Hairless (Hr) Deficiency Mitigates High‐Fat Diet‐Induced Obesity and Insulin Resistance in Mice

Obesity is a significant global health concern linked to excessive dietary energy intake. This research focuses on the mammalian hairless protein (HR), known for its role in skin and hair function, and its impact on metabolism. Examining male wild‐type (Hr+/+) and Hr null (Hr−/−) mice over a 14‐week normal chow diet (NCD) or high‐fat diet (HFD) intervention. This study reveals that HR deficiency exhibited a protective effect against HFD‐induced obesity and insulin resistance. This protective effect is attributed to increased energy expenditure in Hr−/− mice. Moreover, the brown adipose tissue (BAT) of Hr−/− mice displays elevated levels of the thermogenic protein, uncoupling protein 1 (Ucp1), and its key transcriptional regulators (PPARγ and PGC1α), compared to Hr+/+ mice. In summary, the findings underscore the protective role of HR deficiency in countering HFD‐induced adiposity by enhancing insulin sensitivity, raising energy expenditure, and augmenting thermogenic factors in BAT. Further exploration of HR metabolic regulation holds promise for potential therapeutic targets in addressing obesity‐related metabolic disorders.


Introduction
Obesity has emerged as a global non-communicable disease crisis, with projections estimating that 38% of the world's adult population will be overweight and 20% obese by 2030. [1]This metabolic disorder, characterized by disrupted energy homeostasis, significantly contributes to the prevalence of type 2 diabetes, cardiovascular diseases, cancer, inflammation, and other related conditions. [2]The etiology of obesity is complex, influenced by an interplay of genetic and environmental factors, including DOI: 10.1002/adbi.202300635diet. [3]In particular, the long-term consumption of a high-fat diet is a wellstudied environmental contributor to obesity and diabetes, with the C57BL/6J mouse strain serving as a pivotal model for human obesity research due to its diet-sensitive metabolic phenotype. [4]he majority of studies exploring the genetic basis of obesity have traditionally approached the problem through a gene-by-gene analysis, identifying various genes involved in lipid and glucose metabolism that respond to high-fat diets. [5]For instance, alterations in the expression of the acyl-CoA oxidase (Acox) and uncoupling protein-2 genes, along with increased levels of sterol regulatory element-binding protein 1 (SREBP1), highlight the intricate genetic responses to dietary changes. [6]Hairless protein (HR) biological function has been closely associated with skin health and hair follicle development. [7]However, recent discoveries have broadened the scope of HR's relevance, revealing its expression across various tissues, including adipose tissue and the brain. [8]Such findings suggest a pivotal role for HR beyond dermatological significance, implicating it in the complex regulatory networks of metabolic regulation.8c] Despite the preponderance of research focusing on the Hairless role in skin and hair pathology, several studies on HR-deficient mice reveal a notable enhancement in energy metabolism. [9]These observations propose a regulatory capacity of HR in energy balance, marking a significant departure from its traditionally understood functions.Nonetheless, the detailed mechanisms through which HR modulates metabolic pathways, and how these mechanisms influence the pathophysiology of obesity and insulin resistance, remain largely uncharted territories. [10]This gap in knowledge invites a rigorous scientific inquiry to elucidate the specific pathways by which HR contributes to metabolic regulation.Understanding these mechanisms is critical for unveiling novel therapeutic targets for obesity and its associated metabolic disorders, thereby expanding the potential for HR beyond its established roles. [11]his study aims to bridge this gap by investigating the role of HR deficiency in metabolic regulation, with a particular focus on its effects on diet-induced obesity and metabolic complications.By analyzing the metabolic phenotype of HR-deficient mice, we intend to shed light on the underlying mechanisms of its action and evaluate its therapeutic potential in combating obesity.Our research is driven by the urgent need to identify new genetic targets and pathways that could provide novel interventions for the obesity epidemic and its related health challenges.

Experimental Animals
In this study, wild-type (Hr +/+ ) mice and Hr-knockout (Hr −/− ) mice, both possessing a C57BL/6J genetic background, were sourced from the Experimental Animal Center of Zhengzhou University (Henan Province, China).The animals were accommodated and nourished in an environment characterized by alternating 12-h light and 12-h dark cycles, a temperature range of 22-24 °C, relative humidity maintained at 40%-70%, and ad libitum access to food and water.

Schematic Timeline of Experimental Procedures
At Six weeks-of age, male Hr +/+ and Hr −/− mice subjected to diets of either an NCD comprising 11% fat, 61% carbohydrate, and 28% protein (SYSE BIO, Changzhou, China) or an HFD containing 42% fat, 39% carbohydrate, 19% protein (SYSE BIO, Changzhou, China).The fat in the HFD was derived from various sources: lard, olive oil, coconut fat, and fish oil.This dietary regimen was maintained for up to 14 weeks.(refer to Figure 1A).Weekly assessments of body weight were conducted, and at week 8, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed to assess metabolic function.Metabolic rates, food and water intake, and physical activity were evaluated using the TSE PhenoMaster V6.2.0 metabolism monitoring platform (TSE Systems, Germany) at week 10.After undergoing 14 weeks of an HFD, a subset of the mice was subjected to an acute insulin stimulation assay.This involved administering an insulin injection directly into the inferior vena cava at a dose of 0.75 units per kilogram of body weight.Five minutes after the insulin injection, brown adipose tissue (BAT) was collected.Western blot analysis was performed to assess insulin-stimulated phospho-Akt (Ser308 and Ser473), and total Akt and GAPDH were used to normalize.Subsequently, the mice were euthanized, allowing for the retrieval of epididymal white adipose tissue (eWAT).The eWAT was used as an indirect marker of visceral fat.To account for variations in body size, the weight of the eWAT was normalized to the total body weight of each mouse.

Glucose Tolerance Test and Insulin Tolerance Test
GTT and ITT were integral components of the investigation, meticulously executed in accordance with previously established methodologies. [12]Conducted during the 10th week of the experimental framework, these assessments were scheduled on distinct days to mitigate potential confounding stress effects on the subjects.For the GTT, mice were fasted for 6 h (8:00 AM to 2:00 PM), and blood glucose concentration was measured using a glucometer (ACCU-CHEK @ glucose monitor, Roche, Shanghai) by tail snip.Glucose (1 g per kg body weight) was injected intraperitoneally (i.p.) after baseline glucose levels were established in each mouse.Blood glucose levels were measured before glucose administration (0 min) and subsequently at 15-, 30-, 60-, 90-, and 120-min post-injection.For the ITT, mice were fasted for 6 h (8:00 AM to 2:00 PM), and blood glucose levels were measured from the tail vein with a blood glucometer.Human insulin (0.75 U per kg body weight, Humulin R; Novo Nordisk) was injected I.P. after baseline glucose levels were established in each mouse, and blood glucose values were measured at 15-, 30-, 60-, and 90-min post-injection.The area under the curve (AUC) was calculated by the trapezoidal method.

Metabolic Rate and Physical Activity
Oxygen consumption and physical activity were assessed in the 5th week of the study, a pivotal period identified for its relevance in the onset of significant weight gain and metabolic shifts indicative of obesity.This timing aligns with findings from prior research, suggesting that the 5th week of dietary interventions was crucial for detecting the initial manifestations of obesity and its associated metabolic disturbances. [13]Utilizing the TSE PhenoMaster V6.2.0 platform in accordance with previously established methodologies, [14] The activity levels and systemic metabolic rates of subjects across different groups were meticulously recorded.Prior to recording, animals were individually acclimated to the metabolic cage of the TSE system for 24 h.Gas samples were collected and analyzed every 5 min per animal, with half-hourly averages calculated.Output data from the software includes O 2 consumption (VO 2 ) (mL kg −1 min −1 ), CO 2 production (VCO 2 ) (mL kg −1 min −1 ), respiratory quotient (RQ = VCO 2 /VO 2 ), and heat production [heat = CV×VO 2 ; CV = 3.815+(1.232×RQ)].Refer to TSE system∖Equations for energy expenditure for details.(www.tse-systems.com/service/phenotype/).Additionally, the system provided automated recordings of activity parameters, including walking movement and fine movement, alongside the intake of food and water.This comprehensive approach enabled a robust analysis of the physiological and behavioral effects of dietary interventions, contributing valuable insights into the metabolic impact of obesity onset.

Measurement of Rectal Temperature and Serum-Free Triiodothyronine (fT3)
The measurement of rectal temperature and serum-free triiodothyronine (fT3) levels in mice was conducted following methodologies established in previous research. [15]Rectal temperatures of mice were measured using rectal thermometers (Beijing Ji Nuo Tai Technology Development, Beijing, China).The mouse was hand-restrained and placed on a horizontal surface; the tail was then lifted, and the rectal probe (covered with Vaseline) was gently inserted to a fixed depth of up to 2 cm.
After measuring the rectal temperature, mice were anesthetized with 1.5% to 2% sevoflurane.Subsequently, blood samples were collected via cardiac puncture.These samples were allowed to incubate at room temperature for 2 h before being centrifuged at 3000 g for 10 min at a temperature of 4 °C.The supernatant was then collected and stored it at −20 °C for further analysis.
It was well-documented in the literature that there were direct and specific interactions between the Hr gene and the thyroid hormone receptor.These interactions were crucial for the free triiodothyronine (fT3)-mediated enhancement of energy expenditure across various tissues, including the liver and skeletal muscle. [16]In alignment with these findings, the serum concentrations of fT3 in this study was also measured.Specifically, the concentration of fT3 in the serum was determined using a double-adsorption enzyme-linked immunosorbent assay (ELISA) technique.The assay kit used was obtained from Xpress-Bio Company (ELISA, reagent number: XPEM1036, Xpress-Bio Company, Frederick, MD).

Histomorphology of BAT
Histomorphology of BAT was performed as reported previously. [17]Briefly, BAT from the scapula of each mouse was collected, fixed in 4% paraformaldehyde for 24 h, then washed for 10-min in water, dehydrated in a gradient of ethanol solutions of different concentrations (i.e., 50%, 75%, 85%, 95%, and 100% ethanol for 1 h in each concentration, and finally 100% ethanol overnight), and cleared two times in xylene, first for 20 min and then for 2 h.All BAT samples were soaked two times in paraffin solutions (20 min each time) before they were embedded in paraffin and sectioned.BAT sections were subjected to hydration processes (in xylene solutions twice, 10 min each; 100% ethanol twice, 5 min each; 95%, 85%, and 75% ethanol solutions, 5 min each; and ultimately dH 2 O) before hematoxylin-eosin (HE) staining (in hematoxylin staining solution for 5-8 min, washed in water, differentiated with 1% hydrochloric acid alcohol for a few seconds, washed in water, blued in 0.6% ammonia water, washed in water, and stained in eosin solution for 1-3 min).The stained sections were subjected to conventional dehydration (i.e., in 95% ethanol solutions twice and 100% ethanol twice, 5 min each), clearing in xylene twice (5 min each), and mounting with neutral gum before observation, image acquisition, and analysis under a light microscope (RX50 biological microscope, Ningbo Sunny Instruments, Zhejiang Province, China).

Western Blotting
It was adopted the previously described procedure for protein extraction and Western blotting. [18]Stored BAT was cut into small pieces and homogenized on ice using a tissue grinder before immersion in pre-cooled RIPA lysis buffer (Thermal Fisher Scientific, MA) containing protease inhibitor (Roche, Germany), and the lysate was kept on ice for 10 min before centrifugation at 3000 g for 10 min at 4 °C.The supernatant was collected, and then protein concentration was measured using the bicinchoninic acid assay (Thermo Fisher Scientific).Equal amounts of total protein were heated at 99 °C for 5 min and loaded onto a 4%-20% stacking/7.5% separating SDS-polyacrylamide gel (GentScript).The proteins were then electrophoretically transferred onto a polyvinylidene difluoride membrane (Merck Millipore).The membranes were first blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20t for 1 h at room temperature and then incubated at 4 °C overnight with the following primary antibodies: anti-pAKT-Thr308 antibody (1:1000; Abcam), anti-pAKT-Ser473 antibody (1:1000; Abcam), anti-AKT antibody (1:1000; Cell Signaling Technology), anti-PPAR antibodies (1:2000; Abcam), anti-PGC1 antibodies (1:1000; Invitrogen), anti-UCP1 antibodies(1:2000; BioVision, CA), and anti-GAPDH antibodies (1:3000; Abcam).The proteins were detected by horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:3000; Jackson ImmunoResearch) and visualized by western peroxide reagent and luminol/enhancer reagent (Clarity Western ECL Substrate, Bio-Rad) and exposure using ChemiDoc XRS and System with Image Lab software (Bio-Rad).The intensity of blots was quantified with densitometry using Image Lab software (Bio-Rad).The grayscale of target protein bands was analyzed with Image-Pro Plus software.The GAPDH protein served as an internal reference.

Statistical Analysis
Animals were randomly assigned to various treatment groups.Data were presented as mean ± standard error of the mean (SEM).Molecular biology experiments were repeated three or four times.For the analysis of body weight changes over time, a repeated measures two-way analysis of variance (ANOVA) was employed, with 'Hr' and 'diet' as the independent variables.Activity levels, serum fT3 concentration, rectal temperature, and 24 h food intake were analyzed using two-way ANOVA, with 'Hr' and 'diet' as the independent variables.AKT, pAKT-Thr308, and pAKT-Ser473 protein expression levels were assessed using a three-way ANOVA, incorporating 'Hr', 'diet', and 'insulin' as the independent variables.
All statistical analyses and graph preparations were conducted using the GraphPad Prism 8.0 software package (GraphPad Software Inc., San Diego, CA).A p-value of less than 0.05 was considered statistically significant for all analyses.Significant differences between experimental groups were indicated as * p <0.05, ** p <0.01.2

Hr Deficiency Provides a Protective Effect Against Adiposity Induced by HFD
To elucidate the role of HR in energy metabolism, encompassing fat metabolism and adiposity, male mice with Hr +/+ and Hr −/− genotypes were subjected to either NCD or HFD for a 14-week period, commencing at 6 weeks of age.Under NCD conditions, no significant disparities were observed between Hr +/+ and Hr −/− mice in terms of body weight, total eWAT weight, and the eWATto-body weight ratio (Figure B-D).The selection of eWAT, representing abdominal fat, was predicated on its recognized role as a major contributor to obesity and a predictor of metabolic dysfunction.
Commencing at 6 weeks post-initiation of HFD feeding, Hr −/− mice exhibited significantly lower body weight compared to Hr +/+ mice (Figure 1B).Following a 14-week exposure to the HFD, both the total eWAT weight and the eWATto-body weight ratio were reduced in Hr −/− mice compared to Hr +/+ (Figure 1C,D).These findings underscore the impact of HR deficiency in mitigating the effects of HFD-induced adiposity.

Hr Deficiency Ameliorates HFD-Induced Insulin Resistance, Hyperglycemia, and Hyperinsulinemia
Obesity is known to trigger insulin resistance, metabolic syndrome, and type 2 diabetes. [20]To investigate whether the reduced adiposity observed in Hr −/− mice coincides with enhanced metabolic function, including improved insulin sensitivity and decreased hyperglycemia, we conducted GTT and ITT at week 10.The results of GTT and ITT demonstrated that Hr −/− mice exhibited significantly improved glucose tolerance and insulin sensitivity compared to Hr +/+ mice when subjected to HFD (Figure 2A,B).Additionally, although fasting glucose levels remained unchanged, both fed blood glucose and fasting serum insulin were notably lower in Hr −/− mice than in Hr +/+ mice fed the HFD (Figure 2C).
Given that insulin signaling plays a crucial role in determining insulin sensitivity and compromised insulin signaling is common in obese individuals, we conducted an acute insulin response assay at week 10 to assess insulin signaling in BAT. [21]The results revealed a substantial improvement in insulin signaling in Hr −/− mice, as indicated by increased insulin-stimulated phosphorylation of Akt at both Ser473 and Thr308 (Figure 2D-G).These findings further support the systemic enhancement of insulin sensitivity observed in GTT and ITT analyses.

Hr Deficiency Increases Energy Expenditure
Energy consumption serves as a critical marker for energy homeostasis within the body. [22]To elucidate the physiological mechanisms contributing to the reduced HFD-induced obesity in HRdeficient mice, we next investigated the effect of HR deficiency on whole-body energy expenditure, dietary intake, and physical activity.These parameters were assessed in the 5th week following the initiation of the experiment.We observed that energy expenditure for both Hr +/+ and Hr −/− mice was comparable under NCD conditions (Figure 3A,C).However, when subjected to the HFD, Hr −/− mice exhibited higher energy expenditure than their Hr +/+ counterparts on the same diet (Figure 3B,D).Specifically, Hr −/− mice fed the HFD demonstrated elevated energy expenditure per half hour, normalized for weight, compared to both Hr +/+ mice on the same diet and Hr −/− on the NCD (Figure 3C,D).Remarkably, Food intake and activity levels in Hr +/+ and Hr −/− mice were strikingly similar under both dietary conditions (Figure 3E,F).These data suggest that HR deficiency may enhance metabolic rate under HFD conditions, thereby conferring protection against diet-induced adiposity.

Hr Deficiency Results in Elevated Levels of Thermogenic Factors in Brown Adipose Tissue
8b,23] To Figure 3. Impact of Hr deficiency on energy expenditure under HFD.Hr +/+ and Hr −/− mice were exposed to either NCD or HFD for a duration of 10 weeks.A,B) Energy expenditure throughout a 24-h was calculated from the respiratory parameters VO 2 and VCO 2 , utilizing the methodology described in the methods section, employing the TSE PhenoMaster metabolism monitoring platform.C,D) Comprehensive analysis of energy expenditure during daytime, nighttime, and the entire 24-h period.n = 10 mice per group.* P <0.05, ** P <0.01.Repeated measure Two-way ANOVA followed by post hoc Tukey test.E) Caloric intake in mice, determined as daily food intake per mouse multiplied by 5.6 Kcal g −1 .F) Overall, locomotor activity was monitored over a single day.Two-way ANOVA followed by post hoc Tukey test.Error bars, ±SEM.
investigate this, we meticulously compared serum concentrations of fT3 between Hr +/+ mice and Hr −/− mice under both NCD and HFD conditions.Strikingly, no significant differences in serum fT3 concentrations were evident between Hr +/+ and Hr −/− mice, regardless of the NCD or HFD fed (Figure 4A).Furthermore, rectal temperatures exhibited no significant variations between the two groups (Figure 4B).
BAT is pivotal for energy expenditure through heat production.Examination of H&E stained sections from BAT in Hr −/− mice revealed smaller adipocytes compared to Hr +/+ mice after 14 weeks of HFD feeding (Figure 4C).Notably, both mRNA and protein expressions of mitochondrial uncoupling proteins Ucp1 were substantially higher in the BAT of Hr −/− mice compared to Hr −/− mice after 14 weeks of HFD feeding (Figure 4D,G).Crucially, key upstream transcriptional regulators of Ucp1, including PPAR and PGC1, were also elevated in the BAT of Hr −/− mice (Figure 4E-G).Importantly, the levels of Ucp1, PPAR, and PGC1 were not different between Hr +/+ and Hr −/− when mice were on the NCD.HFD feeding specifically increased their levels only in the BAT of Hr −/− mice, with no impact on their levels in Hr +/+ mice (Figure 4D-G).

Discussion
Obesity and its associated comorbidities, for example, diabetes mellitus and hepatic steatosis, contribute to ≈2.5 million deaths annually and are among the most prevalent and challenging con- ditions confronting the medical profession. [24]7b,8c] Despite these findings, the specific involvement of HR in HFD-induced metabolic dysfunction remains unclear.The present study delineates a novel facet of HR function in energy metabolism and glucose homeostasis.In the context of the HFD condition, the lean phe-notype observed in HR-deficient mice primarily results from elevated energy expenditure.This heightened energy expenditure is linked to increased metabolic activity in BAT, independent of variations in food intake and physical activity levels.Furthermore, HR deficiency corresponds to diminished fed glucose levels, lowered fasted insulin levels, and improved insulin responsiveness compared to wild-type mice, collectively these results suggest that HR deficiency protects against HFD-induced obesity and insulin resistance.
The association between obesity induced by HFD and the intricate regulation of energy homeostasis in BAT represents a complex and multifaceted process. [25]BAT plays a pivotal role in energy dissipation and thermogenesis through the activation of UCP1, which converts adenosine triphosphate (ATP) into heat energy.This mechanism is vital for sustaining overall energy metabolism and body temperature. [26]Notably, HR-deficient mice, when subjected to HFD, exhibit heightened energy expenditure without significant alterations in activity or food intake when compared to wild-type mice under equivalent dietary conditions.The augmented energy consumption observed in HR-deficient mice during HFD may be attributed to physiological disparities within BAT.UCP1, a key player in adaptive thermogenesis in BAT, lowers the proton gradient by uncoupling the respiratory chain, thereby promoting thermogenesis capacity through the synthesis of cyclic adenosine monophosphate (cAMP) in mitochondria.Activation of UCP1, therefore, serves as a crucial protective mechanism against obesity and related metabolic diseases. [27]In addition to UCP1, the transcription factor PPAR emerges as a central regulator of mitochondrial fatty acid -oxidation, acting as a sensor for fat metabolism. [28]n response to HFD, PPAR expression increases as an adaptive measure to counteract fat accumulation. [29]The transcriptional coactivator PGC-1, which can bind to targets such as PPAR, coordinates the expression of mitochondrial genes, indirectly influencing fatty acid transport and utilization. [30]Furthermore, PGC-1 upregulates the expression of genes involved in the tricarboxylic acid cycle and mitochondrial fatty acid oxidation pathway. [31]PGC-1 also exerts regulatory control over genes encoding components of the electron transport system and oxidative phosphorylation, establishing its crucial role in BAT thermogenesis. [21]n this study, we observe that HR deficiency up-regulates both mRNA and protein expression of PPAR and PGC-1.Further investigation is warranted to elucidate the molecular mechanisms through which HR modulates the expression of these transcription factors.8c] These pivotal discoveries open up intriguing avenues for further exploration into how HR modulates metabolic pathways and its potential influence in other metabolic tissues.Specifically, targeting the HR protein and its downstream signaling pathways presents a promising area for developing novel therapeutic strategies.Additionally, the possible adverse effects associated with targeting the HR protein represent another critical area for future inquiry.
However, our study has several limitations that warrant acknowledgment.First, despite the well-established role of skeletal muscle in energy expenditure through the regulation of fatty acid -oxidation, [32] we did not investigate the impact of HR deficiency on energy expenditure in skeletal muscle.Second, previous research has demonstrated direct and specific interactions between HR and the thyroid hormone receptor, essential for the fT3-mediated increase in energy expenditure across various tissues and organs, including the liver and skeletal muscle. [33]hile we measured fT3 concentrations in our study, we did not investigate the impact of HR deficiency on thyroid hormone receptor expression and function in tissues and organs associated with energy metabolism.Third, the intricate relationship between weight gain and energy expenditure underscores the importance of nutrient absorption by the gut. [34]Given that HR is widely expressed in the intestines, it is imperative to delve further into whether HR deficiency might impact nutrient absorption.Our study did not explore this aspect, highlighting the need for additional investigations to elucidate the potential influence of HR deficiency on nutrient absorption in the gut.
This study identifies the deficiency of the Hr gene as a protective factor against the detrimental metabolic effects associated with HFD feeding in mice.The observed resistance to weight gain, abdominal obesity, and the fostering of a lean body phenotype are linked to increased BAT activity.This heightened BAT activity not only promotes leanness but also appears to safeguard against the escalation of inflammation and insulin resistance.Furthermore, the reduced fat absorption in the liver afforded by HR deficiency guards against HFD-induced hepatic steatosis.
The implications of these findings suggest that modulating Hr gene activity could serve as an effective strategy to enhance metabolic functions, potentially offering a novel approach to prevent obesity and its associated complications, independent of dietary modifications.However, this promising therapeutic avenue does come with considerations of several potential side effects that merit close scrutiny.These include effects on hair follicle development, which could lead to hair growth abnormalities or alopecia [35] ; impacts on adipocyte differentiation, which must be balanced against healthy adipose tissue function [8c] ; and the implications of modulation on cancer risk, given its role in cell proliferation and tumorigenesis. [36]n summary, targeting the Hr gene and its downstream signaling pathways holds promise as a novel pharmaceutical intervention against obesity.Nonetheless, a thorough understanding and careful examination of its potential side effects are essential to fully harness the benefits while minimizing any risks.