Glucagon blockade restores functional ß-cell mass in type 1 diabetic mice and enhances function of human islets.

We evaluated the potential for a monoclonal antibody antagonist of the glucagon receptor (Ab-4) to maintain glucose homeostasis in type 1 diabetic rodents. We noted durable and sustained improvements in glycemia which persist long after treatment withdrawal. Ab-4 promoted ß-cell survival and enhanced the recovery of insulin+ islet mass with concomitant increases in circulating insulin and C peptide. In PANIC-ATTAC mice, an inducible model of ß-cell apoptosis which allows for robust assessment of ß-cell regeneration following caspase-8-induced diabetes, Ab-4 drove a 6.7-fold increase in ß-cell mass. Lineage tracing suggests that this restoration of functional insulin-producing cells was at least partially driven by α-cell-to-ß-cell conversion. Following hyperglycemic onset in nonobese diabetic (NOD) mice, Ab-4 treatment promoted improvements in C-peptide levels and insulin+ islet mass was dramatically increased. Lastly, diabetic mice receiving human islet xenografts showed stable improvements in glycemic control and increased human insulin secretion.

A monocarboxylate transporter required for hepatocyte secretion of ketone bodies during fasting.

To find new genes that influence liver lipid mass, we performed a genetic screen for zebrafish mutants with hepatic steatosis, a pathological accumulation of fat. The red moon (rmn) mutant develops hepatic steatosis as maternally deposited yolk is depleted. Conversely, hepatic steatosis is suppressed in rmn mutants by adequate nutrition. Adult rmn mutants show increased liver neutral lipids and induction of hepatic lipid biosynthetic genes when fasted. Positional cloning of the rmn locus reveals a loss-of-function mutation in slc16a6a (solute carrier family 16a, member 6a), a gene that we show encodes a transporter of the major ketone body ß-hydroxybutyrate. Restoring wild-type zebrafish slc16a6a expression or introducing human SLC16A6 in rmn mutant livers rescues the mutant phenotype. Radiotracer analysis confirms that loss of Slc16a6a function causes diversion of liver-trapped ketogenic precursors into triacylglycerol. Underscoring the importance of Slc16a6a to normal fasting physiology, previously fed rmn mutants are more sensitive to death by starvation than are wild-type larvae. Our unbiased, forward genetic approach has found a heretofore unrecognized critical step in fasting energy metabolism: hepatic ketone body transport. Since ß-hydroxybutyrate is both a major fuel and a signaling molecule in fasting, the discovery of this transporter provides a new direction for modulating circulating levels of ketone bodies in metabolic diseases.

Specialized insulin is used for chemical warfare by fish-hunting cone snails.

More than 100 species of venomous cone snails (genus Conus) are highly effective predators of fish. The vast majority of venom components identified and functionally characterized to date are neurotoxins specifically targeted to receptors, ion channels, and transporters in the nervous system of prey, predators, or competitors. Here we describe a venom component targeting energy metabolism, a radically different mechanism. Two fish-hunting cone snails, Conus geographus and Conus tulipa, have evolved specialized insulins that are expressed as major components of their venoms. These insulins are distinctive in having much greater similarity to fish insulins than to the molluscan hormone and are unique in that posttranslational modifications characteristic of conotoxins (hydroxyproline, γ-carboxyglutamate) are present. When injected into fish, the venom insulin elicits hypoglycemic shock, a condition characterized by dangerously low blood glucose. Our evidence suggests that insulin is specifically used as a weapon for prey capture by a subset of fish-hunting cone snails that use a net strategy to capture prey. Insulin appears to be a component of the nirvana cabal, a toxin combination in these venoms that is released into the water to disorient schools of small fish, making them easier to engulf with the snail's distended false mouth, which functions as a net. If an entire school of fish simultaneously experiences hypoglycemic shock, this should directly facilitate capture by the predatory snail.

A genetic screen for zebrafish mutants with hepatic steatosis identifies a locus required for larval growth.

In a screen for zebrafish larval mutants with excessive liver lipid accumulation (hepatic steatosis), we identified harvest moon (hmn). Cytoplasmic lipid droplets, surrounded by multivesicular structures and mitochondria whose cristae appeared swollen, are seen in hmn mutant hepatocytes. Whole body triacylglycerol is increased in hmn mutant larvae. When we attempted to raise mutants, which were morphologically normal at the developmental stage that the screen was conducted, to adulthood, we observed that most hmn mutants do not survive to the juvenile period when raised. An arrest in growth occurs in the late larval period without obvious organ defects. Maternal zygotic mutants have no additional defects, suggesting that the mutation affects a late developmental process. The developmental window between embryogenesis and the metamorphosis remains under-studied, and hmn mutants might be useful for exploring the molecular and anatomic processes occurring during this transition period.

Metabolic insights from zebrafish genetics, physiology, and chemical biology.

Metabolic diseases-atherosclerotic cardiovascular disease, type 2 diabetes mellitus, obesity, and non-alcoholic fatty liver disease--have reached pandemic proportions. Across gene, cell, organ, organism, and social-environmental scales, fundamental discoveries of the derangements that occur in these diseases are required to develop effective new treatments. Here we will review genetic, physiological, pathological and chemical biological discoveries in the emerging zebrafish model for studying metabolism and metabolic diseases. We present a synthesis of recent studies using forward and reverse genetic tools to make new contributions to our understanding of lipid trafficking, diabetes pathogenesis and complications, and to ß-cell biology. The technical and physiological advantages and the pharmacological potential of this organism for discovery and validation of metabolic disease targets are stressed by our summary of recent findings. We conclude by arguing that metabolic research using zebrafish will benefit from adoption of conventional blood and tissue metabolite measurements, employment of modern imaging techniques, and development of more rigorous metabolic flux methods.

Lxr-driven enterocyte lipid droplet formation delays transport of ingested lipids.

Liver X receptors (Lxrs) are master regulators of cholesterol catabolism, driving the elimination of cholesterol from the periphery to the lumen of the intestine. Development of pharmacological agents to activate Lxrs has been hindered by synthetic Lxr agonists' induction of hepatic lipogenesis and hypertriglyceridemia. Elucidating the function of Lxrs in regulating enterocyte lipid handling might identify novel aspects of lipid metabolism that are pharmacologically amenable. We took a genetic approach centered on the single Lxr gene nr1h3 in zebrafish to study the role of Lxr in enterocyte lipid metabolism. Loss of nr1h3 function causes anticipated gene regulatory changes and cholesterol intolerance, collectively reflecting high evolutionary conservation of zebrafish Lxra function. Intestinal nr1h3 activation delays transport of absorbed neutral lipids, with accumulation of neutral lipids in enterocyte cytoplasmic droplets. This delay in transport of ingested neutral lipids protects animals from hypercholesterolemia and hepatic steatosis induced by a high-fat diet. On a gene regulatory level, Lxra induces expression of acsl3a, which encodes acyl-CoA synthetase long-chain family member 3a, a lipid droplet-anchored protein that directs fatty acyl chains into lipids. Forced overexpression of acls3a in enterocytes delays, in part, the appearance of neutral lipids in the vasculature of zebrafish larvae. Activation of Lxr in the intestine cell-autonomously regulates the rate of delivery of absorbed lipids by inducting a temporary lipid intestinal droplet storage depot.

Studying non-alcoholic fatty liver disease with zebrafish: a confluence of optics, genetics, and physiology.

Obesity is a public health crisis. New methods for amelioration of its consequences are required because it is very unlikely that the social and economic factors driving it will be reversed. The pathological accumulation of neutral lipids in the liver (hepatic steatosis) is an obesity-related problem whose molecular underpinnings are unknown and whose effective treatment is lacking. Here I review how zebrafish, a powerful model organism long-used for studying vertebrate developmental programs, is being harnessed to uncover new factors that contribute to normal liver lipid handling. Attention is given to dietary models and individual mutants. I speculate on the possible roles of non-hepatocyte residents of the liver, the adipose tissue, and gut microbiome on the development of hepatic steatosis. The highlighted work and future directions may lead to fresh insights into the pathogenesis and treatment of excess liver lipid states.

A Novel INS Mutation in the C-Peptide Region Causing Hyperproinsulinemic Maturity Onset Diabetes of Youth Type 10.

Monogenetic diabetes mellitus (DM) describes a collection of single-gene diseases marked by hyperglycemia presenting in childhood or adulthood and the absence of immunological markers of type 1 DM. Mutations in the human insulin gene INS give rise to two separate clinical syndromes: permanent neonatal DM, type 4 (PNDM4), and maturity-onset diabetes of youth, type 10 (MODY10); the former presents shortly after birth and the latter presents in childhood and adulthood. We describe a 40-year-old man in a kindred with high prevalence of DM who presented with severe hyperglycemia but not ketoacidosis or hypertriglyceridemia. Twelve years after initial presentation, the patient had elevated proinsulin and normal plasma C-peptide when nearly euglycemic on treatment with insulin glargine. A novel INS mutation, Gln65Arg, within the C-peptide region was identified. The INS (p.Gln65Arg) mutation may cause MODY10 by disrupting proinsulin maturation.

Macroprolactinoma-Induced Syndrome of Inappropriate Antidiuresis and Its Reversal with Dopamine Agonist Therapy.

Hyponatremia is an uncommon manifestation of pituitary adenomas. Herein, I report a case of syndrome of inappropriate antidiuresis (SIAD) caused by a macroprolactinoma that rapidly resolved with dopamine agonist therapy. A 29-year-old White woman presented with euvolemic, hypotonic hyponatremia, normal thyroid and glucocorticoid axes, and inappropriately concentrated urine. She was found to have a 1.2-cm sellar mass. Investigation of additional pituitary axes revealed an elevated prolactin level of 193.7 ng/mL. The SIAD experienced by the patient corrected rapidly with initiation of cabergoline. The patient could not tolerate dopamine agonist therapy, and after 1 year, she underwent transsphenoidal resection of the mass after the prolactin began to increase. Pathological examination confirmed the diagnosis of macroprolactinoma. There was no recurrence of the tumor, and the patient continued to have normonatremia and normoprolactinemia 7 years after her operation. To my knowledge, this is the first report in the literature of pathology-confirmed macroprolactinoma marked by SIAD that showed rapid normalization of water metabolism with dopamine agonist therapy.

Identifying Glucocorticoid Insufficiency in Silent Corticotroph Adenoma with Elevated Adrenocorticotropic Hormone.

Silent corticotroph adenoma (SCA) is as an aggressive pituitary tumor. A 48 year old man developed hypogonadotrophic hypogonadism. The basal morning adrenocorticotropic hormone (ACTH) was elevated, but the basal morning and peak after ACTH (1-24) stimulation cortisol were normal. A 3.7 cm sellar mass with evidence of internal hemorrhage, encasement of the right internal carotid artery, and invasion of the right cavernous sinus were identified, resected, and stained positive for ACTH. Over the next 5 years, the basal morning ACTH and cortisol were normal, and imaging revealed the presence of a small residual tumor. One year later, the patient became fatigued and nauseated, with elevated ACTH. An overnight metyrapone stimulation test (OMST) revealed glucocorticoid insufficiency, without further increase in ACTH. Symptoms resolved with hydrocortisone treatment. This case study suggests that SCA can secrete an ACTH precursor that is detected by clinical assays but is not active biologically. Postoperative OMST reveals glucocorticoid insufficiency in this context.

Loss of Dnmt1 catalytic activity reveals multiple roles for DNA methylation during pancreas development and regeneration.

Developmental mechanisms regulating gene expression and the stable acquisition of cell fate direct cytodifferentiation during organogenesis. Moreover, it is likely that such mechanisms could be exploited to repair or regenerate damaged organs. DNA methyltransferases (Dnmts) are enzymes critical for epigenetic regulation, and are used in concert with histone methylation and acetylation to regulate gene expression and maintain genomic integrity and chromosome structure. We carried out two forward genetic screens for regulators of endodermal organ development. In the first, we screened for altered morphology of developing digestive organs, while in the second we screed for the lack of terminally differentiated cell types in the pancreas and liver. From these screens, we identified two mutant alleles of zebrafish dnmt1. Both lesions are predicted to eliminate dnmt1 function; one is a missense mutation in the catalytic domain and the other is a nonsense mutation that eliminates the catalytic domain. In zebrafish dnmt1 mutants, the pancreas and liver form normally, but begin to degenerate after 84 h post fertilization (hpf). Acinar cells are nearly abolished through apoptosis by 100 hpf, though neither DNA replication, nor entry into mitosis is halted in the absence of detectable Dnmt1. However, endocrine cells and ducts are largely spared. Surprisingly, dnmt1 mutants and dnmt1 morpholino-injected larvae show increased capacity for pancreatic beta cell regeneration in an inducible model of pancreatic beta cell ablation. Thus, our data suggest that Dnmt1 is dispensable for pancreatic duct or endocrine cell formation, but not for acinar cell survival. In addition, Dnmt1 may influence the differentiation of pancreatic beta cell progenitors or the reprogramming of cells toward the pancreatic beta cell fate.

Lessons from "lower" organisms: what worms, flies, and zebrafish can teach us about human energy metabolism.

A pandemic of metabolic diseases (atherosclerosis, diabetes mellitus, and obesity), unleashed by multiple social and economic factors beyond the control of most individuals, threatens to diminish human life span for the first time in the modern era. Given the redundancy and inherent complexity of processes regulating the uptake, transport, catabolism, and synthesis of nutrients, magic bullets to target these diseases will be hard to find. Recent studies using the worm Caenorhabditis elegans, the fly Drosophila melanogaster, and the zebrafish Danio rerio indicate that these "lower" metazoans possess unique attributes that should help in identifying, investigating, and even validating new pharmaceutical targets for these diseases. We summarize findings in these organisms that shed light on highly conserved pathways of energy homeostasis.

FOXN3 hyperglycemic risk allele and insulin sensitivity in humans.

Objective: The rs8004664 variation within the FOXN3 gene is significantly and independently associated with fasting blood glucose in humans. We have previously shown that the hyperglycemia risk allele (A) increases FOXN3 expression in primary human hepatocytes; over-expression of human FOXN3 in zebrafish liver increases fasting blood glucose; and heterozygous deletion of the zebrafish ortholog foxn3 decreases fasting blood glucose. Paralleling these model organism findings, we found that rs8004664 A|A homozygotes had blunted glucagon suppression during an oral glucose tolerance test. Here, we test associations between insulin sensitivity and the rs8004664 variation. Research design and methods: 92 participants (49±13 years, body mass index: 32±6 kg/m2, 28 with and 64 without type 2 diabetes mellitus) were genotyped at rs8004664. Insulin sensitivity was measured by the euglycemic-hyperinsulinemic clamp technique. Results: The "A" allele frequency was 59%; the protective (G) allele frequency was 41% (A|A: n=29; G|G: n=12; A|G: n=50). Clamp-measured glucose disposal rate (GDR) was not different by genotype (F=0.046, p=0.96) or by "A" allele carrier (p=0.36). Female G|G homozygotes had better insulin sensitivity compared to female "A" allele carriers (GDR; G|G: 9.9±3.0 vs A|A+A|G: 7.1±3.0 mg/kg fat-free mass+17.7/min; p=0.04). Insulin sensitivity was not different by genotype or by "A" allele carriers. Conclusion: The rs8004664 variation within the FOXN3 gene may modulate insulin sensitivity in women.

FOXN3 controls liver glucose metabolism by regulating gluconeogenic substrate selection.

The FOXN3 gene locus is associated with fasting blood glucose levels in non-diabetic human population genetic studies. The blood glucose-modifying variation within this gene regulates the abundance of both FOXN3 protein and transcript in primary human hepatocytes, with the hyperglycemia risk allele causing increases in both FOXN3 protein and transcript. Using transgenic and knock-out zebrafish models, we showed previously that FOXN3 is a transcriptional repressor that regulates fasting blood glucose by altering liver gene expression of MYC, a  master transcriptional regulator of glucose utilization, and by modulating pancreatic α cell mass and function through an unknown mechanism. Since homozygous Foxn3 null mice die perinatally, and heterozygous carries of the null allele are smaller than wild-type siblings, we examine the metabolic effects of decreasing mouse liver Foxn3 expression in adult life, performing dynamic endocrine tests not feasible in adult zebrafish. Fasting glucose, glucagon, and insulin; and dynamic responses to glucose, insulin, pyruvate, glutamine, and glucagon were measured. Gluconeogenic and amino acid catabolic gene expression was examined in livers, as well. Knocking down liver Foxn3 expression via transduction with adeno-associated virus serotype 8 particles encoding a short hairpin RNA targeting Fonx3 decreases fasting glucose and increases Myc expression, without altering fasting glucagon or fasting insulin. Liver Foxn3 knock-down confers increases glucose tolerance, has no effect on insulin tolerance or response to glucagon challenge, blunts pyruvate and glutamine tolerance, and modulates expression of amino acid transporters and catabolic enzymes. We conclude that liver Foxn3 regulates substrate selection for gluconeogenesis.

Fish-hunting cone snail venoms are a rich source of minimized ligands of the vertebrate insulin receptor.

The fish-hunting marine cone snail Conus geographus uses a specialized venom insulin to induce hypoglycemic shock in its prey. We recently showed that this venom insulin, Con-Ins G1, has unique characteristics relevant to the design of new insulin therapeutics. Here, we show that fish-hunting cone snails provide a rich source of minimized ligands of the vertebrate insulin receptor. Insulins from C. geographus, Conus tulipa and Conus kinoshitai exhibit diverse sequences, yet all bind to and activate the human insulin receptor. Molecular dynamics reveal unique modes of action that are distinct from any other insulins known in nature. When tested in zebrafish and mice, venom insulins significantly lower blood glucose in the streptozotocin-induced model of diabetes. Our findings suggest that cone snails have evolved diverse strategies to activate the vertebrate insulin receptor and provide unique insight into the design of novel drugs for the treatment of diabetes.

Insulin is a hormone critical for maintaining healthy blood sugar levels in humans. When the insulin system becomes faulty, blood sugar levels become too high, which can lead to diabetes. At the moment, the only effective treatment for one of the major types of diabetes are daily insulin injections. However, designing fast-acting insulin drugs has remained a challenge. Insulin molecules form clusters (so-called hexamers) that first have to dissolve in the body to activate the insulin receptor, which plays a key role in regulating the blood sugar levels throughout the body. This can take time and can therefore delay the blood-sugar control. In 2015, researchers discovered that the fish-hunting cone snail Conus geographus uses a specific type of insulin to capture its prey ­ fish. The cone snail releases insulin into the surrounding water and then engulfs its victim with its mouth. This induces dangerously low blood sugar levels in the fish and so makes them an easy target. Unlike the human version, the snail insulin does not cluster, and despite structural differences, can bind to the human insulin receptor. Now, Ahorukomeye, Disotuar et al. ­ including some of the authors involved in the previous study ­ wanted to find out whether other fish-hunting cone snails also make insulins and if they differed from the one previously discovered in C. geographus. The insulin molecules were extracted and analyzed, and the results showed that the three cone snail species had different versions of insulin ­ but none of them formed clusters. Ahorukomeye, Disotuar et al. further revealed that the snail insulins could bind to the human insulin receptors and could also reverse high blood sugar levels in fish and mouse models of the disease. This research may help guide future studies looking into developing fast-acting insulin drugs for diabetic patients. A next step will be to fully understand how snail insulins can be active at the human receptor without forming clusters. Cone snails solved this problem millions of years ago and by understanding how they have done this, researchers are hoping to redesign current diabetic therapeutics. Since the snail insulins do not form clusters and should act faster than currently available insulin drugs, they may lead to better or new diabetes treatments.

The Monocarboxylate Transporter SLC16A6 Regulates Adult Length in Zebrafish and Is Associated With Height in Humans.

When fasted as larvae or fed ketogenic diets as adults, homozygous zebrafish slc16a6a mutants develop hepatic steatosis because their livers cannot export the major ketone body ß-hydroxybutyrate, diverting liver-trapped ketogenic carbon atoms to triacylglycerol. Here, we find that slc16a6a mutants are longer than their wild-type siblings. This effect is largely not sexually dimorphic, nor is it affected by dietary fat content on a pure genetic background. A mixed genetic background alters the proportionality of mass to length modestly. We also observe that non-coding variations in the 5'-untranslated region and first intron, and coding variations within the fifth exon of the orthologous human gene locus SLC16A6 are highly significantly associated with human height. Since both zebrafish and human orthologs of SLC16A6 are expressed in multiple locations, this gene likely regulates height through modulating transport of monocarboxylic acids in several tissues.