Featured ArticlesVolume 4 | No. 4 | April 2015
|Clic4 sensitizes β-cells to apoptosis|
Patel and colleagues show that Clic4 sensitizes beta cells to cytokines- or palmitic acid-induced apoptosis. Reducing Clic4 expression increases beta cell survival. Targeting Clic4 expression or its interaction with specific protein partners may provide a new way to prevent b eta cell apoptosis in the context of diabetes mellitus
Abstract | PDF
Objectives: Chloride intracellular channel protein 4 (Clic4) is a ubiquitously expressed protein involved in multiple cellular processes including cell-cycle control, cell differentiation, and apoptosis. Here, we investigated the role of Clic4 in pancreatic β-cell apoptosis.
Methods: We used βTC-tet cells and islets from β-cell specific Clic4 knockout mice (βClic4KO) and assessed cytokine-induced apoptosis, Bcl2 family protein expression and stability, and identified Clic4-interacting proteins by co-immunoprecipitation and mass spectrometry analysis.
Results: We show that cytokines increased Clic4 expression in βTC-tet cells and in mouse islets and siRNA-mediated silencing of Clic4 expression in βTC-tet cells or its genetic inactivation in islets β-cells, reduced cytokine-induced apoptosis. This was associated with increased expression of Bcl-2 and increased expression and phosphorylation of Bad. Measurement of Bcl-2 and Bad half-lives in βTC-tet cells showed that Clic4 silencing increased the stability of these proteins. In primary islets β-cells, absence of Clic4 expression increased Bcl-2 and Bcl-xL expression as well as expression and phosphorylation of Bad. Mass-spectrometry analysis of proteins co-immunoprecipitated with Clic4 from βTC-tet cells showed no association of Clic4 with Bcl-2 family proteins. However, Clic4 co-purified with proteins from the proteasome suggesting a possible role for Clic4 in regulating protein degradation.
Conclusions: Collectively, our data show that Clic4 is a cytokine-induced gene that sensitizes β-cells to apoptosis by reducing the steady state levels of Bcl-2, Bad and phosphorylated Bad.[Hide abstract]
|GLP-1 receptor signaling in pancreatic β-cells|
Rajan and colleagues demonstrate that chronically elevated glucose acts via PKA to reduce GLP-1R signalling through a SUMO-1-dependent mechanism. Alteration of receptor levels is a likely mechanism for the reduced efficacy of incretin therapies in type 2 diabetes.
Abstract | PDF
Objective: Glucagon-like peptide 1 (GLP-1) enhances insulin secretion and protects β-cell mass. Diabetes therapies targeting the GLP-1 receptor (GLP-1R), expressed in numerous tissues, have diminished dose-response in patients with type 2 diabetes compared with healthy human controls. The aim of this study was to determine the mechanistic causes underlying the reduced efficacy of GLP-1R ligands.
Methods: Using primary mouse islets and the β-cell line MIN6, outcomes downstream of the GLP-1R were analyzed: Insulin secretion; phosphorylation of the cAMP-response element binding protein (CREB); cAMP responses. Signaling systems were studied by immunoblotting and qRT-PCR, and PKA activity was assayed. Cell surface localization of the GLP-1R was studied by confocal microscopy using a fluorescein-tagged exendin-4 and GFP-tagged GLP-1R.
Results: Rodent β-cells chronically exposed to high glucose had diminished responses to GLP-1R agonists including: diminished insulin secretory response; reduced phosphorylation of (CREB); impaired cAMP response, attributable to chronically increased cAMP levels. GLP-1R signaling systems were affected by hyperglycemia with increased expression of mRNAs encoding the inducible cAMP early repressor (ICER) and adenylyl cyclase 8, reduced PKA activity due to increased expression of the PKA-RIα subunit, reduced GLP-1R mRNA expression and loss of GLP-1R from the cell surface. To specifically examine the loss of GLP-1R from the plasma membrane a GLP-1R-GFP fusion protein was employed to visualize subcellular localization. Under low glucose conditions or when PKA activity was inhibited, GLP-1R-GFP was found at the plasma membrane. Conversely high glucose, expression of a constitutively active PKA subunit, or exposure to exendin-4 or forskolin led to GLP-1R-GFP internalization. Mutation of serine residue 301 of the GLP-1R abolished the glucose-dependent loss of the receptor from the plasma membrane. This was associated with a loss of an interaction between the receptor and the small ubiquitin-related modifier (SUMO), an interaction that was found to be necessary for internalization of the receptor.
Conclusions: These data show that glucose acting, at least in part, via PKA leads to the loss of the GLP-1R from the cell surface and an impairment of GLP-1R signaling, which may underlie the reduced clinical efficacy of GLP-1R based therapies in individuals with poorly controlled hyperglycemia.[Hide abstract]
|LKB1 and AMPKα1 are required for the normal regulation of glucagon secretion|
Sun and colleagues show that LKB1 signalling, at least partly mediated via AMPKα1, is essential for the normal stimulation of glucagon secretion at low glucose levels. Moreover, they show that LKB1 plays a limited if any role in the control of alpha cell size or total alpha cell mass.
Abstract | PDF
Aims/Hypothesis: Glucagon release from pancreatic alpha cells is required for normal glucose homoeostasis and is dysregulated in both Type 1 and Type 2 diabetes. The tumour suppressor LKB1 (STK11) and the downstream kinase AMP-activated protein kinase (AMPK), modulate cellular metabolism and growth, and AMPK is an important target of the anti-hyperglycaemic agent metformin. While LKB1 and AMPK have emerged recently as regulators of beta cell mass and insulin secretion, the role of these enzymes in the control of glucagon production in vivo is unclear.
Methods: Here, we ablated LKB1 (αLKB1KO), or the catalytic alpha subunits of AMPK (αAMPKdKO, -α1KO, -α2KO), selectively in ∼45% of alpha cells in mice by deleting the corresponding flox'd alleles with a preproglucagon promoter (PPG) Cre.Results: Blood glucose levels in male αLKB1KO mice were lower during intraperitoneal glucose, aminoimidazole carboxamide ribonucleotide (AICAR) or arginine tolerance tests, and glucose infusion rates were increased in hypoglycemic clamps (p < 0.01). αLKB1KO mice also displayed impaired hypoglycemia-induced glucagon release. Glucose infusion rates were also elevated (p < 0.001) in αAMPKα1 null mice, and hypoglycemia-induced plasma glucagon increases tended to be lower (p = 0.06). Glucagon secretion from isolated islets was sensitized to the inhibitory action of glucose in αLKB1KO, αAMPKdKO, and -α1KO, but not -α2KO islets. Conclusions/Interpretation: An LKB1-dependent signalling cassette, involving but not restricted to AMPKα1, is required in pancreatic alpha cells for the control of glucagon release by glucose.
|FTO is necessary for the induction of leptin resistance by high-fat feeding|
Tung and colleagues report that mice deficient in FTO significantly increase their fat mass in response to HFD and remain sensitive to leptin. They show that genes encoding components of the NFкB signalling pathway are down-regulated in FTO-deficient mice following a HFD. TRIP4, a transcriptional coactivator of NFкB, is identified as a binding partner of FTO.
Abstract | PDF
Objective: Loss of function FTO mutations significantly impact body composition in humans and mice, with Fto-deficient mice reported to resist the development of obesity in response to a high-fat diet (HFD). We aimed to further explore the interactions between FTO and HFD and determine if FTO can influence the adverse metabolic consequence of HFD.
Methods: We studied mice deficient in FTO in two well validated models of leptin resistance (HFD feeding and central palmitate injection) to determine how Fto genotype may influence the action of leptin. Using transcriptomic analysis of hypothalamic tissue to identify relevant pathways affected by the loss of Fto, we combined data from co-immunoprecipitation, yeast 2-hybrid and luciferase reporter assays to identify mechanisms through which FTO can influence the development of leptin resistant states.
Results: Mice deficient in Fto significantly increased their fat mass in response to HFD. Fto+/− and Fto −/− mice remained sensitive to the anorexigenic effects of leptin, both after exposure to a HFD or after acute central application of palmitate. Genes encoding components of the NFкB signalling pathway were down-regulated in the hypothalami of Fto-deficient mice following a HFD. When this pathway was reactivated in Fto-deficient mice with a single low central dose of TNFα, the mice became less sensitive to the effect of leptin. We identified a transcriptional coactivator of NFкB, TRIP4, as a binding partner of FTO and a molecule that is required for TRIP4 dependent transactivation of NFкB.
Conclusions: Our study demonstrates that, independent of body weight, Fto influences the metabolic outcomes of a HFD through alteration of hypothalamic NFкB signalling. This supports the notion that pharmacological modulation of FTO activity might have the potential for therapeutic benefit in improving leptin sensitivity, in a manner that is influenced by the nutritional environment.[Hide abstract]
|TRAP-seq defines markers for novel populations of LepRb neurons|
By elucidating the transcriptome of brainstem and hypothalamic LepRb neurons, the TRAP-seq analysis performed by Allison and colleagues reveals markers for numerous subpopulations of LepRb neurons and genes likely to contribute importantly to central leptin action.
Abstract | PDF
Objective: Leptin acts via its receptor (LepRb) on multiple subpopulations of LepRb neurons in the brain, each of which controls specific aspects of energy balance. Despite the importance of LepRb-containing neurons, the transcriptome and molecular identity of many LepRb subpopulations remain undefined due to the difficulty of studying the small fraction of total cells represented by LepRb neurons in heterogeneous brain regions. Here we sought to examine the transcriptome of LepRb neurons directly and identify markers for functionally relevant LepRb subsets.
Methods: We isolated mRNA from mouse hypothalamic and brainstem LepRb cells by Translating Ribosome Affinity Purification (TRAP) and analyzed it by RNA-seq (TRAP-seq).
Results: TRAP mRNA from LepRb cells was enriched for markers of peptidergic neurons, while TRAP-depleted mRNA from non-LepRb cells was enriched for markers of glial and immune cells. Genes encoding secreted proteins that were enriched in hypothalamic and brainstem TRAP mRNA revealed subpopulations of LepRb neurons that contained neuropeptide-encoding genes (including prodynorphin, Pdyn) not previously used as functional markers for LepRb neurons. Furthermore, Pdyncre-mediated ablation of Leprflox in Pdyn-expressing neurons (LepRbPdynKO mice) blunted energy expenditure to promote obesity during high-fat feeding.
Conclusions: TRAP-seq of CNS LepRb neurons defines the LepRb neuron transcriptome and reveals novel markers for previously unrecognized subpopulations of LepRb neurons.[Hide abstract]
|Therapeutic effects of adropin on glucose tolerance and substrate utilization|
The study of Gao and colleagues provides a molecular basis for the improvements in glucose homeostasis that are observed with adropin treatment. The data suggest that skeletal muscle is a major organ target in mediating these effects. Adropin may be a promising drug target in developing treatments against diet-induced dysregulation of glucose homeostasis and insulin resistance.
Abstract | PDF
Objective: The peptide hormone adropin regulates fuel selection preferences in skeletal muscle under fed and fasted conditions. Here, we investigated whether adropin treatment can ameliorate the dysregulation of fuel substrate metabolism, and improve aspects of glucose homeostasis in diet-induced obesity (DIO) with insulin resistance.
Methods: DIO C57BL/6 mice maintained on a 60% kcal fat diet received five intraperitoneal (i.p.) injections of the bioactive peptide adropin34-76 (450 nmol/kg/i.p.). Following treatment, glucose tolerance and whole body insulin sensitivity were assessed and indirect calorimetry was employed to analyze whole body substrate oxidation preferences. Biochemical assays performed in skeletal muscle samples analyzed insulin signaling action and substrate oxidation.
Results: Adropin treatment improved glucose tolerance, enhanced insulin action and augmented metabolic flexibility towards glucose utilization. In muscle, adropin treatment increased insulin-induced Akt phosphorylation and cell-surface expression of GLUT4 suggesting sensitization of insulin signaling pathways. Reduced incomplete fatty acid oxidation and increased CoA/acetyl-CoA ratio suggested improved mitochondrial function. The underlying mechanisms appear to involve suppressions of carnitine palmitoyltransferase-1B (CPT-1B) and CD36, two key enzymes in fatty acid utilization. Adropin treatment activated pyruvate dehydrogenase (PDH), a rate-limiting enzyme in glucose oxidation, and downregulated PDH kinase-4 (PDK-4) that inhibits PDH. Along with these changes, adropin treatment downregulated peroxisome proliferator-activated receptor-gamma coactivator-1α that regulates expression of Cpt1b, Cd36 and Pdk4.
Conclusions: Adropin treatment of DIO mice enhances glucose tolerance, ameliorates insulin resistance and promotes preferential use of carbohydrate over fat in fuel selection. Skeletal muscle is a key organ in mediating adropin's whole-body effects, sensitizing insulin signaling pathways and altering fuel selection preference to favor glucose while suppressing fat oxidation.[Hide abstract]
|Enhanced insulin signaling in density-enhanced phosphatase-1 (DEP-1) knockout mice|
Krüger and colleagues report that a conventional knockout of DEP-1 results in an improved metabolic phenotype in mice, characterized in particular by enhanced insulin sensitivity and insulin signaling. Knockdown of DEP-1 in skeletal muscle cells leads to an increased insulin-induced glucose uptake. The findings support the notion of DEP-1 as a novel negative regulator of insulin signaling.
Abstract | PDF
Objective: Insulin resistance can be triggered by enhanced dephosphorylation of the insulin receptor or downstream components in the insulin signaling cascade through protein tyrosine phosphatases (PTPs). Downregulating density-enhanced phosphatase-1 (DEP-1) resulted in an improved metabolic status in previous analyses. This phenotype was primarily caused by hepatic DEP-1 reduction.
Methods: Here we further elucidated the role of DEP-1 in glucose homeostasis by employing a conventional knockout model to explore the specific contribution of DEP-1 in metabolic tissues. Ptprj−/− (DEP-1 deficient) and wild-type C57BL/6 mice were fed a low-fat or high-fat diet. Metabolic phenotyping was combined with analyses of phosphorylation patterns of insulin signaling components. Additionally, experiments with skeletal muscle cells and muscle tissue were performed to assess the role of DEP-1 for glucose uptake.
Results: High-fat diet fed-Ptprj−/− mice displayed enhanced insulin sensitivity and improved glucose tolerance. Furthermore, leptin levels and blood pressure were reduced in Ptprj−/− mice. DEP-1 deficiency resulted in increased phosphorylation of components of the insulin signaling cascade in liver, skeletal muscle and adipose tissue after insulin challenge. The beneficial effect on glucose homeostasis in vivo was corroborated by increased glucose uptake in skeletal muscle cells in which DEP-1 was downregulated, and in skeletal muscle of Ptprj−/− mice.
Conclusion: Together, these data establish DEP-1 as novel negative regulator of insulin signaling.[Hide abstract]
|Angiotensin type 1a receptors facilitate leptin-induced weight loss through brown adipose tissue thermogenesis|
Young and colleagues show that the subfornical organ, a tiny forebrain structure dense with AT1aR, and recently implicated as an integrative metabolic center, plays a previously unrecognized role in the control of body weight. They demonstrate an interaction between SFO-AT1aR and CNS leptin in the regulation of brown adipose tissue thermogenic metabolism and body weight.
Abstract | PDF
Objective: Elevations in brain angiotensin-II cause increased energy expenditure and a lean phenotype. Interestingly, the metabolic effects of increased brain angiotensin-II mimic the actions of leptin, suggesting an interaction between the two systems. Here we demonstrate that angiotensin-type 1a receptors (AT1aR) in the subfornical organ (SFO), a forebrain structure emerging as an integrative metabolic center, play a key role in the body weight-reducing effects of leptin via brown adipose tissue (BAT) thermogenesis.
Methods: Cre/LoxP technology coupled with targeted viral delivery to the SFO in a mouse line bearing a conditional allele of the Agtr1a gene was utilized to determine the interaction between leptin and SFO AT1aR in metabolic regulation.
Results: Selective deletion of AT1aR in the SFO attenuated leptin-induced weight loss independent of changes in food intake or locomotor activity. This was associated with diminished leptin-induced increases in core body temperature, blunted upregulation of BAT thermogenic markers, and abolishment of leptin-mediated sympathetic activation to BAT.
Conclusions: These data identify a novel interaction between angiotensin-II and leptin in the control of BAT thermogenesis and body weight, and highlight a previously unrecognized role for the forebrain SFO in metabolic regulation.[Hide abstract]
|The expression of dominant negative TCF7L2 causes impaired glucose homeostasis|
Shao and colleagues demonstrate that TCF7L2DN expression in Ins-1 cells attenuates cell growth, glucose stimulated insulin secretion (GSIS), and β-cell specific gene expression. Its expression during embryonic development significantly impairs the generation of Pdx-1 and Nkx6.1 positive islet cells, β-cell gene expression, and the function of pancreatic islets in response to GLP-1.
Abstract | PDF
Objective: Disruption of TCF7L2 in mouse pancreatic β-cells has generated different outcomes in several investigations. Here we aim to clarify role of β-cell TCF7L2 and Wnt signaling using a functional-knockdown approach.
Methods: Adenovirus-mediated dominant negative TCF7L2 (TCF7L2DN) expression was conducted in Ins-1 cells. The fusion gene in which TCF7L2DN expression is driven by PTRE3G was utilized to generate the transgenic mouse line TCF7L2DNTet. The double transgenic line was created by mating TCF7L2DNTet with Ins2-rtTA, designated as βTCFDN. β-cell specific TCF7L2DN expression was induced in βTCFDN by doxycycline feeding.
Results: TCF7L2DN expression in Ins-1 cells reduced GSIS, cell proliferation and expression of a battery of genes including incretin receptors and β-cell transcription factors. Inducing TCF7L2DN expression in βTCFDN during adulthood or immediately after weaning generated no or very modest metabolic defect, while its expression during embryonic development by doxycycline feeding in pregnant mothers resulted in significant glucose intolerance associated with altered β-cell gene expression and reduced β-cell mass.
Conclusions: Our observations support a cell autonomous role for TCF7L2 in pancreatic β-cells suggested by most, though not all, investigations. βTCFDN is a novel model for further exploring the role of TCF7L2 in β-cell genesis and metabolic homeostasis.[Hide abstract]
|The LXR inverse agonist SR9238 suppresses fibrosis in a model of non-alcoholic steatohepatitis|
Griffett and colleagues show that the LXR inverse agonist SR9238 displays efficacy in reduction of hepatic pathology in a mouse model of diet induced NASH. SR9238 is effective in reducing hepatic steatosis and inflammation and reduces hepatic fibrosis. LXR inverse agonists, such as SR9238, may offer novel therapies to treat NASH.
Abstract | PDF
Objective: Non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, inflammation and fibrosis. There are currently no targeted therapies for NASH. We developed a liver-specific LXR inverse agonist, SR9238, which effectively reduces hepatic lipogenesis in models of obesity and hepatic steatosis. We hypothesized that suppression of lipogenesis, which is pathologically elevated in NASH may suppress progression of hepatic steatosis to NASH.
Methods: NASH was induced in B6 V-lep ob/J (ob/ob) mice using a custom complete rodent diet (HTF) containing high amounts of trans-fat, fructose, and cholesterol. Once NASH was induced, mice were treated with SR9238 for one month by i.p. injection. Plasma lipid levels and liver health were analyzed by clinical chemistry. QPCR, western blot, and immunohistochemistry were used to assess disease severity.
Results: Ob/ob mice are obese and diabetic thus they are commonly used as models for the study of metabolic diseases. These mice quickly developed the NASH phenotype when provided the HTF diet. The mice develop hepatic steatosis, severe hepatic inflammation and fibrosis on the HTF diet. Treatment with SR9238 significantly reduced the severity of hepatic steatosis and most importantly reduced hepatic inflammation and ameliorated hepatic fibrosis.
Conclusions: Here, we demonstrate that an LXR inverse agonist, SR9238, is effective in reduction of hepatic steatosis, inflammation and fibrosis in an animal model of NASH. These results have important implications for the development of therapeutics for treatment NASH in humans.[Hide abstract]