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.

A Hepatocyte FOXN3-α Cell Glucagon Axis Regulates Fasting Glucose.

The common genetic variation at rs8004664 in the FOXN3 gene is independently and significantly associated with fasting blood glucose, but not insulin, in non-diabetic humans. Recently, we reported that primary hepatocytes from rs8004664 hyperglycemia risk allele carriers have increased FOXN3 transcript and protein levels and liver-limited overexpression of human FOXN3, a transcriptional repressor that had not been implicated in metabolic regulation previously, increases fasting blood glucose in zebrafish. Here, we find that injection of glucagon into mice and adult zebrafish decreases liver Foxn3 protein and transcript levels. Zebrafish foxn3 loss-of-function mutants have decreased fasting blood glucose, blood glucagon, liver gluconeogenic gene expression, and α cell mass. Conversely, liver-limited overexpression of foxn3 increases α cell mass. Supporting these genetic findings in model organisms, non-diabetic rs8004664 risk allele carriers have decreased suppression of glucagon during oral glucose tolerance testing. By reciprocally regulating each other, liver FOXN3 and glucagon control fasting glucose.

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.

FOXN3 Regulates Hepatic Glucose Utilization.

A SNP (rs8004664) in the first intron of the FOXN3 gene is associated with human fasting blood glucose. We find that carriers of the risk allele have higher hepatic expression of the transcriptional repressor FOXN3. Rat Foxn3 protein and zebrafish foxn3 transcripts are downregulated during fasting, a process recapitulated in human HepG2 hepatoma cells. Transgenic overexpression of zebrafish foxn3 or human FOXN3 increases zebrafish hepatic gluconeogenic gene expression, whole-larval free glucose, and adult fasting blood glucose and also decreases expression of glycolytic genes. Hepatic FOXN3 overexpression suppresses expression of mycb, whose ortholog MYC is known to directly stimulate expression of glucose-utilization enzymes. Carriers of the rs8004664 risk allele have decreased MYC transcript abundance. Human FOXN3 binds DNA sequences in the human MYC and zebrafish mycb loci. We conclude that the rs8004664 risk allele drives excessive expression of FOXN3 during fasting and that FOXN3 regulates fasting blood glucose.

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.

Polyunsaturated fatty acyl-coenzyme As are inhibitors of cholesterol biosynthesis in zebrafish and mice.

Lipid disorders pose therapeutic challenges. Previously we discovered that mutation of the hepatocyte ß-hydroxybutyrate transporter Slc16a6a in zebrafish causes hepatic steatosis during fasting, marked by increased hepatic triacylglycerol, but not cholesterol. This selective diversion of trapped ketogenic carbon atoms is surprising because acetate and acetoacetate can exit mitochondria and can be incorporated into both fatty acids and cholesterol in normal hepatocytes. To elucidate the mechanism of this selective diversion of carbon atoms to fatty acids, we fed wild-type and slc16a6a mutant animals high-protein ketogenic diets. We find that slc16a6a mutants have decreased activity of the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr), despite increased Hmgcr protein abundance and relative incorporation of mevalonate into cholesterol. These observations suggest the presence of an endogenous Hmgcr inhibitor. We took a candidate approach to identify such inhibitors. First, we found that mutant livers accumulate multiple polyunsaturated fatty acids (PUFAs) and PUFA-CoAs, and we showed that human HMGCR is inhibited by PUFA-CoAs in vitro. Second, we injected mice with an ethyl ester of the PUFA eicosapentaenoic acid and observed an acute decrease in hepatic Hmgcr activity, without alteration in Hmgcr protein abundance. These results elucidate a mechanism for PUFA-mediated cholesterol lowering through direct inhibition of Hmgcr.

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.

Influences of dietary biotin and avidin on growth, survival, deficiency syndrome and hepatic gene expression of juvenile Nile tilapia Oreochromis niloticus.

This study was undertaken to assess the interactive effects of dietary biotin and avidin on growth, feed conversion, survival and deficiency syndrome of tilapia and to determine the influence of dietary biotin deficiency on the expression of key genes related to biotin metabolism in tilapia. Six iso-nitrogenous and iso-energetic diets based on a common purified basal diet (vitamin-free casein as the protein source) were prepared for this study. The six dietary groups were 0 g avidin with 0 mg biotin (A0B0), 0 g avidin with 0.06 mg biotin/kg diet (A0B1), four avidin-supplemented diets incorporating at a incremental concentrations 0.25, 0.5, 1.0 and 2.0 g/kg diet with 0.06 mg biotin/kg diet (A15B1, A30B1, A60B1 and A120B1). Fish were hand-fed three times a day to apparent satiation for 12 weeks. Each diet was fed to three replicate groups of fish. Fish were kept in glass aquaria in a recirculating aquaculture system under standardized environmental conditions. Growth was significantly higher in fish that received the biotin-supplemented diet (A0B1), compared to diets lacking biotin or supplemented with avidin. Tilapia fed higher concentration of avidin-supplemented diets (A60B1 and A120B1) showed significant growth depression and displayed severe deficiency syndromes such as lethargy, anorexia, circular swimming and convulsions, which ultimately lead to death. There was a strong proportional linear relationship between the avidin content of the diet and feed conversion ratio, FCR (y = 0.43x + 0.135; r = 0.960; P < 0.001) and strong inverse relationship with protein efficiency ratio, PER (y = -0.309x + 2.195; r = 0.961; P < 0.0001). Elevated levels of biotinidase, pyruvate carboxylase, propionyl-CoA carboxylase-A and propionyl-CoA carboxylase-B transcripts were noted in fish fed all graded level of avidin-supplemented diets. A broken-line analysis indicated that feeding tilapia a diet with 44.5 times more avidin than the dietary biotin requirement can induce deficiency syndromes including retarded growth, when analyzing the data of percentage weight gain.

Effects of dietary biotin and avidin on growth, survival, feed conversion, biotin status and gene expression of zebrafish Danio rerio.

A study was conducted to investigate the effects of dietary avidin on growth, survival, food conversion, biotin status and gene expression of zebrafish (Danio rerio Hamilton-Buchanan) juveniles (average wet mass 0.178 g) fed 7 purified diets for 12 weeks. Experimental diets were formulated to provide 0×, 1×, 15×, 30×, 60× and 120× excess avidin versus biotin kg(-1) diet, on a molar basis; a control diet contained neither supplemental biotin nor avidin. Fish fed the control diet had the lowest percentage weight gain and the highest mortality, while the highest percentage weight gain and the lowest mortality was observed with the 0× diet (P<0.05). A linear relationship was observed between feed conversion ratio (FCR) and dietary avidin (r=0.876; P<0.0001). Fish fed diets with 120× more avidin than biotin had the highest whole-body biotin content, while the lowest value was obtained with the control and avidin-free diets (P<0.05). Elevated levels of acetyl CoA carboxylase-A (acca), methylcrotonyl CoA carboxylase (mcc) and propionyl CoA carboxylase-A (pcca) transcripts were recorded in fish fed the control diet, in comparison to the other diets. A broken-line analysis indicated that feeding zebrafish a diet with 60 times more avidin than the dietary biotin requirement level will cause biotin deficiency signs.

Tandem duplication of the fabp1b gene and subsequent divergence of the tissue-specific distribution of fabp1b.1 and fabp1b.2 transcripts in zebrafish (Danio rerio).

We describe a fatty acid-binding protein 1 (fabp1b.2) gene and its tissue-specific expression in zebrafish embryos and adults. The 3.5 kb zebrafish fabp1b.2 gene is the paralog of the previously described zebrafish fabp1a and fabp1b genes. Using the LN54 radiation hybrid mapping panel, we assigned the zebrafish fabp1b.2 gene to linkage group 8, the same linkage group to which fabp1b.1 was mapped. fabp1b.1 and fabp1b.2 appear to have arisen by a tandem duplication event. Whole-mount in situ hybridization of a riboprobe to embryos and larvae detected fabp1b.2 transcripts in the diencephalon and as spots in the periphery of the yolk sac. In adult zebrafish, in situ hybridization revealed fabp1b.2 transcripts in the anterior intestine and skin, and reverse transcription PCR (RT-PCR) detected fabp1b.2 transcripts in the intestine, brain, heart, ovary, skin, and eye. By contrast, fabp1b.1 transcripts were detected by RT-PCR in the liver, intestine, heart, testis, ovary, and gills. The tissue-specific distribution of transcripts for the tandemly duplicated fabp1b.1 and fabp1b.2 genes in adult tissues and during development suggests that the duplicated fabp1b genes of zebrafish have acquired additional functions compared with the ancestral fabp1 gene, i.e., by neofunctionalization. Furthermore, these functions were subsequently divided between fabp1b.1 and fabp1b.2 owing to subfunctionalization.

Differential transcriptional modulation of duplicated fatty acid-binding protein genes by dietary fatty acids in zebrafish (Danio rerio): evidence for subfunctionalization or neofunctionalization of duplicated genes.

BACKGROUND: In the Duplication-Degeneration-Complementation (DDC) model, subfunctionalization and neofunctionalization have been proposed as important processes driving the retention of duplicated genes in the genome. These processes are thought to occur by gain or loss of regulatory elements in the promoters of duplicated genes. We tested the DDC model by determining the transcriptional induction of fatty acid-binding proteins (Fabps) genes by dietary fatty acids (FAs) in zebrafish. We chose zebrafish for this study for two reasons: extensive bioinformatics resources are available for zebrafish at zfin.org and zebrafish contains many duplicated genes owing to a whole genome duplication event that occurred early in the ray-finned fish lineage approximately 230-400 million years ago. Adult zebrafish were fed diets containing either fish oil (12% lipid, rich in highly unsaturated fatty acid), sunflower oil (12% lipid, rich in linoleic acid), linseed oil (12% lipid, rich in linolenic acid), or low fat (4% lipid, low fat diet) for 10 weeks. FA profiles and the steady-state levels of fabp mRNA and heterogeneous nuclear RNA in intestine, liver, muscle and brain of zebrafish were determined. RESULT: FA profiles assayed by gas chromatography differed in the intestine, brain, muscle and liver depending on diet. The steady-state level of mRNA for three sets of duplicated genes, fabp1a/fabp1b.1/fabp1b.2, fabp7a/fabp7b, and fabp11a/fabp11b, was determined by reverse transcription, quantitative polymerase chain reaction (RT-qPCR). In brain, the steady-state level of fabp7b mRNAs was induced in fish fed the linoleic acid-rich diet; in intestine, the transcript level of fabp1b.1 and fabp7b were elevated in fish fed the linolenic acid-rich diet; in liver, the level of fabp7a mRNAs was elevated in fish fed the low fat diet; and in muscle, the level of fabp7a and fabp11a mRNAs were elevated in fish fed the linolenic acid-rich or the low fat diets. In all cases, induction of the steady-state level of fabp mRNAs by dietary FAs correlated with induced levels of hnRNA for a given fabp gene. As such, up-regulation of the steady-state level of fabp mRNAs by FAs occurred at the level of initiation of transcription. None of the sister duplicates of these fabp genes exhibited an increase in their steady-state transcript levels in a specific tissue following feeding zebrafish any of the four experimental diets. CONCLUSION: Differential induction of only one of the sister pair of duplicated fabp genes by FAs provides evidence to support the DDC model for retention of duplicated genes in the zebrafish genome by either subfunctionalization or neofunctionalization.

The evolutionary relationship between the duplicated copies of the zebrafish fabp11 gene and the tetrapod FABP4, FABP5, FABP8 and FABP9 genes.

We describe the structure of a fatty acid-binding protein 11 (fabp11b) gene and its tissue-specific expression in zebrafish. The 3.4 kb zebrafish fabp11b is the paralog of the previously described zebrafish fabp11a, with a deduced amino acid sequence for Fabp11B exhibiting 65% identity with that of Fabp11A. Whole mount in situ hybridization of a riboprobe to embryos and larvae showed that zebrafish fabp11b transcripts were restricted solely to the retina and were first detected at 24 h postfertilization. In situ hybridization revealed fabp11b transcripts along the spinal cord in adult zebrafish. However, the highly sensitive RT-PCR assay detected fabp11b transcripts in the brain, heart, ovary and eye in adult tissues. By contrast, fabp11a transcripts had been previously detected in the liver, brain, heart, testis, muscle, ovary and skin of adult zebrafish. Using the LN54 radiation hybrid panel, we assigned zebrafish fabp11b to linkage group 16. Phylogenetic analysis and conserved gene synteny with tetrapod genes indicated that the emergence of two copies of fabp11 in the zebrafish genome may have resulted from a fish-specific whole genome duplication event. Furthermore, we propose that the FABP4-FABP5-FABP8-FABP9 (PERF15) gene cluster on a single chromosome in the tetrapod genome and the fabp11 genes in the zebrafish genome originated from a common ancestral gene, which, following their divergence, gave rise to the fabp11 genes of zebrafish, and the progenitor of the FABP4, FABP5, FABP8 and FABP9 genes in tetrapods after the separation of the fish and tetrapod lineages.