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.
Insufficient insulin secretion to meet metabolic demand results in diabetes. The intracellular flux of Ca
into β-cells triggers insulin release. Since genetics strongly influences variation in islet ...secretory responses, we surveyed islet Ca
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
waveforms to islet protein abundances in the same strains used for the Ca
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
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.
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.
Genetic susceptibility to type 2 diabetes is primarily due to β-cell dysfunction. However, a genetic study to directly interrogate β-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 >1,000-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 QTL from mouse are associated with diabetes-related SNPs in human genome-wide association studies. We report on three genes, Ptpn18, Hunk and Zfp148, where the phenotype predictions from the genetic screen were fulfilled in our studies of transgenic mouse models. These three genes encode a non-receptor type protein tyrosine phosphatase, a serine/threonine protein kinase, and a Krϋppel-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 non-diabetic individuals.
Introduction & Objective: High-risk single-nucleotide polymorphisms (SNPs) account for only ~20% of type 2 diabetes heritability, implying islet genes greatly influence one another to alter diabetes ...risk. Correlative analysis does not reveal conditional dependencies between key players regulating islet function whereas machine learning (ML) may do so. Methods: Using data from 374 genetically diverse mice, we derived models predicting islet function from protein abundance with gradient-boosted decision tree algorithms. Some mice (70%) were used to train the models and the rest were used to validate them. We also applied our models to similar data from other mouse studies and to data from humans to see if the models’ accuracies replicated. We then determined if the models included any known islet regulators, proteins with human orthologues possessing glycemia-related SNPs, and enriched for islet-relevant functional pathways. Finally, using the proteins’ predictive influences as meta-traits, we ran quantitative trait locus (QTL) scans to identify genomic regions altering the influence of proteins on islet function. Results: Our analysis revealed < 150 of the ~ 5000 detected proteins sufficiently modeled any one functional trait, with > 90% of a model’s prediction influenced by < 50 proteins. Some models predicted as well or better in the other mouse sets (R > 0.6) and reasonably well in human data (R > 0.5). ML analysis correctly identified the direction of effect for candidates (e.g. DPP8) where prior studies using correlation alone failed. Models for islet traits enriched for pathways potentially relevant for islet function and several unstudied proteins (e.g. CRELD2) present in multiple models have SNPs for glycemia-related traits. Finally, QTL scans revealed genomic regions that may alter the influence of key proteins on islet function and are far from those proteins’ genomic positions. Conclusions: ML can be used to identify conditional dependencies regulating islet function. Disclosure C. Emfinger: None. E. Freiberger: Employee; AbbVie Inc. M. Rabaglia: None. J. Kolic: None. S. Simonett: Employee; Exact Sciences. M. Shortreed: None. L. Clark: None. D. Stapleton: None. K. Schueler: None. K. Mitok: None. T.R. Price: None. J.J. Coon: Research Support; Agilent, Eli Lilly and Company, Merck & Co., Inc. L. Smith: None. M.J. Merrins: None. J.D. Johnson: None. M. Keller: None. A.D. Attie: None. Funding American Diabetes Association (7-21-PDF-157); National Institutes of Health (R01-DK101573-06); National Institutes of Health (GM070683); National Institutes of Health (P41-GM108538); National Institutes of Health (R35GM126914); National Institutes of Health (R35-GM118110); National Institutes of Health (T32-HL007936); National Institutes of Health (R01-DK113103); National Institutes of Health (R01DK127637); Canadian Institutes for Health Research (CIHR) operating grant 168857 CIHR Team Grant (ASD-179092/5-SRA-2021-1149-S-B); CIHR-JDRF Team (ASD-173663/5-SRA-2020-1059-S-B); CIHR Banting fellowship United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service (I01BX005113)
Neurite formation is a seminal event in the early development of neurons. However, little is known about the mechanisms by which neurons form neurites. F-BAR proteins function in sensing and inducing ...membrane curvature 1, 2. Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family, regulates endocytosis in a variety of cell types 3–9. However, there is little data on how CIP4 functions in neurons 10, 11. Here we show that CIP4 plays a novel role in neuronal development by inhibiting neurite formation. Remarkably, CIP4 exerts this effect not through endocytosis, but by producing lamellipodial protrusions. In primary cortical neurons CIP4 is concentrated specifically at the tips of extending lamellipodia and filopodia, instead of endosomes as in other cell types. Overexpression of CIP4 results in lamellipodial protrusions around the cell body, subsequently delaying neurite formation and enlarging growth cones. These effects depend on the F-BAR and SH3 domains of CIP4 and on its ability to multimerize. Conversely, cortical neurons from CIP4-null mice initiate neurites twice as fast as controls. This is the first study to demonstrate that an F-BAR protein functions differently in neuronal versus nonneuronal cells and induces lamellipodial protrusions instead of invaginations or filopodia-like structures.
► CIP4 localizes to protruding lamellipodia in neurons ► CIP4-induced lamellipodia inhibit neurite formation ► The F-BAR/SH3 domains and multimerization are required for CIP4 function in neurons
We previously positionally cloned Sorcs1 as a diabetes quantitative trait locus. Sorcs1 belongs to the Vacuolar protein sorting-10 (Vps10) gene family. In yeast, Vps10 transports enzymes from the ...trans-Golgi network (TGN) to the vacuole. Whole-body Sorcs1 KO mice, when made obese with the leptin(ob) mutation (ob/ob), developed diabetes. β Cells from these mice had a severe deficiency of secretory granules (SGs) and insulin. Interestingly, a single secretagogue challenge failed to consistently elicit an insulin secretory dysfunction. However, multiple challenges of the Sorcs1 KO ob/ob islets consistently revealed an insulin secretion defect. The luminal domain of SORCS1 (Lum-Sorcs1), when expressed in a β cell line, acted as a dominant-negative, leading to SG and insulin deficiency. Using syncollin-dsRed5TIMER adenovirus, we found that the loss of Sorcs1 function greatly impairs the rapid replenishment of SGs following secretagogue challenge. Chronic exposure of islets from lean Sorcs1 KO mice to high glucose and palmitate depleted insulin content and evoked an insulin secretion defect. Thus, in metabolically stressed mice, Sorcs1 is important for SG replenishment, and under chronic challenge by insulin secretagogues, loss of Sorcs1 leads to diabetes. Overexpression of full-length SORCS1 led to a 2-fold increase in SG content, suggesting that SORCS1 is sufficient to promote SG biogenesis.
Cdc42-interacting protein 4 (CIP4), a member of the F-BAR family of proteins, plays important roles in a variety of cellular events by regulating both membrane and actin dynamics. In many cell types, ...CIP4 functions in vesicle formation, endocytosis and membrane tubulation. However, recent data indicate that CIP4 is also involved in protrusion in some cell types, including cancer cells (lamellipodia and invadopodia) and neurons (ribbed lamellipodia and veils). In neurons, CIP4 localizes specifically to extending protrusions and functions to limit neurite outgrowth early in development. The mechanism by which CIP4 localizes to the protruding edge membrane and induces lamellipodial/veil protrusion and actin rib formation is not known. Here, we show that CIP4 localization to the protruding edge of neurons is dependent on both the phospholipid content of the plasma membrane and the underlying organization of actin filaments. Inhibiting phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production decreases CIP4 at the membrane. CIP4 localization to the protruding edge is also dependent on Rac1/WAVE1, rather than Cdc42/N-WASP. Capping actin filaments with low concentrations of cytochalasin D or by overexpressing capping protein dramatically decreases CIP4 at the protruding edge, whereas inactivating Arp2/3 drives CIP4 to the protruding edge. We also demonstrate that CIP4 dynamically colocalizes with Ena/VASP and DAAM1, two proteins known to induce unbranched actin filament arrays and play important roles in neuronal development. Together, this is the first study to show that the localization of an F-BAR protein depends on both actin filament architecture and phospholipids at the protruding edge of developing neurons.
We previously positionally cloned Sorcs1 as a diabetes quantitative trait locus. Sorcs1 belongs to the Vacuolar protein sorting-10 (Vps10) gene family. In yeast, Vps10 transports enzymes from the ...trans-Golgi network (TGN) to the vacuole. Whole-body Sorcs1 KO mice, when made obese with the leptin.sup.ob mutation (ob/ob), developed diabetes. beta Cells from these mice had a severe deficiency of secretory granules (SGs) and insulin. Interestingly, a single secretagogue challenge failed to consistently elicit an insulin secretory dysfunction. However, multiple challenges of the Sorcs1 KO ob/ob islets consistently revealed an insulin secretion defect. The luminal domain of SORCS1 (Lum-Sorcs7), when expressed in a beta cell line, acted as a dominant-negative, leading to SG and insulin deficiency. Using syncollin-dsRed5TIMER adenovirus, we found that the loss of Sorcs1 function greatly impairs the rapid replenishment of SGs following secretagogue challenge. Chronic exposure of islets from lean Sorcs1 KO mice to high glucose and palmitate depleted insulin content and evoked an insulin secretion defect. Thus, in metabolically stressed mice, Sorcs1 is important for SG replenishment, and under chronic challenge by insulin secretagogues, loss of Sorcs1 leads to diabetes. Overexpression of full-length SORCS1 led to a 2-fold increase in SG content, suggesting that SORCS1 is sufficient to promote SG biogenesis.
PDF
