Promises and Limitations of Current Models for Understanding Barrett's Esophagus and Esophageal Adenocarcinoma.

BACKGROUND & AIMS: This review was developed to provide a thorough and effective update on models relevant to esophageal metaplasia, dysplasia, and carcinogenesis, focusing on the advantages and limitations of different models of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). METHODS: This expert review was written on the basis of a thorough review of the literature combined with expert interpretation of the state of the field. We emphasized advances over the years 2012-2023 and provided detailed information related to the characterization of established human esophageal cell lines. RESULTS: New insights have been gained into the pathogenesis of BE and EAC using patient-derived samples and single-cell approaches. Relevant animal models include genetic as well as surgical mouse models and emphasize the development of lesions at the squamocolumnar junction in the mouse stomach. Rat models are generated using surgical approaches that directly connect the small intestine and esophagus. Large animal models have the advantage of including features in human esophagus such as esophageal submucosal glands. Alternatively, cell culture approaches remain important in the field and allow for personalized approaches, and scientific rigor can be ensured by authentication of cell lines. CONCLUSIONS: Research in BE and EAC remains highly relevant given the morbidity and mortality associated with cancers of the tubular esophagus and gastroesophageal junction. Careful selection of models and inclusion of human samples whenever possible will ensure relevance to human health and disease.

APOL1-mediated monovalent cation transport contributes to APOL1-mediated podocytopathy in kidney disease.

Two coding variants of apolipoprotein L1 (APOL1), called G1 and G2, explain much of the excess risk of kidney disease in African Americans. While various cytotoxic phenotypes have been reported in experimental models, the proximal mechanism by which G1 and G2 cause kidney disease is poorly understood. Here, we leveraged 3 experimental models and a recently reported small molecule blocker of APOL1 protein, VX-147, to identify the upstream mechanism of G1-induced cytotoxicity. In HEK293 cells, we demonstrated that G1-mediated Na+ import/K+ efflux triggered activation of GPCR/IP3-mediated calcium release from the ER, impaired mitochondrial ATP production, and impaired translation, which were all reversed by VX-147. In human urine-derived podocyte-like epithelial cells (HUPECs), we demonstrated that G1 caused cytotoxicity that was again reversible by VX-147. Finally, in podocytes isolated from APOL1 G1 transgenic mice, we showed that IFN-γ-mediated induction of G1 caused K+ efflux, activation of GPCR/IP3 signaling, and inhibition of translation, podocyte injury, and proteinuria, all reversed by VX-147. Together, these results establish APOL1-mediated Na+/K+ transport as the proximal driver of APOL1-mediated kidney disease.

HOPX+ injury-resistant intestinal stem cells drive epithelial recovery after severe intestinal ischemia.

Intestinal ischemia is a life-threatening emergency with mortality rates of 50%-80% due to epithelial cell death and resultant barrier loss. Loss of the epithelial barrier occurs in conditions including intestinal volvulus and neonatal necrotizing enterocolitis. Survival depends on effective epithelial repair; crypt-based intestinal epithelial stem cells (ISCs) are the source of epithelial renewal in homeostasis and after injury. Two ISC populations have been described: 1) active ISC [aISC; highly proliferative; leucine-rich-repeat-containing G protein-coupled receptor 5 (LGR5+)-positive or sex-determining region Y-box 9 -antigen Ki67-positive (SOX9+Ki67+)] and 2) reserve ISC [rISC; less proliferative; homeodomain-only protein X positive (HOPX+)]. The contributions of these ISCs have been evaluated both in vivo and in vitro using a porcine model of mesenteric vascular occlusion to understand mechanisms that modulate ISC recovery responses following ischemic injury. In our previously published work, we observed that rISC conversion to an activated state was associated with decreased HOPX expression during in vitro recovery. In the present study, we wanted to evaluate the direct role of HOPX on cellular proliferation during recovery after injury. Our data demonstrated that during early in vivo recovery, injury-resistant HOPX+ cells maintain quiescence. Subsequent early regeneration within the intestinal crypt occurs around 2 days after injury, a period in which HOPX expression decreased. When HOPX was silenced in vitro, cellular proliferation of injured cells was promoted during recovery. This suggests that HOPX may serve a functional role in ISC-mediated regeneration after injury and could be a target to control ISC proliferation.NEW & NOTEWORTHY This paper supports that rISCs are resistant to ischemic injury and likely an important source of cellular renewal following near-complete epithelial loss. Furthermore, we have evidence that HOPX controls ISC activity state and may be a critical signaling pathway during ISC-mediated repair. Finally, we use multiple novel methods to evaluate ISCs in a translationally relevant large animal model of severe intestinal injury and provide evidence for the potential role of rISCs as therapeutic targets.

Reductive TCA cycle metabolism fuels glutamine- and glucose-stimulated insulin secretion.

Metabolic fuels regulate insulin secretion by generating second messengers that drive insulin granule exocytosis, but the biochemical pathways involved are incompletely understood. Here we demonstrate that stimulation of rat insulinoma cells or primary rat islets with glucose or glutamine + 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (Gln + BCH) induces reductive, "counter-clockwise" tricarboxylic acid (TCA) cycle flux of glutamine to citrate. Molecular or pharmacologic suppression of isocitrate dehydrogenase-2 (IDH2), which catalyzes reductive carboxylation of 2-ketoglutarate to isocitrate, results in impairment of glucose- and Gln + BCH-stimulated reductive TCA cycle flux, lowering of NADPH levels, and inhibition of insulin secretion. Pharmacologic suppression of IDH2 also inhibits insulin secretion in living mice. Reductive TCA cycle flux has been proposed as a mechanism for generation of biomass in cancer cells. Here we demonstrate that reductive TCA cycle flux also produces stimulus-secretion coupling factors that regulate insulin secretion, including in non-dividing cells.

Hepatic HKDC1 Expression Contributes to Liver Metabolism.

Glucokinase (GCK) is the principal hexokinase (HK) in the liver, operating as a glucose sensor to regulate glucose metabolism and lipid homeostasis. Recently, we proposed HK domain-containing 1 (HKDC1) to be a fifth HK with expression in the liver. Here, we reveal HKDC1 to have low glucose-phosphorylating ability and demonstrate its association with the mitochondria in hepatocytes. As we have shown previously that genetic deletion of HKDC1 leads to altered hepatic triglyceride levels, we also explored the influence of overexpression of HKDC1 in hepatocytes on cellular metabolism, observing reduced glycolytic capacity and maximal mitochondrial respiration with concurrent reductions in glucose oxidation and mitochondrial membrane potential. Furthermore, we found that acute in vivo overexpression of HKDC1 in the liver induced substantial changes in mitochondrial dynamics. Altogether, these findings suggest that overexpression of HKDC1 causes mitochondrial dysfunction in hepatocytes. However, its overexpression was not enough to alter energy storage in the liver but led to mild improvement in glucose tolerance. We next investigated the conditions necessary to induce HKDC1 expression, observing HKDC1 expression to be elevated in human patients whose livers were at more advanced stages of nonalcoholic fatty liver disease (NAFLD) and similarly, found high liver expression in mice on diets causing high levels of liver inflammation and fibrosis. Overall, our data suggest that HKDC1 expression in hepatocytes results in defective mitochondrial function and altered hepatocellular metabolism and speculate that its expression in the liver may play a role in the development of NAFLD.

Hepatic hexokinase domain containing 1 (HKDC1) improves whole body glucose tolerance and insulin sensitivity in pregnant mice.

Hexokinase domain containing 1, a recently discovered putative fifth hexokinase, is hypothesized to play key roles in glucose metabolism. Specifically, during pregnancy in a recent genome wide association study (GWAS), a strong correlation between HKDC1 and 2-h plasma glucose in pregnant women from different ethnic backgrounds was shown. Our earlier work also reported diminished glucose tolerance during pregnancy in our whole body HKDC1 heterozygous mice. Therefore, we hypothesized that HKDC1 plays important roles in gestational metabolism, and designed this study to assess the role of hepatic HKDC1 in whole body glucose utilization and insulin action during pregnancy. We overexpressed human HKDC1 in mouse liver by injecting a human HKDC1 adenoviral construct; whereas, for the liver-specific HKDC1 knockout model, we used AAV-Cre constructs in our HKDC1fl/fl mice. Both groups of mice were subjected to metabolic testing before and during pregnancy on gestation day 17-18. Our results indicate that hepatic HKDC1 overexpression during pregnancy leads to improved whole-body glucose tolerance and enhanced hepatic and peripheral insulin sensitivity while hepatic HKDC1 knockout results in diminished glucose tolerance. Further, we observed reduced gluconeogenesis with hepatic HKDC1 overexpression while HKDC1 knockout led to increased gluconeogenesis. These changes were associated with significantly enhanced ketone body production in HKDC1 overexpressing mice, indicating that these mice shift their metabolic needs from glucose reliance to greater fat oxidation and ketone utilization during fasting. Taken together, our results indicate that hepatic HKDC1 contributes to whole body glucose disposal, insulin sensitivity, and aspects of nutrient balance during pregnancy.

Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing.

Genetic manipulation via transgene overexpression, RNAi, or Cas9-based methods is central to biomedical research. Unfortunately, use of these tools is often limited by vector options. We have created a modular platform (pMVP) that allows a gene of interest to be studied in the context of an array of promoters, epitope tags, conditional expression modalities, and fluorescent reporters, packaged in 35 custom destination vectors, including adenovirus, lentivirus, PiggyBac transposon, and Sleeping Beauty transposon, in aggregate >108,000 vector permutations. We also used pMVP to build an epigenetic engineering platform, pMAGIC, that packages multiple gRNAs and either Sa-dCas9 or x-dCas9(3.7) fused to one of five epigenetic modifiers. Importantly, via its compatibility with adenoviral vectors, pMAGIC uniquely enables use of dCas9/LSD1 fusions to interrogate enhancers within primary cells. To demonstrate this, we used pMAGIC to target Sa-dCas9/LSD1 and modify the epigenetic status of a conserved enhancer, resulting in altered expression of the homeobox transcription factor PDX1 and its target genes in pancreatic islets and insulinoma cells. In sum, the pMVP and pMAGIC systems empower researchers to rapidly generate purpose-built, customized vectors for manipulation of gene expression, including via targeted epigenetic modification of regulatory elements in a broad range of disease-relevant cell types.

Syntaxin 4 Expression in Pancreatic ß-Cells Promotes Islet Function and Protects Functional ß-Cell Mass.

Syntaxin 4 (Stx4) enrichment in human and mouse islet grafts improves the success of transplants in reversing streptozotocin (STZ)-induced diabetes in mice, although the underlying molecular mechanisms remain elusive. Toward a further understanding of this, human islets and inducible transgenic mice that selectively overexpress Stx4 in islet ß-cells (ßTG-Stx4) were challenged with proinflammatory stressors in vitro and in vivo. Remarkably, ßTG-Stx4 mice resisted the loss of ß-cell mass and the glucose intolerance that multiple low doses of STZ induce. Under standard conditions, glucose tolerance was enhanced and mice maintained normal fasting glycemia and insulinemia. Conversely, Stx4 heterozygous knockout mice succumbed rapidly to STZ-induced glucose intolerance compared with their wild-type littermates. Human islet ß-cells overexpressing Stx4 exhibited enhanced insulin secretory capability; resilience against proinflammatory cytokine-induced apoptosis; and reduced expression of the CXCL9, CXCL10, and CXCL11 genes coordinate with decreased activation/nuclear localization of nuclear factor-κB. Finding ways to boost Stx4 expression presents a novel potential therapeutic avenue for promoting islet function and preserving ß-cell mass.

Insights into beta cell regeneration for diabetes via integration of molecular landscapes in human insulinomas.

Although diabetes results in part from a deficiency of normal pancreatic beta cells, inducing human beta cells to regenerate is difficult. Reasoning that insulinomas hold the "genomic recipe" for beta cell expansion, we surveyed 38 human insulinomas to obtain insights into therapeutic pathways for beta cell regeneration. An integrative analysis of whole-exome and RNA-sequencing data was employed to extensively characterize the genomic and molecular landscape of insulinomas relative to normal beta cells. Here, we show at the pathway level that the majority of the insulinomas display mutations, copy number variants and/or dysregulation of epigenetic modifying genes, most prominently in the polycomb and trithorax families. Importantly, these processes are coupled to co-expression network modules associated with cell proliferation, revealing candidates for inducing beta cell regeneration. Validation of key computational predictions supports the concept that understanding the molecular complexity of insulinoma may be a valuable approach to diabetes drug discovery.Diabetes results in part from a deficiency of functional pancreatic beta cells. Here, the authors study the genomic and epigenetic landscapes of human insulinomas to gain insight into possible pathways for therapeutic beta cell regeneration, highlighting epigenetic genes and pathways.

A Pdx-1-Regulated Soluble Factor Activates Rat and Human Islet Cell Proliferation.

The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in ß-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a ß-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both α and ß cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor ß (TGF-ß) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and ß-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and ß cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation.

The Brain-to-Pancreatic Islet Neuronal Map Reveals Differential Glucose Regulation From Distinct Hypothalamic Regions.

The brain influences glucose homeostasis, partly by supplemental control over insulin and glucagon secretion. Without this central regulation, diabetes and its complications can ensue. Yet, the neuronal network linking to pancreatic islets has never been fully mapped. Here, we refine this map using pseudorabies virus (PRV) retrograde tracing, indicating that the pancreatic islets are innervated by efferent circuits that emanate from the hypothalamus. We found that the hypothalamic arcuate nucleus (ARC), ventromedial nucleus (VMN), and lateral hypothalamic area (LHA) significantly overlap PRV and the physiological glucose-sensing enzyme glucokinase. Then, experimentally lowering glucose sensing, specifically in the ARC, resulted in glucose intolerance due to deficient insulin secretion and no significant effect in the VMN, but in the LHA it resulted in a lowering of the glucose threshold that improved glucose tolerance and/or improved insulin sensitivity, with an exaggerated counter-regulatory response for glucagon secretion. No significant effect on insulin sensitivity or metabolic homeostasis was noted. Thus, these data reveal novel direct neuronal effects on pancreatic islets and also render a functional validation of the brain-to-islet neuronal map. They also demonstrate that distinct regions of the hypothalamus differentially control insulin and glucagon secretion, potentially in partnership to help maintain glucose homeostasis and guard against hypoglycemia.

Aurora Kinase A is critical for the Nkx6.1 mediated ß-cell proliferation pathway.

Type 1 and type 2 diabetes are ultimately characterized by depleted ß-cell mass. Characterization of the molecular pathways that control ß-cell proliferation could be harnessed to restore these cells. The homeobox ß-cell transcription factor Nkx6.1 induces ß-cell proliferation by activating the orphan nuclear receptors Nr4a1 and Nr4a3. Here, we demonstrate that Nkx6.1 localizes to the promoter of the mitotic kinase AURKA (Aurora Kinase A) and induces its expression. Adenovirus mediated overexpression of AURKA is sufficient to induce proliferation in primary rat islets while maintaining glucose stimulated insulin secretion. Furthermore, AURKA is necessary for Nkx6.1 mediated ß-cell proliferation as demonstrated by shRNA mediated knock down and pharmacological inhibition of AURKA kinase activity. AURKA preferentially induces DNA replication in ß-cells as measured by BrdU incorporation, and enhances the rate of histone H3 phosphorylation in primary ß-cells, demonstrating that AURKA induces the replicative and mitotic cell cycle phases in rat ß-cells. Finally, overexpression of AURKA results in phosphorylation of the cell cycle regulator p53, which targets p53 for degradation and permits cell cycle progression. These studies define a pathway by which AURKA upregulation by Nkx6.1 results in phosphorylation and degradation of p53, thus removing a key inhibitory factor and permitting engagement of the ß-cell proliferation pathway.

Coordinated regulatory variation associated with gestational hyperglycaemia regulates expression of the novel hexokinase HKDC1.

Maternal glucose levels during pregnancy impact the developing fetus, affecting metabolic health both early and later on in life. Both genetic and environmental factors influence maternal metabolism, but little is known about the genetic mechanisms that alter glucose metabolism during pregnancy. Here, we report that haplotypes previously associated with gestational hyperglycaemia in the third trimester disrupt regulatory element activity and reduce expression of the nearby HKDC1 gene. We further find that experimentally reducing or increasing HKDC1 expression reduces or increases hexokinase activity, respectively, in multiple cellular models; in addition, purified HKDC1 protein has hexokinase activity in vitro. Together, these results suggest a novel mechanism of gestational glucose regulation in which the effects of genetic variants in multiple regulatory elements alter glucose homeostasis by coordinately reducing expression of the novel hexokinase HKDC1.

Regulation of skeletal muscle oxidative capacity and insulin signaling by the mitochondrial rhomboid protease PARL.

Type 2 diabetes mellitus (T2DM) and aging are characterized by insulin resistance and impaired mitochondrial energetics. In lower organisms, remodeling by the protease pcp1 (PARL ortholog) maintains the function and lifecycle of mitochondria. We examined whether variation in PARL protein content is associated with mitochondrial abnormalities and insulin resistance. PARL mRNA and mitochondrial mass were both reduced in elderly subjects and in subjects with T2DM. Muscle knockdown of PARL in mice resulted in malformed mitochondrial cristae, lower mitochondrial content, decreased PGC1alpha protein levels, and impaired insulin signaling. Suppression of PARL protein in healthy myotubes lowered mitochondrial mass and insulin-stimulated glycogen synthesis and increased reactive oxygen species production. We propose that lower PARL expression may contribute to the mitochondrial abnormalities seen in aging and T2DM.

The mitochondrial 2-oxoglutarate carrier is part of a metabolic pathway that mediates glucose- and glutamine-stimulated insulin secretion.

Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.

Cooperative transcriptional regulation of the essential pancreatic islet gene NeuroD1 (beta2) by Nkx2.2 and neurogenin 3.

Nkx2.2 and NeuroD1 are two critical regulators of pancreatic beta cell development. Nkx2.2 is a homeodomain transcription factor that is essential for islet cell type specification and mature beta cell function. NeuroD1 is a basic helix-loop-helix transcription factor that is critical for islet beta cell maturation and maintenance. Although both proteins influence beta cell development directly downstream of the endocrine progenitor factor, neurogenin3 (Ngn3), a connection between the two proteins in the regulation of beta cell fate and function has yet to be established. In this study, we demonstrate that Nkx2.2 transcriptional activity is required to facilitate the activation of NeuroD1 by Ngn3. Furthermore, Nkx2.2 is necessary to maintain high levels of NeuroD1 expression in developing mouse and zebrafish islets and in mature beta cells. Interestingly, Nkx2.2 regulates NeuroD1 through two independent promoter elements, one that is bound and activated directly by Nkx2.2 and one that appears to be regulated by Nkx2.2 through an indirect mechanism. Together, these findings suggest that Nkx2.2 coordinately activates NeuroD1 with Ngn3 within the endocrine progenitor cell and also plays a role in the maintenance of NeuroD1 expression to regulate beta cell function in the mature islet. Collectively, these findings further define the conserved regulatory networks involved in islet beta cell formation and function.

Relationship among brain and blood glucose levels and spontaneous and glucoprivic feeding.

Although several studies implicate small declines in blood glucose levels as stimulus for spontaneous meal initiation, no mechanism is known for how these dips might initiate feeding. To assess the role of ventromedial hypothalamus (VMH) (arcuate plus ventromedial nucleus) glucosensing neurons as potential mediators of spontaneous and glucoprivic feeding, meal patterns were observed, and blood and VMH microdialysis fluid were sampled in 15 rats every 10 min for 3.5 h after dark onset and 2 h after insulin (5 U/kg, i.v.) infusion. Blood glucose levels declined by 11% beginning approximately 5 min before 65% of all spontaneous meals, with no fall in VMH levels. After insulin, blood and VMH glucose reached nadirs by 30-40 min, and the same rats ate 60% faster and spent 84% more time eating during the ensuing hypoglycemia. Although 83% of first hypoglycemic meals were preceded by 5 min dips in VMH (but not blood) glucose levels, neither blood nor VMH levels declined before second meals, suggesting that low glucose, rather than changing levels, was the stimulus for glucoprivic meals. Furthermore, altering VMH glucosensing by raising or lowering glucokinase (GK) activity failed to affect spontaneous feeding, body or adipose weights, or glucose tolerance. However, chronic depletion by 26-70% of VMH GK mRNA reduced glucoprivic feeding. Thus, although VMH glucosensing does not appear to be involved in either spontaneous feeding or long-term body-weight regulation, it does participate in glucoprivic feeding, similar to its role in the counter-regulatory neurohumoral responses to glucoprivation.

Small interfering RNA-mediated suppression of proislet amyloid polypeptide expression inhibits islet amyloid formation and enhances survival of human islets in culture.

OBJECTIVE: Islet amyloid, formed by aggregation of the beta-cell peptide islet amyloid polypeptide (IAPP; amylin), is a pathological characteristic of pancreatic islets in type 2 diabetes. Toxic IAPP aggregates likely contribute to the progressive loss of beta-cells in this disease. We used cultured human islets as an ex vivo model of amyloid formation to investigate whether suppression of proIAPP expression would inhibit islet amyloid formation and enhance beta-cell survival and function. RESEARCH DESIGN AND METHODS: Islets from cadaveric organ donors were transduced with a recombinant adenovirus expressing a short interfering RNA (siRNA) designed to suppress human proIAPP (Ad-hProIAPP-siRNA), cultured for 10 days, and then assessed for the presence of islet amyloid, beta-cell apoptosis, and beta-cell function. RESULTS: Thioflavine S-positive amyloid deposits were clearly present after 10 days of culture. Transduction with Ad-hProIAPP-siRNA reduced proIAPP expression by 75% compared with nontransduced islets as assessed by Western blot analysis of islet lysates 4 days after transduction. siRNA-mediated inhibition of IAPP expression decreased islet amyloid area by 63% compared with nontransduced cultured islets. Cell death assessed by transferase-mediated dUTP nick-end labeling staining was decreased by 50% in transduced cultured human islets, associated with a significant increase in islet insulin content (control, 100 +/- 4 vs. +Ad-siRNA, 153 +/- 22%, P < 0.01) and glucose-stimulated insulin secretion (control, 222 +/- 33 vs. +Ad-siRNA, 285 +/- 21 percent basal, P < 0.05). CONCLUSIONS: These findings demonstrate that inhibition of IAPP synthesis prevents amyloid formation and beta-cell death in cultured human islets. Inhibitors of IAPP synthesis may have therapeutic value in type 2 diabetes.

Chronic suppression of acetyl-CoA carboxylase 1 in beta-cells impairs insulin secretion via inhibition of glucose rather than lipid metabolism.

Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60-80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to beta-cells suppressed [(14)C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy.