Dynamic exchange at regulatory elements during chromatin remodeling underlies assisted loading mechanism.

The glucocorticoid receptor (GR), like other eukaryotic transcription factors, regulates gene expression by interacting with chromatinized DNA response elements. Photobleaching experiments in living cells indicate that receptors transiently interact with DNA on the time scale of seconds and predict that the response elements may be sparsely occupied on average. Here, we show that the binding of one receptor at the glucocorticoid response element (GRE) does not reduce the steady-state binding of another receptor variant to the same GRE. Mathematical simulations reproduce this noncompetitive state using short GR/GRE residency times and relatively long times between DNA binding events. At many genomic sites where GR binding causes increased chromatin accessibility, concurrent steady-state binding levels for the variant receptor are actually increased, a phenomenon termed assisted loading. Temporally sparse transcription factor-DNA interactions induce local chromatin reorganization, resulting in transient access for binding of secondary regulatory factors.

Nicotinamide riboside rescues dysregulated glycolysis and fatty acid ß-oxidation in a human hepatic cell model of citrin deficiency.

Citrin deficiency (CD) is an inborn error of metabolism caused by loss-of-function of the mitochondrial aspartate/glutamate transporter, CITRIN, which is involved in both the urea cycle and malate-aspartate shuttle. Patients with CD develop hepatosteatosis and hyperammonemia but there is no effective therapy for CD. Currently, there are no animal models that faithfully recapitulate the human CD phenotype. Accordingly, we generated a CITRIN knockout HepG2 cell line using Clustered Regularly Interspaced Short Palindromic Repeats/Cas 9 genome editing technology to study metabolic and cell signaling defects in CD. CITRIN KO cells showed increased ammonia accumulation, higher cytosolic ratio of reduced versus oxidized form of nicotinamide adenine dinucleotide (NAD) and reduced glycolysis. Surprisingly, these cells showed impaired fatty acid metabolism and mitochondrial activity. CITRIN KO cells also displayed increased cholesterol and bile acid metabolism resembling those observed in CD patients. Remarkably, normalizing cytosolic NADH:NAD+ ratio by nicotinamide riboside increased glycolysis and fatty acid oxidation but had no effect on the hyperammonemia suggesting the urea cycle defect was independent of the aspartate/malate shuttle defect of CD. The correction of glycolysis and fatty acid metabolism defects in CITRIN KO cells by reducing cytoplasmic NADH:NAD+ levels suggests this may be a novel strategy to treat some of the metabolic defects of CD and other mitochondrial diseases.

Fenofibrate rapidly decreases hepatic lipid and glycogen storage in neonatal mice with glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD Ia) is caused by autosomal mutations in glucose-6-phosphatase α catalytic subunit (G6PC) and can present with severe hypoglycemia, lactic acidosis and hypertriglyceridemia. In both children and adults with GSD Ia, there is over-accumulation of hepatic glycogen and triglycerides that can lead to steatohepatitis and a risk for hepatocellular adenoma or carcinoma. Here, we examined the effects of the commonly used peroxisomal proliferated activated receptor α agonist, fenofibrate, on liver and kidney autophagy and lipid metabolism in 5-day-old G6pc -/- mice serving as a model of neonatal GSD Ia. Five-day administration of fenofibrate decreased the elevated hepatic and renal triglyceride and hepatic glycogen levels found in control G6pc -/- mice. Fenofibrate also induced autophagy and promoted ß-oxidation of fatty acids and stimulated gene expression of acyl-CoA dehydrogenases in the liver. These findings show that fenofibrate can rapidly decrease hepatic glycogen and triglyceride levels and renal triglyceride levels in neonatal G6pc -/- mice. Moreover, since fenofibrate is an FDA-approved drug that has an excellent safety profile, our findings suggest that fenofibrate could be a potential pharmacological therapy for GSD Ia in neonatal and pediatric patients as well as for adults. These findings may also apply to non-alcoholic fatty liver disease, which shares similar pathological and metabolic changes with GSD Ia.

Vitamin B12 and folate decrease inflammation and fibrosis in NASH by preventing syntaxin 17 homocysteinylation.

BACKGROUND & AIMS: Several recent clinical studies have shown that serum homocysteine (Hcy) levels are positively correlated, while vitamin B12 (B12) and folate levels are negative correlated, with non-alcoholic steatohepatitis (NASH) severity. However, it is not known whether hyperhomocysteinemia (HHcy) plays a pathogenic role in NASH. METHODS: We examined the effects of HHcy on NASH progression, metabolism, and autophagy in dietary and genetic mouse models, patients, and primates. We employed vitamin B12 (B12) and folate (Fol) to reverse NASH features in mice and cell culture. RESULTS: Serum Hcy correlated with hepatic inflammation and fibrosis in NASH. Elevated hepatic Hcy induced and exacerbated NASH. Gene expression of hepatic Hcy-metabolizing enzymes was downregulated in NASH. Surprisingly, we found increased homocysteinylation (Hcy-lation) and ubiquitination of multiple hepatic proteins in NASH including the key autophagosome/lysosome fusion protein, Syntaxin 17 (Stx17). This protein was Hcy-lated and ubiquitinated, and its degradation led to a block in autophagy. Genetic manipulation of Stx17 revealed its critical role in regulating autophagy, inflammation and fibrosis during HHcy. Remarkably, dietary B12/Fol, which promotes enzymatic conversion of Hcy to methionine, decreased HHcy and hepatic Hcy-lated protein levels, restored Stx17 expression and autophagy, stimulated ß -oxidation of fatty acids, and improved hepatic histology in mice with pre-established NASH. CONCLUSIONS: HHcy plays a key role in the pathogenesis of NASH via Stx17 homocysteinylation. B12/folate also may represent a novel first-line therapy for NASH. LAY SUMMARY: The incidence of non-alcoholic steatohepatitis, for which there are no approved pharmacological therapies, is increasing, posing a significant healthcare challenge. Herein, based on studies in mice, primates and humans, we found that dietary supplementation with vitamin B12 and folate could have therapeutic potential for the prevention or treatment of non-alcoholic steatohepatitis.

Noise in the operating room during induction of anesthesia: impact of a quality improvement initiative.

PURPOSE: Noise in the operating room (OR) is common and associated with negative effects on anesthesiologists, surgeons, and patient outcomes. Induction of anesthesia is among the loudest perioperative periods. Despite its critical nature, there is little data on noise levels during induction, associated patient and anesthesiologist satisfaction, and the effects of noise reduction strategies. METHODS: We conducted a two-part prospective interventional quality improvement project on the care of adult patients receiving general anesthesia for elective noncardiac surgery. For part A, we measured average and peak noise (dB[A]) levels during anesthesia induction in N = 100 cases and administered a satisfaction questionnaire to anesthesiologists. We then applied a multidisciplinary educational program to OR personnel on active noise reduction strategies and subsequently collected data during N = 109 cases in a post-intervention phase. For part B, we administered satisfaction questionnaires to N = 100 patients pre- vs postintervention, respectively. RESULTS: Median [interquartile range] noise levels throughout induction were 66.0 [62.5-68.6] dB(A) preintervention vs 63.5 [60.1-65.4] dB[A] post-intervention (Hodges-Lehmann estimator of the difference, - 2.7 dB[A]; 95% confidence interval [CI], - 4.0 to - 1.5; P < 0.001). Peak noise levels during induction were 87.3 [84.0-90.5] dB(A) preintervention and 86.2 [81.8-89.3] dB(A) postintervention (Hodges-Lehmann estimator of the difference, - 1.8 dB[A]; 95% CI, - 3.3 to - 0.3; P = 0.02). Noise-related anesthesiologist satisfaction postintervention was significantly improved in multiple domains, including assessment of noise having distracted anesthesiologists. Patient satisfaction was high pre-intervention and did not significantly improve further. CONCLUSION: In this quality improvement project, average noise levels during induction of anesthesia, anesthesiologist satisfaction, and anesthesiologists' perceived ability to perform were improved following a multidisciplinary educational program on noise reduction in the OR. STUDY REGISTRATION: www. CLINICALTRIALS: gov (NCT04204785); registered 19 December 2019.

RéSUMé: OBJECTIF: Le bruit en salle d'opération (SOP) est fréquent et associé à des effets négatifs sur les anesthésiologistes, les chirurgiens et les issues des patients. L'induction de l'anesthésie est l'une des périodes périopératoires les plus bruyantes. Malgré sa nature critique, il existe peu de données sur les niveaux sonores pendant l'induction, la satisfaction des patients et des anesthésiologistes qui y est reliée, et les effets des stratégies de réduction du bruit. MéTHODE: Nous avons mené un projet prospectif et interventionnel, en deux parties, d'amélioration de la qualité sur les soins aux patients adultes recevant une anesthésie générale pour une chirurgie non cardiaque non urgente. Dans le cadre de la première partie A, nous avons mesuré les niveaux de bruit moyen et maximaux (dB[A]) pendant l'induction de l'anesthésie dans n = 100 cas et administré un questionnaire de satisfaction aux anesthésiologistes. Nous avons ensuite appliqué un programme de formation multidisciplinaire au personnel de la salle d'opération sur les stratégies de réduction active du bruit et avons ensuite recueilli des données pour n = 109 cas dans une phase post-intervention. Pour la deuxième partie B, nous avons administré des questionnaires de satisfaction à n = 100 patients pré- vs post-intervention, respectivement. RéSULTATS: Les niveaux de bruit médians [écart interquartile] tout au long de l'induction étaient de 66,0 [62,5­68,6] dB(A) avant l'intervention vs 63,5 [60,1­65,4] dB[A] après l'intervention (estimateur de Hodges-Lehmann, − 2,7 dB[A]; intervalle de confiance [IC] 95 %, − 4,0 à − 1,5; P < 0,001). Les niveaux maximaux de bruit pendant l'induction étaient de 87,3 [84,0­90,5] dB(A) avant l'intervention et de 86,2 [81,8­89,3] dB(A) après l'intervention (estimateur de Hodges-Lehmann, − 1,8 dB[A]; IC 95 %, − 3,3 à − 0,3; P = 0,02). La satisfaction des anesthésiologistes par rapport au bruit après l'intervention a été considérablement améliorée dans de nombreux domaines, y compris l'évaluation du bruit ayant distrait les anesthésiologistes. La satisfaction des patients était élevée avant l'intervention et ne s'est pas améliorée de manière significative. CONCLUSION: Dans ce projet d'amélioration de la qualité, les niveaux de bruit moyens lors de l'induction de l'anesthésie, la satisfaction des anesthésiologistes et la capacité perçue des anesthésiologistes à réaliser leurs tâches ont été améliorés à la suite d'un programme de formation multidisciplinaire sur la réduction du bruit en salle d'opération. ENREGISTREMENT DE L'éTUDE: www.ClinicalTrials.gov (NCT04204785); enregistrée le 19 décembre 2019.

PD-linked CHCHD2 mutations impair CHCHD10 and MICOS complex leading to mitochondria dysfunction.

Coiled-coil-helix-coiled-coil-helix domain containing protein 2 (CHCHD2) mutations were linked with autosomal dominant Parkinson's disease (PD) and recently, Alzheimer's disease/frontotemporal dementia. In the current study, we generated isogenic human embryonic stem cell (hESC) lines harboring PD-associated CHCHD2 mutation R145Q or Q126X via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) method, aiming to unravel pathophysiologic mechanism and seek potential intervention strategy against CHCHD2 mutant-caused defects. By engaging super-resolution microscopy, we identified a physical proximity and similar distribution pattern of CHCHD2 along mitochondria with mitochondrial contact site and cristae organizing system (MICOS), a large protein complex maintaining mitochondria cristae. Isogenic hESCs and differentiated neural progenitor cells (NPCs) harboring CHCHD2 R145Q or Q126X mutation showed impaired mitochondria function, reduced CHCHD2 and MICOS components and exhibited nearly hollow mitochondria with reduced cristae. Furthermore, PD-linked CHCHD2 mutations lost their interaction with coiled-coil-helix-coiled-coil-helix domain containing protein 10 (CHCHD10), while transient knockdown of either CHCHD2 or CHCHD10 reduced MICOS and mitochondria cristae. Importantly, a specific mitochondria-targeted peptide, Elamipretide/MTP-131, now tested in phase 3 clinical trials for mitochondrial diseases, was found to enhance CHCHD2 with MICOS and mitochondria oxidative phosphorylation enzymes in isogenic NPCs harboring heterozygous R145Q, suggesting that Elamipretide is able to attenuate CHCHD2 R145Q-induced mitochondria dysfunction. Taken together, our results suggested CHCHD2-CHCHD10 complex may be a novel therapeutic target for PD and related neurodegenerative disorders, and Elamipretide may benefit CHCHD2 mutation-linked PD.

Bezafibrate induces autophagy and improves hepatic lipid metabolism in glycogen storage disease type Ia.

Glucose-6-phosphatase α (G6Pase) deficiency, also known as von Gierke's Disease or Glycogen storage disease type Ia (GSD Ia), is characterized by decreased ability of the liver to convert glucose-6-phosphate to glucose leading to glycogen accumulation and hepatosteatosis. Long-term complications of GSD Ia include hepatic adenomas and carcinomas, in association with the suppression of autophagy in the liver. The G6pc-/- mouse and canine models for GSD Ia were treated with the pan-peroxisomal proliferator-activated receptor agonist, bezafibrate, to determine the drug's effect on liver metabolism and function. Hepatic glycogen and triglyceride concentrations were measured and western blotting was performed to investigate pathways affected by the treatment. Bezafibrate decreased liver triglyceride and glycogen concentrations and partially reversed the autophagy defect previously demonstrated in GSD Ia models. Changes in medium-chain acyl-CoA dehydrogenase expression and acylcarnintine flux suggested that fatty acid oxidation was increased and fatty acid synthase expression associated with lipogenesis was decreased in G6pc-/- mice treated with bezafibrate. In summary, bezafibrate induced autophagy in the liver while increasing fatty acid oxidation and decreasing lipogenesis in G6pc-/- mice. It represents a potential therapy for glycogen overload and hepatosteatosis associated with GSD Ia, with beneficial effects that have implications for non-alcoholic fatty liver disease.

Titin truncations lead to impaired cardiomyocyte autophagy and mitochondrial function in vivo.

Titin-truncating variants (TTNtv) are the most common genetic cause of dilated cardiomyopathy. TTNtv occur in ~1% of the general population and causes subclinical cardiac remodeling in asymptomatic carriers. In rat models with either proximal or distal TTNtv, we previously showed altered cardiac metabolism at baseline and impaired cardiac function in response to stress. However, the molecular mechanism(s) underlying these effects remains unknown. In the current study, we used rat models of TTNtv to investigate the effect of TTNtv on autophagy and mitochondrial function, which are essential for maintaining cellular metabolic homeostasis and cardiac function. In both the proximal and distal TTNtv rat models, we found increased levels of LC3B-II and p62 proteins, indicative of diminished autophagic degradation. The accumulation of autophagosomes and p62 protein in cardiomyocytes was also demonstrated by electron microscopy and immunochemistry, respectively. Impaired autophagy in the TTNtv heart was associated with increased phosphorylation of mTOR and decreased protein levels of the lysosomal protease, cathepsin B. In addition, TTNtv hearts showed mitochondrial dysfunction, as evidenced by decreased oxygen consumption rate in cardiomyocytes, increased levels of reactive oxygen species and mitochondrial protein ubiquitination. We also observed increased acetylation of mitochondrial proteins associated with decreased NAD+/NADH ratio in the TTNtv hearts. mTORC1 inhibitor, rapamycin, was able to rescue the impaired autophagy in TTNtv hearts. In summary, TTNtv leads to impaired autophagy and mitochondrial function in the heart. These changes not only provide molecular mechanisms that underlie TTNtv-associated ventricular remodeling but also offer potential targets for its intervention.

Inhibiting Interleukin 11 Signaling Reduces Hepatocyte Death and Liver Fibrosis, Inflammation, and Steatosis in Mouse Models of Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: We studied the role of interleukin 11 (IL11) signaling in the pathogenesis of nonalcoholic steatohepatitis (NASH) using hepatic stellate cells (HSCs), hepatocytes, and mouse models of NASH. METHODS: We stimulated mouse and human fibroblasts, HSCs, or hepatocytes with IL11 and other cytokines and analyzed them by imaging, immunoblot, and functional assays and enzyme-linked immunosorbent assays. Mice were given injections of IL11. Mice with disruption of the interleukin 11 receptor subunit alpha1 gene (Il11ra1-/-) mice and Il11ra1+/+ mice were fed a high-fat methionine- and choline-deficient diet (HFMCD) or a Western diet with liquid fructose (WDF) to induce steatohepatitis; control mice were fed normal chow. db/db mice were fed with methionine- and choline-deficient diet for 12 weeks and C57BL/6 NTac were fed with HFMCD for 10 weeks or WDF for 16 weeks. Some mice were given intraperitoneal injections of anti-IL11 (X203), anti-IL11RA (X209), or a control antibody at different timepoints on the diets. Livers and blood were collected; blood samples were analyzed by biochemistry and liver tissues were analyzed by histology, RNA sequencing, immunoblots, immunohistochemistry, hydroxyproline, and mass cytometry time of flight assays. RESULTS: HSCs incubated with cytokines produced IL11, resulting in activation (phosphorylation) of ERK and expression of markers of fibrosis. Livers of mice given injections of IL11 became damaged, with increased markers of fibrosis, hepatocyte cell death and inflammation. Following the HFMCD or WDF, livers from Il11ra1-/- mice had reduced steatosis, fibrosis, expression of markers of inflammation and steatohepatitis, compared to and Il11ra1+/+ mice on the same diets. Depending on the time of administration of anti-IL11 or anti-IL11RA antibodies to wild-type mice on the HFMCD or WDF, or to db/db mice on the methionine and choline-deficient diet, the antibodies prevented, stopped, or reversed development of fibrosis and steatosis. Blood samples from Il11ra1+/+ mice fed the WDF and given injections of anti-IL11 or anti-IL11RA, as well as from Il11ra1-/- mice fed WDF, had lower serum levels of lipids and glucose than mice not injected with antibody or with disruption of Il11ra1. CONCLUSIONS: Neutralizing antibodies that block IL11 signaling reduce fibrosis, steatosis, hepatocyte death, inflammation and hyperglycemia in mice with diet-induced steatohepatitis. These antibodies also improve the cardiometabolic profile of mice and might be developed for the treatment of NASH.

Autophagic protein ULK1 regulates FOXM1 signalling in human hepatoma cells.

Hepatocellular cancer (HCC) is one of the leading causes of mortality worldwide. Unfortunately, a limited choice of anti-cancer drugs is available for treatment, owing to their minimal efficacy and development of acquired resistance. Autophagy, a cellular survival pathway, often exhibits a pleiotropic role in HCC progression. Studies show increased autophagy in established HCC, promoting the survival of HCC cells in the tumour microenvironment. Therefore, novel anti-autophagy drugs hold promise for preventing HCC progression. Here, using a non-biased transcriptomics analysis in HepG2 cells we demonstrate the existence of an autophagy-FOXM1 nexus regulating growth in HepG2 cells. Additionally, we show that suppression of autophagy by an Unc-51 Like Autophagy Activating Kinase 1(ULK1) inhibitor not only attenuates the expression of FOXM1 and its transcriptional targets, but also has a synergistic effect on the inhibition of HepG2 growth when combined with FOXM1 inhibitors. Thus, the autophagic protein, ULK1, is a promising candidate for preventing HCC progression. Collectively, our results provide new insight into the role of autophagy in HCC growth and are a proof-of concept for combinatorial therapy using ULK1 and FOXM1 inhibitors.

Links between autophagy and disorders of glycogen metabolism - Perspectives on pathogenesis and possible treatments.

The glycogen storage diseases are a group of inherited metabolic disorders that are characterized by specific enzymatic defects involving the synthesis or degradation of glycogen. Each disorder presents with a set of symptoms that are due to the underlying enzyme deficiency and the particular tissues that are affected. Autophagy is a process by which cells degrade and recycle unneeded or damaged intracellular components such as lipids, glycogen, and damaged mitochondria. Recent studies showed that several of the glycogen storage disorders have abnormal autophagy which can disturb normal cellular metabolism and/or mitochondrial function. Here, we provide a clinical overview of the glycogen storage disorders, a brief description of autophagy, and the known links between specific glycogen storage disorders and autophagy.

Thermogenesis in Adipose Tissue Activated by Thyroid Hormone.

Thermogenesis is the production of heat that occurs in all warm-blooded animals. During cold exposure, there is obligatory thermogenesis derived from body metabolism as well as adaptive thermogenesis through shivering and non-shivering mechanisms. The latter mainly occurs in brown adipose tissue (BAT) and muscle; however, white adipose tissue (WAT) also can undergo browning via adrenergic stimulation to acquire thermogenic potential. Thyroid hormone (TH) also exerts profound effects on thermoregulation, as decreased body temperature and increased body temperature occur during hypothyroidism and hyperthyroidism, respectively. We have termed the TH-mediated thermogenesis under thermoneutral conditions "activated" thermogenesis. TH acts on the brown and/or white adipose tissues to induce uncoupled respiration through the induction of the uncoupling protein (Ucp1) to generate heat. TH acts centrally to activate the BAT and browning through the sympathetic nervous system. However, recent studies also show that TH acts peripherally on the BAT to directly stimulate Ucp1 expression and thermogenesis through an autophagy-dependent mechanism. Additionally, THs can exert Ucp1-independent effects on thermogenesis, most likely through activation of exothermic metabolic pathways. This review summarizes thermogenic effects of THs on adipose tissues.

Estrogen-Related Receptor Alpha: An Under-Appreciated Potential Target for the Treatment of Metabolic Diseases.

The estrogen-related receptor alpha (ESRRA) is an orphan nuclear receptor (NR) that significantly influences cellular metabolism. ESRRA is predominantly expressed in metabolically-active tissues and regulates the transcription of metabolic genes, including those involved in mitochondrial turnover and autophagy. Although ESRRA activity is well-characterized in several types of cancer, recent reports suggest that it also has an important role in metabolic diseases. This minireview focuses on the regulation of cellular metabolism and function by ESRRA and its potential as a target for the treatment of metabolic disorders.

Hepatic Lipid Catabolism via PPARα-Lysosomal Crosstalk.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors which belong to the nuclear hormone receptor superfamily. They regulate key aspects of energy metabolism within cells. Recently, PPARα has been implicated in the regulation of autophagy-lysosomal function, which plays a key role in cellular energy metabolism. PPARα transcriptionally upregulates several genes involved in the autophagy-lysosomal degradative pathway that participates in lipolysis of triglycerides within the hepatocytes. Interestingly, a reciprocal regulation of PPARα nuclear action by autophagy-lysosomal activity also exists with implications in lipid metabolism. This review succinctly discusses the unique relationship between PPARα nuclear action and lysosomal activity and explores its impact on hepatic lipid homeostasis under pathological conditions such as non-alcoholic fatty liver disease (NAFLD).

Novel Transcriptional Mechanisms for Regulating Metabolism by Thyroid Hormone.

The thyroid hormone plays a key role in energy and nutrient metabolisms in many tissues and regulates the transcription of key genes in metabolic pathways. It has long been believed that thyroid hormones (THs) exerted their effects primarily by binding to nuclear TH receptors (THRs) that are associated with conserved thyroid hormone response elements (TREs) located on the promoters of target genes. However, recent transcriptome and ChIP-Seq studies have challenged this conventional view as discordance was observed between TH-responsive genes and THR binding to DNA. While THR association with other transcription factors bound to DNA, TH activation of THRs to mediate effects that do not involve DNA-binding, or TH binding to proteins other than THRs have been invoked as potential mechanisms to explain this discrepancy, it appears that additional novel mechanisms may enable TH to regulate the mRNA expression. These include activation of transcription factors by SIRT1 via metabolic actions by TH, the post-translational modification of THR, the THR co-regulation of transcription with other nuclear receptors and transcription factors, and the microRNA (miR) control of RNA transcript expression to encode proteins involved in the cellular metabolism. Together, these novel mechanisms enlarge and diversify the panoply of metabolic genes that can be regulated by TH.

Loss of prohormone convertase 2 promotes beta cell dysfunction in a rodent transplant model expressing human pro-islet amyloid polypeptide.

AIMS/HYPOTHESIS: A contributor to beta cell failure in type 2 diabetes and islet transplants is amyloid formation by aggregation of the beta cell peptide, islet amyloid polypeptide (IAPP). Similar to the proinsulin processing pathway that generates insulin, IAPP is derived from a prohormone precursor, proIAPP, which requires cleavage by prohormone convertase (PC) 1/3 and PC2 in rodent pancreatic beta cells. We hypothesised that loss of PC2 would promote beta cell death and dysfunction in a rodent model of human beta cell proIAPP overexpression. METHODS: We generated an islet transplant model wherein immune-deficient mouse models of diabetes received islets expressing amyloidogenic human proIAPP and lacking PC2, leading to restoration of normoglycaemia accompanied by increased secretion of human proIAPP. Blood glucose levels were analysed for up to 16 weeks in transplant recipients and grafts were assessed for islet amyloid and beta cell number and death. RESULTS: Hyperglycaemia (blood glucose >16.9 mmol/l) returned in 94% of recipients of islets expressing human proIAPP and lacking PC2, whereas recipients of islets that express human proIAPP and normal PC2 levels remained normoglycaemic for at least 16 weeks. Islet graft failure was accompanied by a ∼20% reduction in insulin-positive cells, yet the degree of amyloid deposition and beta cell apoptosis was similar to those of controls expressing human proIAPP with functional PC2 levels. CONCLUSIONS/INTERPRETATION: PC2 deficiency in transplanted mouse islets expressing human proIAPP promotes beta cell loss and graft failure. Our data suggest that impaired NH2-terminal processing and increased secretion of human proIAPP promote beta cell failure.

Hepatic FOXO1 Target Genes Are Co-regulated by Thyroid Hormone via RICTOR Protein Deacetylation and MTORC2-AKT Protein Inhibition.

MTORC2-AKT is a key regulator of carbohydrate metabolism and insulin signaling due to its effects on FOXO1 phosphorylation. Interestingly, both FOXO1 and thyroid hormone (TH) have similar effects on carbohydrate and energy metabolism as well as overlapping transcriptional regulation of many target genes. Currently, little is known about the regulation of MTORC2-AKT or FOXO1 by TH. Accordingly, we performed hepatic transcriptome profiling in mice after FOXO1 knockdown in the absence or presence of TH, and we compared these results with hepatic FOXO1 and THRB1 (TRß1) ChIP-Seq data. We identified a subset of TH-stimulated FOXO1 target genes that required co-regulation by FOXO1 and TH. TH activation of FOXO1 was directly linked to an increase in SIRT1-MTORC2 interaction and RICTOR deacetylation. This, in turn, led to decreased AKT and FOXO1 phosphorylation. Moreover, TH increased FOXO1 nuclear localization, DNA binding, and target gene transcription by reducing AKT-dependent FOXO1 phosphorylation in a THRB1-dependent manner. These events were associated with TH-mediated oxidative phosphorylation and NAD(+) production and suggested that downstream metabolic effects by TH can post-translationally activate other transcription factors. Our results showed that RICTOR/MTORC2-AKT can integrate convergent hormonal and metabolic signals to provide coordinated and sensitive regulation of hepatic FOXO1-target gene expression.

Renal endoplasmic reticulum stress is coupled to impaired autophagy in a mouse model of GSD Ia.

GSD Ia (von Gierke Disease, Glycogen Storage Disease Type Ia) is a devastating genetic disorder with long-term sequelae, such as non-alcoholic fatty liver disease and renal failure. Down-regulated autophagy is involved in the development of hepatic metabolic dysfunction in GSD Ia; however, the role of autophagy in the renal pathology is unknown. Here we show that autophagy is impaired and endoplasmic reticulum (ER) stress is increased in the kidneys of a mouse model of GSD Ia. Induction of autophagy by rapamycin also reduces this ER stress. Taken together, these results show an additional role for autophagy down-regulation in the pathogenesis of GSD Ia, and provide further justification for the use of autophagy modulators in GSD Ia.