**3. Improvement of pathophysiology in metabolic syndrome**

#### **3.1 Brown and beige adipocytes**

Once T2DM, atherosclerosis, or chronic renal failure develop, their reversal becomes tremendously challenging. Therefore, to prevent MetS, it is imperative to eliminate insulin resistance and chronic inflammation during the pre-metabolic syndrome stage. Here, we discuss brown and beige adipocytes, which have gained prominence as the best approach to improving metabolic syndrome.

Brown adipocytes are so named owing to their brown, muscle-like color; nevertheless, they accumulate and degrade fat rapidly. Beige adipocytes, which materialize in WAT upon cold stimulation, have an excellent ability like that of brown adipocytes but are not as brownish [16]. These adipocytes originate from different developmental lineages. Brown adipocytes are derived from Myf5+/Sca1+/Pax7+ cells in the dermis muscle layer of skeletal muscle and dermal precursors, whereas beige adipocytes are reported to emerge by trans-differentiation of WAT or differentiate from beige adipocyte precursors. Recently, it has been proposed that there may be numerous beige adipocytes depending on the WAT type [17]. Marker genes specific to brown and beige adipocytes have been found, including Cd137 and Cited1, specific for beige adipocytes, and Prdm16, Ucp1, and Pgc-1α are common markers for brown and beige adipocytes [18, 19].

Brown and beige adipocytes are heat-producing cells that generate nonshivering heat through diverse mechanisms [16, 20, 21]. In the mitochondrial inner membrane, energy, including NADH, produced by the TCA circuit is employed to pump protons outside of the membrane, creating an energy difference inside and outside the membrane for ATP synthesis. Brown and beige adipocytes have numerous mitochondria expressing uncoupling protein 1 (UCP1) in their inner membrane. On cold stimulation exposure, UCP1 opens ion channels and conjugates with anions, including long-chain fatty acids, to bring protons into the membrane. The energy lost in this process is converted to thermal energy, producing nonshivering heat. As mitochondria rush to restore the proton gradient via the TCA circuit, fatty acids are positively degraded by β-oxidation to produce acetyl CoA. In this manner, brown and beige adipocytes generate heat in response to cold stimuli, causing rapid fatty acid degradation. Although not deliberated here, mechanisms other than UCP1 that activate heat production have recently been identified and several regulators have been determined [22, 23]. This may be critical for reducing obesity, especially in the elderly and obese populations with few UCP1-positive adipocytes.

#### **3.2 Effectiveness of brown and beige adipocytes on metabolic syndrome**

It is well established that nonshivering heat produced by brown adipose tissue (BAT) is involved in thermoregulation of body temperature when animals awaken from hibernation and in humans in response to alterations in body temperature at birth. Thermogenesis by UCP1 in brown adipocytes has been observed to be closely related to obesity and T2DM [24]. For example, in mice, UCP1 does not function well in individuals prone to obesity, and UCP1 functions well in individuals that are not prone to obesity. In humans, BAT, which is abundant in newborns, drastically decreases as they grow older or develop MetS, although it plays an imperative role in maintaining health in adults. This decline is one of the reasons for the rapid rise in the incidence of MetS in adults over 40 years of age [25]. Therefore, augmenting the number of brown and beige adipocytes is expected to be an effective means of obesity and MetS [26].

Brown adipocytes not only play a role in fat metabolism through thermogenesis but have also been found to secrete cytokines, including FGF-21, follistatin, IL-6, CLCX14, or miRNA (batokines), upon thermogenic activation. Batokines work as paracrine and endocrine molecules and crosstalk with several organs [27, 28]. For example, BMP8 promotes sympathetic innervation and angiogenesis in BAT, whereas NRG4 promotes sympathetic axon elongation and secretion. FGF21 is released from BAT activated by thermogenesis and the liver, protecting against hyperlipidemia and nonalcoholic liver disease. Thermogenesis-activated BAT also secretes IL-6, which helps maintain BAT metabolic homeostasis and enhances gluconeogenesis in the liver. 12,13-dihydroxy-9Z-octadekanoic acid (12,13 diHOMO) improves cardiac function and cardiomyocyte respiration. Additionally, CLCX14 induces M2 macrophages and myostatin regulates skeletal muscle function. Thus, batokines secreted from BAT and beige adipocytes are anticipated to suppress MetS.

#### **3.3 Signals for white adipocytes browning and inducing brown adipocytes**

Activation of beige or brown adipocytes is primarily mediated by a pathway involving the β3-adrenergic receptor (β3-AR) [29]. Noradrenaline binds to β3-AR on adipocytes' surface and stimulates adenylate cyclase, which in turn activates PKA employing cAMP as a second messenger. Activated PKA phosphorylates pJM-JD1A, CEBP, ATF2, and other proteins via p38 MAPK to enhance the expression of PPARγ, PGC1a, PRDM16, NFIA, and others [30]. Enhanced expression of PPARγ and PRDM16 induces browning and PGC1a augments the number of mitochondria, resulting in the induction of UCP1, CIDEAR, COX8b, CDK5, and others.

The most crucial heat-producing factor in BAT is the transcription factor PRDM16, which initiates the brown adipocyte transcriptional program when coexpressed with C/ EBPβ. Experiments in mice in which PRDM16 was disrupted have demonstrated that it is vital for brown adipose tissue maintenance and WAT browning [31]. The transcription factor Zfp516 also plays a pivotal role in BAT development and cold-induced regulation. Deletion of Zfp516 in mice results in BAT development failure, while overexpression results in WAT browning and an increase in tissue oxygen consumption by more than 80% [32]. Additionally, IFR4 ablation in mice drastically decreases thermogenesis and energy consumption [33]. This indicated that IRF4 interacts with PGC1α and is involved in energy expenditure. It is noteworthy that continuous cold stimulation is essential for the differentiation of pre-brown to brown adipocytes and white to beige adipocytes. It has been reported that the modification from white to beige adipocytes is trans-differentiation, which returns to its original state once cold stimulation is stopped [34].

However, pathways have been found to activate BAT or induce beige adipocytes independent of BAT β3-AR signaling [35]. For example, adenosine A2A receptors bind to adenosine released by brown fat cells, and their activation can induce beige adipogenesis and suppress obesity [36]. Additionally, beige adipocytes differentiated from MyoD+ progenitors (glycolytic beige adipocytes) exhibit thermogenesis and energy homeostasis by adapting to cold conditions without β3-AR signaling [37]. It has also been demonstrated that UCP1 knockout mice adapt to cold environments by employing other compensatory pathways [38]. This β3-AR signaling-independent pathway will also be an imperative target for enhancing beige adipocytes in the future.

#### **3.4 Preventive treatment of metabolic syndrome with thermogenic fat**

Beige adipocytes in humans have beneficial effects in alleviating insulin resistance and weight loss [39] and treating metabolic syndrome by converting into beige adipocytes which have been attempted. The most representative approach for WAT browning is cold stimulation. Even in humans, cold exposure at 17°C every 2 h for 6 weeks augments BAT activity and reduces body fat percentage [40]. Additionally, cold stimulation for 10 days in type 2 diabetics boosted insulin sensitivity by 43% and skeletal muscle glucose uptake [41]. Thus, continuous cold stimulation is a simple and effective means to enhance BAT or beige adipocytes.

BAT activation by agonists has also been explored. Because the β3-AR of BAT acts on noradrenaline secreted by cold stimulation, β3-AR agonists also induce thermogenesis. For example, mirabegron, a β3-AR agonist, induces BAT activity and augments resting energy expenditure by up to 5.8% [42]. PPARγ receptor agonists, such as rosiglitazone, also activate SIRT1-PRDM16 and induce beige adipocyte development in mice [43]. However, β3-AR and PPARγ receptors are distributed throughout the body, and their side effects are challenging and have not yet been acknowledged for the treatment of metabolic disorders. Thus, researchers at Columbia University Medical Center are attempting to provide them specifically using a skin patch. The skin patch was equipped with several tiny needles. When applied to mice's abdomen with skin patches coated with rosiglitazone, WAT turned into beige adipocytes, resulting in a 20% fat reduction [44]. Additionally, cell therapy for direct BAT augmentation by transplantation is also being considered, as brown and beige fat cells can be produced from iPS cells and diverse other cells [45, 46].

Moreover, there is also a requirement for foods that can reduce fat and promote the activation of BAT and WAT beige [47]. Active consumption of these foods will help obtain a lean body and prevent metabolic syndrome. Because cold stimuli are received by TRP channels on the vagus nerve, compounds that stimulate TRP channels, including capsaicin, gingerol, and allyl isothiocyanate, are expected to have a cold stimuli-like effect. For example, when ingested, capsaicin or capsinoids activates the exchange nerve through its TRPV1 channel, and adrenaline is secreted [48]. When capsinoids were continuously ingested for 6 weeks, there was an increase in BAT and cold-induced heat production [40]. Additionally, EPA and DHA, or intestinal bacterial metabolites of unsaturated fatty acids, also have TRPV1 activating effects and have been reported to improve cold-induced heat production in BAT [49].

Food components with noradrenaline-like structures can stimulate β3-AR, and their continuous intake may induce brown or beige adipocytes. We reported that *Kikyo*, the constituents of *Bofutsushosan* (Chinese medicine), contain components that transdifferentiate mouse white adipocytes into beige adipocytes [50]. Another group reported that p-synephrine extracted from *Citrus unshiu Marcow* contains a component that converts to beige adipocytes [51].

It has also recently been demonstrated that sirtuins are closely involved in browning [52], SIRT3 is involved in mitochondrial function maintenance, and SIRT5 regulates UCP1. Furthermore, SIRT1 aids in the BAT gene transcription via PPARγ and activates PGC-1a [53]. Because SIRT1 is reduced by aging and age-related diseases, enhancing SIRT1 activity may prevent metabolic syndrome pathogenesis. Intravenous administration of resveratrol-bound nanoparticles targeting adipose stromal cells (ASCs) for 5 weeks significantly induced differentiation into beige adipocytes, reduction in fat mass by 40%, and enhanced glucose homeostasis and inflammation [54].

Additionally, it has been noted that chitosan, although not differentiating into beige adipocytes, acts on adipokines and has an inhibitory effect on obesity [55]. Experiments employing an animal model of diet-induced obesity indicated that chitosan oligosaccharide capsules activated the JAK2-STAT3 signaling pathway to mitigate leptin resistance, inhibit lipogenesis, and reduce lipid accumulation [56].
