Disorganization and degeneration of liver sympathetic innervations in nonalcoholic fatty liver disease revealed by 3D imaging.

Hepatic nerves have a complex role in synchronizing liver metabolism. Here, we used three-dimensional (3D) immunoimaging to explore the integrity of the hepatic nervous system in experimental and human nonalcoholic fatty liver disease (NAFLD). We demonstrate parallel signs of mild degeneration and axonal sprouting of sympathetic innervations in early stages of experimental NAFLD and a collapse of sympathetic arborization in steatohepatitis. Human fatty livers display a similar pattern of sympathetic nerve degeneration, correlating with the severity of NAFLD pathology. We show that chronic sympathetic hyperexcitation is a key factor in the axonal degeneration, here genetically phenocopied in mice deficient of the Rac-1 activator Vav3. In experimental steatohepatitis, 3D imaging reveals a severe portal vein contraction, spatially correlated with the extension of the remaining nerves around the portal vein, enlightening a potential intrahepatic neuronal mechanism of portal hypertension. These fundamental alterations in liver innervation and vasculature uncover previously unidentified neuronal components in NAFLD pathomechanisms.

SNAP-25 Puts SNAREs at Center Stage in Metabolic Disease.

Synaptosomal Associated Protein of 25 kD (SNAP-25) is an essential protein contributing 2 out of 4 α-helices in the formation of the core soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex which mediates regulated membrane fusion. Regulated exocytosis is a strictly controlled event in eukaryotic cells mediating important homeostatic processes and cellular communications. Altered release of neurotransmitters or hormones is usually considered as part of the progressing pathophysiology of central neurological or peripheral metabolic disorders. However, the molecular changes which precede and initiate disturbed secretion of neurotransmitters and hormones are still unclear. We have explored an alternative hypothesis; that a minor modification in the machinery mediating regulated exocytosis, instead, may underlie the origin of the diseases associated with altered secretion of neurotransmitters and hormones. Possibly, certain modifications to genes encoding for SNAREs or proteins affecting SNARE function may increase the susceptibility to develop disease and its progression can be accelerated when combined with aging and life style factors. To test this theory, we genetically manipulated the Snap25 gene to express only one of the two alternatively spliced isoforms, SNAP-25a. SNAP-25b-deficient mice demonstrated alterations in synaptic transmission and increased insulin secretion which, with time, spontaneously progressed into a pronounced metabolic disease, including defects in glucose homeostasis, obesity, liver steatosis and perturbations in central homeostatic signaling. Thus, deregulated function of SNAP-25 and possibly other SNAREs or SNARE-interacting proteins, can, by itself, act as risk factors for the development of metabolic disease. Here, we provide an overview of the peripheral and central consequences of the deregulations in core SNARE complex with focus on SNAP-25.

SNAP-25a and SNAP-25b differently mediate interactions with Munc18-1 and Gßγ subunits.

SNAP-25 is a protein involved in regulated membrane fusion and part of the SNARE complex. It exists as two splicing variants, SNAP-25a and SNAP-25b, which differ in 9 out of 206 amino acids. SNAP-25 together with Syntaxin 1 and VAMP-2 forms the ternary SNARE complex essential for mediating activity-dependent release of hormones and neurotransmitters. The functional difference between SNAP-25a and SNAP-25b is poorly understood as both can participate in SNARE complexes and mediate membrane fusion. However, we recently demonstrated that SNAP-25b-deficiency results in metabolic disease and increased insulin secretion. Here we investigated if SNAP-25a and SNAP-25b differently affect interactions with other SNAREs and SNARE-interacting proteins in mouse hippocampus. Adult mice almost exclusively express the SNAP-25b protein in hippocampus whereas SNAP-25b-deficient mice only express SNAP-25a. Immunoprecipitation studies showed no significant differences in amount of Syntaxin 1 and VAMP-2 co-precipitated with the different SNAP-25 isoforms. In contrast, Munc18-1, that preferentially interacts with SNAP-25 via Syntaxin 1 and/or the trimeric SNARE complex, demonstrated an increased ability to bind protein-complexes containing SNAP-25b. Moreover, we found that both SNAP-25 isoforms co-precipitated the Gßγ subunits of the heterotrimeric G proteins, an interaction known to play a role in presynaptic inhibition. We have identified Gß1 and Gß2 as the interacting partners of both SNAP-25 isoforms in mouse hippocampus, but Gß2 was less efficiently captured by SNAP-25a. These results implicate that the two SNAP-25 isoforms could differently mediate protein interactions outside the ternary SNARE core complex and thereby contribute to modulate neurotransmission.

SNAP-25b-deficiency increases insulin secretion and changes spatiotemporal profile of Ca2+oscillations in ß cell networks.

SNAP-25 is a protein of the core SNARE complex mediating stimulus-dependent release of insulin from pancreatic ß cells. The protein exists as two alternatively spliced isoforms, SNAP-25a and SNAP-25b, differing in 9 out of 206 amino acids, yet their specific roles in pancreatic ß cells remain unclear. We explored the effect of SNAP-25b-deficiency on glucose-stimulated insulin release in islets and found increased secretion both in vivo and in vitro. However, slow photo-release of caged Ca2+ in ß cells within pancreatic slices showed no significant differences in Ca2+-sensitivity, amplitude or rate of exocytosis between SNAP-25b-deficient and wild-type littermates. Therefore, we next investigated if Ca2+ handling was affected in glucose-stimulated ß cells using intracellular Ca2+-imaging and found premature activation and delayed termination of [Ca2+] i elevations. These findings were accompanied by less synchronized Ca2+-oscillations and hence more segregated functional ß cell networks in SNAP-25b-deficient mice. Islet gross morphology and architecture were maintained in mutant mice, although sex specific compensatory changes were observed. Thus, our study proposes that SNAP-25b in pancreatic ß cells, except for participating in the core SNARE complex, is necessary for accurate regulation of Ca2+-dynamics.

Minor differences in the molecular machinery mediating regulated membrane fusion has major impact on metabolic health.

The exocytosis of signaling molecules from neuronal, neuroendocrine and endocrine cells is regulated by membrane fusion involving SNAP-25 and associated SNARE proteins. The importance of this process for metabolic control recently became evident by studies of mouse mutants genetically engineered to only express one of 2 closely related, alternatively-spliced variants of SNAP-25. The results showed that even minor differences in the function of proteins regulating exocytosis are sufficient to provoke metabolic disease, including hyperglycaemia, liver steatosis, adipocyte hypertrophy and obesity. Thus, an imbalance in the dynamics of hormonal and/or neurotransmitter release can cause obesity and type 2 diabetes. This recent discovery highlights the fact that metabolic health requires a perfectly operating interplay between the SNARE protein machinery in excitable cells and the organs responding to these messengers.

Replacing SNAP-25b with SNAP-25a expression results in metabolic disease.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a key molecule in the soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE) complex mediating fast Ca(2+)-triggered release of hormones and neurotransmitters, and both splice variants, SNAP-25a and SNAP-25b, can participate in this process. Here we explore the hypothesis that minor alterations in the machinery mediating regulated membrane fusion can increase the susceptibility for metabolic disease and precede obesity and type 2 diabetes. Thus, we used a mouse mutant engineered to express normal levels of SNAP-25 but only SNAP-25a. These SNAP-25b-deficient mice were exposed to either a control or a high-fat/high-sucrose diet. Monitoring of food intake, body weight, hypothalamic function, and lipid and glucose homeostases showed that SNAP-25b-deficient mice fed with control diet developed hyperglycemia, liver steatosis, and adipocyte hypertrophy, conditions dramatically exacerbated when combined with the high-fat/high-sucrose diet. Thus, modified SNARE function regulating stimulus-dependent exocytosis can increase the vulnerability to and even provoke metabolic disease. When combined with a high-fat/high-sucrose diet, this vulnerability resulted in diabesity. Our SNAP-25b-deficient mouse may represent a diabesity model.

Characterization of secretagogin-immunoreactive amacrine cells in marmoset retina.

The retina contains at least 30 different types of amacrine cells but not many are well characterized. In the present study the calcium-binding protein secretagogin was localized in a population of regular and displaced amacrine cells in the retina of the common marmoset Callithrix jacchus. Irrespective of their soma location, the dendrites of secretagogin amacrine cells occupy strata 2, 3, and 4 of the inner plexiform layer, between the two bands formed by cholinergic amacrine cells. Segretagogin amacrine cells are also immunopositive to antibodies against glutamic acid decarboxylase, suggesting that they use γ-aminobutyric acid (GABA) as their neurotransmitter. The spatial density of secretagogin amacrine cells decreases from a peak of about 400 cells/mm(2) near 1 mm eccentricity to less than 100 cells/mm(2) in peripheral retina; these densities account for about 1% of amacrine cells in the inner nuclear layer and for up to 27% of displaced amacrine cells. The cell bodies form a regular mosaic, suggesting that they constitute a single amacrine cell population. Secretagogin cells have varicose dendrites, which are decorated with small spines. Intracellular injection of DiI into secretagogin cells revealed an average dendritic field diameter of 170 µm and an average coverage factor of 3.2. In summary, secretagogin cells in marmoset retina are medium-field amacrine cells that share their stratification pattern with narrow-field amacrine cells and their neurotransmitter with wide-field amacrine cells. They may mediate spatial inhibition spanning the centralmost (on and off) bands of the inner plexiform layer.

G-protein-gated inwardly rectifying K+ channel 4 (GIRK4) immunoreactivity in chemically defined neurons of the hypothalamic arcuate nucleus that control body weight.

G-protein-gated inwardly rectifying K(+) channels (GIRKs; also called Kir3) are a family of K(+) channels, which are activated (opened) via a signal transduction cascade starting with ligand-stimulated G-protein-coupled receptors (GPCRs). Four GIRK genes have been identified (GIRK1-4). GIRK4 (Kir3.4) has a role in regulating energy homeostasis, since mice with a targeted mutation in the GIRK4 gene exhibit a predisposition to late-onset obesity. GIRK4 mRNA is expressed in hypothalamic regions that harbor neurons involved in the regulation of food intake and body weight. Using goat and rabbit antisera to the GIRK4 protein, the cellular localization and transmitter content of GIRK4-immunoreactive neurons was determined in the hypothalamic arcuate nucleus, a region that contains neurons which are accessible to circulating hormones and is intimately associated with the control of body weight. GIRK4-immunoreactive large cell bodies were demonstrated in the ventrolateral part of the arcuate nucleus, with smaller neuronal cell bodies in the ventromedial part of the nucleus. Double-labeling showed presence of GIRK4 immunoreactivity in large neurons of the ventrolateral arcuate nucleus containing the peptides α-melanocyte-stimulating hormone (α-MSH), a marker for pro-opiomelanocortin (POMC) neurons, and cocaine- and amphetamine-regulated transcript (CART). GIRK4 immunoreactivity was also seen in neurons of the ventromedial part of the arcuate nucleus containing agouti-regulated peptide (AgRP) and neuropeptide Y (NPY). The results suggest that the GIRK4 channel protein plays a role in regulating membrane excitability in chemically defined neurons of the arcuate nucleus that control body weight.