METABOLIC STRESS IN OBESITY

Stress can be explained as a state of threatened or imbalanced homeostasis. Metabolic stress occurs in both obesity and T2D, and it can be explained as a group of stress responses that are dysregulated in metabolically relevant sites. Obesity is considered as one causative factor in the development of MetS, which is known to cover several interlinked detrimental conditions involving metabolic stress. Chronic, metabolically triggered inflammation—often referred to as chronic low-grade inflammation—has been recognized as one of the potential causes to MetS, and it is considered as a risk factor for a cluster of metabolic diseases (Hotamisligil, 2006, Esser et al., 2014). The chronic low-grade inflammation, together with an activation of the immune system, is involved in the pathogenesis of obesity-related insulin resistance and T2D (Esser et al., 2014). However, not all obese individuals develop MetS, and even lean individuals can be resistant (Kotronen & Yki-Järvinen, 2008). Regardless of their BMI, insulin-resistant individuals tend to develop excess, non-alcohol related fat in the liver—a condition defined as non-alcoholic fatty liver disease (NAFLD) (Kotronen & Yki-Järvinen, 2008). Independent of the obesity class, this insulin-resistant fatty liver correlates strongly with MetS, and the prevalence of both MetS and NAFLD is known to increase with obesity (Kotronen & Yki-Järvinen, 2008, Yki-Järvinen, 2014). In addition, the obese individuals with a dysfunctional fatty liver are more likely to have inflammatory changes in adipose tissue, especially in the intra-abdominal area, where surplus of fat is accumulated (Kotronen & Yki-Järvinen, 2008).

2.2.1 Dietary modifications affecting metabolic stress in adipose tissue

The high abundance of fat in the diet can drastically alter the health and is considered the main cause of obesity-related metabolic stress. The development of metabolic diseases is closely associated with weight gain and Western diet, which contains excess of simple carbohydrates and dietary fat, especially saturated

fat. These unhealthy diets are also known to modify intestinal microbiota and induce low-grade inflammation and insulin resistance.

Adipose tissue can react readily and beneficially to different modifications in lifestyle, especially to dietary changes. Beneficial dietary changes, such as increasing the consumption of whole-grain cereals, foods with a low glycemic index and dietary resistant starch in the diet, can improve the clinical outcome and eventually associate with changes in gene expression profiles in adipose tissue (Dahlman et al., 2005, Kallio et al., 2007, Kolehmainen et al., 2008). Adipocytes, and adipose tissue in general, can sense and react to the dietary changes by altering their metabolism. The effect of dietary changes on adipose tissue function has been greatly studied on genetic level and in the light of weight reduction. Kolehmainen and co-workers showed e.g., reduced expression of genes regulating the extracellular matrix and cell death after long-term weight reduction, while another group reported downregulation of genes regulating the production of polyunsaturated fatty acids. (Dahlman et al., 2005, Kolehmainen et al., 2008).

Nordic diet—following Nordic Nutrition recommendations—is generally considered to be beneficial for health. It has been suggested as an alternative to the Mediterranean diet, which is commonly known to improve health and to support the prevention of CVD, certain cancers and T2D (Uusitupa et al., 2013).

The typical Nordic diet is composed of whole grains, rapeseed oil, vegetable oil–based margarines, low-fat or low-fat-free milk products and local berries, fruits, vegetables and fish of Nordic origin. The foundation of the Mediterranean diet is very similar to Nordic diet, favoring the use of vegetables, fruits, herbs, nuts, beans and whole grains with moderate amounts of dairy, poultry, eggs and seafood. Nordic diet has reportedly reduced inflammatory gene expression in subcutaneous adipose tissue (SAT), compared with an isoenergic control diet, independent of body weight change in individuals with features of MetS (Kolehmainen et al., 2015). Especially, the genes expressed in immune-related pathways were differentially expressed in individuals consuming Nordic diet and those on control diet for 18 to 24 weeks (Kolehmainen et al., 2015). Healthy Nordic diet can also have beneficial effect on lipid metabolism and reduce the signs of systemic inflammation, as reported in the same randomized dietary intervention study [Systems Biology in Controlled Dietary Interventions and Cohort Studies (SYSDIET)] (Uusitupa et al., 2013, Brader et al., 2014).

2.2.2 Betaine and metabolic stress in different tissues

Betaine (glycine betaine, trimethylglycine) is a naturally occurring organic osmolyte that can be found in living organisms, such as various plant, animal and microbial species. Important sources of betaine include marine invertebrates (≈1%), wheat germ or bran (≈1%) and spinach (≈0.7%) and sugar beets (Craig, 2004). Dietary intake of betaine is estimated to range from an average of 1 g/d to 2.5 g/d, the latter measured from individuals consuming a diet high in whole wheat and seafood (Craig, 2004). Sugar beet (Beta vulgaris) is especially known for accumulating betaine and the mechanistic extraction of betaine from sugar beet molasses can be executed by a patented chromatographic separation method (Heikkilä et al., 1982).

By the chemical structure, betaine molecule is small (117.2 Da) N-methylated amino acid with three chemically reactive methyl groups attached to the nitrogen atom of a glycine molecule. It is a stable, non-toxic, highly water-soluble (160 g/100 g) and very hygroscopic molecule. In fact, betaine has a tendency to attach to several water molecules (Mathlouthi, 1997). Since betaine can carry water molecules without immobilizing them, it can assist other biological reactions that utilize water.

The diet must provide adequate amount of methyl groups, since they cannot be synthesized in the body. Methyl groups are essential for many metabolic processes, including the synthesis of phospholipids, adrenal hormones, RNA and DNA. Choline, methionine and betaine can donate methyl groups for these reactions using varying biochemical routes (Craig, 2004). The primary methyl donor in most metabolic reactions in the body is activated methionine, adenosylmethionine (AM). adenosylmethionine is converted to adenosylhomocysteine after it donates the methyl group and S-adenosylhomocysteine is then further converted to homocysteine (Craig, 2004). If genetic or nutritional defects occur in the metabolic processes of homocysteine, this can lead to elevated plasma homocysteine levels which are known to be associated with increased risk of CVD (Craig, 2004). However, betaine can re-methylate homocysteine back to methionine via the activity of betaine–homocysteine

S-30

methyltransferase (BHMT), and this will lead to reduced plasma homocysteine (Schwab et al., 2002, Olthof et al., 2003). Thus, the dietary betaine supplementation would benefit those individuals who suffer from homocystinuria—a disease caused by abnormally high levels of homocysteine at birth.

Plant and animal tissues alike are known to benefit from betaine, which is produced in plants against different environmental stresses, such as drought, salinity, extreme temperatures and UV radiation (Ashraf & Foolad, 2007). The bioavailability of betaine to various tissues is a key factor for its functional role in various stress situations. Several tissues contain betaine, and in animal studies, significant concentrations of betaine have been detected especially in the kidney and liver (Kettunen, 2001, Slow et al., 2009). Most of the body’s betaine originates from the aforementioned dietary sources containing both betaine and choline, and a small proportion of dietary choline is converted to betaine in the liver mitochondria (Siljander-Rasi et al., 2003). Liver seems to be the body’s primary betaine storage, and dietary betaine has shown to increase liver betaine concentrations in previous animal studies (Kettunen et al., 2001, Siljander-Rasi et al., 2003). Pekkinen and co-workers used nontargeted metabolite profiling to demonstrate that betaine levels increase in the mouse liver after the animals were supplemented with 1%

(wt/vol) betaine in drinking water for four weeks (Pekkinen et al., 2013). The basal betaine concentration measured in human plasma is approximately 50 µmol/l (Schwab et al., 2006). However, already a 6-g betaine supplementation can increase the plasma betaine concentration to 1 mmol/l, as demonstrated by Schwab and co-workers (Schwab et al., 2006). It has been shown in humans that the free dietary betaine is absorbed quickly into the blood circulation, showing peak values at 60 to 80 minutes after the ingestion (Schwab et al., 2006). The digestion of food products containing endogenous betaine takes more time and may prolong the absorption process of betaine, when compared to pure betaine supplementations.

Betaine can also be synthesized from choline involving a two-step oxidation process including choline dehydrogenase and betaine aldehyde dehydrogenase (Craig, 2004). Even though a small amount of betaine is excreted in the urine, the brush-border of the renal cortical tubules has specific transporters that can recover that small portion of betaine (Broer, 2008).

Betaine has been used as a supplement in animal nutrition, e.g., in fish and pig feed, and in pigs, betaine has reportedly reduced the amount of body fat (Eklund et al., 2005). The effect of dietary betaine has also been evaluated in humans, and Cholewa and co-workers have reported significant increases in lean mass and decreases in fat mass and body fat percentage with betaine consumption, when compared to placebo (Cholewa et al., 2013, Cholewa et al., 2018). There are several other examples of the ability of betaine to alleviate high-fat-driven metabolic changes. Pekkinen and co-workers noted in mice that supplemented dietary betaine decreased hepatic triglycerides and a variety of other lipid species in the liver (Pekkinen et al., 2013), as measured with metabolic profiling. In the same study and with metabolic profiling, it was established that betaine supplementation increased the number of short-chain acylcarnitine species, recognized as carnitine metabolites. Thus, this study suggested that dietary betaine supplementation is able to affect hepatic carnitine metabolism via the transmethylation cycle (Pekkinen et al., 2013). These findings support the view that supplementary betaine is able to reduce the high-fat-diet-induced lipid accumulation to the liver. Another study by Wang et al. (2010) showed that while a high-fat feeding induced mice to developed NAFLD in 12 weeks, simultaneous supplementation of betaine improved the pathological changes observed in the liver and attenuated adipose tissue insulin sensitivity (Wang et al., 2010). Betaine supplementation in vivo has also proven to protect the liver from non-alcoholic steatosis and oxidative stress (Kwon et al., 2009). Supplemented betaine has also shown potential in preventing ER stress response in isolated epididymal adipose tissue in animals fed with high-fat diet (Wang et al., 2010). More recently, preclinical studies have showed that betaine treatment significantly inhibited the proliferation and differentiation of mouse adipocytes in vitro and decreased plasma lipid and lipoprotein levels, reduced intramyocellular lipid accumulation and improved obesity-induced insulin resistance in high-fat-diet-fed mice (Du et al., 2018). These effects seen in adipose tissue could serve as potential mechanisms explaining the beneficial effects of betaine in obesity. Thus, by improving the adipose tissue function, i.e., reducing adipose tissue inflammation and metabolic stress, betaine may be beneficial in preventing the early stages of MetS and other obesity-related disorders.