Original Article AAV-mediated BMP7 gene therapy counteracts insulin resistance and obesity Estefania Casana,1,2,3,4 Veronica Jimenez,1,2,3,4 Claudia Jambrina,1,2,3 Victor Sacristan,1,2 Sergio Muñoz,1,2,3 Jordi Rodo,1,2 Ignasi Grass,1,2 Miquel Garcia,1,2,3 Cristina Mallol,1,2 Xavier León,1,2,3 Alba Casellas,1,2,3 Víctor Sánchez,1,2 Sylvie Franckhauser,1,2,3 Tura Ferré,1,2,3 Sara Marcó,1,2,3 and Fatima Bosch1,2,3 1Center of Animal Biotechnology and Gene Therapy (CBATEG), Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain; 2Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain; 3CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 28029 Madrid, Spain Type 2 diabetes, insulin resistance, and obesity are strongly The continued imbalance between energy intake and expenditure is associated and are a major health problem worldwide. the main cause of obesity. In obese individuals, adipose tissue hyper- Obesity largely results from a sustained imbalance between trophy is associated with accumulation of fat in ectopic compart- energy intake and expenditure. Therapeutic approaches tar- ments, such as the liver, and in development of insulin resistance. geting metabolic rate may counteract body weight gain and Increased energy expenditure, through non-shivering thermogenesis insulin resistance. Bone morphogenic protein 7 (BMP7) mediated by brown adipose tissue (BAT) uncoupling activity and/or has proven to enhance energy expenditure by inducing browning (appearance of beige adipocytes) of subcutaneous white ad- non-shivering thermogenesis in short-term studies in mice ipose tissue (WAT), has been associated with leanness and improved treated with the recombinant protein or adenoviral vectors insulin sensitivity in both obese animal models and humans.6–12 encoding BMP7. To achieve long-term BMP7 effects, the use of adeno-associated viral (AAV) vectors would The bone morphogenetic protein (BMP) family belongs to the trans- provide sustained production of the protein after a single forming growth factor b (TGF-b) superfamily of cytokines that administration. Here, we demonstrated that treatment of regulate an array of fundamental cellular processes including prolifer- high-fat-diet-fed mice and ob/ob mice with liver-directed ation, differentiation, apoptosis and morphogenesis.13,14 Bone AAV-BMP7 vectors enabled a long-lasting increase in circu- morphogenic protein 7 (BMP7) has also been described as promoting lating levels of this factor. This rise in BMP7 concentration white adipogenesis, brown adipocyte differentiation, and thermogen- induced browning of white adipose tissue (WAT) and activa- esis.15,16 Administration of recombinant BMP7 or adenoviral (Ad) tion of brown adipose tissue, which enhanced energy expen- vectors encoding BMP7 to healthy or high-fat diet (HFD)-fed mice diture, and reversed WAT hypertrophy, hepatic steatosis, increases whole-body energy expenditure in short-term studies.16–18 and WAT and liver inflammation, ultimately resulting in Moreover, these treatments also improve insulin sensitivity and normalization of body weight and insulin resistance. This reverse obesity in HFD-fed, ob/ob, and db/db mice.18,19 Nevertheless, study highlights the potential of AAV-BMP7-mediated periodic administration of recombinant BMP7 protein is required,19 gene therapy for the treatment of insulin resistance, type 2 which, in addition, could compromise treatment compliance by pa- diabetes, and obesity. tients. Likewise, the use of Ad-BMP7 vectors is not able to promote long-term expression of the transgene due to the high immunoge- nicity of Ad vectors.20,21 INTRODUCTION Type 2 diabetes (T2D) has become a major health problem because of Here, we hypothesized that adeno-associated viral (AAV) vector- its alarmingly growing prevalence worldwide. Obesity is very strongly mediated gene therapy offers the possibility of a one-time treatment associated with insulin resistance and T2D. These diseases lead to that would lead to sustained production of BMP7 for extended pe- long-term damage, dysfunction, and failure of several key organs, re- riods of time with low toxicity and immunogenicity.22,23 In this study, sulting in a variety of severe complications such as hepatosteatosis, non-alcoholic steatohepatitis (NASH), arthritis, hypertension, car- diovascular disease, retinopathy, nephropathy, or neurodegeneration, among others.1–5 Although conventional pharmacologic treatments Received 7 July 2021; accepted 13 March 2022; https://doi.org/10.1016/j.omtm.2022.03.007. and lifestyle intervention have proven effective for many obese and 4 These authors contributed equally T2D patients, these approaches are not successful in all cases and Correspondence: Fatima Bosch, Center of Animal Biotechnology and Gene could result in undesirable side effects. Therefore, there is an urgent Therapy Edifici H, Universitat Autònoma de Barcelona 08193 Bellaterra, Barcelona, need for novel therapeutic approaches to prevent and reverse the Spain. T2D and obesity epidemic. E-mail: fatima.bosch@uab.es 190 Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 ª 2022 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.moleculartherapy.org we found that treatment of obese HFD-fed or ob/ob mice with AAV cies, including mice, rat, guinea pig, rabbit, and cow.24–28 In agreement, vectors encoding Bmp7 under the control of a liver-specific promoter the lean-chow-fed animals at 4 and 12 weeks after treatment with AAV- induced browning of subcutaneous WAT and activation of BAT, BMP7 showed circulating levels much lower than HFD-fed AAV- which increased energy expenditure and mediated long-term reversal BMP7-treated mice (Figure 1C). As body and liver weight normalized, of body weight gain and insulin resistance. These results highlight the serum BMP7 concentration also decreased in HFD-fed AAV-BMP7- potential of AAV-BMP7-mediated gene therapy for the treatment of treated mice, reaching levels similar to those of chow-fed AAV- T2D and obesity. BMP7-treated mice at the end of the study (Figure 1C). No alteration in the liver damage marker alanine aminotransferase (ALT) was RESULTS observed at any time after AAV-BMP7 treatment (Figure 1D). In Liver-specific BMP7 overexpression mediates persistent contrast, HFD-fed mice administered AAV-null vectors showed pro- reversion of obesity gressively very high serum ALT levels (Figure 1D) in agreement with To determine the anti-obesogenic and anti-insulin resistance effects increased adiposity in the liver (Figure 1B). of AAV-BMP7-mediated gene therapy, 2-month-old C57BL/6 mice were first fed with either chow or an HFD for 6 weeks. During this Because of the hAAT promoter, AAV-BMP7-treated mice showed period, the weight of chow- and HFD-fed animals increased by specific overexpression of codon-optimized BMP7 in the liver, which 19% and 53%, respectively (Figure 1A). Chow-fed and obese HFD- resulted in increased hepatic BMP7 content (Figures 1E and S1I). This fed animals were then treated intravenously (i.v.) with 1  1012 vg agreed with the increased transduction of the liver by AAV8 vectors of AAV vectors of serotype 8 encoding an optimized murine BMP7 (Figure S1J). In contrast, marginal levels of endogenous Bmp7 expres- coding sequence (omBmp7) under the control of a synthetic hybrid sion, compared with the expression of omBmp7, were observed in all liver-specific promoter composed of three copies of the hepatocyte the tissues examined, including the kidney, the main endogenous pro- control region (HCR) enhancer from the apolipoprotein E gene ducer of the factor29 (Figures 1F and 1G). (ApoE) and the human a1-antitrypsin (hAAT) promoter (AAV- BMP7). As controls, another cohort of obese HFD-fed mice and the AAV-BMP7 reverses HFD-associated WAT hypertrophy and cohort of chow-fed mice received the same dose of non-coding inflammation AAV8 null vectors (AAV-null). Following AAV delivery, mice were HFD feeding induced an increase in the size of white adipocytes in maintained on chow or HFD feeding for about 6 months, and body null-treated mice (Figures 2A and 2B). In agreement with decreased weight and metabolic parameters were monitored regularly. adiposity, morphometric analysis of visceral (epididymal, eWAT) and subcutaneous (inguinal, iWAT) WAT revealed that the area of Chow-fed animals treated with AAV-BMP7 maintained their body white adipocytes of BMP7-treated animals was similar to that of an- weight at the time of AAV treatment, which was slightly lower than imals fed a chow diet (Figures 2A and 2B). As a result of the reversion that of chow-fed AAV-null-treated mice, which progressively gained of WAT hypertrophy, serum adiponectin and leptin levels were weight as the animals aged (Figure 1A). HFD-fed animals treated normalized in HFD-fed mice treated with AAV-BMP7 vectors with AAV-BMP7 vectors experienced progressive loss of body weight (Figures 2C and 2D). following gene delivery, which stabilized within a few weeks to a weight similar to that of chow-fed AAV-BMP7-treated mice (Figures 1A and Obesity also causes inflammation of WAT.30 Immunostaining of S1A). This was parallel to normalization of the weight of the liver and eWAT sections for the macrophage-specific marker Mac2 revealed the main adipose depots of HFD-fed AAV-BMP7-treated animals increased presence of “crown-like” structures in HFD-fed null-treated compared with the HFD-fed null-treated mice (Figures 1B and S1B– mice, while HFD-fed animals treated with AAV-BMP7 had no sign of S1H). Moreover, the weight of the main white and brown fat pads of macrophage infiltration (Figures 2E and 2F). This was parallel to the chow- and HFD-fed AAV-BMP7-treated animals was slightly lower normalization of the expression of the macrophage markers Cd68 and than that of chow-fed null-treated mice (Figures 1B and S1B–S1G). F4/80 in BMP7-treated mice (Figure 2E), indicating that BMP7 expression counteracted the inflammation of WAT associated with The decrease in body weight was parallel to increased circulating BMP7 obesity. levels (Figure 1C). In HFD-fed AAV-BMP7-treated mice, serum BMP7 concentration was very high 4 weeks after AAV administration and BMP7 downstream signaling was assessed in WAT by evaluating the gradually decreased, reaching levels similar to those of chow-fed phosphorylation levels of SMAD1/5/8 (pSMAD1/5/8) and p38 AAV-BMP7-treated mice at week 23 post-AAV (Figure 1C). A similar mitogen-activated protein kinase (MAPK) (p-p38MAPK), which serum BMP7 level profile was observed in chow-fed AAV-BMP7- are downstream effectors of the canonical and non-canonical, respec- treated mice (Figure 1C). The changes in BMP7 circulating levels would tively, BMP7 signaling pathways.31,32 Increased pSMAD1/5/8 and be consistent with increased expression of BMP7 from the synthetic unchanged p-p38MAPK were observed in eWAT of HFD-fed HCR/hAAT promoter under conditions of high hepatic fat content, AAV-BMP7-treated mice (Figures 2G and 2H). These observations such as liver steatosis after HFD feeding. In this regard, transcription agreed with previous studies reporting BMP7-mediated increased from the ApoE promoter has already been reported to be induced in pSMAD1/5/8 in white preadipocytes but a lack of enhancement of the liver after high-fat and/or high-cholesterol feeding in different spe- p-p38MAPK in the 3T3-L1 cell line.16 Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 191 Molecular Therapy: Methods & Clinical Development A B C D E F G Figure 1. AAV-mediated liver gene transfer of BMP7 counteracts HFD-induced obesity (A) Body-weight follow-up of C57BL/6 mice fed either chow or an HFD for 6 weeks and then administered intravenously with 1  1012 vg of AAV-BMP7 vector per mouse. Control obese mice and control chow-fed mice were treated with AAV-null vectors (n = 8/group). &p < 0.05 and &&&p < 0.001 versus the HFD-fed null-injected group. $ p < 0.05 and $$$p < 0.001 versus the chow-fed null-injected group. (B) Weight of the liver, epididymal (eWAT) and inguinal (iWAT) white adipose tissue depots, and in- terscapular brown adipose tissue (iBAT) of the same cohorts as in (A) (n = 7–8/group). (C) Circulating levels of BMP7 (n = 8/group). (D) Alanine aminotransferase (ALT) serum levels (n = 3–8/group). (E) Quantitative PCR analysis of Bmp7 expression in the liver, eWAT, iWAT, gastrocnemius, and kidney was performed with primers that specifically detected vector-derived BMP7 mRNA (omBMP7) (n = 7–8/group). (F) Quantitative PCR analysis of endogenous Bmp7 expression in liver, eWAT, iWAT, and gastrocnemius (n = 7–8/group). (G) Quantitative PCR analysis of endogenous Bmp7 expression in kidney (n = 8/group). All values are expressed as the mean ± SEM. ND, not detected; NA, not available; AU, arbitrary units; HFD, high-fat diet. *p < 0.05, **p < 0.01, and ***p < 0.001. Treatment with AAV-BMP7 enhances energy expenditure energy expenditure were evaluated. Weekly measurements of the To elucidate the mechanism(s) underlying the counteraction of number of calories consumed per day revealed that the energy intake obesity in HFD-fed mice treated with AAV-BMP7, food intake and of HFD-fed BMP7-treated animals was slightly reduced (about 12%) 192 Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 www.moleculartherapy.org A B C D E F G H Figure 2. Reversal of WAT hypertrophy and inflammation by AAV-BMP7 treatment (A) Representative images of hematoxylin-eosin staining of eWAT. Scale bars: 200 mm. (B) Morphometric analysis of the area of eWAT and iWAT adipocytes (n = 7–8/group). (C and D) Circulating levels of adiponectin (C) and leptin (D) (n = 7–8/group). (E) Quantitative PCR analysis of the expression of the macrophage markers Cd68 and F4/80 in eWAT (n = 8/group). (F) Immunohistochemistry for the macrophage-specific marker Mac2 in eWAT sections. Scale bars: 200 and 50 mm (inset). (G) Determination of phosphorylated SMAD1/5/8 (p-SMAD1/5/8) protein abundance in eWAT: immunoblot and densitometric analysis (n = 4/group). (H) Determination of phosphorylated p38 (p-p38) protein abundance in eWAT: immunoblot and densitometric analysis (n = 4/group). All values are expressed as the mean ± SEM. HFD, high-fat diet; FC, fold change. *p < 0.05, **p < 0.01, and ***p < 0.001. Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 193 Molecular Therapy: Methods & Clinical Development compared with that of the HFD-fed null-treated cohort (Figures 3A increased glucose uptake in both visceral and subcutaneous WAT and S2A). Nevertheless, normalization of food intake data per body that likely contributed to increased insulin sensitivity and glucose weight revealed that HFD-fed mice treated with AAV-BMP7 ate tolerance (Figure S3D). more than the chow-fed counterparts (Figure 3B). When indirect calorimetry was performed 10 weeks after AAV delivery, BMP7- Treatment of ob/ob mice with AAV-BMP7 treated mice had higher oxygen consumption and energy expenditure To further evaluate the anti-obesogenic and anti-insulin resistance ef- during both the light and the dark cycles than HFD-fed null control fects of AAV-mediated BMP7 gene transfer, ob/ob mice were used, counterparts (Figures 3C, S2B, and S2C). No striking differences in since they are a well-known model of extreme obesity and insulin spontaneous activity or respiratory exchange ratio were observed resistance.33 Eleven-week-old ob/ob mice were treated i.v. with among groups (Figures S2D and S2E). 1  1013 vg AAV-BMP7 vectors. As controls, AAV-null vectors were administered to another cohort of ob/ob animals. Whereas the To evaluate whether non-shivering thermogenesis was responsible for body weight of null-injected ob/ob mice increased as animals aged, the enhancement of energy expenditure, the degree of activation of the body weight of ob/ob animals treated with AAV-BMP7 progres- BAT and browning of subcutaneous iWAT was assessed. Compared sively decreased after AAV administration (Figure 5A). These with HFD-fed null-treated mice, AAV-BMP7-treated mice showed changes resulted from increased circulating levels of BMP7 (Fig- decreased lipid deposition in iBAT and the presence of numerous ure 5B). As expected, AAV-BMP7 treatment led to overexpression multilocular adipocytes in iWAT (Figure 3D). This was parallel to of codon-optimized Bmp7 specifically in the liver, increased hepatic increased expression of the thermogenic markers Ucp1, Cidea, and BMP7 content, and high hepatic transduction by AAV8 vectors Ppargc1a in iWAT of AAV-BMP7-treated mice (Figures 3E and (Figures 5C, 5D, and S4A). Endogenous Bmp7 expression was mainly 3F). No changes in the expression of Ucp1 and Cidea were detected detected in kidney, although at marginal levels (Figure S4B). In agree- in the iBAT of these mice, although other thermogenic markers ment with reduced body weight, the weight of the fat pads was such as Ppargc1a, Dio2, and Elovl3 were upregulated (Figure S2F), decreased in ob/ob BMP7-treated animals (Figures 5E and S4C). in agreement with BAT activation. Moreover, animals treated with AAV-BMP7 vectors showed reduced size of white adipocytes in eWAT and iWAT (Figure 5F). These find- AAV-BMP7 ameliorates hepatic steatosis and counteracts ings were associated with a marked increase in serum adiponectin insulin resistance levels (Figure 5G). In addition, we observed increased pSMAD1/5/8 As expected, HFD-fed null-treated animals showed marked hepatic and unchanged p-p38MAPK in eWAT of AAV-BMP7-treated ob/ steatosis, which was ameliorated after treatment with AAV-BMP7 ob mice (Figures S4D and S4E). vectors (Figure 4A). This finding was parallel to a reduction in total hepatic triglyceride content and a decrease in macrophage infiltration, The decreased adiposity may result, at least in part, from the reduced evidenced by the lower number of Mac2+ cells in liver sections food intake observed in AAV-BMP7-treated ob/ob mice (Figures 5H, (Figures 4A and 4B). Moreover, the expression of the pro-inflamma- S4F, and S4G). In contrast to HFD-fed AAV-BMP7-treated mice, ob/ tory cytokines Ifng, Mcp1, Ccl5, and Tnf1a was normalized in AAV- ob mice treated with AAV-BMP7 vectors showed neither marked BMP7-treated HFD-fed mice (Figure 4C). In agreement with reduction of lipid accumulation in BAT nor multilocular adipocytes normalization of hepatic steatosis, serum cholesterol levels were in iWAT (Figure 5F). However, expression of Ucp1 and Elov3 was up- also normalized (Figure S3A). regulated in both iWAT and iBAT (Figures 5I–5L). Moreover, increased Ppargc1a expression levels were also observed in iBAT (Fig- Obese HFD-fed null-treated mice showed normal fed and fasted gly- ure 5M). These results suggested induction of non-shivering thermo- cemia (Figure 4D) but were hyperinsulinemic (Figure 4E). In genesis in AAV-BMP7-treated ob/ob mice. contrast, HFD-fed AAV-BMP7-treated mice were normoglycemic and normoinsulinemic (Figures 4D and 4E). To evaluate whole- Control ob/ob mice presented marked hepatic steatosis, whereas body insulin sensitivity of AAV-BMP7-treated animals, intraperito- treatment with AAV-BMP7 significantly decreased liver weight and neal insulin tolerance tests (ITTs) were performed 1.5 and 4 months hepatic lipid accumulation (Figures 6A–6C). ob/ob mice treated post-AAV delivery. Feeding with HFD led to insulin resistance in an- with AAV-BMP7 also exhibited markedly reduced expression of imals receiving AAV-null vectors, while treatment with AAV-BMP7 pro-inflammatory cytokines in the liver (Figure 6D). ob/ob animals counteracted HFD-induced insulin resistance (Figures 4F and S3B). treated with AAV-BMP7 showed similar fed glycemia but markedly Moreover, HFD-fed AAV-treated mice showed higher insulin sensi- decreased fed insulinemia (Figures 6E and 6F). Treatment with tivity than chow-fed mice receiving null vectors (Figures 4F and S3B), AAV-BMP7 improved insulin sensitivity in comparison with AAV- indicating that AAV-BMP7 treatment was able to counteract insulin null-treated ob/ob mice (Figure 6G), confirming the anti-insulin resistance. Moreover, similar to chow-fed mice, HFD-fed animals resistance effects of AAV-BMP7 observed in HFD-fed animals. treated with AAV-BMP7 vectors showed increased glucose tolerance BMP7 has been reported to induce hepatic insulin signaling in obese compared with the null-treated obese cohort (Figures 4G and S3C). mice via inhibition of MAPKs and induction of phosphorylation of These results were parallel to increased Glut4 expression in eWAT GSK3b (p-GSK3b).34 In agreement, ob/ob mice treated with AAV- and iWAT of HFD-fed AAV-BMP7-treated mice, suggesting BMP7 showed increased p-GSK3b and reduced p-p38MAPK in the 194 Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 www.moleculartherapy.org A B C D E F Figure 3. AAV-BMP7 increases energy expenditure, decreases fat accumulation in iBAT and iWAT, and induces browning (A) Histogram depicting the mean food intake per day from week 15 to week 36 of age of HFD-fed mice treated with either AAV-BMP7 or AAV-null vectors and of chow-fed mice administered AAV-null vectors (n = 3 cages/group and 2–3 mice/cage). (B) Food intake normalized by body weight from week 15 to week 36 of age (n = 3 cages/group and 2–3 mice/cage). (C) Energy expenditure was measured with an indirect open-circuit calorimeter 10 weeks after AAV vector delivery. Data were taken during the light and dark cycles (n = 7–8/group). (D) Hematoxylin-eosin staining of iBAT and iWAT sections. Scale bars: 200 and 50 mm (inset). (E and F) Quantitative PCR analysis in iWAT of the expression of the thermogenic markers Ucp1 (E) and Cidea and Ppargc1a (F) (n = 6–8/group). All values are expressed as the mean ± SEM. HFD, high-fat diet; FC, fold change. *p < 0.05, **p < 0.01, and ***p < 0.001. Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 195 Molecular Therapy: Methods & Clinical Development A B C D E F G Figure 4. Treatment with BMP7-encoding vectors improves hepatic steatosis and reverses insulin resistance (A) Hematoxylin-eosin staining (top) and immunostaining for the macrophage-specific marker Mac2 (bottom) of liver sections. Red arrows indicate macrophages. Scale bars: 200 mm (hematoxylin-eosin) and 100 mm (Mac2). (B) Hepatic triglyceride content (n = 7–8/group). (C) Quantitative PCR analysis of the expression of the pro-inflammatory cytokines Tnfa, Ifng, Mcp1, and Ccl5 in the liver (n = 7–8/group). (D) Fed and fasted blood glucose levels 19 and 8 weeks after vector administration, respectively (n = 8/group). (E) Fed and fasted serum insulin levels 19 and 20 weeks after vector delivery, respectively (n = 7–8/group). (F) Insulin sensitivity was determined after an (legend continued on next page) 196 Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 www.moleculartherapy.org liver (Figures 6H and 6I). These findings suggested that p-GSK3b and AAV-BMP7-mediated reversal of obesity probably resulted from p-p38MAPK would be involved in the AAV-BMP7-mediated increased non-shivering thermogenesis in both BAT and subcutane- improvement of insulin resistance. ous WAT leading to enhanced energy expenditure. Similarly, BAT activation was described in lean and obese mice treated with recom- DISCUSSION binant BMP7 or Ad-BMP7 vectors.16,17 BMP7 has also been reported In this study, increased liver-derived BMP7 circulating levels increased to upregulate thermogenesis in adipocytes derived from human energy expenditure, normalized WAT hypertrophy and inflammation, BAT.50 Increased expression of UCP1 and multilocular beige adipo- and reversed hepatic steatosis and inflammation, which in turn coun- cytes were also detected in iWAT of C57BL/6J mice treated with teracted obesity and insulin resistance. We proved disease reversal in recombinant BMP7.17 In addition, murine and human adipogenic HFD-fed mice, the model that most closely resembles the metabolic precursor cells from subcutaneous WAT have been reported to differ- characteristics of human obesity and insulin resistance.35 The efficacy entiate into brown-like adipocytes upon stimulation with BMP7 of AAV-mediated BMP7 gene transfer to the liver was further in vitro.51,52 Nevertheless, our results highlighted differences in confirmed in the genetically obese ob/ob mouse model. AAV-BMP7 BMP7-mediated induction of browning between HFD-fed C57BL/6 treatment elicited superior anti-insulin resistance and anti-obesogenic mice and ob/ob mice, which lacked multilocular adipocytes in subcu- therapeutic benefit in comparison with previous studies using Ad- taneous WAT, although this tissue did overexpress thermogenic BMP7 vectors or periodic administration of recombinant BMP7 pro- markers. The propensity for browning varies among mouse strains,53 tein.16–19 Recombinant BMP7 or Ad-BMP7 prevented or partially and browning differences between HFD-fed C57BL/6 and ob/ob mice reduced body weight gain and improved insulin sensitivity in HFD- have previously been described during intermittent fasting, in which fed, ob/ob, and db/db mice, but failed to fully counteract obesity and HFD-fed C57BL/6 but not ob/ob mice exhibited beige adipocytes in insulin resistance.17–19 While these studies demonstrated therapeutic WAT.54–56 Moreover, strains of mice that have a higher propensity benefit for 2–4 weeks,16–19 one-time administration of AAV-BMP7 for browning show a greater degree of protection from diet-induced vectors resulted in long-lasting metabolic efficacy in agreement with obesity.57 Likewise, browning of WAT has been described to play a lack of immunogenicity of AAV vectors.22,23 more important role than BAT activation in promoting weight loss in mice after cold or b-adrenergic stimulation.58 In humans, it has The present study in male mice represents a proof of concept of the been described that BAT comprises both brown and beige adipo- beneficial effects of liver-directed AAV-BMP7-mediated gene therapy cytes,59–61 and browning of WAT has also been documented in hu- to counteract obesity and insulin resistance. Transduction of the liver man adults.62–64 by AAV vectors has been widely reported to be markedly decreased in female mice in comparison with male mice.36–45 This phenomenon is AAV-mediated gene transfer of Bmp4, another member of the BMP not restricted to a particular AAV vector serotype.36–44 This sexual family, to the liver of chow-diet-fed mice or mice treated with AAV- dimorphism has been proved to be independent of the promoter BMP4 prior to HFD feeding also led to browning of subcutaneous and mouse strain used36,37,46 and seems to be androgen driven.36 In WAT and increased whole-body energy expenditure.65 Nevertheless, contrast to the results obtained in rodent models, studies performed in already obese HFD-fed mice, AAV-mediated genetic engineering in non-human primates have revealed similar levels of AAV-medi- of the liver with BMP4 failed to enhance browning of adipose tissue ated liver transduction in male and female animals.47,48 or to reverse obesity.66 BMP4 resistance in WAT of obese HFD-fed mice has been suggested as the underlying cause of this lack of ther- To achieve therapeutic efficacy in obesity and insulin resistance, in the apeutic efficacy.65,67 In this regard, increased mRNA levels of endog- present study, we treated HFD-fed mice with 1  1012 vg of AAV8- enous Bmp4 were detected in subcutaneous WAT of obese HFD-fed hAAT-BMP7 vector per mouse. This dose efficiently mediated high mice compared with lean animals.65,67 Moreover, increased gene and BMP7 secretion into the circulation (Figure 1C), which led to a protein expression of a BMP4 antagonist and decreased BMP4 down- reversal of body weight gain and augmented insulin sensitivity. stream signaling in subcutaneous WAT were also reported.65,67 Nevertheless, previous studies in our laboratory have demonstrated Similar results have been documented in human adipocytes from sub- that higher circulating levels of a therapeutic factor are required in cutaneous WAT of obese patients.68 BMP4 has also been reported to ob/ob mice to mediate similar therapeutic efficacy compared with be secreted by adipocytes68, which may be consistent with the that elicited in HFD-fed mice.49 Therefore, ob/ob mice were treated increased serum BMP4 levels observed in HFD-fed and db/db with a dose of AAV-BMP7 vectors 10-fold higher than that used in mice19 as well as obese humans.69–71 In contrast, age-dependent HFD-fed mice. ob/ob mice treated with 1  1013 vg of AAV8- decreased circulating BMP7 levels have been reported in HFD-fed hAAT-BMP7 per mouse showed markedly decreased body weight and db/db mice.19 Moreover, we found that the expression levels of and improved insulin sensitivity. endogenous Bmp7 were similar in iWAT of chow- and HFD-fed intraperitoneal injection of insulin (0.75 units insulin/kg body weight) 17 weeks post-AAV delivery. Results were calculated as the percentage of initial blood glucose levels (n = 8, chow AAV-null; n = 8, HFD AAV-null; and n = 7, HFD AAV-BMP7). (G) Glucose tolerance was performed 8 weeks post-AAV (1 g glucose/kg body weight) (n = 8/group). Fasted refers to 16 h of food deprivation. All values are expressed as the mean ± SEM. HFD, high-fat diet; FC, fold change. *p < 0.05, **p < 0.01, and ***p < 0.001. &p < 0.05 and &&&p < 0.001 versus the HFD-fed null-injected group. $p < 0.05, $$p < 0.01, and $$$p < 0.001 versus the chow-fed null-injected group. Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 197 Molecular Therapy: Methods & Clinical Development A B C D E F G H I J K L M Figure 5. Reduced obesity in ob/ob mice treated with AAV-BMP7 vectors. ob/ob mice were administered intravenously with 1  1013 vg of either AAV-BMP7 or AAV-null vector per mouse and followed up for 7 weeks (A) Follow-up of the body weight (n = 10, AAV-null, and n = 9, AAV-BMP7). (B) Serum BMP7 levels at 7 weeks after vector administration (n = 8–10/group). (C) Quantitative PCR analysis of omBMP7 expression in the liver, eWAT, gastrocnemius (Gastro), and kidney. The qPCR was performed with primers that specifically detected vector-derived BMP7 mRNA (omBMP7) (n = 6–10/group). (D) Liver BMP7 content 7 weeks after vector administration (n = 6–10/group). (E) Weight of the eWAT, iWAT, mesenteric white adipose tissue (mWAT), and iBAT (n = 6–10/group). (F) Representative images of the hematoxylin-eosin staining of eWAT, iWAT, and iBAT tissue sections obtained from ob/ ob animals injected with either null or BMP7-encoding AAV. Scale bars: 100 mm. (G) Serum adiponectin levels (n = 6–10/group). (H) Histogram depicting the mean food intake per day from week 13 to week 17 of age of ob/ob mice treated with either AAV-BMP7 or AAV-null vectors (n = 4 cages, AAV-null; n = 3 cages, AAV-BMP7; 1–3 mice/cage). (I– M) Quantitative PCR analysis in iWAT (I, J) or iBAT (K, L, M) of the expression of the thermogenic markers Ucp1 (I, K), Elovl3 (J, L), or Ppargc1a (M) (n = 6–10/group). All values are expressed as the mean ± SEM. ND, not detected; FC, fold change; AU, arbitrary units. *p < 0.05, **p < 0.01, and ***p < 0.001. 198 Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 www.moleculartherapy.org A B C D E F G H I Figure 6. Improved insulin sensitivity in ob/ob mice treated with AAV-BMP7 vectors (A) Liver weight (n = 6–10/group). (B) Liver weight normalized by body weight (n = 6–10/group). (C) Representative images of the hematoxylin-eosin staining of liver tissue sections obtained from ob/ob animals injected with either null or BMP7-encoding AAV. Scale bars: 200 mm. (D) Quantitative PCR analysis of the expression of the pro- inflammatory cytokines Tnfa, Ifng, Mcp1, and Ccl5 in liver (n = 6–10/group). (E) Fed blood glucose levels at 5 weeks after vector administration (n = 9–10/group). (F) Fed serum insulin levels at 6 weeks after vector administration (n = 9–10/group). (G) Insulin tolerance test after intraperitoneal injection of insulin at a dose of 0.75 units insulin/kg body weight. Results were calculated as the percentage of initial blood glucose levels (n = 10, AAV-null, and n = 6, AAV-BMP7). (H) Determination of phosphorylated GSK3b (p- GSK3b) protein abundance in the liver: immunoblot and densitometric analysis (n = 4–5/group). (I) Determination of phosphorylated p38 (p-p38) protein abundance in the liver: immunoblot and densitometric analysis (n = 5/group). All values are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. mice. These results suggest that in obesity and insulin resistance con- through leptin-independent mTOR pathways in the hypothalamus ditions BMP7 resistance would not develop. to reduce appetite.18 In contrast, although HFD-fed mice treated with AAV-BMP7 showed lower food intake, when this parameter The effect of AAV-BMP7 on body weight loss in ob/ob mice may also was normalized per body weight, these mice actually ate more than be in part due to a decrease in food intake. Similar results were chow-fed mice. In the Ad-BMP7 study,18 food intake data corrected observed in HFD-fed and ob/ob mice treated with Ad-BMP7.18 In by body weight are missing. Moreover, Ad-derived BMP7 serum this regard, BMP7 has been described as an anorectic factor, acting levels were higher than in AAV-BMP7-treated HFD-fed mice but Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 199 Molecular Therapy: Methods & Clinical Development similar to those exhibited by ob/ob mice treated with AAV-BMP7 and eye.22,23 Indeed, three of the seven gene therapy treatments vectors,18 suggesting that BMP7 may exert anorectic effects only at approved for commercialization are AAV-based therapies.23 Given very high circulating levels. that almost all natural AAV capsids can transduce liver efficiently following systemic administration,23 AAV vectors provide a robust Non-alcoholic fatty liver disease (NAFLD) and NASH are strongly liver-targeting platform to treat a variety of diseases. In this regard, associated with insulin resistance and T2D.72 In the present work, a total of 30 clinical trials for liver-directed AAV-mediated gene ther- HFD-fed AAV-null-treated mice exhibited increased liver weight and apy to treat rare metabolic conditions in lean patients are currently hepatic lipid content, as well as marked inflammation. AAV-BMP7- ongoing.80 It is also worth mentioning that BMP7 protein has already treated HFD-fed and ob/ob mice animals showed counteraction of liver been approved by the FDA for the treatment of bone diseases.14 steatosis and inflammation, as also observed using recombinant BMP7 Hence, the gene therapy strategy developed in this work may be envis- or Ad-BMP7 vectors.17,18 Adiponectin is a key insulin-sensitizing and aged as a novel approach for the future treatment of T2D and obesity. anti-inflammatory adipokine and is also associated with decreased he- patic steatosis.73–75 Improvement of insulin sensitivity in ob/ob and MATERIALS AND METHODS HFD-fed mice overexpressing Bmp7 in the liver could be mediated, Animals at least in part, by increased circulating adiponectin levels. In humans, Eight-week-old male C57BL/6J littermate mice and 11-week-old adiponectin levels are inversely correlated with the development of male B6.V-Lepob/OlaHsd (ob/ob) littermate mice obtained from En- non-alcoholic fatty liver disease and with the degree of insulin resis- vigo RMS SL (Spain) were used. Mice were kept in a specific-path- tance and T2D.76,77 Treatment of HFD-fed mice and rats with recom- ogen-free facility (SER-CBATEG, UAB) and maintained under a binant adiponectin or gene transfer of adiponectin using AAV vectors light-dark cycle of 12 h at 22 C. Mice were fed ad libitum with a decreased hepatic steatosis and increased insulin sensitivity.74,75,78,79 standard diet (standard chow diet, Harlan Teklad Global Diet Similarly, the increased circulating adiponectin levels in AAV-treated 2018, Envigo) or an HFD (TD.88137 Harlan Teklad). The HFD con- ob/ob and HFD-fed mice overexpressing Bmp7 in the liver may play tained 15.2% protein, 42.7% carbohydrate, and 42% fat. The exact an important role in the amelioration of liver and WAT inflammation, formula of the HFD included casein 195.0 g/kg, DL-methionine thus contributing to reduced liver steatosis, which would in turn 3.0 g/kg, sucrose 341.46 g/kg, corn starch 150.0 g/kg, anhydrous improve insulin sensitivity. milk fat 210.0 g/kg, cholesterol 1.5 g/kg, cellulose 50.0 g/kg, mineral mix AIN-76 (170915) 35.0 g/kg, calcium carbonate 4.0 g/kg, vitamin Recently, we have also demonstrated sustained amelioration of insu- mix Teklad (40060) 10.0 g/kg, and ethoxyquin 0.04 g/kg. When lin sensitivity in ob/ob mice upon specific genetic engineering of stated, mice were fasted for 16 h. For tissue sampling, mice were WAT with BMP7-encoding AAV vectors, which resulted in increased anesthetized with inhalational anesthetic isoflurane (IsoFlo, Abbott but low levels of BMP7 in the bloodstream15 compared with the circu- Laboratories, Abbott Park, IL, USA) and decapitated. Tissues of in- lating BMP7 levels observed in the present study or studies using terest were excised and kept at 80 C or in formalin until analysis. Ad-BMP7 vectors.16,18 In our previous study, AAV-mediated gene Animal care and experimental procedures were approved by the transfer of Bmp7 to WAT of ob/ob mice induced white adipogenesis, Ethics Committee in Animal and Human Experimentation of the which led to increased body weight but amelioration of insulin resis- Universitat Autònoma de Barcelona. tance.15 In contrast, in the same study, treatment of ob/ob mice with 5  1011 vg of liver-directed AAV8-hAAT-BMP7 vector per mouse Recombinant AAV vectors did not change adipogenesis despite similar low BMP7 circulating The AAV expression cassette was obtained by cloning, between the in- levels.15 This was consistent with BMP7-mediated induction of verted terminal repeats (ITRs) of AAV2, an optimized murine Bmp7 hyperplasic expansion of WAT in an autocrine/paracrine manner. coding sequence (omBmp7) under the control of a synthetic hybrid Altogether, these results suggest that BMP7 may elicit different effects liver-specific promoter composed of three copies of the HCR enhancer depending on the circulating levels and site of production. Moreover, from ApoE and the hAAT promoter (AAV-BMP7) A non-coding these studies highlight the need of high levels of circulating BMP7 to cassette carrying the synthetic HCR/hAAT promoter, but no transgene, mediate whole-body correction of fat-derived metabolic alterations was used to produce null vectors (AAV-null). Single-stranded AAV and normalization of body weight and insulin resistance in obese vectors of serotype 8 vectors were produced by triple transfection in mice. HEK293 cells and purified using an optimized CsCl gradient-based pu- rification protocol that renders vector preparations of high purity and In summary, all these results demonstrate the relevance of the liver- devoid of empty capsids.81 Viral genome titers were determined by directed AAV-BMP7-mediated gene therapy approach for the treat- PicoGreen using phage lambda DNA as the standard curve. ment of obesity and T2D. In this regard, AAV vectors have been used in clinical trials since the mid-1990s, and no adverse events Administration of AAV vectors related to these vectors have been described so far.22,23 More than For systemic administration, AAV vectors were diluted in 200 mL of 3,000 patients have been enrolled in these studies and administered 0.001% F68 Pluronic (Gibco) in PBS and injected via the tail vein. recombinant AAV vectors at different doses and through different HFD-fed animals were injected with 1  1012 vg/animal. ob/ob routes to target several organs, mainly liver, skeletal muscle, brain, mice were administered 1  1012 vg/animal. 200 Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 www.moleculartherapy.org Immunohistochemistry Rplp0, 50 -TCCCACCTTGTCTCCAGTCT-30 , 50 -ACTGGTCTAGGA Tissues were fixed for 12–24 h in 10% formalin, embedded in paraffin, CCCGAGAAG-30 . and sectioned. Sections were incubated overnight at 4 C with rat anti- Mac2 (CL8942AP; Cedarlane). Biotinylated rabbit anti-rat (E0467; Vector genome copy number Dako) was used as secondary antibody. The ABC peroxidase kit Tissue samples were digested overnight at 56  C in 300 mL of tissue lysis (Pierce) was used for immunodetection, and sections were counter- solution supplemented with proteinase K (0.2 mg/mL). Total DNA was stained in Mayer’s hematoxylin. isolated from supernatants with the MasterPure DNA purification kit (Lucigen). DNA was resuspended in distilled water and quantified using Measurement of the adipocyte area a NanoDrop ND-1000 spectrophotometer (NanoDrop). Vector A morphometric study of adipocyte size was performed in WAT genome copy number in 20 ng of total DNA was determined by quan- sections stained with hematoxylin-eosin. Adipocyte area was deter- titative PCR using LightCycler 480 Probes Master (Roche). Primers and mined as previously described.82 A minimum of seven animals per probes were designed for a specific sequence within the SV40 poly(A) group were used and at least 250 adipocytes per animal were cDNA: forward primer, 50 -AGCAATAGCATCACAAATTTCAC analyzed. AA-30 ; reverse primer, 50 -CAGACATGATAAGATACATTGATGA GTT-30 ; probe, /56-FAM/AGCATTTTTTT/ZEN/CACTGCATTCTA RNA analysis GTTGTGGTTTGTC/3IABkFQ/. A reference standard curve was built Total RNA was obtained from different tissues using an isolation re- from serial dilutions of a linearized plasmid bearing the hAAT pro- agent (Tripure, Roche, for liver and QIAzol, Qiagen, for adipose de- moter and the optimized Bmp7 cDNA spiked into 20 ng/mL non-trans- pots) and an RNeasy Minikit (Qiagen) and treated with DNase I (Qia- duced mouse genomic DNA. gen). One microgram of total RNA was used for cDNA synthesis with the Transcriptor First Strand cDNA synthesis kit (Roche Diagnostics) Western blot analysis using a combination of anchored-oligo(dT)18 and random hexamer eWAT and liver samples were homogenized in protein lysis buffer. priming at 55 C for 30 min according to the manufacturer’s instruc- Proteins were separated by 12% SDS-PAGE and analyzed by immu- tions. For gene expression quantification, retrotranscribed samples noblotting with anti-P38 (#9212; Cell Signaling), anti-P38-P (#9211; were diluted 1/10 and then 2 mL was used in triplicate qPCRs. Primers Cell Signaling), anti-SMAD1 (#6944; Cell Signaling), anti-SMAD1/5/ were used at a final concentration of 200 nM. Real-time qPCR was per- 8-P (#13820; Cell Signaling), anti-GSK3b (#12456; Cell Signaling), formed in a LightCycler 480 II (Roche) using the LightCycler 480 SyBr and anti-GSK3b-P (#5558; Cell Signaling) antibodies. Detection Green I Master Mix (Roche). Manufacturer standard cycling condi- was performed using ECL Plus detection reagent (Amersham tions were used: 10 s at 95 C, 10 s at 90 C, 10 s at 95 C for 45 cycles. Biosciences). Melting curve analysis was performed for each amplification to assess single product amplification. Data were normalized to Rplp0 expres- Hormone and metabolite assays sion. After normalization, relative expression was calculated using Hepatic triglyceride content was determined by chloroform:methanol the 2(DDC(T)) method.83 omBmp7 gene expression was calculated (2:1 vol/vol) extraction of total lipids, as described previously.84 He- after Rplp0 normalization and reported as arbitrary units, since it patic triglycerides and serum cholesterol were quantified spectropho- was not possible to perform relative quantification. The following tometrically using an enzymatic assay (Horiba-ABX) in a Pentra 400 Mus musculus primers were used: omBmp7, 50 -AGAGCATCAACCC analyzer (Horiba-ABX). Glycemia was determined using a Glucome- TAAGCTGG-30 , 50 -TGTTCTGGGATCTCTGCTTGC-30 ; endoge- ter Elite (Bayer) and insulin levels were measured using the Rat Insu- nous Bmp7, 50 -TACATGAACGCCACCAACCA-30 , 50 -ACAGAGAT lin ELISA kit (90010; Crystal Chem). Serum and liver BMP7 were GGCGTTGAGCTG-30 ; Ppargc1a, 50 -ATACCGCAAAGAGCACG determined using the Human BMP7 ELISA (DBP700; R&D Systems). AGAAG-30 , 50 -CTCAAGAGCAGCGAAAGCGTCACAG-30 ; Ucp1, The standard curve was conducted with the recombinant human 50 -GGATTGGCCTCTACGACTCAG-30 , 50 -TGTAGGCTGCCCAA BMP7 provided in the kit. The detection limit specified in the product TGAACA-30 ; Cidea, 50 -CCTACGACATCCGATGCACA-30 , 50 -GTA datasheet was 31.3 pg/mL. Adiponectin and leptin were determined TGTGCCCGCATAGACCA-30 ; Dio2, 50 -AATTATGCCTCGGAGA using the Mouse Adiponectin ELISA kit (80569; Crystal Chem) and AGACCG-30 , 50 -GGCAGTTGCCTAGTGAAAGGT-30 ; Elovl3, 50 -G the Mouse Leptin ELISA kit (90030; Crystal Chem), respectively. GACTTAAGGCCCTTTTTGG-30 , 50 -CCAACAACGATGAGCAAC AG-30 ; Glut4, 50 -TGCCCGAAAGAGTCTAAAGC-30 , 50 -TCCGT Insulin tolerance test TTCTCATCCTTCAGC; F4/80, 50 -CTTTGGCTATGGGCTTCCAG Insulin (Humulin Regular; Eli Lilly) was injected intraperitoneally at a TC-30 , 50 -GCAAGGAGGACAGAGTTTATC-30 ; Cd68, 50 -GGGGC dose of 0.75 IU/kg body weight to fed mice. Glycemia was measured TCTTGGGAACTACAC-30 , 50 -CAAGCCCTCTTTAAGCCCCA-30 ; in tail vein blood samples at the indicated time points. Mcp1, 50 -AACTGCATCTGCCCTAAGGTC-30 , 50 -AAGTGCTTGAG GTGGTTGTG; Tnfa, 50 -CTGTAGCCCACGTCGTAGC-30 , 50 -TT Glucose tolerance test GAGATCCATGCCGTTG-30 ; Ccl5, 50 -GTGCCCACGTCAAGGA Awake mice were fasted overnight (16 h) and administered an intra- GTATT-30 , 50 -CCCACTTCTTCTCTGGGTTGG-30 ; Ifng, 50 -AGAC peritoneal injection of glucose (1 g/kg body weight). Glycemia was AATCAGGCCATCAGCA, 50 -TGGACCTGTGGGTTGTTGAC-30 ; measured in tail vein blood samples at the indicated time points. Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 201 Molecular Therapy: Methods & Clinical Development Indirect calorimetry DECLARATION OF INTERESTS An indirect open circuit calorimeter (Oxylet; Panlab) was used to The authors declare no competing interests. monitor O2 consumption and CO2 production. Mice were individu- alized and acclimated to the metabolic chambers for 24 h. O2 con- REFERENCES sumption and CO2 production data were collected in each cage for 1. Duclos, M. (2016). Osteoarthritis, obesity and type 2 diabetes: the weight of waist 3 min, every 15 min, for 24 h during the light and dark cycles and circumference. Ann. Phys. Rehabil. Med. 59, 157–160. adjusted by body weight. Activity was recorded continuously for 2. Van Gaal, L.F., Mertens, I.L., and De Block, C.E. (2006). Mechanisms linking obesity with cardiovascular disease. Nature 444, 875–880. 24 h during the light and dark cycles. 3. Renehan, A.G., Tyson, M., Egger, M., Heller, R.F., and Zwahlen, M. (2008). Body- mass index and incidence of cancer: a systematic review and meta-analysis of pro- Statistical analysis spective observational studies. Lancet 371, 569–578. Sample size determination was based on previous experience with 4. Kim, B., and Feldman, E.L. (2015). Insulin resistance as a key link for the increased similar studies. Randomization was performed using the Excel func- risk of cognitive impairment in the metabolic syndrome. Exp. Mol. Med. 47, e149. tion Roundup() or by GraphPad QuickCalcs to allocate mice in each 5. Association, A.D. (2018). Classification and diagnosis of diabetes: standards of med- group. In addition, we tested that the mean body weight and the mean ical care in Diabetesd2018. Diabetes Care 41, S13–S27. glycemia were statistically not different for each experimental group 6. van Marken Lichtenbelt, W.D., Vanhommerig, J.W., Smulders, N.M., Drossaerts, prior to assignment to diet and/or treatment groups. All tests (ITT, J.M., Kemerink, G.J., Bouvy, N.D., Schrauwen, P., and Teule, G.J. (2009). Cold-acti- vated brown adipose tissue in healthy men. N. Engl. J. Med. 360, 1500–1508. GTT, etc.) and measurements (for example, adipocyte area) were per- 7. Cypess, A.M., Lehman, S., Williams, G., Tal, I., Rodman, D., Goldfine, A.B., Kuo, F.C., formed by investigators blinded to the treatment. All results are ex- Palmer, E.L., Tseng, Y.-H., Doria, A., et al. (2009). Identification and importance of pressed as the mean ± SEM. The GraphPad Prism 7 software was Brown adipose tissue in adult humans. N. Engl. J. Med. 360, 1509–1517. used for statistical analyses. Data were analyzed by one-way 8. Zhang, Z., Zhang, H., Li, B., Meng, X., Wang, J., Zhang, Y., Yao, S., Ma, Q., Jin, L., ANOVA with Tukey’s post hoc correction, except for those parame- Yang, J., et al. (2014). Berberine activates thermogenesis in white and brown adipose ters involving comparison of only two experimental groups, in which tissue. Nat. Commun. 5, 1–15. case an unpaired Student’s t test was used. Differences were consid- 9. Yin, N., Zhang, H., Ye, R., Dong, M., Lin, J., Zhou, H., Huang, Y., Chen, L., Jiang, X., ered significant when p < 0.05. Nagaoka, K., et al. (2019). Fluvastatin sodium ameliorates obesity through brown fat activation. Int. J. Mol. Sci. 20, 1622. https://doi.org/10.3390/ijms20071622. 10. Lee, P., Swarbrick, M.M., and Ho, K.K.Y. (2013). Brown adipose tissue in adult hu- SUPPLEMENTAL INFORMATION mans: a metabolic renaissance. Endocr. Rev. 34, 413–438. Supplemental information can be found online at https://doi.org/10. 11. Lee, P., Greenfield, J.R., Ho, K.K.Y., and Fulham, M.J. (2010). A critical appraisal of 1016/j.omtm.2022.03.007. the prevalence and metabolic significance of brown adipose tissue in adult humans. Am. J. Physiol. - Endocrinol. Metab. 299, E601–E606. https://doi.org/10.1152/aj- ACKNOWLEDGMENTS pendo.00298.2010. The authors thank Marta Moya, Maria Molas, Jennifer Barrero, Lídia 12. Thamer, C., Machann, J., Staiger, H., Müssig, K., Schwenzer, N., Ludescher, B., Machicao, F., Claussen, C., Fritsche, A., Schick, F., et al. (2010). Interscapular fat is Hernández, and Lorena Noya for technical assistance. This work was strongly associated with insulin resistance. J. Clin. Endocrinol. Metab. 95, 4736–4742. supported by grants from the Ministerio de Economía, Industria y 13. Oxburgh, L. (2009). Control of the bone morphogenetic protein 7 gene in develop- Competitividad (MINECO), the Agencia Estatal de Investigación mental and adult life. Curr. Genomics 10, 223–230. (AEI) and Fondo Europeo de Desarrollo Regional (FEDER, EU), 14. Carreira, A.C.O., Zambuzzi, W.F., Rossi, M.C., Filho, R.A., Sogayar, M.C., and the Programa Estatal de I+D+i Orientada a los Retos de la Sociedad Granjeiro, J.M. (2015). Bone morphogenetic proteins: promising molecules for (SAF2017-86266R), grant PID2020-113864RB-I00 funded by bone healing, bioengineering, and regenerative medicine. In The Bone Morphogenetic Proteins and Their Antagonists. Vitamins and Hormones, 99, MCIN/AEI/10.13039/501100011033, and the Generalitat de Catalu- Gerald Litwack, ed (Elsevier Inc.), pp. 293–322. nya (2017 SGR 1508, ICREA Academia Award to F.B.), Spain, and 15. Casana, E., Jimenez, V., Sacristan, V., Muñoz, S., Jambrina, C., Rodó, J., et al. (2021). the European Foundation for the Study of Diabetes (EFSD/MSD Eu- BMP7 overexpression in adipose tissue induces white adipogenesis and improves in- ropean Research Program on Novel Therapies for Type 2 Diabetes, sulin sensitivity in ob/ob mice. Int. J. Obes. 449–460. 2013). V.J. was the recipient of a post-doctoral research fellowship 16. Tseng, Y.-H., Kokkotou, E., Schulz, T.J., Huang, T.L., Winnay, J.N., Taniguchi, C.M., from EFSD/Lilly. E.C., V.S., and C.M. each received a predoctoral Tran, T.T., Suzuki, R., Espinoza, D.O., Yamamoto, Y., et al. (2008). New role of bone fellowship from the Ministerio de Educación, Cultura y Deporte, morphogenetic protein 7 in brown adipogenesis and energy expenditure. Nature 454, 1000–1004. J.R. from the Ministerio de Economía y Competitividad, and I.G. 17. Boon, M.R., van den Berg, S.A.A., Wang, Y., van den Bossche, J., Karkampouna, S., from Ministerio de Ciencia e Innovación, Spain. V.S. received a pre- Bauwens, M., De Saint-Hubert, M., van der Horst, G., Vukicevic, S., de Winther, doctoral fellowship from the Generalitat de Catalunya, Spain. M.P.J., et al. (2013). BMP7 activates Brown adipose tissue and reduces diet-induced obesity only at subthermoneutrality. PLoS One 8, e74083. AUTHOR CONTRIBUTIONS 18. Townsend, K.L., Suzuki, R., Huang, T.L., Jing, E., Schulz, T.J., Lee, K., Taniguchi, E.C., V.J., and F.B. designed and supervised experiments, analyzed C.M., Espinoza, D.O., McDougall, L.E., Zhang, H., et al. (2012). Bone morphogenetic protein 7 (BMP7) reverses obesity and regulates appetite through a central mTOR data, contributed to discussion, and wrote and edited the manuscript. pathway. FASEB J. 7, 1–10. E.C., V.J., C.J., V.S., S.M., J.R., M.G., I.G., and C.M. generated reagents 19. Chattopadhyay, T., Singh, R.R., Gupta, S., and Surolia, A. (2017). Bone morphoge- and performed experiments. X.L. produced AAV vectors. A.C., V.S., netic protein-7 (BMP-7) augments insulin sensitivity in mice with type II diabetes T.F., and S.M. performed experiments. S.F. contributed to discussion. mellitus by potentiating PI3K/AKT pathway. BioFactors 43, 195–209. 202 Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 www.moleculartherapy.org 20. Romano, G. (2006). The controversial role of adenoviral-derived vectors in gene ther- 41. Ruzo, A., Marcó, S., García, M., Villacampa, P., Ribera, A., Ayuso, E., Maggioni, L., apy programs: where do we stand? Drug News Perspect. 19, 99. Mingozzi, F., Haurigot, V., and Bosch, F. (2012). Correction of pathological accumu- 21. Descamps, D., and Benihoud, K. (2009). Two key challenges for effective adenovirus- lation of glycosaminoglycans in central nervous system and peripheral tissues of mediated liver gene therapy:innate immune responses and hepatocyte-specific trans- MPSIIIA mice through systemic AAV9 gene transfer. Hum. Gene Ther. 23, 1237– duction. Curr. Gene Ther. 9, 115–127. 1246. 22. Kuzmin, D.A., Shutova, M.V., Johnston, N.R., Smith, O.P., Fedorin, V.V., Kukushkin, 42. Haurigot, V., Marcó, S., Ribera, A., Garcia, M., Ruzo, A., Villacampa, P., Ayuso, E., Y.S., van der Loo, J.C.M., and Johnstone, E.C. (2021). The clinical landscape for AAV Añor, S., Andaluz, A., Pineda, M., et al. (2013). Whole body correction of mucopo- gene therapies. Nat. Rev. Drug Discov. 20, 173–174. lysaccharidosis IIIA by intracerebrospinal fluid gene therapy. J. Clin. Invest. 123, 3254–3271. 23. Mendell, J.R., Al-Zaidy, S.A., Rodino-Klapac, L.R., Goodspeed, K., Gray, S.J., Kay, C.N., Boye, S.L., Boye, S.E., George, L.A., Salabarria, S., et al. (2021). Current clinical 43. Nathwani, A.C., Cochrane, M., McIntosh, J., Ng, C.Y.C., Zhou, J., Gray, J.T., and applications of in vivo gene therapy with AAVs. Mol. Ther. 29, 464–488. Davidoff, A.M. (2009). Enhancing transduction of the liver by adeno-associated viral vectors. Gene Ther. 16, 60–69. 24. Petanceska, S.S., DeRosa, S., Sharma, A., Diaz, N., Duff, K., Tint, S.G., Refolo, L.M., and Pappolla, M. (2003). Changes in apolipoprotein E expression in response to di- 44. Berraondo, P., Crettaz, J., Ochoa, L., Pañeda, A., Prieto, J., Trocóniz, I.F., and etary and pharmacological modulation of cholesterol. J. Mol. Neurosci. 20, 395–406. González-Aseguinolaza, G. (2006). Intrahepatic injection of recombinant adeno- associated virus serotype 2 overcomes gender-related differences in liver transduc- 25. Lin-Lee, Y.C., Tanaka, Y., Lin, C.T., and Chan, L. (1981). Effects of an atherogenic diet tion. Hum. Gene Ther. 17, 601–610. on apolipoprotein E biosynthesis in the rat. Biochemistry 20, 6474–6480. 45. Wang, L., and Herzog, R. (2005). AAV-mediated gene transfer for treatment of he- 26. Driscoll, D.M., Mazzone, T., Matsushima, T., and Getz, G.S. (1990). Apoprotein E mophilia. Curr. Gene Ther. 5, 349–360. biosynthesis in the cholesterol-fed Guinea pig. Arteriosclerosis 10, 31–39. 46. Ruzo, A., Marcó, S., García, M., Villacampa, P., Ribera, A., Ayuso, E., Maggioni, L., 27. Garcia, R.E., Beck, D.L., Wright, D.A., Tomlinson, J.E., Nakayama, R., and Holten, D. Mingozzi, F., Haurigot, V., and Bosch, F. (2012). Correction of pathological accumu- (1984). Effect of dietary cholesterol on apolipoprotein E synthesis in the rabbit. lation of glycosaminoglycans in central nervous system and peripheral tissues of Atherosclerosis 51, 199–210. MPSIIIA mice through systemic AAV9 gene transfer. Hum. Gene Ther. 23, 1237– 28. Li, Y., Zou, S., Ding, H., Hao, N., Huang, Y., Tang, J., Cheng, J., Feng, S., Li, J., Wang, 1246. X., et al. (2020). Low expression of sirtuin 1 in the dairy cows with mild fatty liver 47. Binny, C., McIntosh, J., Della Peruta, M., Kymalainen, H., Tuddenham, E.G.D., alters hepatic lipid metabolism. Anim. Open Access J. MDPI 10, 560. Buckley, S.M.K., Waddington, S.N., McVey, J.H., Spence, Y., Morton, C.L., et al. 29. Özkaynak, E., Schnegelsberg, P.N.J., and Oppermann, H. (1991). Murine osteogenic (2012). AAV-mediated gene transfer in the perinatal period results in expression protein (OP-1): high levels of mRNA in kidney. Biochem. Biophys. Res. Commun. of FVII at levels that protect against fatal spontaneous hemorrhage. Blood 119, 179, 116–123. 957–966. 30. Hajer, G.R., van Haeften, T.W., and Visseren, F.L.J. (2008). Adipose tissue dysfunc- 48. Greig, J.A., Limberis, M.P., Bell, P., Chen, S.J., Calcedo, R., Rader, D.J., and Wilson, tion in obesity, diabetes, and vascular diseases. Eur. Heart J. 29, 2959–2971. J.M. (2017). Non-clinical study examining AAV8.TBG.hLDLR vector-associated 31. Townsend, K.L., An, D., Lynes, M.D., Huang, T.L., Zhang, H., Goodyear, L.J., and toxicity in chow-fed wild-type and LDLR +/- rhesus macaques. Hum. Gene Ther. Tseng, Y.H. (2013). Increased mitochondrial activity in BMP7-treated brown adipo- Clin. Dev. 28, 39–50. cytes, due to increased CPT1-and CD36-mediated fatty acid uptake. Antioxid. Redox 49. Jimenez, V., Jambrina, C., Casana, E., Sacristan, V., Muñoz, S., Darriba, S., Rodó, J., Signal 19, 243–257. Mallol, C., Garcia, M., León, X., et al. (2018). FGF21 gene therapy as treatment for 32. Lee, M.J. (2018). Transforming growth factor beta superfamily regulation of adipose obesity and insulin resistance. EMBO Mol. Med. 10, e8791. tissue biology in obesity. Biochim. Biophys. Acta. Mol. Basis Dis. 1864, 1160–1171. 50. Shaw, A., Tóth, B.B., Arianti, R., Csomós, I., Póliska, S., Vámos, A., Bacso, Z., Gy}ory, 33. Fellmann, L., Nascimento, A.R., Tibiriça, E., and Bousquet, P. (2013). Murine models F., Fésüs, L., and Kristóf, E. (2021). Bmp7 increases ucp1-dependent and independent for pharmacological studies of the metabolic syndrome. Pharmacol. Ther. 137, thermogenesis with a unique gene expression program in human neck area derived 331–340. adipocytes. Pharmaceuticals 14, 1078. 34. Ma, H., Yuan, J., Ma, J., Ding, J., Lin, W., Wang, X., Zhang, M., Sun, Y., Wu, R., Liu, 51. Schulz, T.J., Huang, T.L., Tran, T.T., Zhang, H., Townsend, K.L., Shadrach, J.L., C., et al. (2019). BMP7 improves insulin signal transduction in the liver via inhibition Cerletti, M., McDougall, L.E., Giorgadze, N., Tchkonia, T., et al. (2011). of mitogen-activated protein kinases. J. Endocrinol. 243, 97–110. Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat. Proc. Natl. Acad. Sci. U S A 108, 143–148. 35. Fuchs, T., Loureiro, M. de P., Macedo, L.E., Nocca, D., Nedelcu, M., and Costa- Casagrande, T.A. (2018). Animal models in metabolic syndrome. Rev. Col. Bras. 52. Okla, M., Ha, J.H., Temel, R.E., and Chung, S. (2015). BMP7 drives human adipo- Cir. 45, e1975. genic stem cells into metabolically active beige adipocytes. Lipids 50, 111–120. 36. Davidoff, A.M., Ng, C.Y.C., Zhou, J., Spence, Y., and Nathwani, A.C. (2003). Sex 53. Ferrannini, G., Namwanje, M., Fang, B., Damle, M., Li, D., Liu, Q., Lazar, M.A., and significantly influences transduction of murine liver by recombinant adeno-associ- Qiang, L. (2016). Genetic backgrounds determine brown remodeling of white fat in ated viral vectors through an androgen-dependent pathway. Blood 102, 480–488. rodents. Mol. Metab. 5, 948–958. 37. Ruzo, A., Garcia, M., Ribera, A., Villacampa, P., Haurigot, V., Marcó, S., Ayuso, E., 54. Liu, B., Page, A.J., Hutchison, A.T., Wittert, G.A., and Heilbronn, L.K. (2019). Anguela, X.M., Roca, C., Agudo, J., et al. (2012). Liver production of sulfamidase re- Intermittent fasting increases energy expenditure and promotes adipose tissue verses peripheral and ameliorates CNS pathology in mucopolysaccharidosis IIIA browning in mice. Nutrition 66, 38–43. mice. Mol. Ther. 20, 254–266. 55. Kim, Y.H., Lee, J.H., Yeung, J.L.-H., Das, E., Kim, R.Y., Jiang, Y., Moon, J.H., Jeong, 38. Ribera, A., Haurigot, V., Garcia, M., Marcó, S., Motas, S., Villacampa, P., Maggioni, L., H., Thakkar, N., Son, J.E., et al. (2019). Thermogenesis-independent metabolic ben- León, X., Molas, M., Sánchez, V., et al. (2015). Biochemical, histological and func- efits conferred by isocaloric intermittent fasting in ob/ob mice. Sci. Rep. 9, 2479. tional correction of mucopolysaccharidosis type IIIB by intra-cerebrospinal fluid 56. Li, G., Xie, C., Lu, S., Nichols, R.G., Tian, Y., Li, L., Patel, D., Ma, Y., Brocker, C.N., gene therapy. Hum. Mol. Genet. 24, 2078–2095. Yan, T., et al. (2017). Intermittent fasting promotes white adipose browning and de- 39. Guenzel, A.J., Collard, R., Kraus, J.P., Matern, D., and Barry, M.A. (2015). Long-term creases obesity by shaping the gut microbiota. Cell Metab 26, 672–685.e4. sex-biased correction of circulating propionic acidemia disease markers by adeno- 57. Collins, S., Daniel, K.W., Petro, A.E., and Surwit, R.S. (1997). Strain-specific response associated virus vectors. Hum. Gene Ther. 26, 153–160. tob 3 -adrenergic receptor agonist treatment of diet-induced obesity in mice 1. 40. Pañeda, A., Vanrell, L., Mauleon, I., Crettaz, J.S., Berraondo, P., Timmermans, E.J., Endocrinology 138, 405–413. Beattie, S.G., Twisk, J., Van Deventer, S., Prieto, J., et al. (2009). Effect of adeno-asso- 58. Guerra, C., Koza, R.A., Yamashita, H., Walsh, K., and Kozak, L.P. (1998). Emergence ciated virus serotype and genomic structure on liver transduction and biodistribution of brown adipocytes in white fat in mice is under genetic control effects on body in mice of both genders. Hum. Gene Ther. 20, 908–917. weight and adiposity. J. Clin. Invest. 102, 412–420. Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022 203 Molecular Therapy: Methods & Clinical Development 59. Cypess, A.M., Lehman, S., Williams, G., Tal, I., Rodman, D., Goldfine, A.B., Kuo, F.C., 72. Kitade, H., Chen, G., Ni, Y., and Ota, T. (2017). Nonalcoholic fatty liver disease and Palmer, E.L., Tseng, Y.-H., Doria, A., et al. (2009). Identification and importance of insulin resistance: new insights and potential new treatments. Nutrients 9, 387. Brown adipose tissue in adult humans. N. Engl. J. Med. 360, 1509–1517. 73. Kim, J.Y., Van De Wall, E., Laplante, M., Azzara, A., Trujillo, M.E., Hofmann, S.M., 60. Jespersen, N.Z., Larsen, T.J., Peijs, L., Daugaard, S., Homøe, P., Loft, A., De Jong, J., Schraw, T., Durand, J.L., Li, H., Li, G., et al. (2007). Obesity-associated improvements Mathur, N., Cannon, B., Nedergaard, J., et al. (2013). A classical brown adipose tissue in metabolic profile through expansion of adipose tissue. J. Clin. Invest. 117, 2621– mrna signature partly overlaps with brite in the supraclavicular region of adult hu- 2637. mans. Cell Metab 17, 798–805. 74. Shklyaev, S., Aslanidi, G., Tennant, M., Prima, V., Kohlbrenner, E., Kroutov, V., 61. Lidell, M.E., Betz, M.J., and Enerbäck, S. (2014). Two types of brown adipose tissue in Campbell-Thompson, M., Crawford, J., Shek, E.W., Scarpace, P.J., et al. (2003). humans. Adipocyte 3, 63–66. Sustained peripheral expression of transgene adiponectin offsets the development 62. Frontini, A., Vitali, A., Perugini, J., Murano, I., Romiti, C., Ricquier, D., Guerrieri, M., of diet-induced obesity in rats. Proc. Natl. Acad. Sci. U S A 100, 14217–14222. and Cinti, S. (2013). White-to-brown transdifferentiation of omental adipocytes in patients affected by pheochromocytoma. Biochim. Biophys. Acta 1831, 950–959. 75. Long, W., Hui Ju, Z., Fan, Z., Jing, W., and Qiong, L. (2014). The effect of recombi- nant adeno-associated virus-adiponectin (rAAV2/1-Acrp30) on glycolipid dysme- 63. Li, S., Li, Y., Xiang, L., Dong, J., Liu, M., and Xiang, G. (2018). Sildenafil induces tabolism and liver morphology in diabetic rats. Gen. Comp. Endocrinol. 206, 1–7. browning of subcutaneous white adipose tissue in overweight adults. Metabolism 78, 106–117. 76. Cnop, M., Havel, P.J., Utzschneider, K.M., Carr, D.B., Sinha, M.K., Boyko, E.J., 64. Sidossis, L.S., Porter, C., Saraf, M.K., Børsheim, E., Radhakrishnan, R.S., Chao, T., Ali, Retzlaff, B.M., Knopp, R.H., Brunzell, J.D., and Kahn, S.E. (2003). Relationship of adi- A., Chondronikola, M., Mlcak, R., Finnerty, C.C., et al. (2015). Browning of subcu- ponectin to body fat distribution, insulin sensitivity and plasma lipoproteins: evi- taneous white adipose tissue in humans after severe adrenergic stress. Cell Metab. dence for independent roles of age and sex. Diabetologia 46, 459–469. 22, 219–227. 77. Ma, H., Gomez, V., Lu, L., Yang, X., Wu, X., and Xiao, S.Y. (2009). Expression of adi- 65. Hoffmann, J.M., Grünberg, J.R., Church, C., Elias, I., Palsdottir, V., Jansson, J.-O., ponectin and its receptors in livers of morbidly obese patients with non-alcoholic Bosch, F., Hammarstedt, A., Hedjazifar, S., and Smith, U. (2017). BMP4 gene therapy fatty liver disease. J. Gastroenterol. Hepatol. 24, 233–237. in mature mice reduces BAT activation but protects from obesity by browning sub- 78. Xu, A., Wang, Y., Keshaw, H., Xu, L.Y., Lam, K.S.L., and Cooper, G.J.S. (2003). The cutaneous adipose tissue. Cell Rep. 20, 1038–1049. fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver dis- 66. Hoffmann, J.M., Grünberg, J.R., Hammarstedt, A., Kroon, T., Greiner, T.U., Maurer, eases in mice. J. Clin. Invest. 112, 91–100. S., Elias, I., Palsdottir, V., Bosch, F., Boucher, J., et al. (2020). BMP4 gene therapy en- hances insulin sensitivity but not adipose tissue browning in obese mice. Mol. Metab. 79. Ma, H., Cui, F., Dong, J.-J., You, G.-P., Yang, X.-J., Lu, H.-D., and Huang, Y.-L. 32, 15–26. (2014). Therapeutic effects of globular adiponectin in diabetic rats with nonalcoholic fatty liver disease. World J. Gastroenterol. 20, 14950–14957. 67. Hoffmann, J.M., Grünberg, J.R., Hammarstedt, A., Kroon, T., Greiner, T.U., Maurer, S., Elias, I., Palsdottir, V., Bosch, F., Boucher, J., et al. (2020). BMP4 gene therapy en- 80. Piccolo, P., Rossi, A., and Brunetti-Pierri, N. (2021). Liver-directed gene-based ther- hances insulin sensitivity but not adipose tissue browning in obese mice. Mol. Metab. apies for inborn errors of metabolism. Expert Opin. Biol. Ther. 21, 229–240. 32, 15–26. 81. Ayuso, E., Mingozzi, F., Montane, J., Leon, X., Anguela, X.M., Haurigot, V., 68. Gustafson, B., Hammarstedt, A., Hedjazifar, S., Hoffmann, J.M., Svensson, P.A., Edmonson, S.A., Africa, L., Zhou, S., High, K.A., et al. (2010). High AAV vector purity Grimsby, J., Rondinone, C., and Smith, U. (2015). BMP4 and BMP antagonists regu- results in serotype- and tissue-independent enhancement of transduction efficiency. late human white and beige adipogenesis. Diabetes 64, 1670–1681. Gene Ther. 17, 503–510. 69. Son, J.W., Kim, M.K., Park, Y.M., Baek, K.H., Yoo, S.J., Song, K.H., Son, H.S., Yoon, 82. Muñoz, S., Franckhauser, S., Elias, I., Ferré, T., Hidalgo, A., Monteys, A.M., Molas, K.H., Lee, W.C., Cha, B.Y., et al. (2011). Association of serum bone morphogenetic M., Cerdán, S., Pujol, A., Ruberte, J., et al. (2010). Chronically increased glucose up- protein 4 levels with obesity and metabolic syndrome in non-diabetic individuals. take by adipose tissue leads to lactate production and improved insulin sensitivity Endocr. J. 58, 39–46. rather than obesity in the mouse. Diabetologia 53, 2417–2430. 70. Modica, S., Straub, L.G., Balaz, M., Sun, W., Wolfrum, C., Varga, L., Ukropcova, B., Ukropec, J., Varga, L., Stefanicka, P., et al. (2016). Bmp4 promotes a Brown to white- 83. Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data us- like adipocyte shift. Cell Rep 16, 2243–2258. ing real-time quantitative PCR and the 2DDCT method. Methods 25, 402–408. 71. Wang, X., Chen, J., Li, L., Zhu, C.L., Gao, J., Rampersad, S., Bu, L., and Qu, S. (2017). 84. Carr, T.P., Andresen, C.J., and Rudel, L.L. (1993). Enzymatic determination of triglyc- New association of bone morphogenetic protein 4 concentrations with fat distribu- eride, free cholesterol, and total cholesterol in tissue lipid extracts. Clin. Biochem. 26, tion in obesity and Exenatide intervention on it. Lipids Health Dis. 16, 70. 39–42. 204 Molecular Therapy: Methods & Clinical Development Vol. 25 June 2022