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BackgroundAutophagosome, the central organelle in autophagy process, can assemble via canonical pathway mediated by LC3-II, the lipidated form of autophagy-related protein LC3/ATG8, or noncanonical pathway mediated by the small GTPase Rab9. Canonical autophagy is essential for exocrine pancreas homeostasis, and its disordering initiates and drives pancreatitis. The involvement of noncanonical autophagy has not been explored. We examine the role of Rab9 in pancreatic autophagy and pancreatitis severity.MethodsWe measured the effect of Rab9 on parameters of autophagy and pancreatitis responses using transgenic mice overexpressing Rab9 (Rab9TG) and adenoviral transduction of acinar cells. Effect of canonical autophagy on Rab9 was assessed in ATG5-deficient acinar cells.ResultsPancreatic levels of Rab9 and its membrane-bound (active) form decreased in rodent pancreatitis models and in human disease. Rab9 overexpression stimulated noncanonical and inhibited canonical/LC3-mediated autophagosome formation in acinar cells through up-regulation of ATG4B, the cysteine protease that delipidates LC3-II. Conversely, ATG5 deficiency caused Rab9 increase in acinar cells. Inhibition of canonical autophagy in Rab9TG pancreas was associated with accumulation of Rab9-positive vacuoles containing markers of mitochondria, protein aggregates, and trans-Golgi. The shift to the noncanonical pathway caused pancreatitis-like damage in acinar cells and aggravated experimental pancreatitis.ConclusionsThe results show that Rab9 regulates pancreatic autophagy and indicate a mutually antagonistic relationship between the canonical/LC3-mediated and noncanonical/Rab9-mediated autophagy pathways in pancreatitis. Noncanonical autophagy fails to substitute for its canonical counterpart in protecting against pancreatitis. Thus, Rab9 decrease in experimental and human pancreatitis is a protective response to sustain canonical autophagy and alleviate disease severity.

Disordered lysosomal/autophagy pathways initiate and drive pancreatitis, but the underlying mechanisms and links to disease pathology are poorly understood. Here, we show that the mannose-6-phosphate (M6P) pathway of hydrolase delivery to lysosomes critically regulates pancreatic acinar cell cholesterol metabolism. Ablation of the Gnptab gene encoding a key enzyme in the M6P pathway disrupted acinar cell cholesterol turnover, causing accumulation of nonesterified cholesterol in lysosomes/autolysosomes, its depletion in the plasma membrane, and upregulation of cholesterol synthesis and uptake. We found similar dysregulation of acinar cell cholesterol, and a decrease in GNPTAB levels, in both WT experimental pancreatitis and human disease. The mechanisms mediating pancreatic cholesterol dyshomeostasis in Gnptab-/- and experimental models involve a disordered endolysosomal system, resulting in impaired cholesterol transport through lysosomes and blockage of autophagic flux. By contrast, in Gnptab-/- liver the endolysosomal system and cholesterol homeostasis were largely unaffected. Gnptab-/- mice developed spontaneous pancreatitis. Normalization of cholesterol metabolism by pharmacologic means alleviated responses of experimental pancreatitis, particularly trypsinogen activation, the disease hallmark. The results reveal the essential role of the M6P pathway in maintaining exocrine pancreas homeostasis and function, and implicate cholesterol disordering in the pathogenesis of pancreatitis.

Pancreatic beta cells synthesize and secrete the neurotransmitter γ-aminobutyric acid (GABA) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel (VRAC), functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from type 1 and type 2 diabetic patients, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.

Aim: To assess the effects of faecal microbial transplant (FMT) from lean people to subjects with obesity via colonoscopy. Material and Methods: In a double‐blind, randomized controlled trial, subjects with a body mass index ≥ 35 kg/m2 and insulin resistance were randomized, in a 1:1 ratio in blocks of four, to either allogenic (from healthy lean donor; n = 15) or autologous FMT (their own stool; n = 13) delivered in the caecum and were followed for 3 months. The main outcome was homeostatic model assessment of insulin resistance (HOMA‐IR) and secondary outcomes were glycated haemoglobin levels, lipid profile, weight, gut hormones, endotoxin, appetite measures, intestinal microbiome (IM), metagenome, serum/faecal metabolites, quality of life, anxiety and depression scores. Results: In the allogenic versus autologous groups, HOMA‐IR and clinical variables did not change significantly, but IM and metabolites changed favourably (P < 0.05): at 1 month, Coprococcus, Bifidobacterium, Bacteroides and Roseburia increased, and Streptococcus decreased; at 3 months, Bacteroides and Blautia increased. Several species also changed significantly. For metabolites, at 1 month, serum kynurenine decreased and faecal indole acetic acid and butenylcarnitine increased, while at 3 months, serum isoleucine, leucine, decenoylcarnitine and faecal phenylacetic acid decreased. Metagenomic pathway representations and network analyses assessing relationships with clinical variables, metabolites and IM were significantly enhanced in the allogenic versus autologous groups. LDL and appetite measures improved in the allogenic (P < 0.05) but not in the autologous group. Conclusions: Overall, in those with obeisty, allogenic FMT via colonoscopy induced favourable changes in IM, metabolites, pathway representations and networks even though other metabolic variables did not change. LDL and appetite variables may also benefit. [ABSTRACT FROM AUTHOR]

Choi D, Radziszewska A, Schroer SA, Liadis N, Liu Y, Zhang Y, Lam PP, Sheu L, Hao Z, Gaisano HY, Woo M. Deletion of Fas in the pancreatic [beta}-cells leads to enhanced insulin secretion. Am J Physiol Endocrinol Metab 297: E 1304-E 1312, 2009. First published September 15, 2009; doi: 10.1152/ajpendo.00217.2009.--Fas/Fas ligand belongs to the tumor necrosis factor superfamily of receptors/ ligands and is best known for its role in apoptosis. However, recent evidence supports its role in other cellular responses, including proliferation and survival. Although Fas has been implicated as an essential mediator of [beta]-cell death in the pathogenesis of type 1 diabetes, the essential role of Fas specifically in pancreatic [beta]-cells has been found to be controversial. Moreover, the role of Fas on [beta]-cell homeostasis and function is not clear. The objective of this study is to determine the role of Fas specifically in [beta]-cells under both physiological and diabetes models. Mice with Fas deletion specifically in the [beta]-cells were generated using the Cre-loxP system. Cre-mediated Fas deletion was under the control of the rat insulin promoter. Absence of Fas in [beta]-cells leads to complete protection against FasL-induced cell death. However, Fas is not essential in determining [beta]-cell mass or susceptibility to streptozotocin- or HFD-induced diabetes. Importantly, Fas deletion in [beta]-cells leads to increased p65 expression, enhanced glucose tolerance, and glucose-stimulated insulin secretion, with increased exocytosis as manifested by increased changes in membrane capacitance and increased expression of Syntaxin1A, VAMP2, and muncl8a. Together, our study shows that Fas in the [beta]-cells indeed plays an essential role in the canonical death receptor- mediated apoptosis but is not essential in regulating [beta]-cell mass or diabetes development. However, [beta]-cell Fas is critical in the regulation f glucose homeostasis through regulation of the exocytosis machinely. diabetes; apoptosis; exocytosis; islets doi: 10.1152/ajpendo.00217.2009

Synaptosomal protein of 25 kDa (SNAP-25) is important for [Ca.sup.2+]-dependent fusion of large dense core vesicles (LDCVs) in insulin-secreting cells. Exocytosis is further enhanced by cAMP-increasing agents such as glucagon-like peptide-1 (GLP-1), and this augmentation includes interaction with both PKA and cAMP-GEFII. To investigate the coupling between SNAP-25- and cAMP-dependent stimulation of insulin exocytosis, we have used capacitance measurements, protein-binding assays, and Western blot analysis. In insulin-secreting INS-1 cells overexpressing wild-type SNAP-25 ([SNAP-25.sub.WT]), rapid exocytosis was stimulated more than threefold by cAMP, similar to the situation in nontransfected cells. However, cAMP failed to potentiate rapid exocytosis in INS-1 cells overexpressing a truncated form of SNAP-25 ([SNAP-25.sub.1-197]) or Botulinum neurotoxin A (BoNT/A). Close dissection of the exocytotic response revealed that the inability of cAMP to stimulate exocytosis in the presence of a truncated SNAP-25 was confined to the release of primed LDCVs within the readily releasable pool, especially from the immediately releasable pool, whereas cAMP enhanced mobilization of granules from the reserve pool in both [SNAP-25.sub.1-197] (P < 0.01) and [SNAP-25.sub.WT] (P < 0.05) cells. This was supported by hormone release measurements. Augmentation of the immediately releasable pool by cAMP has been suggested to act through the cAMP-GEFII-dependent, PKA-independent pathway. Indeed, we were able to verify an interaction between SNAP-25 with both cAMP-GEFII and RIM2, two proteins involved in the PKA-independent pathway. Thus we hypothesize that SNAP-25 is a necessary partner in the complex mediating cAMP-enhanced rapid exocytosis in insulin-secreting cells. synaptosomal protein of 25 kDa; insulin; INS-1; cAMP-GEFII; Epac; capacitance measurements

Thioredoxin-interacting protein (TxNIP) is an endogenous inhibitor of thioredoxin, a ubiquitous thiol oxidoreductase, that regulates cellular redox status. Diabetic mice exhibit increased expression of TxNIP in pancreatic islets, and recent studies suggest that TxNIP is a proapoptotic factor in [beta]-cells that may contribute to the development of diabetes. Here, we examined the role of TxNIP deficiency in vivo in the development of insulin-deficient diabetes and whether it impacted on pancreatic [beta]-cell mass and/or insulin secretion. TxNIP-deficient (Hcb-19/[TxNIP.sup.-/-]) mice had lower baseline glycemia, higher circulating insulin concentrations, and higher total pancreatic insulin content and [beta]-cell mass than control mice (C3H). Hcb-19/[TxNIP.sup.-/-] did not develop hyperglycemia when injected with standard multiple low doses of streptozotocin (STZ), in contrast to C3H controls. Surprisingly, although [beta]-cell mass remained higher in Hcb-19/[TxNIP.sup.-/-] mice compared with C3H after STZ exposure, the relative decrease induced by STZ was as great or even greater in the TxNIP-deficient animals. Consistently, cultured pancreatic INS-1 cells transfected with small-interfering RNA against TxNIP were more sensitive to cell death induced by direct exposure to STZ or to the combination of inflammatory cytokines interleukin-1[[beta], interferon-[gamma], and tumor necrosis factor-[alpha]. Furthermore, when corrected for insulin content, isolated pancreatic islets from [TxNIP.sup.-/-] mice exhibited reduced glucose-induced insulin secretion. These data indicate that TxNIP functions as a regulator of [beta]-cell mass and influences insulin secretion. In conclusion, the relative resistance of TxNIP-deficient mice to STZ-induced diabetes appears to be because of an increase in [beta]-cell mass. However, TxNIP deficiency is associated with sensitization to STZ- and cytokine-induced [beta]-cell death, indicating complex regulatory roles of TxNIP under different physiological and pathological conditions. thioredoxin interacting protein; pancreatic [beta]-cell; apoptosis; insulin secretion

Both Epac and PKA are effectors of the second messenger cAMP. Utilizing an exchange protein directly activated by cAMP (Epac) pathway-specific cAMP analog (ESCA), we previously reported that Epac signaling regulates proglucagon gene (gcg) expression in the glucagon-like peptide-1 (GLP-1)-producing intestinal endocrine L-cell lines GLUTag and STC-1. We now show that Epac-2 is also expressed in glucagon-producing pancreatic co-cell lines, including PKA-deficient InR1-G9 cells, and that ESCA stimulates gcg promoter and mRNA expression in the InR1-G9 cells. Using a dominant-negative Epac-2 expression plasmid (Epac-2DN), we found that Epac inhibition attenuated forskolin-stimulated gcg promoter expression in the PKA-active STC-1 cell line and blocked forskolin-stimulated gcg promoter expression in the InR1-G9 cells. Consistently, ESCA was shown to stimulate glucagon and GLP-1 production in the InR1-G9 and GLUTag cell lines, respectively. Surprisingly, ESCA treatment did not show a notable stimulation of glucagon or GLP-1 secretion from these two cell lines. This is in contrast to its ability to stimulate insulin secretion from the pancreatic INS-I [beta]-cell line. Our findings suggest that Epac is selectively involved in peptide hormone secretion in pancreatic and intestinal endocrine cells and that distinct signaling cascades are involved in stimulating production vs. secretion of glucagon and GLP-1 in response to cAMP elevation. glucagon; glucagon-like peptide-1; exchange protein directly activated by adenosine 3',5'-cyclic monophosphate; protein kinase A

SNARE proteins, syntaxin-1A (Syn-1A) and SNAP-25, inhibit delayed rectifier [K.sup.+] channels, [K.sub.v]1.1 and [K.sub.v]2.1, in secretory cells. We showed previously that the mutant open conformation of Syn-1A (Syn-1A L165A/ E166A) inhibits [K.sub.v]2.1 channels more optimally than wild-type Syn-1A. In this report we examined whether Syn-1A in its wild-type and open conformations would exhibit similar differential actions on the gating of [K.sub.v]1.2, a major delayed rectifier [K.sup.+] channel in nonsecretory smooth muscle cells and some neuronal tissues. In coexpression and acute dialysis studies, wild-type Syn-1A inhibited [K.sub.v]1.2 current magnitude. Of interest, wild-type Syn-1A caused a right shift in the activation curves of [K.sub.v]1.2 without affecting its steady-state availability, an inhibition profile opposite to its effects on [K.sub.v]2.1 (steady-state availability reduction without changes in voltage dependence of activation). Also, although both wild-type and open-form Syn-1A bound equally well to [K.sub.v]1.2 in an expression system, open-form Syn-1A failed to reduce [K.sub.v]1.2 current magnitude or affect its gating. This is in contrast to the reported more potent effect of open-form Syn-1A on [K.sub.v]2.1 channels in secretory cells. This finding together with the absence of Munc18 and/or 13-1 in smooth muscles suggested that a change to an open conformation Syn-1A, normally facilitated by Munc18/13-1, is not required in nonsecretory smooth muscle cells. Taken together with previous reports, our results demonstrate the multiplicity of gating inhibition of different [K.sub.v] channels by Syn-1A and is compatible with versatility of Syn-1A modulation of repolarization in various secretory and nonsecretory (smooth muscle) cell types. Keywords: ion channels; electrophysiology; SNARE protein doi:10.1152/ajpgi.00473.2006

The lower esophageal sphincter (LES) has a circular muscle component exhibiting spontaneous tone that is relaxed by nitric oxide (NO) and a low-tone sling muscle that contracts vigorously to cholinergic stimulation but with little or no evidence of NO responsiveness. This study dissected the responses of the sling muscle to nitrergic innervation in relationship to its cholinergic innervation and circular muscle responses. Motor responses were induced by electrical field stimulation (EFS; 1-30 Hz) of muscle strips from sling and circular regions of the feline LES in the presence of cholinergic receptor inhibition (atropine) or NO synthase inhibition [[N.sup.G]-nitro-L-arginine (L-NNA) [+ or -] atropine]. This study showed the following. First, sling muscle developed less intrinsic resting tone compared with circular muscle. Second, with EFS, sling muscle contracted (most at [less than or equal to] 10 Hz), whereas circular muscle relaxed >50% by 5 Hz. Third, on neural blockade with atropine or L-NNA [+ or -] atropine, 1) sling muscle, although predominantly influenced by excitatory cholinergic stimulation, had a small neural NO-mediated inhibition, with no significant non-NO-mediated inhibition and 2) circular muscle, although little affected by cholinergic influence, underwent relaxation predominantly by neural release of NO and some non-NO inhibitory influence (at higher EFS frequency). Fourth, the sling, precontracted with bethanecol, could relax with NO and some non-NO inhibition. Finally, the tension range of both muscles is similar. In conclusion, sling muscle has limited NO-mediated inhibition to potentially augment or replace sling relaxation effected by switching off its cholinergic excitation. Differences within the LES sling and circular muscles could provide new directions for therapy of LES disorders. circular muscle; nitric oxide; acetylcholine; sling muscle

Potassium channels are important contributors to membrane excitability in smooth muscles. There are regional differences in resting membrane potential and [K.sup.+]-channel density along the length of the feline circular smooth muscle esophagus. The aim of this study was to assess responses of [K.sup.+]-channel currents to cholinergic (ACh) stimulation along the length of the feline circular smooth muscle esophageal body. Perforated patch-clamp technique assessed [K.sup.+]-channel responses to ACh stimulation in isolated smooth muscle cells from the circular muscle layer of the esophageal body at 2 (distal)- and 4-cm (proximal) sites above the lower esophageal sphincter. Western immunoblots assessed ion channel and receptor expression. ACh stimulation produced a transient increase in outward current followed by inhibition of spontaneous transient outward currents. These ACh-induced currents were abolished by blockers of large-conductance [Ca.sup.2+]-dependent [K.sup.+] channels ([BK.sub.Ca]). Distal cells demonstrated a greater peak current density in outward current than cells from the proximal region and a longerlasting outward current increase. These responses were abolished by atropine and the specific [M.sub.3] receptor antagonist 4-DAMP but not the [M.sub.1] receptor antagonist pirenzipine or the [M.sub.2] receptor antagonist methoctramine. [BK.sub.Ca], expression along the smooth muscle esophagus was similar, but [M.sub.3] receptor expression was greater in the distal region. Therefore, ACh can differentially activate a potassium channel ([BK.sub.Ca],) current along the smooth muscle esophagus. This activation probably occurs through release of intracellular calcium via an [M.sub.3] pathway and has the potential to modulate the timing and amplitude of peristaltic contraction along the esophagus. esophagus; acetylcholine; potassium channel