Lipid nanoparticle delivery of glucagon receptor siRNA improves glucose homeostasis in mouse models of diabetes

Neumann, Ursula; Ho, Jessica S.S.; Chen, Sam; Tam, Yun K.; Cullis, Pieter R.; Kieffer, Timothy J.

doi:10.1016/j.molmet.2017.06.012

Cited by 22 publications

(14 citation statements)

References 52 publications

(89 reference statements)

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“…In the first model, we treated diet-induced obese (DIO) mice with adeno-associated virus-8 (AAV8) in which short hairpin RNA targeting the glucagon receptor (sh-Gcgr) was driven by the H1 promoter, that silences genes in the liver 23, 30 . This treatment lowered hepatic Gcgr levels by ~80% (Figure 1A), and consistent with previous reports 31, 32 , it lowered fasting blood glucose levels from 189 ± 9 mg/dl to 142 ± 7 mg/dl (Figure 1B). Reduced hepatic Gcgr expression did not affect body weight or plasma insulin in the obese mice (Online Figure IA, B).…”

Section: Resultssupporting

confidence: 91%

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Hepatic Glucagon Signaling Regulates PCSK9 and Low-Density Lipoprotein Cholesterol

Spolitu

Okamoto

Dai

et al. 2019

Circulation Research

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Rationale: Glucagon is a key hormone that regulates the adaptive metabolic responses to fasting. In addition to maintaining glucose homeostasis, glucagon participates in the regulation of cholesterol metabolism, however, the molecular pathways underlying this effect are incompletely understood. Objective: We sought to determine the role of hepatic glucagon receptor (Gcgr) signaling in plasma cholesterol regulation and identify its underlying molecular mechanisms. Methods and Results: We show that Gcgr signaling plays an essential role in low-density lipoprotein (LDL) cholesterol homeostasis through regulating the proprotein convertase subtilisin/kexin type 9 (PCSK9) levels. Silencing of hepatic Gcgr or inhibition of glucagon action increased hepatic and plasma PCSK9 and resulted in lower LDL receptor protein and increased plasma LDL-cholesterol. Conversely, treatment of WT mice with glucagon lowered LDL-cholesterol levels, whereas this response was abrogated in Pcsk9−/− and Ldlr−/− mice. Our gain- and loss-of-function studies identified exchange protein activated by cAMP-2 (Epac2) and Ras-related protein-1 (Rap1) as the downstream mediators of glucagon’s action on PCSK9 homeostasis. Moreover, mechanistic studies revealed that glucagon affected the half-life of PCSK9 protein without changing the level of its mRNA, indicating that Gcgr signaling regulates PCSK9 degradation. Conclusions: These findings provide novel insights into the molecular interplay between hepatic glucagon signaling and lipid metabolism and describe a new post-transcriptional mechanism of PCSK9 regulation.

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Section: Resultssupporting

confidence: 91%

“…Various mouse models and human data have provided evidence that blocking glucagon action results in increased plasma LDL-C levels 13, 18, 31, 32 . While one study attributed this increase to elevated rates of lipogenesis, another report suggested that the mechanism is through an increase in intestinal cholesterol absorption 32, 60 .…”

Section: Discussionmentioning

confidence: 99%

Hepatic Glucagon Signaling Regulates PCSK9 and Low-Density Lipoprotein Cholesterol

Spolitu

Okamoto

Dai

et al. 2019

Circulation Research

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“…For example, a LNP with tropism to hepatocytes delivered siRNAs targeting endolysosomal genes; this uncovered how Rab5 influenced endocytosis 9 . Similar approaches have been applied to hypertension 10 , heart disease 11–13 , extracellular matrix signaling 14 , cancer 15–16 , glucose homeostasis 17 , and other phenotypes 18–19 . In addition to its utility as an in vivo scientific reagent, siRNA has treated disease in patients.…”

Section: Introductionmentioning

confidence: 99%

Nanoparticles That Deliver RNA to Bone Marrow Identified by in Vivo Directed Evolution

et al. 2018

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Bone marrow endothelial cells (BMECs) regulate their microenvironment, which includes hematopoietic stem cells. This makes BMECs an important target cell type for siRNA or gene editing (e.g., CRISPR) therapies. However, siRNA and sgRNA have not been delivered to BMECs using systemically administered nanoparticles. Given that in vitro nanoparticle screens have not identified nanoparticles with BMEC tropism, we developed a system to quantify how >100 different nanoparticles deliver siRNA in a single mouse. This is the first barcoding system capable of quantifying functional cytosolic siRNA delivery (where the siRNA drug is active), distinguishing it from in vivo screens that quantify biodistribution (where the drug went). Combining this approach with bioinformatics, we performed in vivo directed evolution, and identified BM1, a lipid nanoparticle (LNP) that delivers siRNA and sgRNA to BMECs. Interestingly, chemical analysis revealed BMEC tropism was not related to LNP size; tropism changed with the structure of poly(ethylene glycol), as well as the presence of cholesterol. These results suggest that significant changes to vascular targeting can be imparted to a LNP by making simple changes to its chemical composition, rather than using active targeting ligands. BM1 is the first nanoparticle to efficiently deliver siRNA and sgRNA to BMECs in vivo, demonstrating that this functional in vivo screen can identify nanoparticles with novel tropism in vivo. More generally, in vivo screening may help reveal the complex relationship between nanoparticle structure and tropism, thereby helping scientists understand how simple chemical changes control nanoparticle targeting.

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“…Next, we sought to knockdown Smad4 in type 2 diabetes with established kidney disease. In this model, hypertensive eNOS À/À mice on the C57BL/ 6J background are placed on a high fat diet (HFD) for 30 weeks with a single STZ injection on week 8 to further increase blood glucose levels to diabetic state while remaining hyperinsulinaemia, a characteristic of type 2 diabetes [19][20][21][22][23][24][25][26][27][28][29][30][31]. This regimen results in hyperglycaemia, hyperinsulinaemia, albuminuria, glomerulosclerosis and reduced kidney function ( Fig EV1).…”

Section: Resultsmentioning

confidence: 99%

Smad4 promotes diabetic nephropathy by modulating glycolysis and OXPHOS

Sun

Chen

et al. 2020

EMBO Reports

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Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. TGF-b1/Smad3 signalling plays a major pathological role in DN; however, the contribution of Smad4 has not been examined. Smad4 depletion in the kidney using anti-Smad4 locked nucleic acid halted progressive podocyte damage and glomerulosclerosis in mouse type 2 DN, suggesting a pathogenic role of Smad4 in podocytes. Smad4 is upregulated in human and mouse podocytes during DN. Conditional Smad4 deletion in podocytes protects mice from type 2 DN, independent of obesity. Mechanistically, hyperglycaemia induces Smad4 localization to mitochondria in podocytes, resulting in reduced glycolysis and oxidative phosphorylation and increased production of reactive oxygen species. This operates, in part, via direct binding of Smad4 to the glycolytic enzyme PKM2 and reducing the active tetrameric form of PKM2. In addition, Smad4 interacts with ATPIF1, causing a reduction in ATPIF1 degradation. In conclusion, we have discovered a mitochondrial mechanism by which Smad4 causes diabetic podocyte injury.

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Lipid nanoparticle delivery of glucagon receptor siRNA improves glucose homeostasis in mouse models of diabetes

Cited by 22 publications

References 52 publications

Hepatic Glucagon Signaling Regulates PCSK9 and Low-Density Lipoprotein Cholesterol

Hepatic Glucagon Signaling Regulates PCSK9 and Low-Density Lipoprotein Cholesterol

Nanoparticles That Deliver RNA to Bone Marrow Identified by in Vivo Directed Evolution

Smad4 promotes diabetic nephropathy by modulating glycolysis and OXPHOS

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