Role of Ubiquilins for Brown Adipocyte Proteostasis and Thermogenesis

The acclimatization of brown adipose tissue (BAT) to sustained cold exposure requires an adaptive increase in proteasomal protein quality control. Ubiquilins represent a recently identified family of shuttle proteins with versatile functions in protein degradation, such as facilitating substrate targeting and proteasomal degradation. However, whether ubiquilins participate in brown adipocyte function has not been investigated so far. Here, we determine the role of ubiquilins for proteostasis and non-shivering thermogenesis in brown adipocytes. We found that Ubqln1, 2 and 4 are highly expressed in BAT and their expression was induced by cold and proteasomal inhibition. Surprisingly, silencing of ubiquilin gene expression (one or multiple in combinations) did not lead to aggravated ER stress or inflammation. Moreover, ubiquitin level and proteasomal activity under basal conditions were not impacted by loss of ubiquilins. Also, non-shivering thermogenesis measured by norepinephrine-induced respiration remained intact after loss of ubiquilins. In conclusion, ubiquilin proteins are highly abundant in BAT and regulated by cold, but they are dispensable for brown adipocyte proteostasis and thermogenesis.


INTRODUCTION
Brown adipose tissue (BAT) is a unique organ that transforms chemical energy from nutrients into heat in response to cold, a process called non-shivering thermogenesis (NST) (1). While acute cold exposure activates the existing thermogenic potential, sustained cold induces the recruitment of thermogenic capacity. This process of cold adaptation requires tremendous remodeling of the cells and tissue, which involves both differentiation of new thermogenic adipocytes and increasing metabolic activity of the existing ones (1). Paradoxically, despite the catabolic nature of NST, longterm adaptation to cold is an anabolic process that involves enhanced de novo synthesis of lipids, proteins and organelles (2). Brown adipocytes engage protein quality control mechanisms to maintain cellular homeostasis under these stressful metabolic conditions (3) and we have demonstrated that NST requires an adaptive increase in proteasomal activity (4). The increase in transcription of proteasomal subunits was regulated by the ERlocalized transcription factor nuclear factor erythroid 2-like 1 (Nfe2l1, also known as Nrf1 or TCF11). Interestingly, even in light of increased proteasomal protein degradation, cold adaptation resulted in enhanced protein ubiquitination (4), suggesting that the overall set point of the ubiquitinproteasome system (UPS) is shifted in BAT after cold acclimatization. This probably also involves the machinery and regulators of ubiquitination itself.
The family of ubiquilin (Ubqln) proteins has recently emerged as versatile components of protein quality control. Ubiquilins are shuttle proteins that assist protein folding as chaperones, as well as facilitate degradation of ubiquitinated substrates through the UPS, endoplasmic reticulum-associated protein degradation (ERAD), and autophagy (5)(6)(7). To this date, five mammalian ubiquilins have been identified, 1, 2, 3, 4 and L, of which Ubqln3 and UbqlnL are exclusively found in the testis (8,9). Ubqln1, 2 and 4 are known to share a similar domain structure, which comprises a carboxy-terminal Ubiquitin-associated domain (UBA), heat-shock-chaperonin-binding motifs and an amino-terminal Ubiquitin-like domain (UBL). The UBA-UBL construct enables the interaction of ubiquilins with ubiquitinated proteins and the proteasome, which allows them to act as multifaceted adaptor molecules (10).
Based on the dynamic nature of UPS regulation in BAT during cold adaptation, it is likely that there are additional mechanisms supporting the function of Nfe2l1. In that vein, the role of ubiquilins in BAT has not been investigated so far. We hypothesized that Ubiquilins, with their function of targeting ubiquitinated proteins and facilitating their degradation by the UPS, could play a significant role in maintaining proteostasis in brown adipocytes. Here we investigate the role of ubiquilins for proteostasis and NST in immortalized and primary brown adipocytes.

Mice
All animal experiments were performed according to procedures approved by the animal welfare committees of the government of Upper Bavaria, Germany (ROB-55.2-2532.Vet_02-20-32) and performed in compliance with German Animal Welfare Laws. Animals were housed in individually ventilated cages at room temperature (22°C) with a 12-h light-dark cycle. All mouse housing and husbandry occurred on standard chow diet (Ssniff). We used 12 weeks old C57BL/6J (Janvier) wild-type mice, which we housed at 30°C for 7 days for thermoneutral acclimatization. For cold exposure and cold adaptation, mice were housed at 4°C for 24 h and 1 week, respectively.

Reverse Transfection and RNAi
For RNA interference experiments, we reverse transfected imBAT and primary brown adipocytes with SMARTpool siRNA (Dharmacon) on the 3 rd day of differentiation. Lipofectamine ™ RNAiMAX transfection reagent (Thermo Fisher Scientific) was used according to manufacturer's instructions. SMARTpool siRNAs for Nfe2l1, Ubqln1, 2 and 4 were used in a final concentration of 30 nM for single knockdowns. For double and triple knockdown of Ubqln1, 2 and 4 SMARTpool siRNAs were mixed in an equimolar ratio with final concentrations of 60 nM and 90 nM, respectively. After 24 h the transfection mix was replaced with the standard medium and the cells were incubated for another 24 h, after which we treated or directly harvested them.

RNA Extraction, cDNA Synthesis and RT-qPCR
To extract total RNA from frozen adipose tissue and cells we used the NucleoSpin ® RNA kit (Macherey-Nagel) according to the manufacturer's instructions. RNA concentrations were measured on a NanoDrop spectrophotometer (Thermo Fisher Scientific). To synthesize complementary DNA (cDNA), we reversetranscribed 500 ng RNA with the Maxima ™ H Master Mix 5x (Thermo Fisher Scientific) in a total volume of 10 ml. cDNA was diluted 1:40 with RNase-free H 2 O. Relative gene expression was quantified using quantitative real time-PCR. Each reaction contained 4 µL cDNA, 5 µL PowerUp ™ SYBR Green Master Mix (Applied Biosystems) and 1 µL of 5 µM primer stock (full primer list in Supplementary Table 1). We used standard run conditions for Applied Biosystems SYBR Green Gene Expression Assays (2:00 min 50°C, 10:00 min 95°C, 40 cycles of 0:15 min 95°C, 1:00 min 60°C). Cycle thresholds (Cts) of genes of interest were normalized to TATA-box binding protein (Tbp) levels by the DDCt-method and displayed as relative copies per Tbp or relative expression normalized to experimental control groups.

Oil Red O Staining
To visualize and quantify lipid content in adipocytes, we used Oil Red O staining. Confluent cells were harvested 48 h after transfection, washed once with DPBS and incubated with zinc formalin solution (Merck) for 5 min at room temperature. Afterwards, zinc formalin was replaced with fresh zinc formalin and the cells were fixed for 48 h at 4°C. Adipocytes were washed with 100% isopropanol and dried at 37°C. Cells were stained with 0.5 ml 60% vol/vol Oil Red O solution (Sigma), 40% vol/vol distilled H 2 O for 1 h at room temperature and immediately washed 4x with distilled H 2 O afterwards. Pictures from each well were taken with a digital camera as well as with a microscope at 200x magnification before ORO stain was eluted with 100% isopropanol and transferred into a clear 96-well plate. To quantify the staining, absorption of the eluates was measured at 500 nm on a Tecan plate reader. Absorption was normalized by subtracting the absorbance of 100% isopropanol and displayed as fold change to Scramble.

Extracellular Flux Analysis (Seahorse)
For extracellular flux analysis, we reverse transfected imBAT and plated them at 25,000 cells per well in a 24-well Seahorse plate in two independent experiments. To ensure even distribution, cells were seeded in 50 µl of medium and kept for 2 h at room temperature. Then, 175 µl medium were added, and the cells were allowed to fully attach at 37°C overnight. On the day before the experiment, Seahorse cartridges with the sensors were equilibrated in XF calibrant solution (Agilent) at 37°C in a non-CO2 chamber overnight. The experiment was performed on the 5th day of differentiation. Cells were treated with 100 nM bortezomib for 6 h or the corresponding amount of DMSO (Sigma-Aldrich) as a control treatment. XF DMEM pH 7.4 medium (Agilent) was supplemented fresh on the day of the experiment with 10 mM glucose, 1 mM pyruvate and 2 mM L-glutamine (Seahorse medium, all reagents from Agilent). Cells were carefully washed 2x with 1 ml Seahorse medium before incubating them with 500 µl of Seahorse medium for approximately 45 min at 37°C without CO 2 . During the measurement norepinephrine (Sigma-Aldrich), oligomycin (Sigma-Aldrich), FCCP (Sigma-Aldrich) and rotenone-antimycin A (Sigma-Aldrich) were injected via the port of the Seahorse cartridge. Final concentrations were 1 µM norepinephrine (in 100% H 2 O), 1 µM oligomycin, 4 µM FCCP and 0.5 µM rotenone-antimycin A (all in 100% DMSO). The protocol was 3 min mix, 2 min wait and 3 min measure for each time point. For normalization of respiration to live cell count, the cell plate was retrieved after the run, and we quantified the cell count using the CyQUANT ® Cell Proliferation Assay Kit (Thermo Fisher Scientific) according to manufacturer's instructions.

Protein Extraction and Western Blotting
We lysed cells in RIPA buffer [150 mM NaCl (Merck), 5 mM EDTA (Merck), 50 mM Tris pH 8 (Merck), 0.1% wt/vol SDS (Carl Roth), 1% wt/vol IGEPAL ® CA-630 (Sigma-Aldrich), 0.5% wt/vol sodium deoxycholate (Sigma-Aldrich)] freshly supplemented with protease inhibitors (Sigma-Aldrich) in a 1:100 ratio. The lysate was centrifuged at 4°C for 30 min at 21,000xg, to clear the lysate from debris and lipids. Protein concentrations were determined using the Pierce BCA Protein Assay (Thermo Fisher Scientific) according to the manufacturer's instructions. For western blotting, lysates were adjusted to a final concentration of 0.85 µg/µl in 1x Bolt ™ LDS Sample buffer (Thermo Fisher Scientific) supplemented with 5% vol/vol 2-mercaptoethanol (Sigma-Aldrich). For SDS-PAGE we used Bolt ™ 4-12% Bis-Tris gels (Thermo Fisher Scientific) with Bolt ™ MOPS SDS running buffer. After separation, proteins were transferred onto a 0.2 µm PVDF membrane (Bio-Rad) using the Trans-Blot ® Turbo ™ system (Bio-Rad) at 12 V, 1.4 A for 16 min. The membrane was briefly stained with Ponceau S (Sigma-Aldrich) to verify successful transfer and blocked in 1x Roti-Block (Carl Roth) for 1 h at room temperature. Primary antibodies (Cell signaling) were used in a 1:1,000 ratio in 1x Roti-Block overnight at 4°C. After washing 4× for 10 min with TBS-T (200 mM Tris (Merck), 1.36 mM NaCl (Merck), 0.1% vol/vol Tween 20 (Sigma)), secondary antibodies (Santa Cruz) were applied in a 1:10,000 ratio in Roti-Block for 1 h at room temperature. Then membranes were washed again 3× for 10 min in TBS-T and developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and a Chemidoc imager (Bio-Rad). We analyzed the digital images with the Image Lab software (Bio-Rad). Uncropped images can be found in (Supplementary Figure 1).

Proteasomal Activity Assay
For the proteasomal activity assay, we lysed cells with a buffer containing 40 mM TRIS pH 7.2 (Merck), 50 mM NaCl (Merck), 5 mM MgCl 2 (hexahydrate) (Merck) and 10% vol/vol glycerol (Sigma-Aldrich) freshly supplemented with 2 mM b-mercaptoethanol (Sigma-Aldrich) and 2 mM ATP (Sigma-Aldrich). To measure chemotrypsin-like, trypsin-like and caspase-like activity, we used the Proteasome Activity Fluorometric Assay (UBP Bio) according to the manufacturer's instructions. For the normalization of proteasomal activity to total protein content, we used the Bio-Rad Protein Assay Kit II to determine protein concentration in our samples.

Statistics
Data were expressed as the mean ± standard error of the mean (SEM). For comparing three and more groups we used one-way ANOVA with Bonferroni post-hoc test as indicated in the figure legends. For comparing two variables we used two-way ANOVA with Bonferroni post-hoc test. Analysis was performed using R, Microsoft Excel and/or GraphPad Prism. P <0.05 was considered significant, as indicated by asterisks and letters in the figures legends.

Ubqln1 Is Expressed in Adipose Tissue and Induced by Cold and Proteasome Inhibition
As nothing is known about ubiquilins in BAT, we first investigated their gene expression levels in primary and immortalized adipocytes as well as in BAT ex vivo and second how these are impacted by cold and proteasome inhibition. In primary cells from intrascapular brown, inguinal subcutaneous and gonadal white adipose tissue (WAT), Ubqln1, 2 and 4 expression was found to be expressed whereas Ubqln3 was undetectable ( Figure 1A). Ubqln2 and Ubqln4 were expressed at similar levels in all three adipocyte types. Interestingly, Ubqln1 was approximately higher expressed compared to Ubqln2 and 4 with highest expression in adipocytes from the subcutaneous WAT. In BAT from 12-week-old mice, which were kept at 4°C for 24 h (cold exposure) and 7 days (cold adaptation), we found that Ubqln1 and Ubqln4 expression was higher compared to mice kept at 30°C (thermoneutrality) ( Figure 1B). We confirmed that the cold challenge was successful by increased expression of thermogenic markers Ucp1 and Ppargc1a ( Supplementary Figures 2A, B). Compared to primary brown adipocytes, we found a similar expression pattern in imBAT, which remained unchanged upon stimulation with beta3-agonist CL316,243 ( Figure 1C). Proteasome inhibition with epoxomicin, MG132 or bortezomib respectively, resulted in higher Ubqln1 expression compared to DMSO treated controls ( Figure 1D). We were unable to detect Ubqln3 mRNA in any of our samples, which is in line with a previous report that Ubqln3 is specific to testis (9). Thus, Ubqln1, 2 and 4 are expressed in adipose tissue, yet only Ubqln1 is positively regulated by cold and proteasome inhibition.

Silencing of Ubqln1, 2 and 4 Does Not Cause ER Stress or Inflammation
Next, we determined the significance of ubiquilins in brown adipocyte homeostasis. We used RNA interference to silence the gene expression of Ubqln1, 2 and 4 in immortalized and primary adipocytes to investigate their functional relevance. We explored established surrogate markers of stress responses and adipocyte function linked to BAT proteostasis (4). While our RNAi strategy was efficient, knockdown of any of the Ubqln genes did not result in changes in the mRNA expression of surrogate stress markers, Ddit3 and Ccl2 (Figures 2A, B). Knockdown of Ubqln2 resulted in lower Adipoq expression in both immortalized and primary brown adipocytes, which suggests that adipocyte health is affected under these conditions. Since Ubqln1, 2 and 4 share the same domain structure and their sequences are highly similar, we hypothesized that they could compensate for each other (12). This was also supported by upregulation of Ubqln2 after knockdown of Ubqln1 in primary brown adipocytes ( Figure 2B). To circumvent this effect, we studied all possible combinations of Ubqln1, 2 and 4 siRNAs in single, double and triple Ubqln knockdown experiments in imBAT ( Figure 2C).

This resulted in lower Adipoq expression in conditions with
Ubqln2 knockdown compared to the control cells ( Figure 2D). Ddit3 and Ccl2 expression remained largely unaffected. Hence, loss of Ubqln1, 2 or 4 did not increase ER stress or inflammation marker genes, alone or in combination.

Silencing of Ubiquilins Does Not Impair Adipocyte Health and Non-Shivering Thermogenesis
As we observed a reduction in Adipoq mRNA levels in both immortalized and primary brown adipocytes, we next investigated how loss of ubiquilins impacts adipocyte health. To our surprise, the expression levels of other adipocyte marker genes such as Pparg, Plin1, Fabp4, Fasn and Cebpa remained unchanged by Ubqln single or triple knockdown ( Figure 3A. However, compared to the control cells we observed a lower lipid content in imBAT (Figures 3B, C and Supplementary Figure 3). Next, we investigated if loss of ubiquilins impacts NST. We analyzed norepinephrine-induced respiration and mitochondrial stress tests as the gold standard of measuring NST in cells. Neither single nor triple knockdown affected basal cellular respiration, the response to norepinephrine stimulation or maximal respiratory capacity (Figures 3D, E). To investigate if ubiquilins participate in the coordinated cellular response to proteasome inhibition, which has been shown to impair mitochondrial function in brown adipocytes (4), we treated imBAT cells with the compound bortezomib. While pretreatment with bortezomib led to lower levels of respiration, this response was independent of single or triple knockdown of ubiquilins ( Figures 3F, G).

Ubiquilins Are Dispensable for Proteostasis in Brown Adipocytes
As we found that Ubqln1 expression in brown adipocytes is strongly induced by proteasome inhibition, we hypothesized that Ubiquilins are involved in maintaining proteostasis. Indeed, knockdown of Ubqln1, 2 and 4 resulted in higher Atf3 gene expression, albeit the effect was small but significant ( Figure 4A). Other stress markers related to accumulation of misfolded proteins or ER stress such as Atf4, Atf6 and sXbp1 were unchanged and Hspa5 was only higher in the triple knockdown condition. Another important player for brown fat proteostasis is the transcription factor Nfe2l1, which activates the transcription of proteasomal subunits in a bounce-back-mechanism in response to proteasomal inhibition and cold (4). It is conceivable that Nfe2l1 counteracts accumulation of misfolded proteins and ER stress as a consequence of diminished Ubqln expression through increased proteasome formation. However, we neither observed differences in Nfe2l1 expression nor in the expression of two of its downstream targets Psma1 and Psmb1. We also tested whether, the induction of Atf3 in response to Ubqln knockdown was mediated by Nfe2l1, but Atf3 expression was unchanged by Ubqln triple knockdown in combination with Nfe2l1 knockdown (Supplementary Figure 4). In line with these findings, single Ubqln knockdown (Supplementary Figure 5) or triple Ubqln knockdown neither affected the levels of ubiquitinated proteins nor Nfe2l1 protein levels, neither under basal conditions nor after proteasome inhibition ( Figure 4B). Triple knockdown did also not result in lower proteasomal activity ( Figure 4C). Thus, ubiquilins are dispensable to maintain proteostasis in brown adipocytes under basal and proteasome inhibited conditions and their absence is not linked to Nfe2l1 activation.

DISCUSSION
The adaptation to sustained cold is a challenging process for thermogenic adipocytes. These cells undergo extensive remodeling for enhanced and sustainable oxidative metabolism, which involves the synthesis of new proteins, lipids and entire organelles (2). We have previously shown that this process requires enhanced proteasomal protein quality control, transcriptionally mediated by Nfe2l1 (4). However, even with increased proteasomal degradation, cold adaptation still results in increased protein ubiquitination (4). The mechanisms dynamically coupling ubiquitination of proteins to proteasomal degradation in brown adipocytes remain elusive. Here, we investigated the role of ubiquilins for proteostasis and NST in brown adipocytes.
Ubiquilins are multifaceted shuttling proteins, which have an amino-terminal UBL and a carboxy-terminal UBA domain, which enable them to interact with both mono-and poly-ubiquitinated cargo as well as facilitate their degradation through the UPS. This role seems to be their predominant function, as it is conserved from yeast to humans (10). We show that Ubqln1, 2 and 4 are highly expressed in BAT and that their expression, most notably of Ubqln1 is induced by cold and proteasomal inhibition. Ubqln1, 2 and 4 are highly homologous and share a similar domain structure. It is therefore likely that they share similar functions and might compensate for the loss of each other (13). However, silencing of one or the combination of ubiquilins by RNAi did neither impact ubiquitin levels nor proteasomal activity under basal conditions or after treatment with proteasome inhibitors. Consequently, except for mildly upregulated expression of Atf3, a stress marker in response to various physiological stressors, no changes in ER stress or inflammation were observed under these conditions.
We could speculate that Nfe2l1 as the major regulator of proteostasis in brown adipocytes, is involved in upregulation of Atf3 upon knockdown of ubiquilins. However, we did not investigate this in an epistasis-type of experiment, since we did not find any changes in Nfe2l1 gene and protein expression. Another limitation of this study is that we only silenced Ubqln1, 2 and 4 by RNAi. Presumably, knockout of one or all ubiquilins would have a stronger effect on proteostasis and NST. In mice, transgenic knockout of Ubqln1 in neurons aggravated brain injury and delayed functional recovery after ischemic stroke. Ubiquitous overexpression of Ubqln1, however, resulted in reduced neuronal damage (14). Similarly, cardiomyocyte-specific knockout of Ubqln1 in a mouse model of myocardial ischemia-reperfusion injury resulted in enlarged infarct size and late-onset cardiomyopathy, whereas overexpression of Ubqln1 resulted in reduced infarct size (15). Moreover, in humans, mutations in Ubqln1, 2 and 4 are strongly associated with the onset of neurodegenerative diseases A B D C FIGURE 2 | Silencing of Ubqln1, 2 and 4 in brown adipocytes does not cause ER stress or inflammation. Ubiquilins were silenced by siRNA-mediated knockdown in imBAT and primary brown adipocytes. Relative gene expression measured by qPCR in: (A), imBAT (n = 3 technical replicates). (B), primary brown adipocytes (n = 5-6 technical replicates from 2 independent experiments). (C, D), imBAT (n = 3 technical replicates). Throughout, data are mean ± SEM. Statistical testing was done by two-way ANOVA with Bonferroni post-hoc test. Different small letters indicate differences in mean compared to Scramble with at least P adj < 0.05. such as dementia, Alzheimer's disease and amyotrophic lateral sclerosis (12,(16)(17)(18). These data strongly suggest that impairment or loss of function of ubiquilins are associated with numerous pathologies in both humans and rodents.
In Drosophila loss of Ubqn, the homologue of mammalian ubiquilins, results in ER expansion and activation of the PKRlike ER kinase (PERK) pathway, one branch of the unfolded protein response (5). According to our original hypothesis that ubiquilins might be involved in ubiquitination of proteins and their proteasomal degradation during NST, we measured norepinephrine-induced respiration but NST was intact despite the loss of ubiquilins. However, we found reduced lipid content compared to control cells. Lipogenesis and lipid droplet biosynthesis takes place in the ER and ER stress, even if driven by proteotoxic stressors disrupt lipid homeostasis (3). In light of the absence of overt ER stress upon loss of ubiquilins, the changes in lipid content point to a more specific role of ubiquilins in lipid metabolism beyond proteostasis. While this warrants further investigation, we did not observe any functional change in NST. Yeast has only one ubiquilin isoform Dsk2, but other UBL/ UBA proteins such as Rad23. None of these individual proteins are essential in yeast, but combined loss of Rad23, Dsk2 and the proteasome ubiquitin receptor Rpn10/S5a results in mitotic arrest (19). This suggests that, at least in yeast, UBL/UBA proteins are redundant and may have overlapping substrate specificity.
In future, it will be important finding those UBL/UBA proteins that participate in ubiquitination in brown adipocytes. In summary, in this study, we investigated the role of ubiquilins for brown adipocyte proteostasis and thermogenesis. We found that despite high expression levels of Ubqln 1, 2 and 4, these ubiquilins are only minor regulators of brown adipocyte function.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
CM performed experiments and analyzed data. SK provided samples and assisted with experiments. CM and AB conceptually designed the study, interpreted the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.