Adipose tissues are dynamic tissues that play crucial physiological roles in maintaining health and homeostasis. The conversion of white adipose tissue (WAT) to brown adipose tissue (BAT), also known as "fat browning," is considered a promising approach to increasing energy expenditure and improving metabolic health. This process involves the upregulation of uncoupling protein 1 (UCP1), a molecule that uncouples the respiratory chain from adenosine triphosphate (ATP) synthesis, producing heat. This article explores the role of the ketogenic diet (KD) and beta-hydroxybutyrate (β-HB) in promoting WAT browning and its potential benefits for metabolic health.
Adipose Tissue: A Brief Overview
Adipose tissues (ATs) are endocrine and dynamic organs that display high morphological and functional plasticity. White AT (WAT) was named that way because it presents white adipocytes in its composition. In contrast, brown AT (BAT) has as its main integrant the brown adipocytes [1]. Both ATs play various physiological roles, including energy storage, endocrine regulation, and thermogenesis.
White Adipose Tissue (WAT)
WAT, the most abundant AT in the body, contains the white adipocytes, which present unilocular lipid droplets, scarce mitochondria, and lipid storage capacity [23]. Since the discovery of the adipokines, WAT is also recognized as an important endocrine organ, actively participating in the regulation of physiologic and pathologic processes, including immunity and inflammation [24, 25]. Widely distributed throughout the body, there are two main representative types of WATs, the visceral WAT (vWAT) and the subcutaneous WAT (scWAT). While one is distributed around organs and provides protective padding, the other is located under the skin and provides insulation against heat or cold, respectively [26]. WAT primarily engages in energy storage and is central to metabolic diseases associated with obesity.
Brown Adipose Tissue (BAT)
In contrast, brown adipocytes display multilocular lipid droplets, a large number of mitochondria, and thermogenic capacity due to elevated uncoupling protein 1 (UCP1) amounts anchored in its mitochondrial inner membrane [27]. The BAT utilizes this high mitochondrial content and elevated UCP1 amounts to uncouple oxidative phosphorylation from adenosine triphosphate (ATP) synthesis to dissipate chemical energy as heat [28]. Thus, BAT affects the metabolism of the entire body, being able to alter insulin sensitivity and modify the susceptibility to increase weight. BAT, rich in mitochondria and containing uncoupling protein 1 (UCP1), is responsible for heat production that positively affects obesity and metabolic diseases. For a long time, BAT was only considered an energy-producing organ in rodents and newborns, undergoing involution with age. However, BAT has also been identified in human adults near the aorta and within the supraclavicular region of the neck. Nevertheless, the origin of BAT is still under debate [26, 29].
Beige Adipose Tissue
Recently, a type of AT showing intermediary characteristics between that of white and brown adipocytes, which has mixed structural features of both, was identified as beige AT [29]. This type of AT was reported as a set of adipocytes in WAT that might acquire a thermogenic phenotype with higher UCP1 expression, similar to brown adipocytes after enough stimulus [29, 30]. Under certain conditions, WAT can convert to beige fat by mitochondrial biogenesis and upregulation of UCP1, which has similar functions to brown fat (2). This process is referred to as fat browning, and it is considered a promising approach to increasing energy expenditure and improving metabolic health.
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There are two major mechanisms described related to beige cells arising: de novo differentiation which occurs from a progenitor resident cell and transdifferentiation which consist of differentiation of a mature white adipocyte through a molecular mechanism. The first theory is based on that beige adipocytes come from progenitor cells differentiation induced by adipogenic stimulation such as cold exposure, adrenergic signaling, exercise, natriuretic peptides, thyroid hormones, diets, and food components [31, 32]. Currently, several specific cell markers were identified in various types of progenitor cells such as in smooth muscle-like cells (Myh11+), preadipocytes (Pdgfrb+, SMA+), adipocytes progenitor cells (Sca-1+ Pdgfra+ CD81+) [33,34,35]. These adipogenic stimulation actives transcriptional machinery of browning that is characterized by the expression of Ucp1, Prdm16, Zfp516, and Pgc1a genes that will promote a beige differentiation [36].
On the other hand, the transdifferentiation hypothesis proposes beige cells arise from mature white adipocytes, in a reversible process, after adipogenic stimulus without the participation of a progenitor-like state of cells [37]. The underlying molecular mechanisms for transdifferentiation are under intensive research, but some studies already show that this plasticity process occurs mainly in scWAT depots [29]. Known as browning, this process has gained increasing attention in the research area as an alternative method of energy stimulation. UCP1 expression can be stimulated when white adipocytes are exposed to stimuli, previously referred as to adipogenic stimulus, [20, 27, 29], driven by a set of molecules known as browning markers.
The Role of UCP1 in Thermogenesis
The non-shivering thermogenesis is a phenomenon that occurs in brown and beige ATs due mostly to the action of UCP1 [38]. UCPs are transmembrane proteins that belong to the mitochondrial anion carrier family (MACF), i.e., mediate specific metabolite exchanges between the cell cytoplasm and the mitochondrial matrix and thus enable the activation of essential biochemical pathways [39, 40]. The UCPs exhibit 5 isoforms, ranging from UCP1 to UCP5 are present in several tissues [41, 42]. UCP1 is the main isoform associated with thermogenesis, it is widely and selectively expressed in the inner mitochondrial membrane of the adipocyte, representing about 10% of the total mitochondrial protein in human epicardial AT [43,44,45,46].
UCP1 protein is described as participating in thermogenesis by interfering in proton leakage within the chemiosmotic gradient during the mitochondrial oxidative phosphorylation by the translocating fatty acids (FAs). This gradient is obtained from the oxidation of substrates and provides the required force to induce the respiratory machinery to produce ATP. Once UCP1 promotes proton leakage, the energy obtained cannot be stored in the form of ATP and is alternatively dissipated as heat [47, 48]. Thus, it is evident that direct regulation of UCP1 protein activity is one of the means of regulating thermogenesis, and that occurs in opposite ways by cytosolic purine nucleotides and long-chain fatty acids (LCFA), promoting inhibition or activation of UCP1, respectively [49].
The other form of regulating UCP1 is at the transcriptional level. UCP1 gene is transcribed only in brown and beige adipocytes, associates with the differentiation state of these cells, and is quantitatively regulated in response to many physiological signals [9]. These characteristics are consequences of the transcriptional control mediated by trans-acting factors on regulatory regions found in the 5’ non-coding region of the UCP1 gene. The proximal regulatory region, which is found immediately upstream of the transcription start site, contains cAMP response element-binding protein (CREB) [50, 51] and CCAAT-enhancer-binding protein (C/EBP) [52] binding sites. Also in the proximity of the site of transcription start, activating transcription factor-2 (ATF2)-binding site interacts with transcriptional coregulators, such as PGC-1α, impacting UCP1 gene transcription [9]. In opposition to these proximal regulatory sites, a strong enhancer region is placed more than 2 kb upstream of the transcription initiation site [50, 53] and contains a cluster of response elements for nuclear hormone receptors [7, 54].
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UCP1 gene activation and repression depend on which trans-acting factors bind to the regulatory region. For example, CREB binding sites mediate a positive transcriptional response to cAMP [50] and a negative response to AP2 (c-Jun/c- Fos) complexes [51]. Another example is the PPARγ binding site found in the distal enhancer region, which associates with gene activation after binding to its main ligand but represses UCP1 transcription when interacting with liver X receptor (LXR) and its corepressor receptor-interacting protein 140 (RIP140) [55]. RIP140 inhibits UCP1 gene transcription by enabling the assembly of DNA and histone methyltransferases on the UCP1 gene, altering the methylation status of CpG islands in the promoter region and histones, impacting gene expression through transcription machinery accessibility [56].
Although some epigenetic modifications are associated with repressed UCP1 gene expression, as in H3K9 demethylation marks, chromatin modifications indicative of activation of this gene also occur, such as in the case of H3K4 trimethylated marks, which are enriched in BAT [57]. Also participating in fine-tuning of gene expression, microRNAs (miRs)…
Ketone Bodies and β-Hydroxybutyrate (β-HB)
During fasting, energy balance is maintained by breaking down triglyceride (TG) in white adipose tissue (WAT) into fatty acids, producing ketone bodies (β-hydroxybutyrate, acetoacetate and acetone). Among these, β-HB stands out as the most abundant ketone body synthesized predominantly during glucose shortage, formed in the liver from β-oxidation of fatty acids that are produced via lipolysis of adipose tissues (1). It serves as a crucial indicator of energy metabolism, utilized as a significant energy source in the brain, skeletal muscle, heart and other peripheral tissues.
β-HB, the most abundant ketone body, is an essential energy carrier produced from fatty acids during glucose depletion (1, 17). Interestingly, both β-HB and lactate, metabolic intermediates produced during fasting or exercise, have been shown to promote fat browning through redox-dependent adaptations in murine WAT (18).
The Ketogenic Diet and Fat Browning
The objective of this study is to investigate the roles of β-HB in regulating the fat browning program and adipomyokine expression in vitro and WAT in rats on a low-carbohydrate/high-fat ketogenic diet (KD) and/or undergoing aerobic exercise training. By understanding the underlying mechanisms of β-HB-induced fat browning and its potential as a non-pharmacological intervention, important insights can be gained into the development of effective therapeutic strategies against metabolic diseases.
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Experimental Evidence
In adipocyte culture experiments, β-HB reduced intracellular lipid accumulation by enhancing lipolysis and stimulated the expression of thermogenic and fat browning genes like uncoupling protein 1 (UCP1), PR domain containing 16 (PRDM16), and adipokines such as fibroblast growth factor 21 (FGF21) and Fibronectin type III domain-containing protein 5 (FDNC5). Additionally, β-HB activated the AMPK-SIRT1-PGC-1α pathway, with UCP1 and PRDM16 upregulation mediated by β-HB intracellular action and SIRT1 activity.
In animal experiments, KE group raised β-HB levels, decreasing body weight and blood lipids. β-HB induction via KD and/or EX shows potential in promoting WAT browning by activating mitochondrial biogenesis, lipolysis, and thermogenesis, suggesting that dietary and physical intervention inducing β-HB may benefit metabolic health.
To increase circulating levels of β-HB, rats were treated with aerobic exercise and/or KD. Eight-week-old (230-250 g) male Sprague Dawley (SD) rats that were maintained at temperature ranged 22-25°C, 40-60% of humidity, with a 12 h light/dark cycle were randomly divided into four groups (n = 8 per group): sedentary control (CON), control with moderate-intensity exercise (EX), a sedentary group with KD (KD), and a group with moderate intensity exercise and KD (KE). The levels of β-HB in spot blood were measured before exercise (Pre-exercise baseline; Pre), immediately after exercise (Post-exercise; Post) and 1 h after exercise recovery time (RE) on the final day of exercise intervention. To exclude the acute effects of exercise on whole body metabolism, the rat was sacrificed for collecting adipose tissue and blood 3 days after 8-week exercise intervention, under anesthesia with intraperitoneal injection of Zoletil (50 mg·kg−1) and intramuscular Rompun (5 ~ 10 mg·kg−1).
After 1 week of acclimation, EX and KE groups were adapted to the treadmill exercise without inclination at 5-10 m/min (gradually increasing speed, 20 min/day and 5 days/week) for 1 week. After adaptation, the animals underwent aerobic exercise training for 8 weeks. The exercise intensity was gradually increased over the study period. In the initial 4 weeks, the animals performed a 5-min warm-up at a speed of 8 m/min, followed by 22 min at a speed of 15 m/min, and 3-min cool-down (5 m/min) 5 times per week. The animals rested for 3 days after 8-week exercise intervention to exclude the carry-over confounding effects of the last-day exercise on the biological effects of the chronic exercise training. The intensity of exercise was moderate with VO2max of 60-75%, following a previous study (22).
Molecular Mechanisms
Despite evidence suggesting that metabolic intermediates like β-HB influence white adipose tissue (WAT) metabolism, the precise molecular mechanisms remain unclear. The aim of this study was to investigate the impact of beta-hydroxybutyrate (β-HB) on the fat browning program and to explore the underlying molecular mechanisms using both in vitro and in vivo models.
β-HB as an HDAC Inhibitor
Researchers have found that one way to nudge white adipocytes toward the beige or brown phenotype is via inhibition of a type of histone deacetylase, histone deacetylase 11 (HDAC11). Beta-hydroxybutyrate (BOHB), the darling of the ketogenic research world, is a natural, endogenously produced HDAC inhibitor. All that’s required is a very low carbohydrate intake. BOHB is more than just a byproduct of a ketogenic diet. It’s a signaling molecule that influences a broad range of metabolic processes, and may serve as a link between diet and gene expression.
Additional Factors Influencing Fat Browning
Exercise
Physical exercising copes with increased levels of specific molecules, including Beta-Aminoisobutyric acid, irisin, and Fibroblast growth factor 21 (FGF21), which induce adipose tissue browning. FGF21 is a stress-responsive hormone that interacts with beta-klotho.
Hormones and Circadian Rhythm
The central roles played by hormones in the browning process highlight the relevance of the individual lifestyle, including circadian rhythm and diet. Circadian rhythm involves the sleep-wake cycle and is regulated by melatonin, a hormone associated with UCP1 level upregulation.
Diet and Gut Microbiome
In contrast to the pro-inflammatory and adipose tissue disrupting effects of the western diet, specific food items, including capsaicin and n-3 polyunsaturated fatty acids, and dietary interventions such as calorie restriction and intermittent fasting, favor white adipose tissue browning and metabolic efficiency. The intestinal microbiome has also been pictured as a key factor in regulating white tissue browning, as it modulates bile acid levels, important molecules for the thermogenic program activation.