Fibroblastic reticular cells in lymph node potentiate white adipose tissue beiging through neuro-immune crosstalk in male mice.

Lymph nodes (LNs) are always embedded in the metabolically-active white adipose tissue (WAT), whereas their functional relationship remains obscure. Here, we identify fibroblastic reticular cells (FRCs) in inguinal LNs (iLNs) as a major source of IL-33 in mediating cold-induced beiging and thermogenesis of subcutaneous WAT (scWAT). Depletion of iLNs in male mice results in defective cold-induced beiging of scWAT. Mechanistically, cold-enhanced sympathetic outflow to iLNs activates ß1- and ß2-adrenergic receptor (AR) signaling in FRCs to facilitate IL-33 release into iLN-surrounding scWAT, where IL-33 activates type 2 immune response to potentiate biogenesis of beige adipocytes. Cold-induced beiging of scWAT is abrogated by selective ablation of IL-33 or ß1- and ß2-AR in FRCs, or sympathetic denervation of iLNs, whereas replenishment of IL-33 reverses the impaired cold-induced beiging in iLN-deficient mice. Taken together, our study uncovers an unexpected role of FRCs in iLNs in mediating neuro-immune interaction to maintain energy homeostasis.

ß-Klotho promotes glycolysis and glucose-stimulated insulin secretion via GP130.

Impaired glucose-stimulated insulin secretion (GSIS) is a hallmark of type-2 diabetes. However, cellular signaling machineries that control GSIS remain incompletely understood. Here, we report that ß-klotho (KLB), a single-pass transmembrane protein known as a co-receptor for fibroblast growth factor 21 (FGF21), fine tunes GSIS via modulation of glycolysis in pancreatic ß-cells independent of the actions of FGF21. ß-cell-specific deletion of Klb but not Fgf21 deletion causes defective GSIS and glucose intolerance in mice and defective GSIS in islets of type-2 diabetic mice is mitigated by adenovirus-mediated restoration of KLB. Mechanistically, KLB interacts with and stabilizes the cytokine receptor subunit GP130 by blockage of ubiquitin-dependent lysosomal degradation, thereby facilitating interleukin-6-evoked STAT3-HIF1α signaling, which in turn transactivates a cluster of glycolytic genes for adenosine triphosphate production and GSIS. The defective glycolysis and GSIS in Klb-deficient islets are rescued by adenovirus-mediated replenishment of STAT3 or HIF1α. Thus, KLB functions as a key cell-surface regulator of GSIS by coupling the GP130 receptor signaling to glucose catabolism in ß-cells and represents a promising therapeutic target for diabetes.

The adaptor protein APPL2 controls glucose-stimulated insulin secretion via F-actin remodeling in pancreatic ß-cells.

Filamentous actin (F-actin) cytoskeletal remodeling is critical for glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cells, and its dysregulation causes type 2 diabetes. The adaptor protein APPL1 promotes first-phase GSIS by up-regulating soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein expression. However, whether APPL2 (a close homology of APPL1 with the same domain organization) plays a role in ß-cell functions is unknown. Here, we show that APPL2 enhances GSIS by promoting F-actin remodeling via the small GTPase Rac1 in pancreatic ß-cells. ß-cell specific abrogation of APPL2 impaired GSIS, leading to glucose intolerance in mice. APPL2 deficiency largely abolished glucose-induced first- and second-phase insulin secretion in pancreatic islets. Real-time live-cell imaging and phalloidin staining revealed that APPL2 deficiency abolished glucose-induced F-actin depolymerization in pancreatic islets. Likewise, knockdown of APPL2 expression impaired glucose-stimulated F-actin depolymerization and subsequent insulin secretion in INS-1E cells, which were attributable to the impairment of Ras-related C3 botulinum toxin substrate 1 (Rac1) activation. Treatment with the F-actin depolymerization chemical compounds or overexpression of gelsolin (a F-actin remodeling protein) rescued APPL2 deficiency-induced defective GSIS. In addition, APPL2 interacted with Rac GTPase activating protein 1 (RacGAP1) in a glucose-dependent manner via the bin/amphiphysin/rvs-pleckstrin homology (BAR-PH) domain of APPL2 in INS-1E cells and HEK293 cells. Concomitant knockdown of RacGAP1 expression reverted APPL2 deficiency-induced defective GSIS, F-actin remodeling, and Rac1 activation in INS-1E cells. Our data indicate that APPL2 interacts with RacGAP1 and suppresses its negative action on Rac1 activity and F-actin depolymerization thereby enhancing GSIS in pancreatic ß-cells.

Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling-dependent manner.

Fibroblast growth factor 21 (FGF21) is important in glucose, lipid homeostasis and insulin sensitivity. However, it remains unknown whether FGF21 is involved in insulin expression and secretion that are dysregulated in type 2 diabetes mellitus (T2DM). In this study, we found that FGF21 was down-regulated in pancreatic islets of db/db mice, a mouse model of T2DM, along with decreased insulin expression, suggesting the possible involvement of FGF21 in maintaining insulin homeostasis and islet ß-cell function. Importantly, FGF21 knockout exacerbated palmitate-induced islet ß-cell failure and suppression of glucose-stimulated insulin secretion (GSIS). Pancreatic FGF21 overexpression significantly increased insulin expression, enhanced GSIS, improved islet morphology and reduced ß-cell apoptosis in db/db mice. Mechanistically, FGF21 promoted expression of insulin gene transcription factors and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, the major regulators of insulin secretion, as well as activating phosphatidylinositol 3-kinase (PI3K)/Akt signaling in islets of db/db mice. In addition, pharmaceutical inhibition of PI3K/Akt signaling effectively suppressed FGF21-induced expression of insulin gene transcription factors and SNARE proteins, suggesting an essential role of PI3K/Akt signaling in FGF21-induced insulin expression and secretion. Taken together, our results demonstrate a protective role of pancreatic FGF21 in T2DM mice through inducing PI3K/Akt signaling-dependent insulin expression and secretion.

Hypothalamic AMPK as a Mediator of Hormonal Regulation of Energy Balance.

As a cellular energy sensor and regulator, adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a pivotal role in the regulation of energy homeostasis in both the central nervous system (CNS) and peripheral organs. Activation of hypothalamic AMPK maintains energy balance by inducing appetite to increase food intake and diminishing adaptive thermogenesis in adipose tissues to reduce energy expenditure in response to food deprivation. Numerous metabolic hormones, such as leptin, adiponectin, ghrelin and insulin, exert their energy regulatory effects through hypothalamic AMPK via integration with the neural circuits. Although activation of AMPK in peripheral tissues is able to promote fatty acid oxidation and insulin sensitivity, its chronic activation in the hypothalamus causes obesity by inducing hyperphagia in both humans and rodents. In this review, we discuss the role of hypothalamic AMPK in mediating hormonal regulation of feeding and adaptive thermogenesis, and summarize the diverse underlying mechanisms by which central AMPK maintains energy homeostasis.

The Dysfunctional MDM2-p53 Axis in Adipocytes Contributes to Aging-Related Metabolic Complications by Induction of Lipodystrophy.

Profound loss and senescence of adipose tissues are hallmarks of advanced age, but the underlying cause and their metabolic consequences remain obscure. Proper function of the murine double minute 2 (MDM2)-p53 axis is known to prevent tumorigenesis and several metabolic diseases, yet its role in regulation of adipose tissue aging is still poorly understood. In this study, we show that the proximal p53 inhibitor MDM2 is markedly downregulated in subcutaneous white and brown adipose tissues of mice during aging. Genetic disruption of MDM2 in adipocytes triggers canonical p53-mediated apoptotic and senescent programs, leading to age-dependent lipodystrophy and its associated metabolic disorders, including type 2 diabetes, nonalcoholic fatty liver disease, hyperlipidemia, and energy imbalance. Surprisingly, this lipodystrophy mouse model also displays premature loss of physiological integrity, including impaired exercise capacity, multiple organ senescence, and shorter life span. Transplantation of subcutaneous fat rejuvenates the metabolic health of this aging-like lipodystrophy mouse model. Furthermore, senescence-associated secretory factors from MDM2-null adipocytes impede adipocyte progenitor differentiation via a non-cell-autonomous manner. Our findings suggest that tight regulation of the MDM2-p53 axis in adipocytes is required for adipose tissue dynamics and metabolic health during the aging process.

FGF21 Prevents Angiotensin II-Induced Hypertension and Vascular Dysfunction by Activation of ACE2/Angiotensin-(1-7) Axis in Mice.

Fibroblast growth factor 21 (FGF21) is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. However, the role of FGF21 in hypertension remains elusive. Here we show that FGF21 deficiency significantly exacerbates angiotensin II-induced hypertension and vascular dysfunction, whereas such negative effects are reversed by replenishment of FGF21. Mechanistically, FGF21 acts on adipocytes and renal cells to promote induction of angiotensin-converting enzyme 2 (ACE2), which in turn converts angiotensin II to angiotensin-(1-7), then inhibits hypertension and reverses vascular damage. In addition, ACE2 deficiency strikingly abrogates these beneficial effects of FGF21 in mice, including alleviation of angiotensin II-associated hypertension and vascular damage. Otherwise, pharmaceutical inhibition of angiotensin-(1-7) attenuates the protective effect of FGF21 on angiotensin II-induced vascular dysfunction, but not on hypertension. Thus, FGF21 protects against angiotensin II-induced hypertension and vascular impairment by activation of the ACE2/angiotensin-(1-7) axis via fine-tuning the multi-organ crosstalk between liver, adipose tissue, kidney, and blood vessels.

Integrated Metabolomics and Morphogenesis Reveal Volatile Signaling of the Nematode-Trapping Fungus Arthrobotrys oligospora.

The adjustment of metabolic patterns is fundamental to fungal biology and plays vital roles in adaptation to diverse ecological challenges. Nematode-trapping fungi can switch their lifestyle from saprophytic to pathogenic by developing specific trapping devices induced by nematodes to infect their prey as a response to nutrient depletion in nature. However, the chemical identity of the specific fungal metabolites used during the switch remains poorly understood. We hypothesized that these important signal molecules might be volatile in nature. Gas chromatography-mass spectrometry was used to carry out comparative analysis of fungal metabolomics during the saprophytic and pathogenic lifestyles of the model species Arthrobotrys oligospora Two media commonly used in research on this species, cornmeal agar (CMA) and potato dextrose agar (PDA), were chosen for use in this study. The fungus produced a small group of volatile furanone and pyrone metabolites that were associated with the switch from the saprophytic to the pathogenic stage. A. oligospora fungi grown on CMA tended to produce more traps and employ attractive furanones to improve the utilization of traps, while fungi grown on PDA developed fewer traps and used nematode-toxic furanone metabolites to compensate for insufficient traps. Another volatile pyrone metabolite, maltol, was identified as a morphological regulator for enhancing trap formation. Deletion of the gene AOL_s00079g496 in A. oligospora led to increased amounts of the furanone attractant (2-fold) in mutants and enhanced the attractive activity (1.5-fold) of the fungus, while it resulted in decreased trap formation. This investigation provides new insights regarding the comprehensive tactics of fungal adaptation to environmental stress, integrating both morphological and metabolomic mechanisms.IMPORTANCE Nematode-trapping fungi are a unique group of soil-living fungi that can switch from the saprophytic to the pathogenic lifestyle once they come into contact with nematodes as a response to nutrient depletion. In this study, we investigated the metabolic response during the switch and the key types of metabolites involved in the interaction between fungi and nematodes. Our findings indicate that A. oligospora develops multiple and flexible metabolic tactics corresponding to different morphological responses to nematodes. A. oligospora can use similar volatile furanone and pyrone metabolites with different ecological functions to help capture nematodes in the fungal switch from the saprophytic to the pathogenic lifestyle. Furthermore, studies with A. oligospora mutants with increased furanone and pyrone metabolites confirmed the results. This investigation reveals the importance of volatile signaling in the comprehensive tactics used by nematode-trapping fungi, integrating both morphological and metabolomic mechanisms.

Activation of hypothalamic RIP-Cre neurons promotes beiging of WAT via sympathetic nervous system.

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat-insulin-promoter-Cre (RIP-Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT Genetic ablation of APPL2 in RIP-Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP-Cre neurons, inactivation of VMH AMPK, or treatment with a ß3-adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP-Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP-Cre neurons, in which the APPL2-AMPK signaling axis is crucial for this defending mechanism to cold and obesity.

FGF21 ameliorates diabetic cardiomyopathy by activating the AMPK-paraoxonase 1 signaling axis in mice.

The aim of the present study is to explore the molecular mechanism of fibroblast growth factor 21 (FGF21) in protecting against diabetic cardiomyopathy (DCM). Streptozotocin/high-fat diet (STZ/HFD) was used to induced diabetes in FGF21-deficient mice and their wild-type littermates, followed by evaluation of the difference in DCM between the two genotypes. Primary cultured cardiomyocytes were also used to explore the potential molecular mechanism of FGF21 in the protection of high glucose (HG)-induced cardiomyocyte injury. STZ/HFD-induced cardiomyopathy was exacerbated in FGF21 knockout mice, which was accompanied by a significant reduction in cardiac AMP-activated protein kinase (AMPK) activity and paraoxonase 1 (PON1) expression. By contrast, adeno-associated virus (AAV)-mediated overexpression of FGF21 in STZ/HFD-induced diabetic mice significantly enhanced cardiac AMPK activity, PON1 expression and its biological activity, resulting in alleviated DCM. In cultured cardiomyocytes, treatment with recombinant mouse FGF21 (rmFGF21) counteracted HG-induced oxidative stress, mitochondrial dysfunction, and inflammatory responses, leading to increased AMPK activity and PON1 expression. However, these beneficial effects of FGF21 were markedly weakened by genetic blockage of AMPK or PON1. Furthermore, inactivation of AMPK also markedly blunted FGF21-induced PON1 expression but significantly increased HG-induced cytotoxicity in cardiomyocytes, the latter of which was largely reversed by adenovirus-mediated PON1 overexpression. These findings suggest that FGF21 ameliorates DCM in part by activation of the AMPK-PON1 axis.

The MDM2-p53-pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic ß-cells.

Mitochondrial metabolism is pivotal for glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cells. However, little is known about the molecular machinery that controls the homeostasis of intermediary metabolites in mitochondria. Here we show that the activation of p53 in ß-cells, by genetic deletion or pharmacological inhibition of its negative regulator MDM2, impairs GSIS, leading to glucose intolerance in mice. Mechanistically, p53 activation represses the expression of the mitochondrial enzyme pyruvate carboxylase (PC), resulting in diminished production of the TCA cycle intermediates oxaloacetate and NADPH, and impaired oxygen consumption. The defective GSIS and mitochondrial metabolism in MDM2-null islets can be rescued by restoring PC expression. Under diabetogenic conditions, MDM2 and p53 are upregulated, whereas PC is reduced in mouse ß-cells. Pharmacological inhibition of p53 alleviates defective GSIS in diabetic islets by restoring PC expression. Thus, the MDM2-p53-PC signalling axis links mitochondrial metabolism to insulin secretion and glucose homeostasis, and could represent a therapeutic target in diabetes.

High Trap Formation and Low Metabolite Production by Disruption of the Polyketide Synthase Gene Involved in the Biosynthesis of Arthrosporols from Nematode-Trapping Fungus Arthrobotrys oligospora.

A group of morphology regulatory arthrosporol metabolites have been recently characterized from carnivorous fungus Arthrobotrys oligospora that can develop trapping networks to capture their prey. A combination of genetic manipulation and chemical analyses was applied to characterize the function of one polyketide synthase (PKS) gene AOL_s00215g283 in A. oligospora, which was putatively involved in the production of 6-methylsalicylic acid. High-performance liquid chromatography analysis showed that the disruption of the PKS gene not only led to the total loss of the arthrosporol A but also resulted in significant reduction in the production of secondary metabolites in the cultural broth of the mutant ΔAOL_s00215g283 strain. Interestingly, the mutant strain displayed significant increases in the trap formation and the nematicidal activity by 10 and 2 times, respectively, higher than the wild-type strain. These findings revealed a pathogenicity-related biosynthetic gene of this agriculturally important biological agent and have implications for establishment of efficient fungal biocontrol agents.

Yellow Pigment Aurovertins Mediate Interactions between the Pathogenic Fungus Pochonia chlamydosporia and Its Nematode Host.

Nematophagous fungi are globally distributed soil fungi and well-known natural predators of soil-dwelling nematodes. Pochonia chlamydosporia can be found in diverse nematode-suppressive soils as a parasite of nematode eggs and is one of the most studied potential biological control agents of nematodes. However, little is known about the functions of small molecules in the process of infection of nematodes by this parasitic fungus or about small-molecule-mediated interactions between the pathogenic fungus and its host. Our recent study demonstrated that a P. chlamydosporia strain isolated from root knots of tobacco infected by the root-knot nematode Meloidogyne incognita produced a class of yellow pigment metabolite aurovertins, which induced the death of the free-living nematode Panagrellus redivevus. Here we report that nematicidal P. chlamydosporia strains obtained from the nematode worms tended to yield a total yellow pigment aurovertin production exceeding the inhibitory concentration shown in nematicidal bioassays. Aurovertin D was abundant in the pigment metabolites of P. chlamydosporia strains. Aurovertin D showed strong toxicity toward the root-knot nematode M. incognita and exerted profound and detrimental effects on the viability of Caenorhabditis elegans even at a subinhibitory concentration. Evaluation of the nematode mutation in the ß subunit of F1-ATPase, together with the application of RNA interference in screening each subunit of F1FO-ATPase in the nematode worms, demonstrated that the ß subunit of F1-ATPase might not be the specific target for aurovertins in nematodes. The resistance of C. elegans daf-2(e1370) and the hypersensitivity of C. elegans daf-16(mu86) to aurovertin D indicated that DAF-16/FOXO transcription factor in nematodes was triggered in response to the aurovertin attack. These findings advance our understanding of the roles of aurovertin production in the interactions between nematodes and the pathogen fungus P. chlamydosporia.

Characterization of thermolide biosynthetic genes and a new thermolide from sister thermophilic fungi.

Prior chemical analysis of obligate thermophilic fungus Talaromyces thermophilus led to the discovery of thermolides A-F, six previously undescribed members of the lactam-bearing macrolactone class. A combination of chemical screening, genome analyses, and genetic manipulation led to the identification of the thermolide biosynthetic genes from sister thermophilic fungi T. thermophilus and Thermomyces lanuginosus and a new thermolide. The biosynthetic locus for the thermolides' mixed polyketide/amino acid structure encodes a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS). Our results reveal the first fungal hybrid iterative PKS-NRPS genes involved in the biosynthesis of bacterial-like hybrid macrolactones instead of typical fungal tetramic acids-containing metabolites. The finding provides an insight into the convergent biosynthetic end products that bridge the gap between the modular and iterative PKS-NRPS hybrids.

The adaptor protein APPL2 inhibits insulin-stimulated glucose uptake by interacting with TBC1D1 in skeletal muscle.

Insulin stimulates glucose uptake by promoting the trafficking of GLUT4 to the plasma membrane in muscle cells, and impairment of this insulin action contributes to hyperglycemia in type 2 diabetes. The adaptor protein APPL1 potentiates insulin-stimulated Akt activation and downstream actions. However, the physiological functions of APPL2, a close homolog of APPL1, in regulating glucose metabolism remain elusive. We show that insulin-evoked plasma membrane recruitment of GLUT4 and glucose uptake are impaired by APPL2 overexpression but enhanced by APPL2 knockdown. Likewise, conditional deletion of APPL2 in skeletal muscles enhances insulin sensitivity, leading to an improvement in glucose tolerance. We identified the Rab-GTPase-activating protein TBC1D1 as an interacting partner of APPL2. Insulin stimulates TBC1D1 phosphorylation on serine 235, leading to enhanced interaction with the BAR domain of APPL2, which in turn suppresses insulin-evoked TBC1D1 phosphorylation on threonine 596 in cultured myotubes and skeletal muscle. Substitution of serine 235 with alanine diminishes APPL2-mediated inhibition on insulin-dependent TBC1D1 phosphorylation on threonine 596 and the suppressive effects of TBC1D1 on insulin-induced glucose uptake and GLUT4 translocation to the plasma membrane in cultured myotubes. Therefore, the APPL2-TBC1D1 interaction is a key step to fine tune insulin-stimulated glucose uptake by regulating the membrane recruitment of GLUT4 in skeletal muscle.

Signaling mechanisms underlying the insulin-sensitizing effects of adiponectin.

Adiponectin is an insulin-sensitizing adipokine with protective effects against a cluster of obesity-related metabolic and cardiovascular disorders. The adipokine exerts its insulin-sensitizing effects by alleviation of obesity-induced ectopic lipid accumulation, lipotoxicity and chronic inflammation, as well as by direct cross-talk with insulin signaling cascades. Adiponectin and insulin signaling pathways converge at the adaptor protein APPL1. On the one hand, APPL1 interacts with adiponectin receptors and mediates both metabolic and vascular actions of adiponectin through activation of AMP-activated protein kinase and p38 MAP kinase. On the other hand, APPL1 potentiates both the actions and secretion of insulin by fine-tuning the Akt activity in multiple insulin target tissues. In obese animals, reduced APPL1 expression contributes to both insulin resistance and defective insulin secretion. This review summarizes recent advances on the molecular mechanisms by which adiponectin sensitizes insulin actions, and discusses the roles of APPL1 in regulating both adiponectin and insulin signaling cascades.

TRAF6-mediated ubiquitination of APPL1 enhances hepatic actions of insulin by promoting the membrane translocation of Akt.

Insulin inhibits hepatic glucose production through activation of the protein kinase Akt, and any defect in this pathway causes fasting hyperglycaemia in Type 2 diabetes. APPL1 [adaptor protein, phosphotyrosine interaction, PH (pleckstrin homology) domain and leucine zipper containing 1] sensitizes hepatic insulin action on suppression of gluconeogenesis by binding to Akt. However, the mechanisms underlying the insulin-sensitizing actions of APPL1 remain elusive. In the present study we show that insulin induces Lys63-linked ubiquitination of APPL1 in primary hepatocytes and in the livers of C57 mice. Lys160 located within the BAR (Bin/amphiphysin/Rvs) domain of APPL1 is the major site for its ubiquitination. Replacement of Lys160 with arginine abolishes insulin-dependent ubiquitination and membrane localization of APPL1, and also diminishes membrane recruitment and activation of Akt, thereby abrogating the effects of APPL1 on alleviation of hepatic insulin resistance and glucose intolerance in obese mice. Further analysis identified TRAF6 (tumour-necrosis-factor-receptor-associated factor 6) as an E3 ubiquitin ligase for APPL1 ubiquitination. Suppression of TRAF6 expression attenuates insulin-mediated ubiquitination and membrane targeting of APPL1, leading to an impairment of insulin-stimulated Akt activation and inhibition of gluconeogenesis in hepatocytes. Thus TRAF6-mediated ubiquitination of APPL1 is a vital step for the hepatic actions of insulin through modulation of membrane trafficking and activity of Akt.