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The neuropeptide Urocortin 2 (UCN2) acts as a ligand for the G protein-coupled receptor (GPCR) corticotropin-releasing hormone receptor 2 (CRHR2) expressed in the brain and peripheral metabolic tissues. UCN2 has been reported to improve or worsen insulin sensitivity and glucose uptake in skeletal muscle and have opposing effects on glucose tolerance in vivo. In this report, we examined the acute and chronic effect of UCN2 on glucose metabolism and signaling pathways downstream of CRHR2. Consistent with previous reports, we found that acute dosing of UCN2 induced systemic insulin resistance and hyperglycemia in mice and skeletal muscle. Inversely, chronic elevation of UCN2 by injection with adenovirus encoding UCN2 (UCN2.AAV) resolved metabolic complications, improving glucose tolerance. Phosphoproteomic analysis of acutely treated skeletal muscle revealed dephosphorylation of IRS1 and AKT1S1, which was entirely reversed in UCN2.AAV skeletal muscle. Interestingly, pharmacological studies showed that all human isoforms of CRHR2 recruit Gs, as well as Gi and β-Arrestin, in response to stimulation with UCN2. However, Gi and β-Arrestin recruitment occurs at UCN2 concentrations 10-fold higher than Gs recruitment. Furthermore, pre-treating cells with UCN2 led to internalization of CRHR2 and dampened ligand-dependent increases in cAMP. Consistent with the in vivo results, treatment of mouse soleus muscle with UCN2 ex vivo showed AKT1S1 and IRS1 dephosphorylation and decreased glucose uptake in response to insulin; these responses were blunted when the muscle was pre-incubated with UCN2. These studies demonstrate that exposure to high, chronic concentrations of UCN2 desensitizes CRHR2, thus blocking the effects of acute UCN2, and improving insulin sensitivity, in skeletal muscle and systemically. On the other hand, acute treatment with UCN2 activates CRHR2 through recruitment of Gs which leads to blunted insulin signaling and glucose uptake. These results provide mechanistic insights into how UCN2 regulates insulin sensitivity and glucose metabolism in skeletal muscle and in vivo. Importantly, a working model was derived from these results that unifies the contradictory metabolic effects of UCN2.

ObjectivesTo use human genetic variants that proxy angiotensin-converting enzyme (ACE) inhibitor drug effects and cardiovascular risk factors to provide insight into how these exposures affect lung ACE2 and TMPRSS2 gene expression and circulating ACE2 levels.DesignTwo-sample Mendelian randomization (MR) analysis.SettingSummary-level genetic association data.ParticipantsParticipants were predominantly of European ancestry. Variants that proxy ACE inhibitor drug effects and cardiometabolic risk factors (body mass index, chronic obstructive pulmonary disease, lifetime smoking index, low-density lipoprotein cholesterol, systolic blood pressure and type 2 diabetes mellitus) were selected from publicly available genome-wide association study data (sample sizes ranging from 188,577 to 898,130 participants). Genetic association estimates for lung expression of ACE2 and TMPRSS2 were obtained from the Gene-Tissue Expression (GTEx) project (515 participants) and the Lung eQTL Consortium (1,038 participants). Genetic association estimates for circulating plasma ACE2 levels were obtained from the INTERVAL study (4,947 participants).Main outcomes and measuresLung ACE2 and TMPRSS2 expression and plasma ACE2 levels.ResultsThere were no association of genetically proxied ACE inhibition with any of the outcomes considered here. There was evidence of a positive association of genetic liability to type 2 diabetes mellitus with lung ACE2 gene expression in GTEx (p = 4×10−4) and with circulating plasma ACE2 levels in INTERVAL (p = 0.03), but not with lung ACE2 expression in the Lung eQTL Consortium study (p = 0.68). There were no associations between genetically predicted levels of the other cardiometabolic traits with the outcomes.ConclusionsThis study does not provide evidence to support that ACE inhibitor antihypertensive drugs affect lung ACE2 and TMPRSS2 expression or plasma ACE2 levels. In the current COVID-19 pandemic, our findings do not support a change in ACE inhibitor medication use without clinical justification.Summary boxesWhat is already known on this topicSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current coronavirus disease 2019 (COVID-19) pandemic.Serine protease TMPRSS2 is involved in priming the SARS-CoV-2 spike protein for cellular entry through the angiotensin-converting enzyme 2 (ACE2) receptor.Expression of ACE2 and TMPRSS2 in the lung epithelium might have implications for risk of SARS-CoV-2 infection and severity of COVID-19.What this study addsWe used human genetic variants that proxy ACE inhibitor drug effects and cardiometabolic risk factors to provide insight into how these exposures affect lung ACE2 and TMPRSS2 expression and circulating ACE2 levels.Our findings do not support the hypothesis that ACE inhibitors have effects on ACE2 expression.We found some support for an association of genetic liability to type 2 diabetes mellitus with higher lung ACE2 expression and plasma ACE2 levels, but evidence was inconsistent across studies.

Circulating increases in branched chain amino acid (BCAA) levels have long been associated with type II diabetes and metabolic syndrome. Emerging data also suggest that impaired BCAA catabolism may play a role in heart failure progression. BCAA are catabolized via the branched chain ketoacid (BCKA) dehydrogenase enzyme complex (BCKDH). BCKD kinase (BCKDK) is a negative regulator of BCAA catabolism through its inhibitory phosphorylation of the BCKDHE1a subunit, and the phosphatase PPM1k dephosphorylates this same site to activate BCAA catabolism. Using an inhibitor of BCKDK (BT2), BCAA catabolism is increased in vivo. Here, we utilized metabolomics to evaluate the contribution of BCAA catabolism to substrate preference in heart and skeletal muscle. Surprisingly, BCKDK inhibition with BT2 had no effect on incorporation of glucose into TCA cycle intermediates in heart or skeletal muscle. Because others have recently shown that the primary site of BCAA catabolism is skeletal muscle, we knocked down BCKDK and PPM1k in human skeletal myocytes to further investigate how BCKDK loss or inhibition affects substrate utilization. Similar to our in vivo observations, knockdown of BCKDK and PPM1k had no effect on glucose and pyruvate utilization in a mitochondrial function assay. However, an increase in maximal respiration was observed after BCKDK knockdown when fatty acids were used. To evaluate the mechanisms underlying this increase we then performed RNAseq in these cells after BCKDK and PPM1K knockdown and observed changes in a number of genes that may explain these alterations in substrate utilization. Finally, we performed C13 BCAA metabolomics in human skeletal myocytes after BT2 treatment or knockdown of BCKDK and PPM1k. Using BT2, we observed a dose-responsive reduction in BCKA production from C13 BCAA by the muscle cells as expected; however, though BCKA production was increased after PPM1k was knocked down, we surprisingly did not observe a decrease in BCKA production after BCKDK knockdown. Collectively these data suggest that BCKDK inhibition may improve metabolism and cardiac function by altering substrate preference in skeletal myocytes.

Circulating increases in branched chain amino acid (BCAA) levels have long been associated with type 2 diabetes and metabolic syndrome. Emerging data also suggest that BCAA catabolism may play a role in heart failure progression. It is hypothesized that decreased catabolism, rather than increased consumption of BCAAs, is responsible for these correlations. Branched chain ketoacid (BCKA) dehydrogenase (BCKDH) kinase (BCKDK) is a negative regulator of BCAA catabolism through its inhibitory phosphorylation of the BCKDH E1a subunit. Using the BCKDK inhibitor molecule BT2, we demonstrate here a reduction of BCAA, BCKA and tissue p-BCKDH levels concomitant with improved glucose tolerance and reduced insulin levels in diet-induced obese mice after only one day of treatment. To investigate the mechanisms underlying this protection, we assessed plasma biomarkers and tissue gene expression after fasting and re-feeding in glucose intolerant high fat diet-fed animals. Remarkably, BT2 treated animals demonstrated reduced plasma glucose levels after re-feed, which was accompanied by dramatic reductions in plasma insulin levels, reduced activation of Akt in peripheral tissues, and a failure to suppress plasma free fatty acids and lipolytic machinery in adipose tissue. RNAseq was performed in liver to assess changes in gene expression profiles, and while over 400 genes were differentially regulated in vehicle treated re-fed mice compared with vehicle treated fasted mice, there were no differentially regulated genes in BT2 re-fed animals compared with BT2 fasted animals. These data suggest that activation of BCAA catabolism with the BCKDK inhibitor BT2 impairs the systemic response to feeding. Disclosure E. Bollinger: Employee; Self; Pfizer Inc. C.P. Siddall: Employee; Self; Pfizer Inc. Employee; Spouse/Partner; Vertex Pharmaceuticals. T. Greizer: None. J.L. Libera: None. G. Hariri: None. E.E. Pashos: Employee; Self; Pfizer Inc. A. Shipstone: None. A. Hadjipanayis: None. Z. Sun: None. G. Xing: None. M.F. Clasquin: Employee; Spouse/Partner; Merck & Co., Inc. Employee; Self; Pfizer Inc. B.B. Zhang: Employee; Spouse/Partner; Janssen Pharmaceuticals, Inc. Employee; Self; Pfizer Inc. Other Relationship; Self; Eli Lilly and Company. R.A. Miller: Employee; Self; Pfizer Inc. R. Roth Flach: Employee; Self; Pfizer Inc.

Genome-wide association studies (GWAS) have identified 95 loci in the human genome that harbor common variants associated with plasma lipid traits. Of the 95 loci, 17 harbor genes known to cause monogenic lipid disorders and collectively a third of them contain genes with characterized roles in lipid metabolism. Therefore in the majority of loci the causal genes are unknown. We selected 32 genes, not previously implicated in lipid metabolism and representing a total of 26 loci, to test for their ability to modify plasma lipid concentrations upon somatic overexpression in vivo. We utilized adeno-associated virus serotype 8 (AAV8) to overexpress the selected genes specifically in the livers of both C57BL/6 mice and in an appropriate humanized mouse model (either mice expressing human apolipoprotein A-I for HDL loci or Apobec1-knockout, Ldlr haploinsufficient mice expressing human apolipoprotein B-100 for triglyceride and LDL loci). Approximately half of the genes tested reproducibly affected plasma lipids. For 13 of the interrogated loci the lipid-associated variants also correlated with expression variations of the respective genes in liver (liver expression quantitative trait loci-eQTLs). We demonstrate a causal role for 7 of these 13 genes. The overexpression of these 7 genes not only affected the predicted lipid class, but additionally exerted its effect in the predicted direction in 6 of 7 cases (Tmem57, Slc39a8, Ppp1r3b, Vkorc1, Tbkbp1 and Ube2l3). Additionally for a subset of the examined genes we proceeded to develop small interfering RNA (siRNA) nanoparticles that were particularly targeted to the liver. We were able to obtain robust knockdown for a significant number of genes and, in several cases, observe reciprocal effects on plasma lipids from our overexpression and knockdown studies. This work has identified several novel lipid regulators, whose further investigation can uncover novel mechanisms and pathways controlling plasma lipids.