Circulating α-Klotho Regulates Metabolism via Distinct Central and Peripheral Mechanisms

Abstract

Emerging evidence implicates the circulating α-klotho protein as a prominent regulator of energy balance and substrate metabolism, with diverse, tissue-specific functions. Despite its well-documented ubiquitous role inhibiting insulin signaling, α-klotho elicits potent antidiabetic and anti-obesogenic effects. α-Klotho facilitates insulin release and promotes β cell health in the pancreas, stimulates lipid oxidation in liver and adipose tissue, attenuates hepatic gluconeogenesis, and increases whole-body energy expenditure. The mechanisms underlying α-klotho’s peripheral functions are multifaceted, including hydrolyzing transient receptor potential channels, stimulating integrin β1→focal adhesion kinase signaling, and activating PPARα via inhibition of insulin-like growth factor receptor 1. Moreover, until recently, potential metabolic roles of α-klotho in the central nervous system remained unexplored; however, a novel α-klotho→fibroblast growth factor receptor→PI3kinase signaling axis in the arcuate nucleus of the hypothalamus has been identified as a critical regulator of energy balance and glucose metabolism. Overall, the role of circulating α-klotho in the regulation of metabolism is a new focus of research, but accumulating evidence identifies this protein as an encouraging therapeutic target for Type 1 and 2 Diabetes and obesity. This review analyzes the new literature investigating α-klotho-mediated regulation of metabolism and proposes impactful future directions to progress our understanding of this complex metabolic protein.

Introduction

Approximately 24 years ago the α-klotho gene was discovered when a study revealed mutation of a single gene within Chromosome 13 results in a rapid, premature aging phenotype [1]. α-Klotho overexpression has since been researched for its ability to prolong the lifespan 20–30% in rodents [2], as well as its therapeutic potential in various neurological [3–7], metabolic [8–15], cardiorespiratory [16–25], osmolar, [26–29] and cancer-related diseases/disorders [30]. More recently, in an attempt to combat the unrelenting obesity and diabetes epidemics, studies have begun to elucidate the specific roles of α-klotho in the regulation of energy and substrate metabolism. Increasing evidence demonstrates reduced cerebrospinal fluid (CSF) and plasma α-klotho concentrations in humans with Type 2 Diabetes and obesity, suggesting decreased α-klotho bioavailability may be involved in the pathology of these disorders. However, the role of α-klotho in metabolic regulation is complex, in part due to α-klotho’s diverse physiological functions and paradoxical effects on insulin activity [2,9–11,14]. Additionally, most studies utilize models involving whole-body α-klotho manipulation, which lack tissue-specific insight, especially considering α-klotho’s inability to cross the blood-brain barrier (BBB) [3,31]. The following review analyzes the literature investigating α-klotho-mediated regulation of metabolism, highlights the distinct differences between α-klotho circulating in the blood vs. CSF, and proposes impactful experiments to determine the cell-specific effectors of α-klotho.

Structure of the α-klotho protein-

RNA-splice variants of the α-klotho gene encode either a transmembrane or secreted α-klotho protein [32,33]. Transmembrane α-klotho has 1014 amino acids with a molecular weight of 130kD and is comprised of a signal sequence at its N-terminus, a single transmembrane helix domain near the C-terminus, two internal repeats (KL1 and KL2) between the two, and a small intracellular domain. Secreted α-klotho consists of only the KL1 domain and N-terminus, weighs 65kD, and has 550 amino acids. Both transmembrane and secreted α-klotho exhibit enzymatic activity due to KL1 and KL2 domains possessing identical beta-glucuronidase and sialidase homology. Notably, the rodent α-klotho protein has ~86% homology with that of humans [32–34].

Transmembrane α-klotho can also undergo proteolytic cleaving between the KL1 domain and the transmembrane domain (α cut) or between the KL1 and KL2 domains (β cut) to release full or short form α-klotho into the circulation, respectively [35–37]. Similar to tumor necrosis factor α (TNFα) and amyloid precursor protein (APP), α-klotho is cleaved by members of the ADAM family (a disintegrin and metalloprotease) 10 and 17, as well as β APP converting enzyme 1 (BACE-1) [35,37,38]. While no specific α-klotho receptor has been discovered, both cleaved and secreted α-klotho exhibit diverse hormonal function in various tissues [36,39]. Little is known about what regulates the cleaving of transmembrane α-klotho, but insulin can augment this process via PI3kinase signaling [35]. Considering one of the first discovered metabolic functions of α-klotho was to inhibit insulin signaling [2,30,40,41], these findings may suggest a physiologically relevant negative feedback loop.

Regulation of α-klotho expression-

α-Klotho is primarily expressed in the kidneys and the choroid plexus of the brain; however low levels of expression are detected in pituitary gland, placenta, skeletal muscle, adipose tissue, bladder, pancreas, testis, ovary, colon, and lungs [1,42]. Considering the diverse physiological functions of α-klotho, there are likely many redundant mechanisms involved in its regulation; however, currently little is known about tissue-specific regulation of α-klotho expression. Rodent and cell culture studies identify PPARγ as a prominent transcription factor upregulating α-klotho gene expression in adipocytes and kidney cells [43,44], while negative regulators of kidney α-klotho expression include estradiol [45], HMG-CoA reductase [20], and angiotensin II (via an angiotensin 1 receptor) [39]. Moreover, studies investigating regulation of circulating α-klotho levels are largely observational and produce mixed results. For example, one study determined plasma α-klotho concentrations fluctuate diurnally [46], with high concentrations around noon and low concentrations at midnight, while another observed no diurnal variations [47]. Sex differences in serum α-klotho concentrations are equally equivocal, with one study suggesting females have higher α-klotho levels [48] and another observing no differences [49]. Pedersen et. al. compared two popular commercially available α-klotho ELISA kits (Immuno-Biological Laboratories, Japan vs. Casabo, China) and discovered drastic variability between the two [48], suggesting the variability in results throughout the literature is likely due to unreliable α-klotho-detecting assays.

More consistently observed in the literature is that plasma α-klotho concentrations inversely correlate with age in humans [17,46,48–50]. Furthermore, α-klotho expression ubiquitously exhibits a negative relationship with oxidative stress [11,21,51,52], endothelial dysfunction [53,54], and atherosclerosis [1] in rodents. Plasma α-klotho concentrations are also reduced in many human disease states, including cardiovascular disease [17] and Type 2 Diabetes [55–57], suggesting possible involvement in the pathology of these disorders. Additionally, human plasma α-klotho concentrations are positively correlated with HDL [17] and phosphate levels [49], and negatively correlated with triglycerides [50] and fibroblast growth factor (FGF) 23 concentrations [49]. Due to the correlative nature of these studies, the causal relationship between α-klotho concentrations and many of these circulating factors is unclear, and further investigation is needed to determine if α-klotho is the driving factor in these changes or a compensatory mechanism.

α-Klotho functions as two independent pools in the CSF and the blood, with no correlation between the two, and significantly less α-klotho in the CSF [58]. The few studies that have examined CSF α-klotho concentrations have determined no diurnal variations [59], decreased levels in females [58,59], an inverse correlation with age [59], and a positive correlation with FGF23 [58]. Additionally, human CSF α-klotho concentrations are reduced in neurological disorders such as Alzheimer’s disease [59] and Multiple Sclerosis [60].

Functions of α-klotho-

While no specific α-klotho receptor has been identified, a variety of α-klotho functions have been discovered throughout the body. Perhaps the most well-documented function of α-klotho is its role in the kidney regulating Vitamin D, phosphate, and calcium homeostasis. α-Klotho is a critical non-enzymatic scaffolding protein that tethers FGF23 to tyrosine kinase FGF receptors (FGFR’s), without which FGF23→FGFR affinity is very low [61–63]. In the proximal renal tubule, downstream α-klotho→FGF23→FGFR signaling decreases Vitamin D levels by decreasing gene expression of 25-hydroxyvitamin D 1-α-hydroxylase and increasing expression of 1,25-dihydroxyvitamin D 24-hydroxylase, the enzymes responsible for the synthesis and degradation of the bioactive form of Vitamin D, respectively [26,64]. This signaling mechanism is a negative feedback loop in response to elevated Vitamin D levels, and may also be responsible for α-klotho-mediated downregulation of phosphate reabsorption and upregulation of calcium reabsorption [27,45,65–67]. Interestingly, FGF23 knockout mice exhibit a similar phenotype to α-klotho knockout mice, including growth impairment and premature death, highlighting the importance of α-klotho→FGF23→FGFR-mediated regulation of mineral homeostasis [68]. Furthermore, ablation of the enzyme responsible for Vitamin D synthesis, overexpression of the enzyme responsible for Vitamin D degradation, or dietary restriction of Vitamin D and phosphate markedly rescues the signature premature aging phenotype in these mutants [2,26,52,64].

Another mechanism through which α-klotho is involved in the aging process is via inhibition of various Wnt ligands in liver, kidney, fibroblasts, and endothelial cells [51,69–71]. Wnt signaling is associated with stem cell proliferation, but overactivity of Wnt’s is associated with aging and tumorigenesis [69]. α-Klotho is an important competitive antagonist of many Wnt ligands and has even been shown to act as a tumor suppressor in human hepatocellular carcinoma and breast cancer cells via this mechanism [30,51,69–71].

α-Klotho also possesses β-glucuronidase enzymatic capabilities, which allow it to hydrolyze sugar residues on members of the “transient receptor potential cation channel subfamily V” (TRPV channels) and stabilize them on cell membranes [34,72]. Through this mechanism in TRPV5 channels, α-klotho increases calcium reabsorption in the distal renal tubules to promote calcium homeostasis [65–67,72]. Recently, α-klotho has also been observed to similarly regulate TRPV2 channels to promote calcium influx and insulin release in pancreatic beta cells [9,10].

Lastly, α-klotho is ubiquitously involved in multiple reactive oxygen species (ROS) buffering systems. α-Klotho interferes with insulin receptor autophosphorylation which relieves antagonism of forkhead box (FOXO) transcription factors and increases transcription of antioxidative enzymes [52]. For example, α-klotho increases FOXO1, 3a, and 4 activity, augments superoxide dismutase and catalase transcription, and attenuates oxidative stress [15,52,73]. α-Klotho also upregulates the thioredoxin/peroxiredoxin (trx/prx) ROS buffering system and overall has been shown to improve resistance to the toxic effects of paraquat, streptozotocin, glutamate, and hydrogen peroxide [11,52,74].

Overall, circulating α-klotho is involved in diverse hormonal functions throughout many tissues and many molecular mechanisms mediating α-klotho’s non-metabolic roles may also be associated with metabolic regulation. For example, insulin signaling, FGF’s, and TRPV channels are all essential to homeostatic regulation of energy balance and substrate metabolism. Thus, these previously established mechanisms of α-klotho provide a strong foundation facilitating discoveries into α-klotho-mediated regulation of metabolism, a relatively new focus of research.

Peripheral α-Klotho and Metabolism

Peripheral α-klotho inhibits insulin signaling, but does not result in clinical insulin resistance-

One of the first-identified metabolic functions of α-klotho was its ability, via unknown mechanisms, to interfere with autophosphorylation of the insulin/IGF1 receptor in myoblasts, adipocytes, and hepatocytes [2,30,40,41,75]. Consequently, α-klotho-knockout mice exhibit improved glucose clearance and GLUT4 expression, but also drastically stunted growth and premature death [1,12,40,75]. On the contrary, α-klotho overexpressing mice experience slight insulin resistance and prolonged lifespans with no differences in food intake, energy expenditure, or body weight [2]. Since α-klotho-overexpressing mice do not experience hyperglycemia, adiposity, or hyperphagia associated with clinical insulin resistance [2,75], α-klotho is likely an important homeostatic modulator of insulin/IGF1 signaling to prevent hypoglycemia, regulate apoptosis, and enhance ROS buffering [5,6].

Peripheral α-klotho facilitates insulin release and glucose uptake-

Despite α-klotho’s role as a negative regulator of insulin signaling, α-klotho function is critical to homeostatic glucose clearance. Impaired α-klotho function exacerbates insulin resistance, hyperglycemia, β-cell apoptosis, and retinopathies in various models of Type 1 and 2 Diabetes and obesity [11,14,75–77], and many studies observe plasma α-klotho concentrations to be suppressed in humans with these disorders [55,57,78,79]. This documented inverse relationship between plasma α-klotho concentrations and incidence of Type 2 Diabetes and obesity suggests reduced α-klotho bioavailability may be directly involved in the development of these disorders, although some studies refute these findings and observe no correlation [80,81]. These mixed findings may be due to differing degrees of Type 2 Diabetes in the populations studied or, similar to leptin and insulin, α-klotho insensitivity may develop in some diabetic states. Nevertheless, recent studies have demonstrated encouraging therapeutic potential of α-klotho for Type 1 and 2 Diabetes and obesity [8–11,14,73,75].

Studies using the MIN6 β-cell line and α-klotho overexpression specifically in the pancreas have identified a prominent role of pancreatic α-klotho to increase insulin secretion, improve glucose tolerance, and reduce fasting glucose levels in mouse models of Type 1 and 2 diabetes [9–11]. Specifically the secreted/short form (~65kD, KL1 domain only) α-klotho hydrolyzes TRPV2 channels in pancreatic β-cells to increase calcium entry and facilitate insulin release [2,9,10,40]. Moreover, intraperitoneal (IP) injection with full length recombinant α-klotho also attenuates hyperglycemia, independent from insulin secretion [14,73,75], likely due to α-klotho’s additional pancreatic role to preserve β-cell and islet health [9,11,14]. More specifically, α-klotho in the pancreas enhances β-cell proliferation, autophagy, and expression of insulin transcription factors, while attenuating oxidative stress, endoplasmic reticulum stress, and apoptosis [9,11]. α-Klotho inhibits caspase-3-mediated apoptosis in β-cells via integrin β1→focal adhesion kinase→AKT signaling [11], while antioxidative action of α-klotho via inhibition of phosphorylated rac-1 and subsequent suppression of NADPH oxidase activity may also be involved [9]. Interestingly, there are no apparent effects of pancreatic α-klotho overexpression on insulin sensitivity in diabetic mice, and there is no glucoregulatory phenotype in lean mice [9,11].

A recent model using genetic overexpression or downregulation of soluble α-klotho in DIO mice reveals additional glucoregulatory functions of α-klotho in the liver [75]. Both in vivo and in vitro experiments demonstrate α-klotho increases hepatic glucokinase mRNA and decreases phosphoenolpyruvate carboxykinase mRNA, resulting in improved hepatic glucose uptake and glycogen storage, as well as reduced gluconeogenesis [12,75]. Consequently, while DIO mice experience reduced plasma α-klotho concentrations, further transgenic suppression of α-klotho exacerbates hyperglycemia, impaired insulin secretion, and insulin resistance [75]. Strikingly, rescuing α-klotho concentrations has opposite effects, although it is not clear if improved insulin sensitivity is indirectly a result of weight loss [75]. Mechanistically, α-klotho increases PPARα transcriptional activity by directly antagonizing insulin-like growth factor receptor 1 (IGFR1) and subsequently inhibiting downstream PI3kinase→AKT→mTORC1 signaling. Pharmacological inhibitor experiments have revealed this signaling pathway is critical to α-klotho-mediated glucose-lowering and modulation of hepatic gene expression [75].

Peripheral α-klotho increases energy expenditure and increases lipid oxidation-

Increasing circulating α-klotho concentrations via IP injection or genetic manipulation attenuates weight gain and improves body composition in mouse models of obesity-induced insulin resistance, revealing a novel role of α-klotho in energy balance [8,73,75]. Food intake is unchanged in these models, and α-klotho-knockout mice experience impaired thermogenesis, suggesting α-klotho is specifically involved in regulating energy expenditure [8,12,73,75]. Supporting this hypothesis, 4-weeks IP injection with α-klotho increases oxygen consumption in DIO mice; however, no changes in thermogenic gene expression are observed in brown adipose tissue (BAT) or epididymal white adipose tissue (eWAT) [8]. Consequently, the mechanisms underlying α-klotho-mediated increases in energy expenditure require further research, including investigation into browning of other prominent WAT depots, such as inguinal WAT (iwat), and deciphering the molecular mechanisms involved.

While reduced lipid accumulation and improved body composition in DIO mice with experimentally increased soluble α-klotho levels is, in part, due to increased energy expenditure, α-klotho also has a direct role in lipid metabolism in both liver and adipose tissue. α-Klotho modulates liver and WAT gene expression in DIO mice to favor lipid oxidation and decrease lipogenesis via a similar IGFR1→PI3kinase→AKT→mTORC1→PPARα mechanism described above [8,75]. Paradoxically, one report using 3T3-L1 adipocytes observed α-klotho promotes adipocyte differentiation by inducing C/EBPα- and PPARγ gene expression [82]. One possible explanation for these findings is that DIO mice are in a status of chronic energy surplus compared to healthy 3T3-L1 adipocytes; thus, α-klotho’s predominant role in lipid metabolism may be dynamic and fluctuate with energy status. Overall, α-klotho’s role in lipid metabolism is likely complex and requires further investigation.

Future directions: The α-klotho→FGF23→FGFR complex-

The therapeutic potential of α-klotho in the treatment or prevention of Type 1 and 2 Diabetes and obesity is encouraging; however, the molecular mechanisms involved, ranging from target tissues, to cell surface receptor and intracellular signaling, remain poorly understood. The involvement of FGFR’s in peripheral α-klotho’s metabolic functions may be a promising direction for future research. For example, emerging evidence suggests potent antidiabetic and anti-obesogenic effects of FGFR activation in diverse metabolically active tissues, albeit via liver-derived FGF21 and intestine-derived FGF19 ligands [83–90]. More specifically, downstream FGF19/FGF21→FGFR signaling in kidney, WAT, BAT, and/or liver stimulates uncoupling protein 1-mediated thermogenesis, reduces gastrointestinal lipid absorption, improves systemic insulin sensitivity, and suppresses gene expression related to lipogenesis. Notably, studies primarily focus on the metabolic functions of FGF19 and FGF21, which are tethered to FGFR’s by the exclusively transmembrane β-klotho protein, and the potential metabolic role of α-klotho tethering FGF23 to FGFR’s remains unexplored [91–93].

Furthermore, the differences in downstream FGFR signaling and subsequent physiological effects in response to different FGF ligands are poorly understood. Consequently, α-klotho may elicit some of its metabolic effects by also activating FGFR’s either via FGF23 or independently. This would shed significant light on the molecular mechanisms underlying α-klotho-mediated metabolic regulation, especially considering many of the effects of increased FGFR activity are similar to the previously described roles of peripheral α-klotho, including elevated energy expenditure, increased lipid oxidation, and improved glucose regulation [85–90,94–98]. Future studies utilizing Crispr-mediated gene editing or inducible CreLoxP to perform tissue- or cell-specific genetic manipulation of different FGFR isoforms known to interact with α-klotho (FGFR’s 1c, 3c, and 4 [62]) would be extremely valuable to deciphering the complex mechanisms underlying homeostatic and disordered α-klotho function in metabolism.

Summary of α-klotho and peripheral metabolism-

In summary, cell culture studies identify α-klotho as an important, ubiquitous, negative regulator of insulin signaling, but emerging evidence reveals α-klotho function is critical to homeostatic glucose clearance. For example, in the pancreas, α-klotho facilitates insulin release via TRPV channels and attenuates β cell apoptosis via integrin β1→focal adhesion kinase→AKT signaling (Figure 1). Furthermore, α-klotho promotes glucose uptake and glycogen synthesis, as well as suppresses gluconeogenesis in liver via IGFR1→PI3kinase→AKT→mTORC1→PPARα signaling (Figure 2). Additional metabolic roles of peripheral α-klotho include increasing lipid oxidation in liver and WAT via similar IGFR1→PPARα pathways and increasing whole-body energy expenditure through unknown mechanisms. Notably, blood α-klotho concentrations are reduced in metabolic syndrome models, and the most marked metabolic effects of α-klotho are observed in these disorders. This phenomenon suggests a possible direct role of impaired α-klotho bioavailability in the etiologies of Type 2 Diabetes and obesity. Overall, many questions surround peripheral α-klotho’s metabolic functions and molecular mechanisms of action; however accumulating evidence implicates this circulating factor as a promising therapeutic target and preclinical marker in Type 2 Diabetes and obesity.

Figure 1. Metabolic roles of α-klotho in the pancreas.

(A) α-Klotho protects pancreatic β cells from oxidative stress by inhibiting Rac1 phosphorylation and NAPDH oxidase activity via an unidentified receptor. α-Klotho also increases transcription of genes related to insulin, autophagy, and β-cell proliferation, while decreasing genes related to apoptosis. (B) α-Klotho facilitates insulin secretion by increasing membrane localization of TRPV2 channels and subsequent calcium influx. (C) α-Klotho activates integrin β and subsequent FAK→AKT signaling to inhibit caspase 3-mediated apoptosis. Ras1 = Ras-related C3 botulinum toxin substrate 1; NADPH = nicotinamide adenine dinucleotide phosphate; pdx-1 = pancreatic and duodenal homeobox 1; LC3 = Microtubule-associated protein 1A/1B-light chain 3; DNAJC3 = DNAJ homolog subfamily member 3 precursor; PCNA = proliferating cell nuclear antigen; TRPV = transient receptor potential cation channel subfamily V; FAK = focal adhesion kinase; AKT = protein kinase B. Created with BioRender.com.

Figure 2. Metabolic roles of α-klotho in the liver and adipose tissue.

(A) α-Klotho is a negative regulator of insulin signaling. (B) α-Klotho inhibits IGFR1 signaling to activate PPARα which increases genes related to lipid oxidation and decreases lipogenic gene expression. In liver only, α-klotho decreases gluconeogenic gene expression and increases glucokinase mRNA. (C) α-Klotho may also regulate liver/adipose metabolism via FGFR’s. INSR = insulin receptor; IRS = insulin receptor substrate; PI3K = phosphoinositide 3-kinase; PIP3 = phosphatidylinositol 3,4,5-trisphosphate; PDK = phosphatidylinositol 3,4,5-trisphosphate-dependent kinase; AKT = protein kinase B; AS160 = AKT substrate 160kD; GLUT4 = glucose transporter 4; IGFR1 = insulin-like growth factor receptor 1; mTORC1 = mammalian target of rapamycin complex 1; PPAR; peroxisome proliferator-activator receptor; ACC = acetyl-CoA carboxylase; SREPB1 = sterol regulatory element binding protein 1; SCD1 = stearoyl-CoA desaturase 1; CD36 = cluster of differentiation 36; ACOX1 = acyl-CoA oxidase 1; CPT1 = carnitine palmitoyltransferase 1; PEPCK = phosphoenolpyruvate carboxykinase; GK = glucokinase; FGFR = fibroblast growth factor receptor. Created with BioRender.com.

Central Nervous System α-Klotho and Metabolism

CSF α-klotho regulates energy balance-

Although high levels of α-klotho expression were observed in the choroid plexus during its discovery, minimal investigation has been made into its function in the brain [1]. To date, identified roles of central nervous system (CNS) α-klotho include regulation of baroreflex in rats [99], myelination in cultured oligodendrocytes [6,100–102], and synaptic remodeling [103] and ROS buffering in hippocampal cells [5]. Recently, a strong inverse correlation was discovered between CSF α-klotho and body weight in adults, providing the first evidence that CSF α-klotho may be involved in energy balance regulation [104]. Supporting this hypothesis, 7 days intracerebroventricular (ICV) administration of α-klotho reduces food intake by 12% in lean mice [105], while an even greater phenotype is observed in mouse models of Type 1 and 2 Diabetes and obesity. For example, 7 days ICV α-klotho treatment in DIO and STZ-treated mice suppresses food intake 15–28%, resulting in significant reductions in body weight [105]. Additionally, acute ICV α-klotho treatment in DIO mice increases energy expenditure, possibly due to increased thermogenic gene expression in BAT and iWAT [104]. While the mechanisms underlying α-klotho-mediated increases in energy expenditure are currently unknown, the appetite-suppressing effects of α-klotho are abolished by FGFR or PI3kinase inhibition. Coupled with in vitro data in hypothalamic and hippocampal cells revealing PI3kinase is downstream of the α-klotho→FGFR complex [103,105], these findings identify a physiologically significant CNS α-klotho→FGFR→PI3kinase signaling mechanism in the regulation of food intake and body weight. Surprisingly, central α-klotho inhibition using an anti-α-klotho antibody in lean mice results in weight loss and has no effects on food intake [105]. Whole body α-klotho knockout mice also experience drastic weight loss, primarily due to atrophy of metabolic organs, resulting in premature death [1,12]. Overall, these findings reveal complex and diverse metabolic functions of central α-klotho and highlight the need for further research into its roles regulating energy balance.

CSF α-klotho improves glucose regulation in mouse models of Type 1 and 2 Diabetes and obesity-

Acute ICV α-klotho treatment before an overnight fast improves glucose tolerance in DIO mice and attenuates hyperglycemia in pair-fed STZ-treated mice [105]. Furthermore, acute central α-klotho inhibition via ICV anti-α-klotho antibody treatment before an overnight fast impairs glucose tolerance in lean mice, suggesting a potent glucoregulatory role of α-klotho in the CSF independent from changes in body weight [105]. These glucoregulatory effects have been shown to be independent from insulin sensitivity; however, central α-klotho does promote insulin release and suppress hepatic gluconeogenic gene expression [105]. Notably, ICV α-klotho has no effects on fasting insulin or glucose levels in DIO mice, suggesting central α-klotho’s effects on insulin secretion may be glucose-dependent [105]. Similar to α-klotho-mediated suppression of food intake, FGFR or PI3kinase inhibition blunts these glucoregulatory effects, implicating the CNS α-klotho→FGFR→PI3kinase signaling axis in the regulation of glucose metabolism [105].

α-Klotho regulates diverse cell populations in the arcuate nucleus of the hypothalamus-

Similar to previous studies in cultured oligodendrocytes and hippocampal cells, α-klotho was recently observed to induce phosphorylation of AKT^ser473, ERK^{thr202/tyr204}, and FOXO1^ser256 via FGFR’s in the immortal hypothalamic GT1–7 cell line, revealing a novel role for α-klotho to regulate hypothalamic function [6,100,101,105]. Further investigation using patch clamp electrophysiology and immunohistochemical detection of cFOS demonstrates a potent ability of CSF α-klotho to regulate activity of neurons in the arcuate nucleus (ARC) of the hypothalamus. For example, α-klotho directly inhibits ARC neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons and decreases AgRP gene expression via FGFR→PI3kinase signaling [104,105]. Through sympathetic and parasympathetic innervation of diverse metabolically active tissues, NPY/AgRP neuron activity promotes food intake, decreases energy expenditure, and stimulates catabolic glucose release into the blood, which, when dysregulated, can resemble metabolic syndrome [106–110]. α-Klotho also exhibits a complex relationship regulating ARC proopiomelanocortin (POMC) neurons, which deliver opposite efferent signals to peripheral tissues compared to NPY/AgRP neurons. During patch clamp electrophysiology experiments, approximately 44% of ARC POMC neurons experience excitatory effects and 56% experience inhibitory effects in response to α-klotho [104]. Furthermore, while no obvious effects in response to acute ICV α-klotho treatment are observed in ARC POMC neurons of fed mice, a net excitatory effect occurs in the fasted status via FGFR→PI3kinase signaling [104]. These varying effects of α-klotho on POMC neurons highlight the heterogeneity of the POMC neuron population, which vary in receptor and neuropeptide expression [111]. Lastly, experiments have also determined no obvious role of α-klotho to regulate the orexigenic ARC tyrosine hydroxylase-expressing neuron population [104]. The direct connection between α-klotho-mediated regulation of ARC neurons and the therapeutic effects of ICV α-klotho in peripheral metabolism has not yet been investigated; however, the ICV α-klotho phenotype greatly resembles phenotypes observed in response to inhibition of NPY/AgRP neurons or stimulation of POMC neurons including: weight loss, suppression of appetite, improved glucose tolerance, improved insulin release, and reduced hepatic gluconeogenic gene expression [106–110]. Thus, ARC NPY/AgRP and POMC neurons, via their sympathetic and parasympathetic connections to metabolically active tissues, are likely important mediators of central α-klotho’s beneficial effects on peripheral metabolism.

α-Klotho also has been shown to have additional roles in the ARC independent from neuronal activity. For example, 12 days ICV α-klotho treatment in DIO mice increases active progenitor cells in the ARC, despite no short-term effects on neurogenesis. It is possible longer duration α-klotho treatment may reveal effects on ARC neurogenesis, or these results may implicate central α-klotho in the generation of new glial cells [104]. Additionally, acute ICV α-klotho treatment increases phosphorylated ERK^{thr202/tyr204} in ARC astrocytes via FGFR’s [104]. ARC astrocytes are critical to nutrient sensing and hormonal transport, in part via an ERK-gated mechanism, and α-klotho→FGFR→ERK signaling has been shown to regulate astrocyte metabolism in hippocampal cells [112–117]. These findings provide evidence for a novel role of central α-klotho to regulate ARC astrocyte function. α-Klotho also increases phosphorylated STAT3^tyr705 specifically in NPY/AgRP neurons [104]. STAT3 is an important downstream mediator of the anorexigenic hormone leptin, and an inhibitory signaling molecule in NPY/AgRP neurons [118,119], further indicating that central α-klotho may be involved in hormonal sensitizing or transport; however, at this time it is unclear if α-klotho-mediated STAT3 signaling is dependent or independent of leptin.

Future directions: α-Klotho and FGFR’s

Similar to peripheral tissues, central FGFR’s have recently been popular subjects of metabolism research in the brain. ICV FGF19 and FGF21 elicit similar therapeutic effects to ICV α-klotho via FGFR’s, including decreased food intake, reduced weight, improved glucose clearance, inhibited NPY/AgRP neuron activity, and suppressed hepatic gluconeogenic gene expression [120–124]. In fact, brain-specific β-klotho knockout even abolishes many of the weight-reducing and glucose-lowering effects of peripheral FGF19/21 administration [90,124,125]. Since β-klotho’s role is to stabilize FGF19/21 interaction with FGFR’s [91–93], this strongly indicates that brain FGFR’s are the primary mediators of peripheral and central FGF19/21 metabolic function. FGFR’s appear to be equally important to many of the metabolic effects of central α-klotho [104,105], identifying FGFR’s as promising targets of both future research and pharmacological intervention in Type 1 and 2 Diabetes and obesity.

Despite the encouraging evidence observing FGFR activation to elicit therapeutic effects in Type 1 and 2 Diabetes and obesity, studies investigating central FGFR involvement in the pathology of diabetes and obesity produce equivocal results depending on animal model and experimental approach. ICV treatment with the popular FGFR inhibitor PD173074 impairs glucose clearance in healthy rats, but is described as stress-related [126,127], and ICV PD173074 in DIO mice elicits no phenotype [120,128]. Furthermore, antibody-mediated inhibition of FGFR1 in rodents and monkeys has therapeutic effects by increasing energy expenditure, decreasing food intake, and reducing body weight, while genetic deletion of FGFR1 in NPY/AgRP neurons results in no obvious metabolic phenotype [129–131]. Future studies should investigate the specific roles of FGFR’s, their isoforms, and their neuronal effectors in central regulation of metabolism by performing selective deletion of FGFR isoforms in specific neurons of mature mice.

Future directions: Alternative mechanisms of CNS α-klotho-

A novel brain α-klotho/FGFR/PI3kinase signaling axis has been identified in the regulation of ARC neurons and whole-body metabolism; however, there are likely other molecular mechanisms involved in the vital functions of central α-klotho. For example, at this time, the importance of α-klotho-induced phosphorylated ERK in the hypothalamus is not known. ERK signaling negatively regulates NPY/AgRP neurons, possibly via kruppel-like factor 4, and is involved in hypothalamic FGF1- and FGF19-mediated glucose lowering [116,120,132]. α-Klotho’s ability to hydrolyze and increase activity of TRPV channels may also be important to CNS regulation of metabolism. TRPV channels are important to stimulating POMC neuron depolarization and subsequent thermogenesis and satiety [133,134]. Lastly, α-klotho may have direct effects on metabolism via trx interacting protein (TXNIP) [5]. TXNIP, which has been shown to be inhibited by α-klotho and trx, has been identified as a mediator of overactive NPY/AgRP neuron pathologies, including hyperphagia, adiposity, reduced energy expenditure, and leptin resistance [18,135,136]. Overall, these documented physiological effectors of α-klotho in peripheral tissues provide additional signaling pathways that may be promising foci of future investigations into central α-klotho’s metabolic functions.

Summary of CNS α-klotho and metabolism-

CSF α-klotho exhibits promising therapeutic potential in Type 1 and 2 Diabetes and obesity by decreasing food intake, increasing energy expenditure, improving insulin secretion, and reducing hepatic gluconeogenic gene expression. Furthermore, reduced CSF α-klotho concentrations are observed in overweight populations, suggesting impaired CSF α-klotho bioavailability may be involved in the pathophysiology of metabolic disease and potentially identifying CSF α-klotho as a preclinical marker of these disorders. Mechanistically, α-klotho→FGFR→PI3kinase signaling modulates ARC NPY/AgRP and POMC neuron activity, which is likely essential to central α-klotho’s effects on peripheral metabolism (Figure 3). Central α-klotho also regulates ARC astrocyte function via α-klotho→FGFR→ERK signaling, strongly implicating the α-klotho→FGFR signaling axis in homeostatic CNS regulation of metabolism.

Figure 3. Metabolic roles of α-klotho in the brain.

(A) α-Klotho may upregulate TRX protein activity by inhibiting TXNIP. (B) α-Klotho regulates hypothalamic neuron activity and gene expression via FGFR→AKT signaling. (C) α-Klotho may regulate calcium flux and subsequent membrane potential via TRPV channels. (D) α-Klotho activates STAT3 signaling via un unidentified receptor. TXNIP = thioredoxin interacting protein; TRX = thioredoxin; FGFR = fibroblast growth factor receptor; AKT = protein kinase B; ERK = extracellular-regulated kinase; FOXO1 = forkhead box 01; TRPV = transient receptor potential cation channel subfamily V; STAT3 = signal transducer and activator of transcription 3. Created with BioRender.com.

In summary, investigation into central α-klotho-mediated regulation of energy balance and glucose metabolism is a very new topic of research. Only α-klotho’s function in the ARC has been investigated and additional roles in other regions of the brain are likely. Moreover, while the evidence implicating α-klotho as a therapeutic target and preclinical marker of Type 2 Diabetes and obesity is encouraging, many additional experiments are needed to determine dose-response and effects of long term α-klotho administration. Doses used in ICV treatment studies are supraphysiological and, similar to leptin and insulin, developed resistances to α-klotho should be considered. Development of new tools to specifically manipulate CSF α-klotho levels within physiological ranges would also be extremely valuable to thoroughly elucidating this protein’s diverse and complex metabolic functions.

Closing Remarks-

Accumulating evidence demonstrates α-klotho is critical to homeostatic regulation of lipid metabolism, glucose metabolism, and energy balance (Figure 4). Furthermore, impaired function of this circulating factor is associated with Type 1 and 2 Diabetes, dyslipidemia, and obesity. α-Klotho may be a promising new therapeutic target in these diseases, as well as a preclinical marker; however, studies investigating its therapeutic potential in humans is lacking. Thorough dose-response and time course experiments in animal models are needed to determine the potential for adverse side-effects or developing resistances to α-klotho function. Moreover, many of α-klotho’s tissue-specific functions and intracellular mechanisms remain unexplored. To date, α-klotho’s potential metabolic functions in skeletal muscle, where the majority of glucose uptake occurs, are unclear, and the only investigations into α-klotho’s metabolic roles in the CNS focus on the ARC of the hypothalamus. Future investigations using tissue-specific gain- and loss-of-function approaches would be very impactful in further deciphering this complex protein’s diverse metabolic actions.

Figure 4.

Summary of α-klotho-mediated regulation of metabolism.

HIGHLIGHTS:

• Reduced α-klotho concentrations are associated with diabetes and obesity.

• α-Klotho facilitates insulin secretion and protects pancreatic β cells.

• α-Klotho facilitates glucose update and glycogen synthesis in liver.

• α-Klotho promotes lipid oxidation in adipose and liver.

• Central α-klotho modulates hypothalamic neurons and peripheral metabolism.

ABBREVIATIONS:

BBB

blood-brain barrier

CSF

cerebrospinal fluid

TNFα

tumor necrosis factor α

APP

amyloid precursor protein

ADAM

a disintegrin and metalloprotease

BACE-1

β amyloid precursor protein converting enzyme 1

FGF

fibroblast growth factor

FGFR

fibroblast growth factor receptor

TRPV

Transient receptor potential cation channel subfamily V

IGFR

insulin-like growth factor receptor

DIO

diet-induced obesity

IP

intraperitoneal

BAT

brown adipose tissue

WAT

white adipose tissue

ROS

reactive oxygen species

ICV

intracerebroventricular

NPY/AgRP

neuropeptide Y/agouti-related peptide

POMC

proopiomelanocortin

STZ

streptozotocin

CNS

central nervous system

ARC

arcuate nucleus

ERK

extracellular-regulate kinase

STAT3

signal transducer and activator of transcription 3

TXNIP

thioredoxin interacting protein