Under specific conditions, white adipose tissue (WAT) depots are readily converted to a brown-like state, which is associated with weight loss. The transcription factor Krüppel-like factor 15 (KLF15) has emerged as a critical regulator of systemic homeostasis in the face of varying metabolic demands. This article explores the multifaceted role of KLF15 in adipose tissue metabolism, its impact on thermogenesis, and its potential as a therapeutic target for obesity.
Understanding Adipose Tissue and Browning
Brown adipose tissue (BAT) is a specialized metabolic organ responsible for non-shivering thermogenesis. Recently, its activity has been shown to be critical in systemic metabolic health through its utilization and consumption of macronutrients. BAT's ability to generate heat via non-shivering thermogenesis allows endothermic organisms to maintain a narrow body temperature range, a phenomenon crucial to individual survival and the evolutionary success of mammals at large. Both circulating and BAT-intrinsic lipids, glucose, and branched chain amino acids (BCAA) serve as critical thermogenic fuel sources, and BAT utilization of these macronutrients is important for systemic metabolic health. In humans, increased BAT activity is associated with improved insulin sensitivity and decreased circulating lipids, whereas, BAT dysfunction or absence is linked to the development of obesity, insulin resistance, and dyslipidemia.
White adipose tissue (WAT), on the other hand, stores energy. However, under specific conditions, WAT depots can be converted to a brown-like state, a process known as "browning." This conversion is associated with weight loss and improved metabolic health. Subcutaneous WAT contains heterogeneous adipocytes, including white and 'beige' adipocytes, which share features with brown adipocytes.
Allicin and Brown-like Adipocyte Formation
One potential method for inducing browning is through diet-derived factors. Allicin, a major component of garlic, has been investigated for its effects on brown-like adipocyte formation in inguinal WAT (iWAT) and its ability to prevent obesity and related complications in animal models. Allicin significantly increased mRNA and/or protein expression of brown adipocyte markers including uncoupling protein 1 (UCP1) in differentiated mouse embryonic fibroblast cell line 3T3-L1 and differentiated iWAT stromal vascular cells (SVC), suggesting that allicin induced brown-like adipocyte formation in vitro. Concomitantly, allicin markedly enhanced the protein expression of KLF-15 and its interaction with UCP-1 promoter region. Such changes were absent in cells lacking KLF-15, suggesting the critical role of KLF15 in allicin action. Allicin also induced brown-like adipogenesis in vivo along with the appearance of multilocular adipocytes, increased UCP1 expression and increased lipid oxidation.
KLF15's Role in BAT Metabolic Flexibility
In the face of energetically demanding states, metabolic flexibility and systemic coordination of nutrient partitioning is requisite for health and survival. Krüppel-like factor 15 (KLF15) plays a critical role in BAT metabolic flexibility. BAT-specific loss of KLF15 results in widespread changes in circulating metabolites and severely compromised thermogenesis in response to high energy demands, indicative of impaired nutrient utilization and metabolic flexibility.
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Maintaining body temperature in cold exposure requires systemic coordination of nutrient mobilization and selective utilization to ensure survival. To gain a comprehensive understanding of the transcriptional adaptations that must occur for successful non-shivering thermogenesis in cold, RNA-sequencing (RNA-seq) studies were performed on BAT isolated from control mice at room temperature (RT, fed ad libitum) and after a chronic cold challenge (10 days at 4°C, fed ad libitum). RNA-seq analysis of BAT comparing RT versus cold revealed 3219 differentially expressed genes (DEGs). This upregulation of lipid metabolism pathways is reminiscent of BAT adaptations in states of fasting where survival again is dependent on appropriate and selective nutrient mobilization and utilization.
To allow for time-of-day changes in nutrient availability, BAT activity and core body temperature exhibits circadian rhythmicity. Klf15 expression increases throughout the light phase and decreases throughout the dark phase, suggesting that KLF15 function in BAT is more analogous to that in these highly oxidative tissues than to nutrient storing tissues such as white adipose tissue (WAT). Concordant with the pattern observed during the light phase (i.e., when food intake and activity are decreased in mice), Klf15 expression acutely increases in BAT in response to fasting. Additionally, mice that are chronically cold challenged at 4°C show significant upregulation of BAT Klf15 expression as well.
Impact of KLF15 Knockout on BAT Metabolism
To explore the role of KLF15 in BAT metabolism, a BAT-specific KLF15 knockout (K15-BKO) mouse was generated. Expression of Ucp1, which encodes the key protein responsible for BAT non-shivering thermogenesis, was unchanged between K15 F/F controls and K15-BKO BAT. Measurement of core body temperature using implanted temperature telemetry devices showed appropriate oscillation of temperature throughout the 12 h day/night cycle with no significant differences between K15 F/F and K15-BKO mice. Interestingly, despite these findings, K15-BKO mice demonstrated a significant deviation from the transcriptional adaptions seen in response to cold and to fasting in K15 F/F BAT.
RNA-seq analysis comparing K15 F/F and K15-BKO BAT at RT identified 579 DEGs. Pathway analysis of downregulated genes in K15-BKO BAT revealed a strong signature for metabolic pathways, fatty acid (FA) degradation, and PPAR signaling pathway, suggesting a fundamental defect in energy utilization pathways in K15-BKO BAT. Indeed, a number of genes involved in lipid uptake into the cell and transport of long-chain acylcarnitines (LCACs) into the mitochondria (e.g. Cd36, Slc25a20, and Cpt1a) were downregulated in K15-BKO BAT. These impairments in lipid uptake and LCAC mitochondrial transport were reflected in plasma acylcarnitine analysis using tandem mass spectrometry: K15-BKO mice demonstrated a significant increase in many circulating acylcarnitine species, particularly LCACs, which are a major fuel source for BAT thermogenesis.
In addition to lipids, recent studies have demonstrated that BCAA metabolism plays a significant role in BAT thermogenesis capabilities and to systemic BCAA clearance. BCAA metabolic pathways (valine, leucine, and isoleucine degradation) were also significantly enriched from K15 F/F vs K15-BKO RT RNA-seq studies. In K15-BKO BAT, confirmatory qPCR analysis showed significant downregulation of genes involved in BCAA catabolism, including Bcat2, Aldh6a1, and Mut.
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Thermogenesis and Fuel Consumption in K15-BKO Mice
To assess BAT non-shivering thermogenesis capability, K15 F/F and K15-BKO mice were exposed to a chronic cold challenge for 10 days at 4°C. K15-BKO mice were surprisingly able to maintain body temperature comparable to K15 F/F. Given that K15-BKO mice had increased food intake throughout the day, mice were subjected to chronic cold challenge for 10 days followed by an overnight fast to explore BAT activity while excluding the potential effects of feeding. Strikingly, K15-BKO mice were incapable of maintaining body temperature in the absence of exogenous fuel sources and quickly succumbed to severe hypothermia within 12 h of fasting.
To gain further functional insights on how KLF15 affects BAT fuel consumption, Seahorse assays were performed in both cultured brown adipocytes and brown adipose tissue isolated from K15 F/F and K15-BKO mice. Loss of KLF15 resulted in significantly decreased oxygen consumption rates in both primary cells and whole tissue, reflecting an impaired ability to oxidize macronutrients. Beiging or browning of white adipose tissue has been shown to have a profound impact on thermoregulation; however, subcutaneous white adipose tissue isolated from K15 F/F and K15-BKO did not reveal any significant differences in expression of browning genes, Klf15, or lipid metabolism genes.
Transcriptional Changes in Cold Exposure and Fasting
RNA-seq analysis resulted in 2913 DEGs, with significant upregulation of genes related to FA and BCAA metabolism. Principal component analysis revealed that the transcriptional response to C + F is much more like that of cold challenge alone than to fasting, further supported by a comparison of K15 F/F RNA-seq data between cold and C + F which only returned 302 DEGs. Thus, when an animal is confronted with both cold and fasting stimuli, a state that is akin to field conditions, BAT thermogenic transcriptional programs predominate over those of fasting. This prioritization of macronutrient uptake and catabolism in BAT is necessary for stable body temperature and survival.
In response to cold, K15-BKO mice demonstrate a significant decrease in overall expression of genes involved in both BCAA and FA metabolism pathways compared to K15 F/F mice. In K15-BKO mice, there is significant downregulation of numerous BCAA and lipid metabolism genes in response to C + F in BAT.
Both BAT-intrinsic nutrients (e.g., lipids through lipolysis) and those taken up from the circulation enter the TCA cycle and are necessary for non-shivering thermogenesis. Interestingly, numerous TCA cycle genes (e.g. Idh3b, Ogdh, Suclg1, Suclg2, and Sdhb) were significantly downregulated in K15-BKO compared to K15 F/F. Additionally, the expression of several amino acid catabolism genes that participate in TCA cycle anaplerosis was also decreased in K15-BKO BAT.
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Systemic Metabolic Shifts in K15-BKO Mice
Under the C + F condition, substantial shifts in K15-BKO systemic metabolism in response to deficient BAT function were observed. In the liver, K15-BKO animals had significant elevations in genes related to acylcarnitine metabolism (e.g. Cpt1a, Cpt1b, and Cpt2) and in numerous amino acid catabolic genes (e.g. Acaa1, Acaa2, Alt2, Fah, Glud1, Got1, Got2, Hmgcl, Mut, and Sds), likely as compensation for insufficient BAT function. Interestingly, the most dramatic increases in gene expression occurred with respect to glucogenic amino acid catabolic genes (Alt2, Sds, and Got1). Subsequent qPCR analyses revealed upregulation of both Pcx and Pepck, in addition to other key gluconeogenesis genes (Fbp1 and G6pc) in K15-BKO liver. Ldha expression, encoding lactate dehydrogenase, was also upregulated, suggesting that there may be increased utilization of lactate for glucose generation in K15-BKO mice. These data together suggest that K15-BKO mice require increased hepatic gluconeogenesis to maintain blood glucose levels with cold exposure and fasting.
KLF15 and White Adipocyte Properties
Klf15 expression is higher in WAT compared with BAT and is suppressed by β-adrenergic signaling. Klf15 expression levels were surveyed across 3 major types of adipose: visceral, subcutaneous, and intrascapular brown in WT mice. Strikingly, the expression level of Klf15 is approximately 75% lower in brown adipose tissue (BAT) than in white (WAT). These results suggest physiological implications for these differences, including raising the possibility of a requirement to downregulate Klf15 levels for proper brown fat function.
Brown fat responds to β-adrenergic signaling with the induction of thermogenesis, and this is a primary physiological function of this tissue. Exposing WAT to the β-adrenergic stimulant isoproterenol in tissue culture resulted in downregulation of Klf15 expression. Injecting WT mice with the β-adrenergic agonist CL-316243 results in an approximately 50% downregulation of Klf15 expression levels in WAT.
There are 3 different adrenergic receptor family members (ADRB 1-3). Adrb1 is a particularly interesting, and perhaps distinct, adrenergic receptor; for example, overexpression of Adrb1 in white adipocytes is sufficient to generate constitutive activity and mimics the effect of systemic infusion of agonists, even in the absence of additional agonists.
KLF15 Deletion and Adipocyte Browning
Deletion of Klf15 disrupts maintenance of white adipocyte properties and induces Adrb1 expression. A Klf15-floxed mouse line was generated using CRISPR/Cas9. White adipocytes from these mice were harvested and infected them with an adenovirus that expresses Cre recombinase (cleaving the loxP sites, knocking out Klf15) or control adenovirus. Cre-infected adipocytes had lower expression levels of Klf15 compared with controls, demonstrating that Cre exposure properly results in efficient deletion of Klf15 in the cells. Deletion of Klf15 substantially induces the expression of genes that are critical to brown fat identity and function, including the expression of the canonical brown fat gene Ucp1.
When the expression levels of β-adrenergic receptors in response to deletion of Klf15 were measured, Adrb1 was upregulated, while the other adrenergic receptors were unaffected or down regulated. β1AR protein levels also increase with Klf15 deletion. These findings are intriguing, in part, because overexpression of Adrb1 in subcutaneous white adipose tissue (iWAT) is sufficient to induce Ucp1 expression and beiging of this white fat depot. Further, β1AR is more sensitive to stimuli than other adrenergic receptors. Deletion of Klf15 leads to a marked increase in the levels of phosphorylated p38 in mature adipocytes.
The antagonist propranolol diminishes, but does not abolish, the induction of Ucp1 expression in Klf15-deleted adipocytes. Reciprocally, the addition of an agonist enhances the induction of Ucp1 in Klf15-deleted adipocytes.
In Vivo Studies of KLF15 Deletion
To investigate this activity of KLF15 selectively in mature adipocytes in vivo, Klf15-floxed mice were crossed with transgenic mice expressing Cre recombinase under the control of the adiponectin promoter and regulatory elements (Adipoq-Cre). Adipoq-Cre mice have been verified to selectively express Cre in mature adipocytes, enabling conditional deletion of Klf15 in mice that have both floxed Klf15 and the Adipoq-Cre transgene (Adipo-Klf15-cKO mice) in their genomes. Klf15 is efficiently and selectively knocked-out in mature adipocytes and not in other tissues nor in the adipose stromal vascular fraction (SVF) of Adipo-Klf15-cKO mice. When the WAT from Adipo-Klf15-cKO mice was harvested, the iWAT had a browner appearance than iWAT from either Adipoq-Cre or Klf15-floxed mice. In addition, the mass of the iWAT was less in Adipo-Klf15-cKO mice compared with littermate controls.
Consistent with findings in tissue culture, the in vivo studies revealed that only Adrb1 was selectively upregulated in the iWAT of Adipo-Klf15-cKO mice.
To rigorously verify these results, the ratio of expression levels of Adrb1 to Adrb3 in Adipo-Klf15-cKO mice against sex-matched littermates as individual paired replicates was directly compared. The results of these orthogonal testing of the findings confirmed that Adrb1 expression in iWAT is selectively upregulated in Adipo-Klf15-cKO mice. Finally, it was tested if the expression change translates into altered protein levels of β1AR. Together, these results indicate that deletion of Klf15 in the Adipo-Klf15-cKO mice disrupts maintenance of white adipocyte properties and induces beiging selectively in the mature adipocytes within the iWAT depot.
To investigate this further, as well as test the findings using an independent approach, a second mouse line was generated by crossing Klf15-floxed mice with transgenic mice expressing Cre under the regulation of Prx1 promoter and regulatory element (Prx1-Cre). Mice generated by this cross that have both floxed Klf15 and the Prx1-Cre transgene (Prx1-Klf15 cKO) have substantially lower levels of Klf15 expression selectively in the iWAT compared with other tissues and with littermate controls. In addition, the iWAT depots specifically weigh less, are browner, and have smaller adipocytes than iWAT depots in control mice.
Notably, the Prx1-Klf15-cKO iWAT has decreased expression of some white adipocyte marker genes as well as significantly higher expression levels of multiple brown fat marker genes. Further, the expression level of Ardb1, but not Ardb2 or Ardb3, is upregulated in Prx1-Klf15-cKO iWAT as quantified in absolute levels as well as relative to Adrb3 expression in littermate controls.
Functional Implications and Energetics
Having established, using 2 mouse model systems, that deletion of Klf15 in iWAT induces β1AR and disrupts mature white adipocyte maintenance, the functional implications of these findings were investigated. The energetics of primary iWAT harvested from Prx1-Klf15 cKO compared with littermate controls in response to the selective β1AR agonist Xamoterol as well as the adrenergic agonist Isoproterenol were measured. The above results indicate that deletion of Klf15 stimulates upregulation of the β1AR and results in adipocytes with enhanced responsiveness to adrenergic stimulation.
By monitoring singly housed mice in metabolic cages, it was found that Prx1-Klf15 cKO mice have higher energy expenditure per total body mass compared with littermate controls, and expenditure becomes significantly further induced by acute cold exposure, a physiologically relevant stimuli. Of note, the Prx1-Klf15-cKO mice are also more competent at maintaining their body temperature during cold exposure, and this occurs without any change locomotor activity.