot exclude the possibility that longer-term differences in food intake or food preference may have influenced the metabolic outcomes of the current study. Unfortunately, we did not record objective data on appetite following l-arginine supplementation.

In conclusion, we show that 6 weeks of l-arginine supplementation does not affect BMR, [¹⁸F]FDG uptake by BAT or skeletal muscle mitochondrial respiration in Europid and South Asian overweight and obese prediabetic men. However, l-arginine improves glucose tolerance in Europid men but not in South Asian men. Furthermore, we show for the first time that South Asian men have lower skeletal muscle respiratory capacity compared with Europid men.