Sortilin is a post-Golgi trafficking receptor homologous to the yeast vacuolar protein sorting receptor 10 (VPS10). The VPS10 motif on sortilin is a 10-bladed β-propeller structure capable of binding ...more than 50 proteins, covering a wide range of biological functions including lipid and lipoprotein metabolism, neuronal growth and death, inflammation, and lysosomal degradation. Sortilin has a complex cellular trafficking itinerary, where it functions as a receptor in the trans-Golgi network, endosomes, secretory vesicles, multivesicular bodies, and at the cell surface. In addition, sortilin is associated with hypercholesterolemia, Alzheimer’s disease, prion diseases, Parkinson’s disease, and inflammation syndromes. The 1p13.3 locus containing SORT1, the gene encoding sortilin, carries the strongest association with LDL-C of all loci in human genome-wide association studies. However, the mechanism by which sortilin influences LDL-C is unclear. Here, we review the role sortilin plays in cardiovascular and metabolic diseases and describe in detail the large and often contradictory literature on the role of sortilin in the regulation of LDL-C levels.
Common noncoding variants at the human 1p13.3 locus associated with SORT1 expression are among those most strongly associated with low-density lipoprotein cholesterol (LDL-C) in human genome-wide ...association studies. However, validation studies in mice and cell lines have produced variable results regarding the directionality of the effect of SORT1 on LDL-C. This, together with the fact that the 1p13.3 variants are associated with expression of several genes, has raised the question of whether SORT1 is the causal gene at this locus. Using whole exome sequencing in members of an Amish population, we identified coding variants in SORT1 that are associated with increased (rs141749679, K302E) and decreased (rs149456022, Q225H) LDL-C. Further, analysis of plasma lipoprotein particle subclasses by ion mobility in a subset of rs141749679 (K302E) carriers revealed higher levels of large LDL particles compared to noncarriers. In contrast to the effect of these variants in the Amish, the sortilin K302E mutation introduced into a C57BL/6J mouse via CRISPR/Cas9 resulted in decreased non-high-density lipoprotein cholesterol, and the sortilin Q225H mutation did not alter cholesterol levels in mice. This is indicative of different effects of these mutations on cholesterol metabolism in the two species. To our knowledge, this is the first evidence that naturally occurring coding variants in SORT1 are associated with LDL-C, thus supporting SORT1 as the gene responsible for the association of the 1p13.3 locus with LDL-C.
Dense-core vesicle (DCV) exocytosis is a SNARE (soluble
-ethylmaleimide-sensitive fusion attachment protein receptor)-dependent anterograde trafficking pathway that requires multiple proteins for ...regulation. Several C2 domain-containing proteins are known to regulate Ca
-dependent DCV exocytosis in neuroendocrine cells. In this study, we identified others by screening all (∼139) human C2 domain-containing proteins by RNA interference in neuroendocrine cells. 40 genes were identified, including several encoding proteins with known roles (CAPS calcium-dependent activator protein for secretion 1, Munc13-2, RIM1, and SYT10) and many with unknown roles. One of the latter, BAIAP3, is a secretory cell-specific Munc13-4 paralog of unknown function. BAIAP3 knockdown caused accumulation of fusion-incompetent DCVs in BON neuroendocrine cells and lysosomal degradation (crinophagy) of insulin-containing DCVs in INS-1 β cells. BAIAP3 localized to endosomes was required for Golgi trans-Golgi network 46 (TGN46) recycling, exhibited Ca
-stimulated interactions with TGN SNAREs, and underwent Ca
-stimulated TGN recruitment. Thus, unlike other Munc13 proteins, BAIAP3 functions indirectly in DCV exocytosis by affecting DCV maturation through its role in DCV protein recycling. Ca
rises that stimulate DCV exocytosis may stimulate BAIAP3-dependent retrograde trafficking to maintain DCV protein homeostasis and DCV function.
Genetic susceptibility to type 2 diabetes is primarily due to beta cell dysfunction. However, a genetic study to directly interrogate beta cell function ex vivo has never been previously performed. ...We isolated 233,447 islets from 483 Diversity Outbred (DO) mice maintained on a Western-style diet, and measured insulin secretion in response to a variety of secretagogues. Insulin secretion from DO islets ranged greater than 1000-fold even though none of the mice were diabetic. The insulin secretory response to each secretagogue had a unique genetic architecture; some of the loci were specific for one condition, whereas others overlapped. Human loci that are syntenic to many of the insulin secretion quantitative trait loci (QTL) from mice are associated with diabetes- related SNPs in human genome-wide association studies. We report on 3 genes, Ptpn18, Hunk, and Zfp148, where the phenotype predictions from the genetic screen were fulfilled in our studies of transgenic mouse models. These 3 genes encode a nonreceptor type protein tyrosine phosphatase, a serine/threonine protein kinase, and a Kruppel-type zinc-finger transcription factor, respectively. Our results demonstrate that genetic variation in insulin secretion that can lead to type 2 diabetes is discoverable in nondiabetic individuals.
The etiology of diabetic nephropathy in type 2 diabetes is multifactorial. Sustained hyperglycemia is a major contributor, but additional contributions come from the hypertension, obesity, and ...hyperlipidemia that are also commonly present in patients with type 2 diabetes and nephropathy. The leptin deficient BTBR ob/ob mouse is a model of type 2 diabetic nephropathy in which hyperglycemia, obesity, and hyperlipidemia, but not hypertension, are present. We have shown that reversal of the constellation of these metabolic abnormalities with leptin replacement can reverse the morphologic and functional manifestations of diabetic nephropathy. Here we tested the hypothesis that reversal specifically of the hypertriglyceridemia, using an antisense oligonucleotide directed against ApoC-III, an apolipoprotein that regulates the interactions of VLDL (very low density lipoproteins) with the LDL receptor, is sufficient to ameliorate the nephropathy of Type 2 diabetes. Antisense treatment resulted in reduction of circulating ApoC-III protein levels and resulted in substantial lowering of triglycerides to near-normal levels in diabetic mice versus controls. Antisense treatment did not ameliorate proteinuria or pathologic manifestations of diabetic nephropathy, including podocyte loss. These studies indicate that pathologic manifestations of diabetic nephropathy are unlikely to be reduced by lipid-lowering therapeutics alone, but does not preclude a role for such interventions to be used in conjunction with other therapeutics commonly employed in the treatment of diabetes and its complications.
The impact of lowering lipids alone on the pathologic complications of Type 2 diabetes is unknown. Treatment with a novel antisense oligonucleotide directed against ApoC-III lowered triglycerides significantly in the leptin deficient BTBR ob/ob murine model of type 2 diabetes, but did not ameliorate hyperglycemia or improve the nephropathy.
G6PC2 is predominantly expressed in pancreatic islet β-cells where it encodes a glucose-6-phosphatase catalytic subunit that modulates the sensitivity of insulin secretion to glucose by opposing the ...action of glucokinase, thereby regulating fasting blood glucose (FBG). Prior studies have shown that the G6pc2 promoter alone is unable to confer sustained islet-specific gene expression in mice, suggesting the existence of distal enhancers that regulate G6pc2 expression. Using information from both mice and humans and knowledge that single nucleotide polymorphisms (SNPs) both within and near G6PC2 are associated with variations in FBG in humans, we identified several putative enhancers 3' of G6pc2. One region, herein referred to as enhancer I, resides in the 25th intron of Abcb11 and binds multiple islet-enriched transcription factors. CRISPR-mediated deletion of enhancer I in C57BL/6 mice had selective effects on the expression of genes near the G6pc2 locus. In isolated islets, G6pc2 and Spc25 expression were reduced ∼50%, and Gm13613 expression was abolished, whereas Cers6 and nostrin expression were unaffected. This partial reduction in G6pc2 expression enhanced islet insulin secretion at basal glucose concentrations but did not affect FBG or glucose tolerance in vivo, consistent with the absence of a phenotype in G6pc2 heterozygous C57BL/6 mice.
Insufficient insulin secretion to meet metabolic demand results in diabetes. The intracellular flux of Ca2+ into β-cells triggers insulin release. Since genetics strongly influences variation in ...islet secretory responses, we surveyed islet Ca2+ dynamics in eight genetically diverse mouse strains. We found high strain variation in response to four conditions: (1) 8 mM glucose; (2) 8 mM glucose plus amino acids; (3) 8 mM glucose, amino acids, plus 10 nM glucose-dependent insulinotropic polypeptide (GIP); and (4) 2 mM glucose. These stimuli interrogate β-cell function, α- to β-cell signaling, and incretin responses. We then correlated components of the Ca2+ waveforms to islet protein abundances in the same strains used for the Ca2+ measurements. To focus on proteins relevant to human islet function, we identified human orthologues of correlated mouse proteins that are proximal to glycemic-associated single-nucleotide polymorphisms in human genome-wide association studies. Several orthologues have previously been shown to regulate insulin secretion (e.g. ABCC8, PCSK1, and GCK), supporting our mouse-to-human integration as a discovery platform. By integrating these data, we nominate novel regulators of islet Ca2+ oscillations and insulin secretion with potential relevance for human islet function. We also provide a resource for identifying appropriate mouse strains in which to study these regulators.
Insulin secretion from pancreatic β cells is essential for glucose homeostasis. An insufficient response to the demand for insulin results in diabetes. We previously showed that β cell-specific ...deletion of Zfp148 (β-Zfp148KO) improves glucose tolerance and insulin secretion in mice. Here, we performed Ca2+ imaging of islets from β‑Zfp148KO and control mice fed both a chow and a Western-style diet. β-Zfp148KO islets demonstrated improved sensitivity and sustained Ca2+ oscillations in response to elevated glucose levels. β-Zfp148KO islets also exhibited elevated sensitivity to amino acid-induced Ca2+ influx under low glucose conditions, suggesting enhanced mitochondrial phosphoenolpyruvate-dependent (PEP-dependent), ATP-sensitive K+ channel closure, independent of glycolysis. RNA-Seq and proteomics of β-Zfp148KO islets revealed altered levels of enzymes involved in amino acid metabolism (specifically, SLC3A2, SLC7A8, GLS, GLS2, PSPH, PHGDH, and PSAT1) and intermediary metabolism (namely, GOT1 and PCK2), consistent with altered PEP cycling. In agreement with this, β-Zfp148KO islets displayed enhanced insulin secretion in response to l-glutamine and activation of glutamate dehydrogenase. Understanding pathways controlled by ZFP148 may provide promising strategies for improving β cell function that are robust to the metabolic challenge imposed by a Western diet.
Insufficient insulin secretion to meet metabolic demand results in diabetes. The intracellular flux of Ca
2+
into β-cells triggers insulin release. Since genetics strongly influences variation in ...islet secretory responses, we surveyed islet Ca
2+
dynamics in eight genetically diverse mouse strains. We found high strain variation in response to four conditions: (1) 8 mM glucose; (2) 8 mM glucose plus amino acids; (3) 8 mM glucose, amino acids, plus 10 nM glucose-dependent insulinotropic polypeptide (GIP); and (4) 2 mM glucose. These stimuli interrogate β-cell function, α- to β-cell signaling, and incretin responses. We then correlated components of the Ca
2+
waveforms to islet protein abundances in the same strains used for the Ca
2+
measurements. To focus on proteins relevant to human islet function, we identified human orthologues of correlated mouse proteins that are proximal to glycemic-associated single-nucleotide polymorphisms in human genome-wide association studies. Several orthologues have previously been shown to regulate insulin secretion (e.g. ABCC8, PCSK1, and GCK), supporting our mouse-to-human integration as a discovery platform. By integrating these data, we nominate novel regulators of islet Ca
2+
oscillations and insulin secretion with potential relevance for human islet function. We also provide a resource for identifying appropriate mouse strains in which to study these regulators.
Using genetic linkage in an Amish population, we identified a coding mutation (K269E) in Sortilin associated with increased low-density lipoprotein cholesterol (LDL-C) and homeostatic model ...assessment of insulin resistance (HOMA-IR), and decreased C-peptide/insulin. This is the first time a naturally-occurring coding mutation in Sortilin has been linked to physiological function. Sortilin (SORT1) is a member of the vacuolar protein sorting 10 (VPS10) family of receptors that are involved in post-Golgi protein trafficking. We introduced the Sortilin K269E variant into mice and found that Western diet-fed, male Sortilin K269E mice had elevated LDL, insulin, and HOMA-IR, and decreased C-peptide/insulin, indicating that the variant is causal for these phenotypes in the humans. We show that mice harboring the Sortilin K269E variant had decreased clearance of both LDL and insulin, and our results indicate that mistrafficking of the LDL and insulin receptors in hepatocytes may be the underlying cause, ultimately resulting in elevated levels of LDL and insulin in the plasma. The Sortilin K269E mice had additional metabolic phenotypes, including increased adiposity, hepatic steatosis, gluconeogenesis, and plasma ketones, consistent with a broad role of Sortilin in mammalian metabolism and the increased insulin resistance. The phenotypes described above were observed in Western diet fed male homozygous-bred WT and Sortilin K269E mice. Subsequently, we bred mice heterozygous for the variant to obtain WT, Sortilin K269E heterozygous, and Sortilin K269E homozygous co-housed littermates. Surprisingly, upon measurement of plasma LDL, insulin, and HOMA-IR, and body weight, we found that all Western diet fed male heterozygous-bred mice, including the WT mice, had levels that were more similar to that of the homozygous-bred Sortilin K269E mice than the homozygous-bred WT mice. This unexpected result implicates a much more complex role of Sortilin K269E in metabolism. These data suggest that the phenotypes elicited by the Sortilin K269E mutation may be through an epigenetic parent-of-origin effect, or through a factor that is excreted in the feces of the mice, and due to the mice being coprophagic, is transmitted to the WT mice when co-housed with the Sortilin K269E mice. We are currently investigating these possibilities. Overall, this work has unveiled a novel site in Sortilin important for its role in lipid and insulin metabolism.
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