High-throughput mediation analysis of human proteome and metabolome identifies mediators of post-bariatric surgical diabetes control

To improve the power of mediation in high-throughput studies, here we introduce High-throughput mediation analysis (Hitman), which accounts for direction of mediation and applies empirical Bayesian linear modeling. We apply Hitman in a retrospective, exploratory analysis of the SLIMM-T2D clinical trial in which participants with type 2 diabetes were randomized to Roux-en-Y gastric bypass (RYGB) or nonsurgical diabetes/weight management, and fasting plasma proteome and metabolome were assayed up to 3 years. RYGB caused greater improvement in HbA1c, which was mediated by growth hormone receptor (GHR). GHR’s mediation is more significant than clinical mediators, including BMI. GHR decreases at 3 months postoperatively alongside increased insulin-like growth factor binding proteins IGFBP1/BP2; plasma GH increased at 1 year. Experimental validation indicates (1) hepatic GHR expression decreases in post-bariatric rats; (2) GHR knockdown in primary hepatocytes decreases gluconeogenic gene expression and glucose production. Thus, RYGB may induce resistance to diabetogenic effects of GH signaling

GPR180 is a component of TGFβ signalling that promotes thermogenic adipocyte function and mediates the metabolic effects of the adipocyte-secreted factor CTHRC1

Activation of thermogenic brown and beige adipocytes is considered as a strategy to improve metabolic control. Here, we identify GPR180 as a receptor regulating brown and beige adipocyte function and whole-body glucose homeostasis, whose expression in humans is associated with improved metabolic control. We demonstrate that GPR180 is not a GPCR but a component of the TGF beta signalling pathway and regulates the activity of the TGF beta receptor complex through SMAD3 phosphorylation. In addition, using genetic and pharmacological tools, we provide evidence that GPR180 is required to manifest Collagen triple helix repeat containing 1 (CTHRC1) action to regulate brown and beige adipocyte activity and glucose homeostasis. In this work, we show that CTHRC1/GPR180 signalling integrates into the TGF beta signalling as an alternative axis to fine-tune and achieve low-grade activation of the pathway to prevent pathophysiological response while contributing to control of glucose and energy metabolism.Activation of thermogenic adipocytes is a strategy to combat metabolic diseases. Here the authors report that GPR180 is a component of TGF beta signalling that promotes thermogenic adipocyte function and mediates the metabolic effects of the adipocyte-secreted factor CTHRC1, and contributes to the regulation of glucose and energy metabolism

SRF and MKL1 Independently Inhibit Brown Adipogenesis

<div><p>Active brown adipose tissue is responsible for non-shivering thermogenesis in mammals which affects energy homeostasis. The molecular mechanisms underlying this activation as well as the formation and activation of brite adipocytes have gained increasing interest in recent years as they might be utilized to regulate systemic metabolism. We show here that the transcriptional regulators SRF and MKL1 both act as repressors of brown adipogenesis. Loss-of-function of these transcription factors leads to a significant induction of brown adipocyte differentiation, increased levels of UCP1 and other thermogenic genes as well as increased respiratory function, while SRF induction exerts the opposite effects. Interestingly, we observed that knockdown of MKL1 does not lead to a reduced expression of typical SRF target genes and that the SRF/MKL1 inhibitor CCG-1423 had no significant effects on brown adipocyte differentiation. Contrary, knockdown of MKL1 induces a significant increase in the transcriptional activity of PPARγ target genes and MKL1 interacts with PPARγ, suggesting that SRF and MKL1 independently inhibit brown adipogenesis and that MKL1 exerts its effect mainly by modulating PPARγ activity.</p></div

Loss of the SRF cofactor MKL1 induces brown adipogenesis.

<p>(A) Relative mRNA expression of SRF cofactors in preadipocytes and mature brown adipocytes. Data are shown as mean + SD, n = 4. (B-H) Brown preadipocytes were with the respective siRNA pools 2 days prior to differentiation, to knockdown SRF and SRF cofactors. Analysis of (B) fluorescent staining was used to calculate (C) percentage of lipid-positive cells and (D) percentage of UCP1-positive cells at day 7. Mean + SEM, n = 6. (E) Protein levels of SRF and MKL1 after siRNA-mediated knockdown in brown adipocytes. (F) Western blot analysis of UCP1 protein levels of mature brown adipocytes upon siRNA-mediated knockdown of MKL1 (G) Seahorse analysis of OCR measurements of mature brown adipocytes upon siRNA-mediated knockdown of MKL1. Data are presented as mean + SEM, n = 6. (H) Effect of knockdown of SRF cofactors on relative mRNA expression of thermogenic genes. Mean + SD, n = 6. * denotes p-value <0.05, ** denotes p-value <0.01 and *** denotes p-value <0.005 vs. control.</p

Loss of the SRF cofactor MKL1 induces brown adipogenesis from white adipocyte precursors.

<p>(A) SRF and MKL1 are downregulated upon induction of differentiation of subcutaneous and epididymal derived cell lines. (B-G) White immortalized preadipocytes derived from epididymal white adipose tissue were treated with the respective siRNA pools 2 days prior to differentiation, to knockdown SRF and SRF cofactors. Analysis of (B) fluorescent staining was used to calculate (C) percentage of lipid-positive cells and (D) percentage of UCP1-positive cells at day 7. Data are shown as mean + SEM, n = 6. (E) Effect of knockdown of SRF cofactors on relative mRNA expression of indicated thermogenic genes normalized to 36B4 measured by qPCR. Data are shown as mean + SD, n = 6. (F) Effect of knockdown of MKL1 and SRF on relative mRNA expression of indicated thermogenic genes normalized to 36B4. Data are shown as mean + SD, n = 6. (G) Seahorse analysis of OCR measurements of mature white adipocytes upon siRNA-mediated knockdown of SRF and MKL1. Data are shown as mean + SEM, n = 6. * denotes p-value <0.05, ** denotes p-value <0.01 and *** denotes p-value <0.005 vs. control.</p

MKL1 effect on brown/white adipocyte differentiation is not mediated through SRF but rather by regulating PPARγ transcriptional activity.

<p>(A-B) Relative mRNA expression of typical SRF target genes upon siRNA-mediated knockdown of SRF and MKL1 in (A) brown and (B) white immortalized adipocytes. Data are shown as mean + SD, n = 6–8. (C) Luciferase activity from an SRE-Luc construct upon SRF and Mkl1 knockdown. Data are shown as mean + SD, n = 8. (D) Treatment of brown and white pre-adipocytes with indicated concentrations of CCG-1423 from day -2 to day 2 of differentiation. Evaluation of percentage of differentiated cells (left panel) and UCP1-positive cells (right panel). Data are shown as mean ± SD, n = 6. (E) Upregulation of typical PPARγ target genes upon MKL1 knockdown. Data are shown as mean + SD, n = 6. (F) Luciferase activity from a PPRE-containing reporter upon Mkl1 knockdown. Data are shown as mean + SD, n = 6. (G) Co-immunoprecipitation of MKL1 and PPARγ from nuclear extracts of immortalized brown adipocytes on day 4 of differentiation. (H) Co-immunoprecipitation of MKL1 and PPARγ from nuclear extracts of 3T3-L1 adipocytes on day 4 of differentiation. * denotes p-value <0.05, ** denotes p-value <0.01 and *** denotes p-value <0.005 vs. control.</p

SRF inhibits brown adipogenesis from brown pre-adipocytes.

<p>(A) Cumulative distribution function (CDF) and (B) Heat Map demonstrating that mRNA expression of the targets activated by SRF is differentially regulated in brown adipose tissue stromal vascular fraction compared to mature brown adipocytes. (C-G) Brown preadipocytes were treated with siRNA pools against SRF 2 days prior to differentiation, to knockdown SRF. Analysis of (C) fluorescent staining was used to calculate (D) percentage of lipid-positive cells and (E) percentage of UCP1-positive cells at day 7. Data are shown as mean + SEM, n = 6. (F) Western blot analysis of UCP1 protein levels of mature brown adipocytes, HSP90 served as loading control. (G) Seahorse analysis of OCR measurements of mature brown adipocytes upon siRNA-mediated knockdown of SRF. Data are presented as mean + SEM, n = 6. (H) mRNA expression level of SRF during <i>in vitro</i> differentiation of immortalized brown preadipocytes. Data are shown as mean ± SEM, n = 6. (I-K) SRF gain-of-function analysis using adenovirus-mediated overexpression in primary brown preadipocytes 2 days prior to differentiation inhibits brown adipocyte formation. (I) Percentage of differentiated lipid-positive cells. (J) Percentage of UCP1-positive cells. (K) mRNA expression levels of thermogenic genes. Data are shown as mean + SD, n = 6. * denotes p-value <0.05, ** denotes p-value <0.01 and *** denotes p-value <0.005 vs. control.</p

Mapping the transcriptional landscape of human white and brown adipogenesis using single-nuclei RNA-seq.

Adipogenesis is key to maintaining organism-wide energy balance and healthy metabolic phenotype, making it critical to thoroughly comprehend its molecular regulation in humans. By single-nuclei RNA-sequencing (snRNA-seq) of over 20,000 differentiating white and brown preadipocytes, we constructed a high-resolution temporal transcriptional landscape of human white and brown adipogenesis. White and brown preadipocytes were isolated from a single individual's neck region, thereby eliminating inter-subject variability across two distinct lineages. These preadipocytes were also immortalized to allow for controlled, in&nbsp;vitro differentiation, allowing sampling of distinct cellular states across the spectrum of adipogenic progression. Pseudotemporal cellular ordering revealed the dynamics of ECM remodeling during early adipogenesis, and lipogenic/thermogenic response during late white/brown adipogenesis. Comparison with adipogenic regulation in murine models Identified several novel transcription factors as potential targets for adipogenic/thermogenic drivers in humans. Among these novel candidates, we explored the role of TRPS1 in adipocyte differentiation and showed that its knockdown impairs white adipogenesis in&nbsp;vitro. Key adipogenic and lipogenic markers revealed in our analysis were applied to analyze publicly available scRNA-seq datasets; these confirmed unique cell maturation features in recently discovered murine preadipocytes, and revealed inhibition of adipogenic expansion in humans with obesity. Overall, our study presents a comprehensive molecular description of both white and brown adipogenesis in humans and provides an important resource for future studies of adipose tissue development and function in both health and metabolic disease state

Maternal overnutrition programs hedonic and metabolic phenotypes across generations through sperm tsRNAs

There is a growing body of evidence linking maternal overnutri-tion to obesity and psychopathology that can be conserved acrossmultiple generations. Recently, we demonstrated in a maternalhigh-fat diet (HFD; MHFD) mouse model that MHFD inducedenhanced hedonic behaviors and obesogenic phenotypes thatwere conserved across three generations via the paternal lineage,which was independent of sperm methylome changes. Here, weshow that sperm tRNA-derived small RNAs (tsRNAs) partly contrib-ute to the transmission of such phenotypes. We observe increasedexpression of sperm tsRNAs in the F1 male offspring born to HFD-exposed dams. Microinjection of sperm tsRNAs from the F1-HFDmale into normal zygotes reproduces obesogenic phenotypes andaddictive-like behaviors, such as increased preference of palatablefoods and enhanced sensitivity to drugs of abuse in the resultantoffspring. The expression of several of the differentially expressedsperm tsRNAs predicted targets such asCHRNA2and GRIN3A, whichhave been implicated in addiction pathology, are altered in themesolimbic reward brain regions of the F1-HFD father and the re-sultant HFD-tsRNA offspring. Together, our findings demonstratethat sperm tsRNA is a potential vector that contributes to the trans-mission of MHFD-induced addictive-like behaviors and obesogenicphenotypes across generations, thereby emphasizing its role in di-verse pathological outcomes.ISSN:0027-8424ISSN:1091-649

Maternal overnutrition programs hedonic and metabolic phenotypes across generations through sperm tsRNAs

There is a growing body of evidence linking maternal overnutrition overnutrition to obesity and psychopathology that can be conserved across multiple generations. Recently Recently, we demonstrated in a maternal high-fat diet (HFD; MHFD) mouse model that MHFD induced enhanced enhanced hedonic behaviors and obesogenic phenotypes that were conserved across three generations via the paternal lineage, which was independent of sperm methylome changes. Here, we show that sperm sperm tRNA-derived small RNAs (tsRNAs) partly contribute to the transmission of such phenotypes. We observe observe increased expression of sperm tsRNAs in the F1 male offspring born to HFD-exposed dams. Microinjection Microinjection of sperm tsRNAs from the F1-HFD male into normal zygotes reproduces obesogenic phenotypes phenotypes and addictive-like behaviors, such as increased preference of palatable foods and enhanced enhanced sensitivity to drugs of abuse in the resultant offspring. The expression of several of the differentially differentially expressed sperm tsRNAs predicted targets such as CHRNA2 and GRIN3A, which have been implicated implicated in addiction pathology, are altered in the mesolimbic reward brain regions of the F1-HFD father father and the resultant HFD-tsRNA offspring. Together, our findings demonstrate that sperm tsRNA is is a potential vector that contributes to the transmission of MHFD-induced addictive-like behaviors and obesogenic phenotypes across generations, thereby emphasizing its role in diverse pathological outcomes outcomes