Missense mutation of ISL1 (E283D) is associated with the development of type 2 diabetes.

AIMS/HYPOTHESIS: Mutations in Isl1, encoding the insulin enhancer-binding protein islet-1 (ISL1), may contribute to attenuated insulin secretion in type 2 diabetes mellitus. We made an Isl1E283D mouse model to investigate the disease-causing mechanism of diabetes mellitus. METHODS: The ISL1E283D mutation (c. 849A>T) was identified by whole exome sequencing on an early-onset type 2 diabetes family and then the Isl1E283D knockin (KI) mouse model was created and an IPGTT and IPITT were conducted. Glucose-stimulated insulin secretion (GSIS), expression of Ins2 and other ISL1 target genes and interacting proteins were evaluated in isolated pancreas islets. Transcriptional activity of Isl1E283D was evaluated by cell-based luciferase reporter assay and electrophoretic mobility shift assay, and the expression levels of Ins2 driven by Isl1 wild-type (Isl1WT) and Isl1E283D mutation in rat INS-1 cells were determined by RT-PCR and western blotting. RESULTS: Impaired GSIS and elevated glucose level were observed in Isl1E283D KI mice while expression of Ins2 and other ISL1 target genes Mafa, Pdx1, Slc2a2 and the interacting protein NeuroD1 were downregulated in isolated islets. Transcriptional activity of the Isl1E283D mutation for Ins2 was reduced by 59.3%, and resulted in a marked downregulation of Ins2 expression when it was overexpressed in INS-1 cells, while overexpression of Isl1WT led to an upregulation of Ins2 expression. CONCLUSIONS/INTERPRETATION: Isl1E283D mutation reduces insulin expression and secretion by regulating insulin and other target genes, as well as its interacting proteins such as NeuroD1, leading to the development of glucose intolerance in the KI mice, which recapitulated the human diabetic phenotype. This study identified and highlighted the Isl1E283D mutation as a novel causative factor for type 2 diabetes, and suggested that targeting transcription factor ISL1 could offer an innovative avenue for the precise treatment of human type 2 diabetes.

Identification and management of GCK-MODY complicating pregnancy in Chinese patients with gestational diabetes.

Precise differentiation of glucokinase (GCK) monogenic diabetes from gestational diabetes mellitus (GDM) is critical for accurate management of the pregnancy outcome. We screened GCK-MODY complicating pregnancies in Chinese GDM patients, explored the pathogenesis of novel GCK mutations, and evaluated the patients' pregnancy outcome and management. The GCK gene from 411 GDM patients was screened with PCR-direct sequencing and multiplex ligation-dependent probe amplification (MLPA) and 15 GCK mutations were identified. We also retrospectively analyzed a total of 65 pregnancies from 21 GCK-MODY families, wherein 41 were from 15 maternal families and 24 were from six paternal families. Bioinformatic analysis and biochemical functional study were conducted to identify novel GCK mutations. In total, we identified 21 GCK mutations: 15 from the 411 GDM patients and six from 24 fathers. Of th Asp78Asn (GAC → AAC), Met87Arg (ATG → AGG), Leu451Val (CTT → GTT), Leu451Pro (CTG → CCG) and 1019 + 20G > A e mutations, five, i.e., were novel and deleterious, with markedly decreased enzyme activity and thermal stability. The unaffected offspring of GCK mutation-affected mothers were heavier than affected offspring (p < 0.001). Of 21 insulin-treated affected mothers, 10 had maternal hypoglycemia (47.6%) and seven had perinatal complications (33.3%), and the affected offspring of the insulin-treated affected mothers had significantly lower birth weights than that of the 20 diet-control affected mothers (p = 0.031). In this study, the prevalence of GCK-MODY complicating pregnancy in Chinese GDM patients was 3.6% (15/411). The defective GCK may contribute to the hyperglycemia in GCK-MODY. Insulin therapy is not beneficial for GCK-MODY complicating pregnancy and therefore should not be recommended.

Decreased 11ß-Hydroxysteroid Dehydrogenase Type 2 Expression in the Kidney May Contribute to Nicotine/Smoking-Induced Blood Pressure Elevation in Mice.

[Figure: see text].

Identification of Ala2Thr mutation in insulin gene from a Chinese MODY10 family.

More than 80% of maturity-onset diabetes of the young (MODY) in Chinese is genetically unexplained. To investigate whether the insulin gene (INS) mutation is responsible for some Chinese MODY, we screened INS mutations causing MODY10 in MODY pedigrees and explored the potential pathogenic mechanisms. INS mutations were screened in 56 MODY familial probands. Structure-function characterization and clinical profiling of identified INS mutations were conducted. An INS mutation, at the position 2 alanine-to-threonine substitution (A2T), was identified and co-segregated with hyperglycemia in a MODY pedigree. The A2T mutation converted an α-helix into a ß-sheet at the N-terminal of the signal peptide (SP) of preproinsulin. The A2T mutation did not affect preproinsulin translocation across endoplasmic reticulum (ER) membrane, but impaired its SP cleavage within the ER. In INS-1 cells transfected with an A2T mutant, glucose-stimulated insulin secretion (GSIS) was significantly decreased, while BiP luciferase activities were significantly increased compared to that of wild type (WT). We identified an INS-A2T mutation cosegregating with diabetes in a Chinese MODY pedigree. This mutation severely impaired SP cleavage and thus blocked the formation of proinsulin, resulting in enhanced ER stress, which may be responsible for decreased insulin secretion and subsequently, the onset of MODY10.

Lack of adipose-specific hexose-6-phosphate dehydrogenase causes inactivation of adipose glucocorticoids and improves metabolic phenotype in mice.

Excessive glucocorticoid (GC) production in adipose tissue promotes the development of visceral obesity and metabolic syndrome (MS). 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is critical for controlling intracellular GC production, and this process is tightly regulated by hexose-6-phosphate dehydrogenase (H6PDH). To better understand the integrated molecular physiological effects of adipose H6PDH, we created a tissue-specific knockout of the H6PDH gene mouse model in adipocytes (adipocyte-specific conditional knockout of H6PDH (H6PDHAcKO) mice). H6PDHAcKO mice exhibited almost complete absence of H6PDH expression and decreased intra-adipose corticosterone production with a reduction in 11ß-HSD1 activity in adipose tissue. These mice also had decreased abdominal fat mass, which was paralleled by decreased adipose lipogenic acetyl-CoA carboxylase (ACC) and ATP-citrate lyase (ACL) gene expression and reduction in their transcription factor C/EBPα mRNA levels. Moreover, H6PDHAcKO mice also had reduced fasting blood glucose levels, increased glucose tolerance, and increased insulin sensitivity. In addition, plasma free fatty acid (FFA) levels were decreased with a concomitant decrease in the expression of lipase adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) in adipose tissue. These results indicate that inactivation of adipocyte H6PDH expression is sufficient to cause intra-adipose GC inactivation that leads to a favorable pattern of metabolic phenotypes. These data suggest that H6PDHAcKO mice may provide a good model for studying the potential contributions of fat-specific H6PDH inhibition to improve the metabolic phenotype in vivo. Our study suggests that suppression or inactivation of H6PDH expression in adipocytes could be an effective intervention for treating obesity and diabetes.

Stimulation of Hair Growth by Small Molecules that Activate Autophagy.

Hair plays important roles, ranging from the conservation of body heat to the preservation of psychological well-being. Hair loss or alopecia affects millions worldwide, but methods that can be used to regrow hair are lacking. We report that quiescent (telogen) hair follicles can be stimulated to initiate anagen and hair growth by small molecules that activate autophagy, including the metabolites α-ketoglutarate (α-KG) and α-ketobutyrate (α-KB), and the prescription drugs rapamycin and metformin, which impinge on mTOR and AMPK signaling. Stimulation of hair growth by these agents is blocked by specific autophagy inhibitors, suggesting a mechanistic link between autophagy and hair regeneration. Consistently, increased autophagy is detected upon anagen entry during the natural hair follicle cycle, and oral α-KB prevents hair loss in aged mice. Our finding that anagen can be pharmacologically activated in telogen skin when natural anagen-inducing signal(s) are absent has implications for the treatment of hair loss patients.

GNAI1 and GNAI3 Reduce Colitis-Associated Tumorigenesis in Mice by Blocking IL6 Signaling and Down-regulating Expression of GNAI2.

BACKGROUND & AIMS: Interleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans. METHODS: B6;129 wild-type (control) or mice with disruption of Gnai1, Gnai2, and/or Gnai3 or conditional disruption of Gnai2 in CD11c+ or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression of Gnai2 messenger RNA by CD11c+ cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses. RESULTS: GNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+ DCs and MDSCs; and decreased numbers of CD8+ DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption of Gnai2 in CD11c+ cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs. Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public human colorectal tumor DNA microarray database (GSE39582) showed that low Gani1 and Gnai3 messenger RNA expression and high Gnai2 messenger RNA expression were significantly associated with decreased relapse-free survival. CONCLUSIONS: GNAI1;3 suppresses DSS-plus-AOM-induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+ cells and IL6 in CD4+/CD11b+ DCs appears to promote these effects. Strategies to induce GNAI1;3, or block GNAI2 and IL6, might be developed for the prevention or therapy of CAC in patients.

Insights into pathogenesis of five novel GCK mutations identified in Chinese MODY patients.

OBJECTIVE: Heterozygous inactivating mutations in GCK are associated with defects in pancreatic insulin secretion and/or hepatic glycogen synthesis leading to mild chronic hyperglycaemia of maturity onset diabetes of young type 2 (MODY2). However, the effect of naturally occurring GCK mutations on the pathogenesis for MODY2 hyperglycaemia remains largely unclear, especially in the Asian population. The aim of this study is to explore the potential pathogenicity of novel GCK mutations related to MODY2. METHODS: Genetic screening for GCK mutations from 96 classical MODY families was performed, and structure-function characterization and clinical profile of identified GCK mutations were conducted. RESULTS: Five novel (F195S, I211T, V222D, E236G and K458R) and five known (T49N, I159V, R186X, A188T and M381T) mutations were identified and co-segregated with hyperglycaemia in their pedigrees. R186X generates non-functional truncated form and V222D and E236G fully inactivate glucokinase due to severe structure disruptions. The other seven GCK mutations exhibited marked reductions in catalytic efficiency and thermo-stability; notably, the interaction with GKRP was significantly enhanced in I211T, I159V, T49N and K458R, reduced in F195S and M381T, and completely lost with A188T. 31% (17/55) of MODY2 patients showed signs of insulin resistance. Conventional hypoglycaemia treatment did not improve the HbA1C in MODY2 patients when insulin resistance is not present. CONCLUSIONS: Five novel GCK mutations have been identified in Chinese MODY. The defects in enzymatic activity and protein stability, together with alteration of GKRP binding on GCK mutants may synergistically contribute to the development of MODY2 hyperglycaemia. No treatment should be prescribed to MODY2 patients when insulin resistance is not present.

KIF20A/MKLP2 regulates the division modes of neural progenitor cells during cortical development.

Balanced symmetric and asymmetric divisions of neural progenitor cells (NPCs) are crucial for brain development, but the underlying mechanisms are not fully understood. Here we report that mitotic kinesin KIF20A/MKLP2 interacts with RGS3 and plays a crucial role in controlling the division modes of NPCs during cortical neurogenesis. Knockdown of KIF20A in NPCs causes dislocation of RGS3 from the intercellular bridge (ICB), impairs the function of Ephrin-B-RGS cell fate signaling complex, and leads to a transition from proliferative to differentiative divisions. Germline and inducible knockout of KIF20A causes a loss of progenitor cells and neurons and results in thinner cortex and ventriculomegaly. Interestingly, loss of function of KIF20A induces early cell cycle exit and precocious neuronal differentiation without causing substantial cytokinesis defect or apoptosis. Our results identify a RGS-KIF20A axis in the regulation of cell division and suggest a potential link of the ICB to regulation of cell fate determination.

Enhanced hexose-6-phosphate dehydrogenase expression in adipose tissue may contribute to diet-induced visceral adiposity.

BACKGROUND: Visceral fat accumulation increases the risk of developing type 2 diabetes and metabolic syndrome, and is associated with excessive glucocorticoids (GCs). Fat depot-specific GC action is tightly controlled by 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) coupled with the enzyme hexose-6-phosphate dehydrogenase (H6PDH). Mice with inactivation or activation of H6PDH genes show altered adipose 11ß-HSD1 activity and lipid storage. We hypothesized that adipose tissue H6PDH activation is a leading cause for the visceral obesity and insulin resistance. Here, we explored the role and possible mechanism of enhancing adipose H6PDH in the development of visceral adiposity in vivo. METHODS: We investigated the potential contribution of adipose H6PDH activation to the accumulation of visceral fat by characterization of visceral fat obese gene expression profiles, fat distribution, adipocyte metabolic molecules, and abdominal fat-specific GC signaling mechanisms underlying the diet-induced visceral obesity and insulin resistance in H6PDH transgenic mice fed a standard of high-fat diet (HFD). RESULTS: Transgenic H6PDH mice display increased abdominal fat accumulation, which is paralleled by elevated lipid synthesis associated with induction of lipogenic transcriptor C/EBPα and PPARγ mRNA levels within adipose tissue. Transgenic H6PDH mice fed a high-fat diet (HFD) gained more abdominal visceral fat mass coupled with activation of GSK3ß and induction of XBP1/IRE1α, but reduced pThr308 Akt/PKB content and browning gene CD137 and GLUT4 mRNA levels within the visceral adipose tissue than WT controls. HFD-fed H6PDH transgenic mice also had impaired insulin sensitivity and exhibited elevated levels of intra-adipose GCs with induction of adipose 11ß-HSD1. CONCLUSION: These data provide the first in vivo mechanistic evidence for the adverse metabolic effects of adipose H6PDH activation on visceral fat distribution, fat metabolism, and adipocyte function through enhancing 11ß-HSD1-driven intra-adipose GC action.

A large Rab GTPase encoded by CRACR2A is a component of subsynaptic vesicles that transmit T cell activation signals.

More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed thatCRACR2A(Ca(2+) release-activated Ca(2+) channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca(2+) and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.

2-Hydroxyglutarate Inhibits ATP Synthase and mTOR Signaling.

We discovered recently that the central metabolite α-ketoglutarate (α-KG) extends the lifespan of C. elegans through inhibition of ATP synthase and TOR signaling. Here we find, unexpectedly, that (R)-2-hydroxyglutarate ((R)-2HG), an oncometabolite that interferes with various α-KG-mediated processes, similarly extends worm lifespan. (R)-2HG accumulates in human cancers carrying neomorphic mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes. We show that, like α-KG, both (R)-2HG and (S)-2HG bind and inhibit ATP synthase and inhibit mTOR signaling. These effects are mirrored in IDH1 mutant cells, suggesting a growth-suppressive function of (R)-2HG. Consistently, inhibition of ATP synthase by 2-HG or α-KG in glioblastoma cells is sufficient for growth arrest and tumor cell killing under conditions of glucose limitation, e.g., when ketone bodies (instead of glucose) are supplied for energy. These findings inform therapeutic strategies and open avenues for investigating the roles of 2-HG and metabolites in biology and disease.

Gαi1 and Gαi3 regulate macrophage polarization by forming a complex containing CD14 and Gab1.

Heterotrimeric G proteins have been implicated in Toll-like receptor 4 (TLR4) signaling in macrophages and endothelial cells. However, whether guanine nucleotide-binding protein G(i) subunit alpha-1 and alpha-3 (Gαi1/3) are required for LPS responses remains unclear, and if so, the underlying mechanisms need to be studied. In this study, we demonstrated that, in response to LPS, Gαi1/3 form complexes containing the pattern recognition receptor (PRR) CD14 and growth factor receptor binding 2 (Grb2)-associated binding protein (Gab1), which are required for activation of PI3K-Akt signaling. Gαi1/3 deficiency decreased LPS-induced TLR4 endocytosis, which was associated with decreased phosphorylation of IFN regulatory factor 3 (IRF3). Gαi1/3 knockdown in bone marrow-derived macrophage cells (Gαi1/3 KD BMDMs) exhibited an M2-like phenotype with significantly suppressed production of TNF-α, IL-6, IL-12, and NO in response to LPS. The altered polarization coincided with decreased Akt activation. Further, Gαi1/3 deficiency caused LPS tolerance in mice. In vitro studies revealed that, in LPS-tolerant macrophages, Gαi1/3 were down-regulated partially by the proteasome pathway. Collectively, the present findings demonstrated that Gαi1/3 can interact with CD14/Gab1, which modulates macrophage polarization in vitro and in vivo.

11ß-Hydroxysteroid dehydrogenase type 1 shRNA ameliorates glucocorticoid-induced insulin resistance and lipolysis in mouse abdominal adipose tissue.

Long-term glucocorticoid exposure increases the risk for developing type 2 diabetes. Prereceptor activation of glucocorticoid availability in target tissue by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) coupled with hexose-6-phosphate dehydrogenase (H6PDH) is an important mediator of the metabolic syndrome. We explored whether the tissue-specific modulation of 11ß-HSD1 and H6PDH in adipose tissue mediates glucocorticoid-induced insulin resistance and lipolysis and analyzed the effects of 11ß-HSD1 inhibition on the key lipid metabolism genes and insulin-signaling cascade. We observed that corticosterone (CORT) treatment increased expression of 11ß-HSD1 and H6PDH and induced lipase HSL and ATGL with suppression of p-Thr(172) AMPK in adipose tissue of C57BL/6J mice. In contrast, CORT induced adipose insulin resistance, as reflected by a marked decrease in IR and IRS-1 gene expression with a reduction in p-Thr(308) Akt/PKB. Furthermore, 11ß-HSD1 shRNA attenuated CORT-induced 11ß-HSD1 and lipase expression and improved insulin sensitivity with a concomitant stimulation of pThr(308) Akt/PKB and p-Thr(172) AMPK within adipose tissue. Addition of CORT to 3T3-L1 adipocytes enhanced 11ß-HSD1 and H6PDH and impaired p-Thr(308) Akt/PKB, leading to lipolysis. Knockdown of 11ß-HSD1 by shRNA attenuated CORT-induced lipolysis and reversed CORT-mediated inhibition of pThr(172) AMPK, which was accompanied by a parallel improvement of insulin signaling response in these cells. These findings suggest that elevated adipose 11ß-HSD1 expression may contribute to glucocorticoid-induced insulin resistance and adipolysis.

Expression of the HSF4 DNA binding domain-EGFP hybrid gene recreates early childhood lamellar cataract in transgenic mice.

PURPOSE: The clinical management of cataracts in infancy involves surgical removal of the lens to ensure transmission of light to the retina, which is essential for normal neural development of the infant. This surgery, however, entails a lifelong follow-up and impaired vision. To our knowledge, no animal models recapitulate human lamellar opacities, the most prevalent form of early childhood cataracts. We present data on the recreation of the human lamellar cataract phenotype in transgenic mice. METHODS: Mutations in the DNA binding domain (DBD) of the heat shock transcription factor 4 (HSF4) are known to be associated with early childhood autosomal dominant lamellar cataract. We used bacterial artificial chromosome (BAC) transgenesis to express a hybrid gene: Hsf4 (DBD)-enhanced green fluorescent protein (EGFP), by recombineering EGFP sequences into the DBD of the Hsf4 gene, to interfere with the DNA binding properties of Hsf4. RESULTS: We recapitulated the human lamellar cataract, in its temporal as well as spatial presentation, within the transgenic mouse lens. This phenotype was reproduced faithfully using four different BACs, indicating that EGFP can be used to target transcription factor function in transgenic mice. Molecular and cell biological examination of early postnatal transgenic lens reveals impairment of secondary fiber cell differentiation. CONCLUSIONS: Recreation of the human lamellar cataract phenotype in mice allows investigation of this human pathology at a level not possible previously and points to the relevance of fiber cell heterogeneity dictated by fiber cell-specific gene activity in the biogenesis of the lamellar cataract.

HSF4 mutation p.Arg116His found in age-related cataracts and in normal populations produces childhood lamellar cataract in transgenic mice.

The p.Arg116His mutation in the heat shock transcription factor-4 (HSF4) has been associated with age-related cataracts, but it is also seen in 2% of the normal population, indicating either reduced penetrance or that the normal subjects were not old enough to express the phenotype. Based on the proximity of p.Arg116His to two known mutations in the DNA-binding domain of HSF4, namely, p.Leu114Pro and p.Arg119Cys, which segregate with childhood lamellar cataract, we tested the possibility that this phenotype may have been missed by the ophthalmologist and/or that it did not spread to the visual axis so as to affect vision significantly. Here, we demonstrate via BAC (bacterial artificial chromosome) transgenesis that p.Arg116His recreates the childhood lamellar cataract in mice suggesting that incomplete penetrance associated with early cataracts may not be an absence but a limitation of the detection of the phenotype.

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit ß is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit ß and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

G(i)α proteins exhibit functional differences in the activation of ERK1/2, Akt and mTORC1 by growth factors in normal and breast cancer cells.

BACKGROUND: In a classic model, G(i)α proteins including G(i1)α, G(i2)α and G(i3)α are important for transducing signals from G(i)α protein-coupled receptors (G(i)αPCRs) to their downstream cascades in response to hormones and neurotransmitters. Our previous study has suggested that G(i1)α, G(i2)α and G(i3)α are also important for the activation of the PI3K/Akt/mTORC1 pathway by epidermal growth factor (EGF) and its family members. However, a genetic role of these G(i)α proteins in the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) by EGF is largely unknown. Further, it is not clear whether these G(i)α proteins are also engaged in the activation of both the Akt/mTORC1 and ERK1/2 pathways by other growth factor family members. Additionally, a role of these G(i)α proteins in breast cancer remains to be elucidated. RESULTS: We found that Gi1/3 deficient MEFs with the low expression level of G(i2)α showed defective ERK1/2 activation by EGFs, IGF-1 and insulin, and Akt and mTORC1 activation by EGFs and FGFs. Gi1/2/3 knockdown breast cancer cells exhibited a similar defect in the activations and a defect in in vitro growth and invasion. The G(i)α proteins associated with RTKs, Gab1, FRS2 and Shp2 in breast cancer cells and their ablation impaired Gab1's interactions with Shp2 in response to EGF and IGF-1, or with FRS2 and Grb2 in response to bFGF. CONCLUSIONS: G(i)α proteins differentially regulate the activation of Akt, mTORC1 and ERK1/2 by different families of growth factors. G(i)α proteins are important for breast cancer cell growth and invasion.

Transgenic overexpression of hexose-6-phosphate dehydrogenase in adipose tissue causes local glucocorticoid amplification and lipolysis in male mice.

The prereceptor activation of glucocorticoid production in adipose tissue by NADPH-dependent 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) has emerged as a potential mechanism in the pathogenesis of visceral obesity and metabolic syndrome. Hexose-6-phosphate dehydrogenase (H6PDH) is an endoplasmic reticulum lumen-resident enzyme that generates cofactor NADPH and thus mediates 11ß-HSD1 activity. To determine the role of adipose H6PDH in the prereceptor modulation of 11ß-HSD1 and metabolic phenotypes, we generated a transgenic (Tg) mouse model overexpressing H6PDH under the control of the enhancer-promoter region of the adipocyte fatty acid-binding protein (aP2) gene (aP2/H6PDH Tg mice). Transgenic aP2/H6PDH mice exhibited relatively high expression of H6PDH and elevated corticosterone production with induction of 11ß-HSD1 activity in adipose tissue. This increase in corticosterone production in aP2-H6PDH Tg mice resulted in mild abdominal fat accumulation with induction of C/EBP mRNA expression and slight weight gain. Transgenic aP2/H6PDH mice also exhibited fasting hyperglycemia and glucose intolerance with insulin resistance. In addition, the aP2/H6PDH Tg mice have elevated circulating free fatty acid levels with a concomitant increased adipose lipolytic action associated with elevated HSL mRNA and Ser(660) HSL phosphorylation within abdominal fat. These results suggest that increased H6PDH expression specifically in adipose tissue is sufficient to cause intra-adipose glucocorticoid production and adverse metabolic phenotypes. These findings suggest that the aP2/H6PDH Tg mice may provide a favorable model for studying the potential impact of H6PDH in the pathogenesis of human metabolic syndrome.

c-Met-dependent multipotent labyrinth trophoblast progenitors establish placental exchange interface.

The placenta provides the interface for gas and nutrient exchange between the mother and the fetus. Despite its critical function in sustaining pregnancy, the stem/progenitor cell hierarchy and molecular mechanisms responsible for the development of the placental exchange interface are poorly understood. We identified an Epcam(hi) labyrinth trophoblast progenitor (LaTP) in mouse placenta that at a clonal level generates all labyrinth trophoblast subtypes, syncytiotrophoblasts I and II, and sinusoidal trophoblast giant cells. Moreover, we discovered that hepatocyte growth factor/c-Met signaling is required for sustaining proliferation of LaTP during midgestation. Loss of trophoblast c-Met also disrupted terminal differentiation and polarization of syncytiotrophoblasts, leading to intrauterine fetal growth restriction, fetal liver hypocellularity, and demise. Identification of this c-Met-dependent multipotent LaTP provides a landmark in the poorly defined placental stem/progenitor cell hierarchy and may help us understand pregnancy complications caused by a defective placental exchange.