Endocrine-Exocrine Signaling Drives Obesity-Associated Pancreatic Ductal Adenocarcinoma.

Obesity is a major modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC), yet how and when obesity contributes to PDAC progression is not well understood. Leveraging an autochthonous mouse model, we demonstrate a causal and reversible role for obesity in early PDAC progression, showing that obesity markedly enhances tumorigenesis, while genetic or dietary induction of weight loss intercepts cancer development. Molecular analyses of human and murine samples define microenvironmental consequences of obesity that foster tumorigenesis rather than new driver gene mutations, including significant pancreatic islet cell adaptation in obesity-associated tumors. Specifically, we identify aberrant beta cell expression of the peptide hormone cholecystokinin (Cck) in response to obesity and show that islet Cck promotes oncogenic Kras-driven pancreatic ductal tumorigenesis. Our studies argue that PDAC progression is driven by local obesity-associated changes in the tumor microenvironment and implicate endocrine-exocrine signaling beyond insulin in PDAC development.

TNFα increases the degradation of pyruvate dehydrogenase kinase 4 by the Lon protease to support proinflammatory genes.

The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism; however, the function and regulation of elevated mitochondrial metabolism in EC in response to proinflammatory cytokines remain unclear. Studies using high-resolution metabolomics and 13C-glucose and 13C-glutamine labeling flux techniques showed that pyruvate dehydrogenase activity (PDH) and oxidative tricarboxylic acid cycle (TCA) flux are elevated in human umbilical vein ECs in response to overnight (16 h) treatment with TNFα (10 ng/mL). Mechanistic studies indicated that TNFα mediated these metabolic changes via mitochondrial-specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB-dependent mechanism. Using RNA sequencing following siRNA-mediated knockdown of the catalytically active subunit of PDH, PDHE1α (PDHA1 gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are up-regulated by TNFα stimulation. Notably, TNFα-induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Metabolomics and ChIP sequencing for acetylated modification on lysine 27 of histone 3 (H3K27ac) showed that TNFα-induced PDH flux promotes histone acetylation of specific gene loci via citrate accumulation and ATP-citrate lyase-mediated generation of acetyl CoA. Together, these results uncover a mechanism by which TNFα signaling increases oxidative TCA flux of glucose to support TNFα-induced gene transcription through extramitochondrial acetyl CoA generation and histone acetylation.

Phosphoenolpyruvate carboxykinase-2 (PCK2) is a therapeutic target in triple-negative breast cancer.

PURPOSE: Metabolic rewiring in malignant transformation is often accompanied by altered expression of metabolic isozymes. Phosphoenolpyruvate carboxykinase-2 (PCK2) catalyzes the rate-limiting step of gluconeogenesis and is the dominant isoform in many cancers including triple-negative breast cancer (TNBC). Our goal was to identify small molecule inhibitors of PCK2 enzyme activity. METHODS: We assessed the impact of PCK2 down regulation with shRNA on TNBC cell growth in vitro and used AtomNet® deep convolutional neural network software to identify potential small molecule inhibitors of PCK2-based structure. We iteratively tested candidate compounds in an in vitro PCK-2 enzyme assay. The impact of the top hit on metabolic flux and cell viability was also assessed. RESULTS: PCK2 downregulation decreased growth of BT-549 and MDA-MB-231 cells and reduced metabolic flux through pyruvate carboxylase. The first AtomNet® in silico structural screen of 7 million compounds yielded 86 structures that were tested in PCK2 enzyme assay in vitro. The top hit (IC50 = 2.4 µM) was used to refine a second round of in silico screen that yielded 82 candidates to be tested in vitro, which resulted in 45 molecules with inhibition > 20%. In the second in vitro screen we also included 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, previously suggested to be PCK2 inhibitor based on structure, which emerged as the top hit. The specificity of this compound was tested in PCK1 and PCK2 enzymatic assays and showed IC50 of 500 nM and 3.5-27 nM for PCK1 and PCK2, respectively. CONCLUSION: 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate is a high affinity PCK2 enzyme inhibitor that also has significant growth inhibitory activity in breast cell lines in vitro and represents a potential therapeutic lead compound.

Acetate mediates a microbiome-brain-ß-cell axis to promote metabolic syndrome.

Obesity, insulin resistance and the metabolic syndrome are associated with changes to the gut microbiota; however, the mechanism by which modifications to the gut microbiota might lead to these conditions is unknown. Here we show that increased production of acetate by an altered gut microbiota in rodents leads to activation of the parasympathetic nervous system, which, in turn, promotes increased glucose-stimulated insulin secretion, increased ghrelin secretion, hyperphagia, obesity and related sequelae. Together, these findings identify increased acetate production resulting from a nutrient-gut microbiota interaction and subsequent parasympathetic activation as possible therapeutic targets for obesity.

N-acyl taurines are endogenous lipid messengers that improve glucose homeostasis.

Fatty acid amide hydrolase (FAAH) degrades 2 major classes of bioactive fatty acid amides, the N-acylethanolamines (NAEs) and N-acyl taurines (NATs), in central and peripheral tissues. A functional polymorphism in the human FAAH gene is linked to obesity and mice lacking FAAH show altered metabolic states, but whether these phenotypes are caused by elevations in NAEs or NATs is unknown. To overcome the problem of concurrent elevation of NAEs and NATs caused by genetic or pharmacological disruption of FAAH in vivo, we developed an engineered mouse model harboring a single-amino acid substitution in FAAH (S268D) that selectively disrupts NAT, but not NAE, hydrolytic activity. The FAAH-S268D mice accordingly show substantial elevations in NATs without alterations in NAE content, a unique metabolic profile that correlates with heightened insulin sensitivity and GLP-1 secretion. We also show that N-oleoyl taurine (C18:1 NAT), the most abundant NAT in human plasma, decreases food intake, improves glucose tolerance, and stimulates GPR119-dependent GLP-1 and glucagon secretion in mice. Together, these data suggest that NATs act as a class of lipid messengers that improve postprandial glucose regulation and may have potential as investigational metabolites to modify metabolic disease.

Pathogenesis of hypothyroidism-induced NAFLD is driven by intra- and extrahepatic mechanisms.

Hypothyroidism, a metabolic disease characterized by low thyroid hormone (TH) and high thyroid-stimulating hormone (TSH) levels in the serum, is strongly associated with nonalcoholic fatty liver disease (NAFLD). Hypothyroidism-induced NAFLD has generally been attributed to reduced TH signaling in the liver with a consequent decrease in lipid utilization. Here, we found that mildly hypothyroid mice develop NAFLD without down-regulation of hepatic TH signaling or decreased hepatic lipid utilization. NAFLD was induced by impaired suppression of adipose tissue lipolysis due to decreased insulin secretion and to a reduced response of adipose tissue itself to insulin. This condition leads to increased shuttling of fatty acids (FAs) to the liver, where they are esterified and accumulated as triglycerides. Lipid accumulation in the liver induces hepatic insulin resistance, which leads to impaired suppression of endogenous glucose production after feeding. Hepatic insulin resistance, synergistically with lowered insulin secretion, increases serum glucose levels, which stimulates de novo lipogenesis (DNL) in the liver. Up-regulation of DNL also contributes to NAFLD. In contrast, severely hypothyroid mice show down-regulation of TH signaling in their livers and profound suppression of adipose tissue lipolysis, which decreases delivery of FAs to the liver. The resulting lack of substrates for triglyceride esterification protects severely hypothyroid mice against NAFLD. Our findings demonstrate that NAFLD occurs when TH levels are mildly reduced, but, paradoxically, not when they are severely reduced. Our results show that the pathogenesis of hypothyroidism-induced NAFLD is both intra- and extrahepatic; they also reveal key metabolic differences between mild and severe hypothyroidism.

Imeglimin lowers glucose primarily by amplifying glucose-stimulated insulin secretion in high-fat-fed rodents.

Imeglimin is a promising new oral antihyperglycemic agent that has been studied in clinical trials as a possible monotherapy or add-on therapy to lower fasting plasma glucose and improve hemoglobin A1c (1-3, 9). Imeglimin was shown to improve both fasting and postprandial glycemia and to increase insulin secretion in response to glucose during a hyperglycemic clamp after 1-wk of treatment in type 2 diabetic patients. However, whether the ß-cell stimulatory effect of imeglimin is solely or partially responsible for its effects on glycemia remains to be fully confirmed. Here, we show that imeglimin directly activates ß-cell insulin secretion in awake rodents without affecting hepatic insulin sensitivity, body composition, or energy expenditure. These data identify a primary amplification rather than trigger the ß-cell mechanism that explains the acute, antidiabetic activity of imeglimin.

CALHM2 is a mitochondrial protein import channel that regulates fatty acid metabolism.

For mitochondrial metabolism to occur in the matrix, multiple proteins must be imported across the two (inner and outer) mitochondrial membranes. Classically, two protein import channels, TIM/TOM, are known to perform this function, but whether other protein import channels exist is not known. Here, using super-resolution microscopy, proteomics, and electrophysiological techniques, we identify CALHM2 as the import channel for the ECHA subunit of the mitochondrial trifunctional protein (mTFP), which catalyzes ß-oxidation of fatty acids in the mitochondrial matrix. We find that CALHM2 sits specifically at the inner mitochondrial and cristae membranes and is critical for membrane morphology. Depletion of CALHM2 leads to a mislocalization of ECHA outside of the mitochondria leading to severe cellular metabolic defects. These defects include cytosolic accumulation of fatty acids, depletion of tricarboxylic acid cycle enzymes and intermediates, and reduced cellular respiration. Our data identify CALHM2 as an essential protein import channel that is critical for fatty acid- and glucose-dependent aerobic metabolism.

Loss of ZNF148 enhances insulin secretion in human pancreatic ß cells.

Insulin secretion from pancreatic ß cells is essential to the maintenance of glucose homeostasis. Defects in this process result in diabetes. Identifying genetic regulators that impair insulin secretion is crucial for the identification of novel therapeutic targets. Here, we show that reduction of ZNF148 in human islets, and its deletion in stem cell-derived ß cells (SC-ß cells), enhances insulin secretion. Transcriptomics of ZNF148-deficient SC-ß cells identifies increased expression of annexin and S100 genes whose proteins form tetrameric complexes involved in regulation of insulin vesicle trafficking and exocytosis. ZNF148 in SC-ß cells prevents translocation of annexin A2 from the nucleus to its functional place at the cell membrane via direct repression of S100A16 expression. These findings point to ZNF148 as a regulator of annexin-S100 complexes in human ß cells and suggest that suppression of ZNF148 may provide a novel therapeutic strategy to enhance insulin secretion.

Endocrine pancreas-specific Gclc gene deletion causes a severe diabetes phenotype.

Reduced glutathione (GSH) is an abundant antioxidant that regulates intracellular redox homeostasis by scavenging reactive oxygen species (ROS). Glutamate-cysteine ligase catalytic (GCLC) subunit is the rate-limiting step in GSH biosynthesis. Using the Pax6-Cre driver mouse line, we deleted expression of the Gclc gene in all pancreatic endocrine progenitor cells. Intriguingly, Gclc knockout (KO) mice, following weaning, exhibited an age-related, progressive diabetes phenotype, manifested as strikingly increased blood glucose and decreased plasma insulin levels. This severe diabetes trait is preceded by pathologic changes in islet of weanling mice. Gclc KO weanlings showed progressive abnormalities in pancreatic morphology including: islet-specific cellular vacuolization, decreased islet-cell mass, and alterations in islet hormone expression. Islets from newly-weaned mice displayed impaired glucose-stimulated insulin secretion, decreased insulin hormone gene expression, oxidative stress, and increased markers of cellular senescence. Our results suggest that GSH biosynthesis is essential for normal development of the mouse pancreatic islet, and that protection from oxidative stress-induced cellular senescence might prevent abnormal islet-cell damage during embryogenesis.

Suppression of angiopoietin-like 4 reprograms endothelial cell metabolism and inhibits angiogenesis.

Angiopoietin-like 4 (ANGPTL4) is known to regulate various cellular and systemic functions. However, its cell-specific role in endothelial cells (ECs) function and metabolic homeostasis remains to be elucidated. Here, using endothelial-specific Angptl4 knock-out mice (Angptl4iΔEC), and transcriptomics and metabolic flux analysis, we demonstrate that ANGPTL4 is required for maintaining EC metabolic function vital for vascular permeability and angiogenesis. Knockdown of ANGPTL4 in ECs promotes lipase-mediated lipoprotein lipolysis, which results in increased fatty acid (FA) uptake and oxidation. This is also paralleled by a decrease in proper glucose utilization for angiogenic activation of ECs. Mice with endothelial-specific deletion of Angptl4 showed decreased pathological neovascularization with stable vessel structures characterized by increased pericyte coverage and reduced permeability. Together, our study denotes the role of endothelial-ANGPTL4 in regulating cellular metabolism and angiogenic functions of EC.

UCP2-dependent redox sensing in POMC neurons regulates feeding.

Paradoxically, glucose, the primary driver of satiety, activates a small population of anorexigenic pro-opiomelanocortin (POMC) neurons. Here, we show that lactate levels in the circulation and in the cerebrospinal fluid are elevated in the fed state and the addition of lactate to glucose activates the majority of POMC neurons while increasing cytosolic NADH generation, mitochondrial respiration, and extracellular pyruvate levels. Inhibition of lactate dehydrogenases diminishes mitochondrial respiration, NADH production, and POMC neuronal activity. However, inhibition of the mitochondrial pyruvate carrier has no effect. POMC-specific downregulation of Ucp2 (Ucp2PomcKO), a molecule regulated by fatty acid metabolism and shown to play a role as transporter in the malate-aspartate shuttle, abolishes lactate- and glucose-sensing of POMC neurons. Ucp2PomcKO mice have impaired glucose metabolism and are prone to obesity on a high-fat diet. Altogether, our data show that lactate through redox signaling and blocking mitochondrial glucose utilization activates POMC neurons to regulate feeding and glucose metabolism.

Dyrk1b promotes hepatic lipogenesis by bypassing canonical insulin signaling and directly activating mTORC2 in mice.

Mutations in Dyrk1b are associated with metabolic syndrome and nonalcoholic fatty liver disease in humans. Our investigations showed that DYRK1B levels are increased in the liver of patients with nonalcoholic steatohepatitis (NASH) and in mice fed with a high-fat, high-sucrose diet. Increasing Dyrk1b levels in the mouse liver enhanced de novo lipogenesis (DNL), fatty acid uptake, and triacylglycerol secretion and caused NASH and hyperlipidemia. Conversely, knockdown of Dyrk1b was protective against high-calorie-induced hepatic steatosis and fibrosis and hyperlipidemia. Mechanistically, Dyrk1b increased DNL by activating mTORC2 in a kinase-independent fashion. Accordingly, the Dyrk1b-induced NASH was fully rescued when mTORC2 was genetically disrupted. The elevated DNL was associated with increased plasma membrane sn-1,2-diacylglyerol levels and increased PKCε-mediated IRKT1150 phosphorylation, which resulted in impaired activation of hepatic insulin signaling and reduced hepatic glycogen storage. These findings provide insights into the mechanisms that underlie Dyrk1b-induced hepatic lipogenesis and hepatic insulin resistance and identify Dyrk1b as a therapeutic target for NASH and insulin resistance in the liver.

ß-cell deletion of the PKm1 and PKm2 isoforms of pyruvate kinase in mice reveals their essential role as nutrient sensors for the KATP channel.

Pyruvate kinase (PK) and the phosphoenolpyruvate (PEP) cycle play key roles in nutrient-stimulated KATP channel closure and insulin secretion. To identify the PK isoforms involved, we generated mice lacking ß-cell PKm1, PKm2, and mitochondrial PEP carboxykinase (PCK2) that generates mitochondrial PEP. Glucose metabolism was found to generate both glycolytic and mitochondrially derived PEP, which triggers KATP closure through local PKm1 and PKm2 signaling at the plasma membrane. Amino acids, which generate mitochondrial PEP without producing glycolytic fructose 1,6-bisphosphate to allosterically activate PKm2, signal through PKm1 to raise ATP/ADP, close KATP channels, and stimulate insulin secretion. Raising cytosolic ATP/ADP with amino acids is insufficient to close KATP channels in the absence of PK activity or PCK2, indicating that KATP channels are primarily regulated by PEP that provides ATP via plasma membrane-associated PK, rather than mitochondrially derived ATP. Following membrane depolarization, the PEP cycle is involved in an 'off-switch' that facilitates KATP channel reopening and Ca2+ extrusion, as shown by PK activation experiments and ß-cell PCK2 deletion, which prolongs Ca2+ oscillations and increases insulin secretion. In conclusion, the differential response of PKm1 and PKm2 to the glycolytic and mitochondrial sources of PEP influences the ß-cell nutrient response, and controls the oscillatory cycle regulating insulin secretion.

ß Cell-specific deletion of Zfp148 improves nutrient-stimulated ß cell Ca2+ responses.

Insulin secretion from pancreatic ß cells is essential for glucose homeostasis. An insufficient response to the demand for insulin results in diabetes. We previously showed that ß cell-specific deletion of Zfp148 (ß-Zfp148KO) improves glucose tolerance and insulin secretion in mice. Here, we performed Ca2+ imaging of islets from ß­Zfp148KO and control mice fed both a chow and a Western-style diet. ß-Zfp148KO islets demonstrated improved sensitivity and sustained Ca2+ oscillations in response to elevated glucose levels. ß-Zfp148KO islets also exhibited elevated sensitivity to amino acid-induced Ca2+ influx under low glucose conditions, suggesting enhanced mitochondrial phosphoenolpyruvate-dependent (PEP-dependent), ATP-sensitive K+ channel closure, independent of glycolysis. RNA-Seq and proteomics of ß-Zfp148KO islets revealed altered levels of enzymes involved in amino acid metabolism (specifically, SLC3A2, SLC7A8, GLS, GLS2, PSPH, PHGDH, and PSAT1) and intermediary metabolism (namely, GOT1 and PCK2), consistent with altered PEP cycling. In agreement with this, ß-Zfp148KO islets displayed enhanced insulin secretion in response to l-glutamine and activation of glutamate dehydrogenase. Understanding pathways controlled by ZFP148 may provide promising strategies for improving ß cell function that are robust to the metabolic challenge imposed by a Western diet.

Glucose Response by Stem Cell-Derived ß Cells In Vitro Is Inhibited by a Bottleneck in Glycolysis.

Stem cell-derived ß (SC-ß) cells could provide unlimited human ß cells toward a curative diabetes treatment. Differentiation of SC-ß cells yields transplantable islets that secrete insulin in response to glucose challenges. Following transplantation into mice, SC-ß cell function is comparable to human islets, but the magnitude and consistency of response in vitro are less robust than observed in cadaveric islets. Here, we profile metabolism of SC-ß cells and islets to quantify their capacity to sense glucose and identify reduced anaplerotic cycling in the mitochondria as the cause of reduced glucose-stimulated insulin secretion in SC-ß cells. This activity can be rescued by challenging SC-ß cells with intermediate metabolites from the TCA cycle and late but not early glycolysis, downstream of the enzymes glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. Bypassing this metabolic bottleneck results in a robust, bi-phasic insulin release in vitro that is identical in magnitude to functionally mature human islets.

Multi-Tissue Acceleration of the Mitochondrial Phosphoenolpyruvate Cycle Improves Whole-Body Metabolic Health.

The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to regulate glucose homeostasis. Here, small molecule PK activators accelerated the PEP cycle to improve islet function, as well as metabolic homeostasis, in preclinical rodent models of diabetes. In contrast, treatment with a PK activator did not improve insulin secretion in pck2-/- mice. Unlike other clinical secretagogues, PK activation enhanced insulin secretion but also had higher insulin content and markers of differentiation. In addition to improving insulin secretion, acute PK activation short-circuited gluconeogenesis to reduce endogenous glucose production while accelerating red blood cell glucose turnover. Four-week delivery of a PK activator in vivo remodeled PK phosphorylation, reduced liver fat, and improved hepatic and peripheral insulin sensitivity in HFD-fed rats. These data provide a preclinical rationale for PK activation to accelerate the PEP cycle to improve metabolic homeostasis and insulin sensitivity.

Pyruvate Kinase Controls Signal Strength in the Insulin Secretory Pathway.

Pancreatic ß cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies ß cell sensing of both glycolytic and mitochondrial fuels. Plasma membrane-localized PK is sufficient to close KATP channels and initiate calcium influx. Small-molecule PK activators increase the frequency of ATP/ADP and calcium oscillations and potently amplify insulin secretion. PK restricts respiration by cyclically depriving mitochondria of ADP, which accelerates PEP cycling until membrane depolarization restores ADP and oxidative phosphorylation. Our findings support a compartmentalized model of ß cell metabolism in which PK locally generates the ATP/ADP required for insulin secretion. Oscillatory PK activity allows mitochondria to perform synthetic and oxidative functions without any net impact on glucose oxidation. These findings suggest a potential therapeutic route for diabetes based on PK activation that would not be predicted by the current consensus single-state model of ß cell function.

Dehydration and insulinopenia are necessary and sufficient for euglycemic ketoacidosis in SGLT2 inhibitor-treated rats.

Sodium-glucose transport protein 2 (SGLT2) inhibitors are a class of anti-diabetic agents; however, concerns have been raised about their potential to induce euglycemic ketoacidosis and to increase both glucose production and glucagon secretion. The mechanisms behind these alterations are unknown. Here we show that the SGLT2 inhibitor (SGLT2i) dapagliflozin promotes ketoacidosis in both healthy and type 2 diabetic rats in the setting of insulinopenia through increased plasma catecholamine and corticosterone concentrations secondary to volume depletion. These derangements increase white adipose tissue (WAT) lipolysis and hepatic acetyl-CoA content, rates of hepatic glucose production, and hepatic ketogenesis. Treatment with a loop diuretic, furosemide, under insulinopenic conditions replicates the effect of dapagliflozin and causes ketoacidosis. Furthermore, the effects of SGLT2 inhibition to promote ketoacidosis are independent from hyperglucagonemia. Taken together these data in rats identify the combination of insulinopenia and dehydration as a potential target to prevent euglycemic ketoacidosis associated with SGLT2i.

Mitochondrial GTP Links Nutrient Sensing to ß Cell Health, Mitochondrial Morphology, and Insulin Secretion Independent of OxPhos.

Mechanisms coordinating pancreatic ß cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of ß cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and ß cell health.