Volume 66 | December 2022

Cellular senescence is a cell fate involving cell cycle arrest, resistance against apoptosis, and the development of a secretome that can be pro-inflammatory. In aging and obesity, senescent cells accumulate in many tissues, including adipose tissue, brain, kidney, pancreas, and liver. These senescent cells and their downstream effects appear to perpetuate inflammation and have been implicated in the pathogenesis of metabolic dysfunction. Senescent cells are cleared in part by the immune system, a process that is diminished in obesity and aging, likely due in part to senescence of immune cells themselves. Targeting senescent cells or their products improves metabolic function in both aging and in animal models of obesity. Novel therapeutics to target senescent cells are on the horizon and are currently being investigated in clinical trials in humans for multiple diseases. Early evidence suggests that senolytic drugs, which transiently disarm the anti-apoptotic defenses of pro-inflammatory senescent cells, are effective in causing depletion of senescent cells in humans. Senescence-targeting therapeutics, including senolytic drugs and strategies to increase immune clearance of senescent cells, hold significant promise for treating metabolic dysfunction in multiple tissues and disease states.

Background

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic insulin-producing β cells are specifically destroyed by the immune system. Understanding the initiation and progression of human T1D has been hampered by the lack of appropriate models that can reproduce the complexity and heterogeneity of the disease. The development of platforms combining multiple human pluripotent stem cell (hPSC) derived tissues to model distinct aspects of T1D has the potential to provide critical novel insights into the etiology and pathogenesis of the human disease.

Scope of review

In this review, we summarize the state of hPSC differentiation approaches to generate cell types and tissues relevant to T1D, with a particular focus on pancreatic islet cells, T cells, and thymic epithelium. We present current applications as well as limitations of using these hPSC-derived cells for disease modeling and discuss efforts to optimize platforms combining multiple cell types to model human T1D. Finally, we outline remaining challenges and emphasize future improvements needed to accelerate progress in this emerging field of research.

Major conclusions

Recent advances in reprogramming approaches to create patient-specific induced pluripotent stem cell lines (iPSCs), genome engineering technologies to efficiently modify DNA of hPSCs, and protocols to direct their differentiation into mature cell types have empowered the use of stem cell derivatives to accurately model human disease. While challenges remain before complex interactions occurring in human T1D can be modeled with these derivatives, experiments combining hPSC-derived β cells and immune cells are already providing exciting insight into how these cells interact in the context of T1D, supporting the viability of this approach.

Background

The dynamics of the cellular glycolysis pathway underpin cellular function and dysfunction, and therefore ultimately health, disease, diagnostic and therapeutic strategies. Evolving our understanding of this fundamental process and its dynamics remains critical.

Scope of review

This paper reviews the medical relevance of glycolytic pathway in depth and explores the current state of the art for monitoring and modelling the dynamics of the process. The future perspectives of label free, vibrational microspectroscopic techniques to overcome the limitations of the current approaches are considered.

Major conclusions

Vibrational microspectroscopic techniques can potentially operate in the niche area of limitations of other omics technologies for non-destructive, real-time, in vivolabel-free monitoring of glycolysis dynamics at a cellular and subcellular level.

Background

Adipose tissue macrophages (ATMs) are a well characterized regulator of adipose tissue inflammatory tone. Previously defined by the M1 vs M2 classification, we now have a better understanding of ATM diversity that departs from the old paradigm and reports a spectrum of ATM function and phenotypes in both brown and white adipose tissue.

Scope of Review

This review provides an updated overview of ATM activation and function, ATM diversity in humans and rodents, and novel ATM functions that contribute to metabolic homeostasis and disease.

Major Conclusions

While the paradigm that resident ATMs predominate in the lean state and obesity leads to the accumulation of lipid-associated and inflammatory ATMs still broadly remains rigorously supported, the details of this model continue to be refined and single cell data provide new insight into ATM subtypes and states.

Objective

Chronic liver diseases often involve metabolic damage to the skeletal system. The underlying mechanism of bone loss in chronic liver diseases remains unclear, and appropriate therapeutic options, except for orthotopic liver transplantation, have proved insufficient for these patients. This study aimed to investigate the efficacy and mechanism of transplantation of immature hepatocyte-like cells converted from stem cells from human exfoliated deciduous teeth (SHED-Heps) in bone loss of chronic liver fibrosis.

Methods

Mice that were chronically treated with CCl₄ received SHED-Heps, and trabecular bone density, reactive oxygen species (ROS), and osteoclast activity were subsequently analyzed in vivo and in vitro. The effects of stanniocalcin 1 (STC1) knockdown in SHED-Heps were also evaluated in chronically CCl₄ treated mice.

Results

SHED-Hep transplantation (SHED-HepTx) improved trabecular bone loss and liver fibrosis in chronic CCl₄-treated mice. SHED-HepTx reduced hepatic ROS production and interleukin 17 (Il-17) expression under chronic CCl₄ damage. SHED-HepTx reduced the expression of both Il-17 and tumor necrosis factor receptorsuperfamily 11A (Tnfrsf11a) and ameliorated the imbalance of osteoclast and osteoblast activities in the bone marrow of CCl₄-treated mice. Functional knockdown of STC1 in SHED-Heps attenuated the benefit of SHED-HepTx including anti-bone loss effect by suppressing osteoclast differentiation through TNFSF11–TNFRSF11A signaling and enhancing osteoblast differentiation in the bone marrow, as well as anti-fibrotic and anti-ROS effects in the CCl₄-injured livers.

Conclusions

These findings suggest that targeting hepatic ROS provides a novel approach to treat bone loss resulting from chronic liver diseases.

Objective

Perfluoroalkyl substances (PFAS) are man-made chemicals with demonstrated endocrine-disrupting properties. Exposure to perfluorooctanoic acid (PFOA) has been linked to disturbed metabolism via the liver, although the exact mechanism is not clear. Moreover, information on the metabolic effects of the new PFAS alternative GenX is limited. We examined whether exposure to low-dose PFOA and GenX induces metabolic disturbances in mice, including NAFLD, dyslipidemia, and glucose tolerance, and studied the involvement of PPARα.

Methods

Male C57BL/6J wildtype and PPARα^−/− mice were given 0.05 or 0.3 mg/kg body weight/day PFOA, or 0.3 mg/kg body weight/day GenX while being fed a high-fat diet for 20 weeks. Glucose and insulin tolerance tests were performed after 18 and 19 weeks. Plasma metabolite levels were measured next to a detailed assessment of the liver phenotype, including lipid content and RNA sequencing.

Results

Exposure to high-dose PFOA decreased body weight and increased liver weight in wildtype and PPARα^−/− mice. High-dose but not low-dose PFOA reduced plasma triglycerides and cholesterol, which for triglycerides was dependent on PPARα. PFOA and GenX increased hepatic triglycerides in a PPARα-dependent manner. RNA sequencing showed that the effects of GenX on hepatic gene expression were entirely dependent on PPARα, while the effects of PFOA were mostly dependent on PPARα. In the absence of PPARα, the involvement of PXR and CAR became more prominent.

Conclusion

Overall, we show that long-term and low-dose exposure to PFOA and GenX disrupts hepatic lipid metabolism in mice. Whereas the effects of PFOA are mediated by multiple nuclear receptors, the effects of GenX are entirely mediated by PPARα. Our data underscore the potential of PFAS to disrupt metabolism by altering signaling pathways in the liver.

Objective

Obesity-associated nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver failure and death. However, the pathogenesis of NAFLD and its severe form, nonalcoholic steatohepatitis (NASH), is poorly understood. The energy sensor, AMP-activated protein kinase (AMPK), has decreased activity in obesity and NAFLD, but the mechanisms are unclear. Here, we examined whether obesity-induced miR-802 has a role in promoting NASH by targeting AMPK. We also investigated whether miR-802 and AMPK have roles in modulating beneficial therapeutic effects mediated by obeticholic acid (OCA), a promising clinical agent for NASH.

Methods

Immunoblotting, luciferase assays, and RNA-protein interaction studies were performed to test whether miR-802 directly targets AMPK. The roles of miR-802 and AMPK in NASH were examined in mice fed a NASH-promoting diet.

Results

Hepatic miR-802 and AMPK levels were inversely correlated in both NAFLD patients and obese mice. MicroRNA in silico analysis, together with biochemical studies in hepatic cells, suggested that miR-802 inhibits hepatic expression of AMPK by binding to the 3’ untranslated regions of both human AMPKα1 and mouse Ampkβ1. In diet-induced NASH mice, OCA treatment reduced hepatic miR-802 levels and improved AMPK activity, ameliorating steatosis, inflammation, and apoptosis, but these OCA-mediated beneficial effects on NASH pathologies, particularly reducing apoptosis, were reversed by overexpression of miR-802 or downregulation of AMPK.

Conclusions

These results indicate that miR-802 inhibits AMPK by directly targeting Ampkβ1, promoting NAFLD/NASH in mice. The miR-802-AMPK axis that modulates OCA-mediated beneficial effects on NASH may represent a new therapeutic target.

Objective

Pancreatic islets of Langerhans secrete hormones to regulate systemic glucose levels. Emerging evidence suggests that islet cells are functionally heterogeneous to allow a fine-tuned and efficient endocrine response to physiological changes. A precise description of the molecular basis of this heterogeneity, in particular linking animal models to human islets, is an important step towards identifying the factors critical for endocrine cell function in physiological and pathophysiological conditions.

Methods

In this study, we used single-cell RNA sequencing to profile more than 50′000 endocrine cells isolated from healthy human, pig and mouse pancreatic islets and characterize transcriptional heterogeneity and evolutionary conservation of those cells across the three species. We systematically delineated endocrine cell types and α- and β-cell heterogeneity through prior knowledge- and data-driven gene sets shared across species, which altogether capture common and differential cellular properties, transcriptional dynamics and putative driving factors of state transitions.

Results

We showed that global endocrine expression profiles correlate, and that critical identity and functional markers are shared between species, while only approximately 20% of cell type enriched expression is conserved. We resolved distinct human α- and β-cell states that form continuous transcriptional landscapes. These states differentially activate maturation and hormone secretion programs, which are related to regulatory hormone receptor expression, signaling pathways and different types of cellular stress responses. Finally, we mapped mouse and pig cells to the human reference and observed that the spectrum of human α- and β-cell heterogeneity and aspects of such functional gene expression are better recapitulated in the pig than mouse data.

Conclusions

Here, we provide a high-resolution transcriptional map of healthy human islet cells and their murine and porcine counterparts, which is easily queryable via an online interface. This comprehensive resource informs future efforts that focus on pancreatic endocrine function, failure and regeneration, and enables to assess molecular conservation in islet biology across species for translational purposes.

Objective

Disturbances in NAD⁺ metabolism have been described as a hallmark for multiple metabolic and age-related diseases, including type 2 diabetes. While alterations in pancreatic β-cell function are critical determinants of whole-body glucose homeostasis, the role of NAD⁺ metabolism in the endocrine pancreas remains poorly explored. Here, we aimed to evaluate the role of nicotinamide riboside (NR) metabolism in maintaining NAD⁺ levels and pancreatic β-cell function in pathophysiological conditions.

Methods

Whole body and pancreatic β-cell-specific NRK1 knockout (KO) mice were metabolically phenotyped in situations of high-fat feeding and aging. We also analyzed pancreatic β-cell function, β-cell mass and gene expression.

Results

We first demonstrate that NRK1, the essential enzyme for the utilization of NR, is abundantly expressed in pancreatic β-cells. While NR treatment did not alter glucose-stimulated insulin secretion in pancreatic islets from young healthy mice, NRK1 knockout mice displayed glucose intolerance and compromised β-cells response to a glucose challenge upon high-fat feeding or aging. Interestingly, β cell dysfunction stemmed from the functional failure of other organs, such as liver and kidney, and the associated changes in circulating peptides and hormones, as mice lacking NRK1 exclusively in β-cells did not show altered glucose homeostasis.

Conclusions

This work unveils a new physiological role for NR metabolism in the maintenance of glucose tolerance and pancreatic β-cell function in high-fat feeding or aging conditions.

Objective

Adipose tissue is a very dynamic metabolic organ that plays an essential role in regulating whole-body glucose homeostasis. Dysfunctional adipose tissue hypertrophy with obesity is associated with fibrosis and type 2 diabetes. Yes-associated protein 1 (YAP) is a transcription cofactor important in the Hippo signaling pathway. However, the role of YAP in adipose tissue and glucose homeostasis is unknown.

Methods

To study the role of YAP with metabolic stress, we assessed how increased weight and insulin resistance impact YAP in humans and mouse models. To further investigate the in vivo role of YAP specifically in adipose tissue and glucose homeostasis, we developed adipose tissue-specific YAP knockout mice and placed them on either chow or high fat diet (HFD) for 12–14 weeks. To further study the direct role of YAP in adipocytes we used 3T3-L1 cells.

Results

We found that YAP protein levels increase in adipose tissue from humans with type 2 diabetes and mouse models of diet-induced obesity and insulin resistance. This suggests that YAP signaling may contribute to adipocyte dysfunction and insulin resistance under metabolic stress conditions. On an HFD, adipose tissue YAP knockout mice had improved glucose tolerance compared to littermate controls. Perigonadal fat pad weight was also decreased in knockout animals, with smaller adipocyte size. Adipose tissue fibrosis and gene expression associated with fibrosis was decreased in vivo and in vitro in 3T3-L1 cells treated with a YAP inhibitor or siRNA.

Conclusions

We show that YAP is increased in adipose tissue with weight gain and insulin resistance. Disruption of YAP in adipocytes prevents glucose intolerance and adipose tissue fibrosis, suggesting that YAP plays an important role in regulating adipose tissue and glucose homeostasis with metabolic stress.

Objective

Insulin-like peptide 5 (INSL5) signalling, through its cognate receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), has been reported to be orexigenic, and the high fat diet (HFD) preference observed in wildtype mice is altered in Rxfp4 knock-out mice. In this study, we used a new Rxfp4-Cre mouse model to investigate the mechanisms underlying these observations.

Methods

We generated transgenic Rxfp4-Cre mice and investigated central expression of Rxfp4 by RT-qPCR, RNAscope and intraparenchymal infusion of INSL5. Rxfp4-expressing cells were chemogenetically manipulated in global Cre-reporter mice using designer receptors exclusively activated by designer drugs (DREADDs) or after stereotactic injection of a Cre-dependent AAV-DIO-Dq-DREADD targeting a population located in the ventromedial hypothalamus (RXFP4^VMH). Food intake and feeding motivation were assessed in the presence and absence of a DREADD agonist. Rxfp4-expressing cells in the hypothalamus were characterised by single-cell RNA-sequencing (scRNAseq) and the connectivity of RXFP4^VMH cells was investigated using viral tracing.

Results

Rxfp4-Cre mice displayed Cre-reporter expression in the hypothalamus. Active expression of Rxfp4 in the adult mouse brain was confirmed by RT-qPCR and RNAscope. Functional receptor expression was supported by cyclic AMP-responses to INSL5 application in ex vivo brain slices and increased HFD and highly palatable liquid meal (HPM), but not chow, intake after intra-VMH INSL5 infusion. scRNAseq of hypothalamic RXFP4 neurons defined a cluster expressing VMH markers, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4^VMH neural projections to nuclei implicated in hedonic feeding behaviour. Whole body chemogenetic inhibition (Di-DREADD) of Rxfp4-expressing cells, mimicking physiological INSL5-RXFP4 Gi-signalling, increased intake of the HFD and HPM, but not chow, whilst activation (Dq-DREADD), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and motivation to work for the HPM.

Conclusion

These findings identify RXFP4^VMH neurons as regulators of food intake and preference, and hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.

Objective

Metabolomics as an approach to solve biological problems is exponentially increasing in use. Thus, this a pivotal time for the adoption of best practices. It is well known that disrupted tissue oxygen supply rapidly alters cellular energy charge. However, the speed and extent to which delayed mouse tissue freezing after dissection alters the broad metabolome is not well described. Furthermore, how tissue genotype may modulate such metabolomic drift and the degree to which traced ¹³C-isotopologue distributions may change have not been addressed.

Methods

By combined liquid chromatography (LC)- and gas chromatography (GC)-mass spectrometry (MS), we measured how levels of 255 mouse liver metabolites changed following 30-second, 1-minute, 3-minute, and 10-minute freezing delays. We then performed test-of-concept delay-to-freeze experiments evaluating broad metabolomic drift in mouse heart and skeletal muscle, differential metabolomic change between wildtype (WT) and mitochondrial pyruvate carrier (MPC) knockout mouse livers, and shifts in ¹³C-isotopologue abundances and enrichments traced from ¹³C-labled glucose into mouse liver.

Results

Our data demonstrate that delayed mouse tissue freezing after dissection leads to rapid hypoxia-driven remodeling of the broad metabolome, induction of both false-negative and false-positive between-genotype differences, and restructuring of ¹³C-isotopologue distributions. Notably, we show that increased purine nucleotidedegradation products are an especially high dynamic range marker of delayed liver and heart freezing.

Conclusions

Our findings provide a previously absent, systematic illustration of the extensive, multi-domain metabolomic changes occurring within the early minutes of delayed tissue freezing. They also provide a novel, detailed resource of mouse liver ex vivo, hypoxic metabolomic remodeling.

Objective

Glycerol-3-phosphate (Gro3P) phosphatase (G3PP) hydrolyzes Gro3P to glycerol that exits the cell, thereby operating a “glycerol shunt”, a metabolic pathway that we identified recently in mammalian cells. We have investigated the role of G3PP and the glycerol shunt in the regulation of glucose metabolism and lipogenesis in mouse liver.

Methods

We generated hepatocyte-specific G3PP-KO mice (LKO), by injecting AAV8-TBG-iCre to male G3PP^fl/fl mice. Controls received AAV8-TBG-eGFP. Both groups were fed chow diet for 10 weeks. Hyperglycemia (16–20 mM) was induced by glucose infusion for 55 h. Hepatocytes were isolated from normoglycemic mice for ex vivostudies and targeted metabolomics were measured in mice liver after glucose infusion.

Results

LKO mice showed no change in body weight, food intake, fed and fasted glycemia but had increased fed plasma triglycerides. Hepatic glucose production from glycerol was increased in fasted LKO mice. LKO mouse hepatocytes displayed reduced glycerol production, elevated triglyceride and lactate production at high glucose concentration. Hyperglycemia in LKO mice led to increased liver weight and accumulation of triglycerides, glycogen and cholesterol together with elevated levels of Gro3P, dihydroxyacetone phosphate, acetyl-CoA and some Krebs cycle intermediates in liver. Hyperglycemic LKO mouse liver showed elevated expression of proinflammatory cytokines and M1-macrophage markers accompanied by increased plasma triglycerides, LDL/VLDL, urea and uric acid and myocardial triglycerides.

Conclusions

The glycerol shunt orchestrated by G3PP acts as a glucose excess detoxificationpathway in hepatocytes by preventing metabolic disturbances that contribute to enhanced liver fat, glycogen storage, inflammation and lipid build-up in the heart. We propose G3PP as a novel therapeutic target for hepatic disorders linked to nutrient excess.

Objective

Branched chain amino acid (BCAA) catabolic defects are implicated to be causal determinates of multiple diseases. This work aimed to better understand how enhancing BCAA catabolism affected metabolic homeostasis as well as the mechanisms underlying these improvements.

Methods

The rate limiting step of BCAA catabolism is the irreversible decarboxylation by the branched chain ketoacid dehydrogenase (BCKDH) enzyme complex, which is post-translationally controlled through phosphorylation by BCKDH kinase (BDK). This study utilized BT2, a small molecule allosteric inhibitor of BDK, in multiple mouse models of metabolic dysfunction and NAFLD including the high fat diet (HFD) model with acute and chronic treatment paradigms, the choline deficient and methionine minimal high fat diet (CDAHFD) model, and the low-density lipoprotein receptor null mouse model (Ldlr^−/−). shRNA was additionally used to knock down BDK in liver to elucidate liver-specific effects of BDK inhibition in HFD-fed mice.

Results

A rapid improvement in insulin sensitivity was observed in HFD-fed and lean mice after BT2 treatment. Resistance to steatosis was assessed in HFD-fed mice, CDAHFD-fed mice, and Ldlr^−/− mice. In all cases, BT2 treatment reduced steatosis and/or inflammation. Fasting and refeeding demonstrated a lack of response to feeding-induced changes in plasma metabolites including insulin and beta-hydroxybutyrate and hepatic gene changes in BT2-treated mice. Mechanistically, BT2 treatment acutely altered the expression of genes involved in fatty acid oxidation and lipogenesis in liver, and upstream regulator analysis suggested that BT2 treatment activated PPARα. However, BT2 did not directly activate PPARα in vitro. Conversely, shRNA-AAV-mediated knockdown of BDK specifically in liver in vivo did not demonstrate any effects on glycemia, steatosis, or PPARα-mediated gene expression in mice.

Conclusions

These data suggest that BT2 treatment acutely improves metabolism and liver steatosis in multiple mouse models. While many molecular changes occur in liver in BT2-treated mice, these changes were not observed in mice with AAV-mediated shRNA knockdown of BDK. All together, these data suggest that systemic BDK inhibition is required to improve metabolism and steatosis by prolonging a fasting signature in a paracrine manner. Therefore, BCAA may act as a “fed signal” to promote nutrient storage and reduced systemic BCAA levels as shown in this study via BDK inhibition may act as a “fasting signal” to prolong the catabolic state.

Objective

Adipose tissue is the largest endocrine organ. When activated by cancer cells, adipocytes secrete adipocytokines and release fatty acids, which are then transferred to cancer cells and used for structural and biochemical support. How this metabolic symbiosis between cancer cells and adipocytes affects skeletal muscle and thermogenesis during cancer cachexia is unknown. Cancer cachexia is a multiorgan syndrome and how the communication between tissues is established has yet to be determined. We investigated adipose tissue secretory factors and explored their role in crosstalk of adipocytes, muscle, and tumor during pancreatic cancer cachexia.

Methods

We used a pancreatic cancer cachexia mouse model generated by syngenic implantation of pancreatic ductal adenocarcinoma (PDAC) cells (KPC) intraperitoneally into C57BL/6 mice and Lcn2-knockout mice. For in vitro studies, adipocytes (3T3-L1 and primary adipocytes), cachectic cancer cells (Panc0203), non-cachectic cancer cells (Du145 cells), and skeletal muscle cells (C2C12 myoblasts) were used.

Results

To identify molecules involved in the crosstalk of adipose tissue with muscle and tumors, we treated 3T3-L1 adipocytes with conditioned medium (CM) from cancer cells. Upon screening the secretomes from PDAC-induced adipocytes, several adipocytokines were identified, including lipocalin 2 (Lcn2). We investigated Lcn2 as a potential mediator of cachexia induced by adipocytes in response to PDAC. During tumor progression, mice exhibited a decline in body weight gain, which was accompanied by loss of adipose and muscle tissues. Tumor-harboring mice developed drastic hypothermia because of a dramatic loss of fat in brown adipose tissue (BAT) and suppression of the thermogenesis pathway. We inhibited Lcn2 with an anti-Lcn2 antibody neutralization or genomic ablation in mice. Lcn2 deficiency significantly improved body temperature in tumor-bearing mice, which was supported by the increased expression of Ucp1 and β3-adrenergic receptor in BAT. In addition, Lcn2 inhibition abrogated the loss of fat and muscle in tumor-bearing mice. In contrast to tumor-bearing WT mice, the corresponding Lcn2-knockout mice showed reduced ATGL expression in iWAT and decreased the expression of muscle atrophy molecular markers MuRF-1 and Fbx32.

Conclusions

This study showed that Lcn2 is causally involved in the dysregulation of adipose tissue–muscle-tumor crosstalk during pancreatic cancer cachexia. Therapeutic targets that suppress Lcn2 may minimize the progression of cachexia.

Objective

Exercise enhances the sensitivity of mammalian target of rapamycin complex 1 (mTORC1) to amino acids, in particular leucine. How long this enhanced sensitivity lasts, and which mechanisms control enhanced leucine-mediated mTORC1 activation following exercise is currently unknown.

Methods

C57BL/6J mice were exercised for one night in a resistance-braked running wheel after a 12-day acclimatization period. Mice were gavaged with a submaximal dose of l-leucine or saline acutely or 48 h after exercise cessation, following 3 h food withdrawal. Muscles were excised 30 min after leucine administration. To study the contribution of mTORC1, we repeated those experiments but blocked mTORC1 activation using rapamycin immediately before the overnight running bout and one hour before the first dose of leucine. mTORC1 signaling, muscle protein synthesis and amino acid sensing machinery were assessed using immunoblot and qPCR. Leucine uptake was measured using L-[¹⁴C(U)]-leucine tracer labeling.

Results

When compared to sedentary conditions, leucine supplementation more potently activated mTORC1 and protein synthesis in acutely exercised muscle. This effect was observed in m. soleus but not in m. tibialis anterior nor m. plantaris. The synergistic effect in m. soleus was long-lasting as key downstream markers of mTORC1 as well as protein synthesis remained higher when leucine was administered 48 h after exercise. We found that exercise enhanced the expression of amino acid transporters and promoted uptake of leucine into the muscle, leading to higher free intramuscular leucine levels. This coincided with increased expression of activating transcription factor 4 (ATF4), a main transcriptional regulator of amino acid uptake and metabolism, and downstream activation of amino acid genes as well as leucyl-tRNA synthetase (LARS), a putative leucine sensor. Finally, blocking mTORC1 using rapamycin did not reduce expression and activation of ATF4, suggesting that the latter does not act downstream of mTORC1. Rather, we found a robust increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, suggesting that the integrated stress response pathway, rather than exercise-induced mTORC1 activation, drives long-term ATF4 expression in skeletal muscle after exercise.

Conclusions

The enhanced sensitivity of mTORC1 to leucine is maintained at least 48 h after exercise. This shows that the anabolic window of opportunity for protein ingestionis not restricted to the first hours immediately following exercise. Increased mTORC1 sensitivity to leucine coincided with enhanced leucine influx into muscle and higher expression of genes involved in leucine sensing and amino acid metabolism. Also, exercise induced an increase in ATF4 protein expression. Altogether, these data suggest that muscular contractions switch on a coordinated program to enhance amino acid uptake as well as intramuscular sensing of key amino acids involved in mTORC1 activation and the stimulation of muscle protein synthesis.

Objective

The Allan-Herndon-Dudley syndrome (AHDS) is a severe disease caused by dysfunctional central thyroid hormone transport due to functional loss of the monocarboxylate transporter 8 (MCT8). In this study, we assessed whether mice with concomitant deletion of the thyroid hormone transporters Mct8 and the organic anion transporting polypeptide (Oatp1c1) represent a valid preclinical model organism for the AHDS.

Methods

We generated and metabolically characterized a new CRISPR/Cas9 generated Mct8/Oatp1c1 double-knockout (dKO) mouse line for the clinical features observed in patients with AHDS.

Results

We show that Mct8/Oatp1c1 dKO mice mimic key hallmarks of the AHDS, including decreased life expectancy, central hypothyroidism, peripheral hyperthyroidism, impaired neuronal myelination, impaired motor abilities and enhanced peripheral thyroid hormone action in the liver, adipose tissue, skeletal muscle and bone.

Conclusions

We conclude that Mct8/Oatp1c1 dKO mice are a valuable model organism for the preclinical evaluation of drugs designed to treat the AHDS.

Objectives

Mesenchymal stromal/stem cell (MSC)-based therapy has become a promising approach to periodontal tissue repair. Adipose-derived stromal/stem cells (ASCs), compared with other dental or non-dental MSCs, serve as promising candidates for MSC therapy due to non-invasive acquisition and abundant sources. This study aimed to explore the effects of ASC therapy in experimental periodontitis and the underlying mechanism.

Methods

Micro-CT was performed to evaluate the alveolar bone parameters following local injection of ASCs. Immunohistochemistry and immunofluorescence were employed to detect the expression of IL-1β, osteocalcin (OCN), nuclear factor (erythroid-derived 2)-like 2 (NRF2), and surface markers of macrophage polarization. Afterward, multiple reaction monitoring (MRM)-based targeted tryptophanmetabolomic analysis was used to examine the ASC metabolites. Chromatin immunoprecipitation (ChIP)-qPCR assay was performed to investigate the direct binding of aryl hydrocarbon receptor (AhR) and NRF2.

Results

Alveolar bone loss was reduced, and the ratio of iNOS⁺/CD206⁺ macrophages was significantly decreased after ASC injection in the rat models of periodontitis. ASCs promoted NRF2 expression and activation in macrophages, while NRF2 silencing in macrophages blocked the regulation of ASCs on macrophages. Furthermore, the expression of indoleamine 2,3-dioxygenase (IDO) of ASCs in the inflammatory condition was high. The inhibitor of IDO, 1-methyltryptophan (1-MT), impaired the therapeutic effects of ASCs in experimental periodontitis and regulation of macrophage polarization. Mechanistically, kynurenine (Kyn), a metabolite of ASCs catalyzed by IDO, activated AhR and enhanced its binding to the promoter of NRF2, which stimulated M2 macrophage polarization.

Conclusions

These findings suggested that ASCs can alleviate ligature-induced periodontitis through modulating macrophage polarization by the IDO-dependent Kyn-AhR-NRF2 pathway, uncovering a novel mechanism and providing a scientific basis for ASC-based therapy in experimental periodontitis.

Objective

Adipose tissue thermogenesis has been suggested as a new therapeutic target to promote energy metabolism for obesity and metabolic disease. Cold-inducible thermogenic adipocytes, called beige adipocytes, have attracted significant attention for their potent anti-obesity activity in adult humans. In this study, we identified the mechanisms underlying beige adipocyte recruitment, so-called adipocyte browning, by different stimuli.

Methods

We generated a new adipocyte cell line with enhanced browning potentials and determined its transcriptomic and epigenomic responses following cAMP (forskolin, FSK) versus PPARγ activation (rosiglitazone). We performed time-course RNA-seq and compared the treatments and in vivo adipocyte browning. We also developed an improved protocol for Assay for Transposase Accessible Chromatin-sequencing (ATAC-seq) and defined changes in chromatin accessibility in a time course. The RNA-seq and ATAC-seq data were integrated to determine the kinetics of their coordinated regulation and to identify a transcription factor that drives these processes. We conducted functional studies using pharmacological and genetic approaches with specific inhibitors and shRNA-mediated knockdown, respectively.

Results

FSK, not rosiglitazone, resulted in a biphasic transcriptomic response, resembling the kinetics of in vivo cold-induced browning. FSK promoted tissue remodeling first and subsequently shifted energy metabolism, concluding with a transcriptomic profile similar to that induced by rosiglitazone. The thermogenic effects of FSK were abolished by PPARγ antagonists, indicating PPARγ as a converging point. ATAC-seq uncovered that FSK leads to a significant chromatin remodeling that precedes or persists beyond transcriptomic changes, whereas rosiglitazone induces minimal changes. Motif analysis identified nuclear factor, interleukin 3 regulated (NFIL3) as a transcriptional regulator connecting the biphasic response of FSK-induced browning, as indicated by disrupted thermogenesis with NFIL3 knockdown.

Conclusions

Our findings elucidated unique dynamics of the transcriptomic and epigenomic remodeling in adipocyte browning, providing new mechanistic insights into adipose thermogenesis and molecular targets for obesity treatment.

Objective

SGLT2 inhibitors increase urinary glucose excretion and have beneficial effects on cardiovascular and renal outcomes; the underlying mechanism may be metabolic adaptations due to urinary glucose loss. Here, we investigated the cellular and molecular effects of 5 weeks of dapagliflozin treatment on skeletal muscle metabolism in type 2 diabetes patients.

Methods

Twenty-six type 2 diabetes mellitus patients were randomized to a 5-week double-blind, cross-over study with 6-8-week wash-out. Skeletal muscle acetylcarnitine levels, intramyocellular lipid (IMCL) content and phosphocreatine (PCr) recovery rate were measured by magnetic resonance spectroscopy (MRS). Ex vivomitochondrial respiration was measured in skeletal muscle fibers using high resolution respirometry. Intramyocellular lipid droplet and mitochondrial network dynamics were investigated using confocal microscopy. Skeletal muscle levels of acylcarnitines, amino acids and TCA cycle intermediates were measured. Expression of genes involved in fatty acid metabolism were investigated.

Results

Mitochondrial function, mitochondrial network integrity and citrate synthase and carnitine acetyltransferase activities in skeletal muscle were unaltered after dapagliflozin treatment. Dapagliflozin treatment increased intramyocellular lipid content (0.060 (0.011, 0.110) %, p = 0.019). Myocellular lipid droplets increased in size (0.03 μm² (0.01–0.06), p < 0.05) and number (0.003 μm⁻² (−0.001–0.007), p = 0.09) upon dapagliflozin treatment. CPT1A, CPT1B and malonyl CoA-decarboxylase mRNA expression was increased by dapagliflozin. Fasting acylcarnitine species and C4–OH carnitine levels (0.4704 (0.1246, 0.8162) pmoles∗mg tissue⁻¹, p < 0.001) in skeletal muscle were higher after dapagliflozin treatment, while acetylcarnitine levels were lower (−40.0774 (−64.4766, −15.6782) pmoles∗mg tissue⁻¹, p < 0.001). Fasting levels of several amino acids, succinate, alpha-ketoglutarate and lactate in skeletal muscle were significantly lower after dapagliflozin treatment.

Conclusion

Dapagliflozin treatment for 5 weeks leads to adaptive changes in skeletal muscle substrate metabolism favoring metabolism of fatty acid and ketone bodies and reduced glycolytic flux.

Objective

RGS2 is a GTPase activating protein that modulates GPCR-G_α signaling and mice lacking RGS2 globally exhibit metabolic alterations. While RGS2 is known to be broadly expressed throughout the body including the brain, the relative contribution of brain RGS2 to metabolic homeostasis remains unknown. The purpose of this study was to characterize RGS2 expression in the paraventricular nucleus of hypothalamus (PVN) and test its role in metabolic homeostasis.

Methods

We used a combination of RNAscope in situ hybridization (ISH), immunohistochemistry, and bioinformatic analyses to characterize the pattern of Rgs2 expression in the PVN. We then created mice lacking Rgs2 either prenatally or postnatally in the PVN and evaluated their metabolic consequences.

Results

RNAscope ISH analysis revealed a broad but regionally enriched Rgs2 mRNA expression throughout the mouse brain, with the highest expression being observed in the PVN along with several other brain regions, such as the arcuate nucleus of hypothalamus and the dorsal raphe nucleus. Within the PVN, we found that Rgs2 is specifically enriched in CRH⁺ endocrine neurons and is further increased by calorie restriction. Functionally, although Sim1-Cre-mediated prenatal deletion of Rgs2 in PVN neurons had no major effects on metabolic homeostasis, AAV-mediated adult deletion of Rgs2 in the PVN led to significantly increased food intake, body weight (both fat and fat-free masses), body length, and blood glucose levels in both male and female mice. Strikingly, we found that prolonged postnatal loss of Rgs2 leads to neuronal cell death in the PVN, while rapid body weight gain in the early phase of viral-mediated PVN Rgs2 deletion is independent of PVN neuronal loss.

Conclusions

Our results provide the first evidence to show that PVN Rgs2 expression is not only sensitive to metabolic challenge but also critically required for PVN endocrine neurons to function and maintain metabolic homeostasis.

Objective

Identifying the transcripts which mediate genetic association signals for type 2 diabetes (T2D) is critical to understand disease mechanisms. Studies in pancreatic islets support the transcription factor ZMIZ1 as a transcript underlying a T2D GWAS signal, but how it influences T2D risk is unknown.

Methods

β-Cell-specific Zmiz1 knockout (Zmiz1^βKO) mice were generated and phenotypically characterised. Glucose homeostasis was assessed in Zmiz1^βKO mice and their control littermates on chow diet (CD) and high fat diet (HFD). Islet morphology and function were examined by immunohistochemistry and in vitro islet function was assessed by dynamic insulin secretion assay. Transcript and protein expression were assessed by RNA sequencing and Western blotting. In islets isolated from genotyped human donors, we assessed glucose-dependent insulin secretion and islet insulin content by static incubation assay.

Results

Male and female Zmiz1^βKO mice were glucose intolerant with impaired insulin secretion, compared with control littermates. Transcriptomic profiling of Zmiz1^βKOislets identified over 500 differentially expressed genes including those involved in β-cell function and maturity, which we confirmed at the protein level. Upon HFD, Zmiz1^βKO mice fail to expand β-cell mass and become severely diabetic. Human islets from carriers of the ZMIZ1-linked T2D-risk alleles have reduced islet insulin content and glucose-stimulated insulin secretion.

Conclusions

β-Cell Zmiz1 is required for normal glucose homeostasis. Genetic variation at the ZMIZ1 locus may influence T2D-risk by reducing islet mass expansion upon metabolic stress and the ability to maintain a mature β-cell state.

Objective

Lifelong insulin replacement remains the mainstay of type 1 diabetes treatment. Genetic FoxO1 ablation promotes enteroendocrine cell (EECs) conversion into glucose-responsive β-like cells. Here, we tested whether chemical FoxO1 inhibitors can generate β-like gut cells.

Methods

We used Ngn3-or Villin-driven FoxO1 ablation to capture the distinctive developmental effects of FoxO1 on EEC pool. We combined FoxO1 ablation with Notch inhibition to enhance the expansion of EEC pool. We tested the ability of an orally available small molecule of FoxO1 inhibitor, Cpd10, to phenocopy genetic ablation of FoxO1. We evaluated the therapeutic impact of genetic ablation or chemical inhibition of FoxO1 on insulin-deficient diabetes in Ins2^Akita/+ mice.

Results

Pan-intestinal epithelial FoxO1 ablation expanded the EEC pool, induced β-like cells, and improved glucose tolerance in Ins2^Akita/+ mice. This genetic effect was phenocopied by Cpd10. Cpd10 induced β-like cells that released insulin in response to glucose in gut organoids, and this effect was enhanced by the Notch inhibitor, DBZ. In Ins2^Akita/+ mice, a five-day course of either Cpd10 or DBZ induced intestinal insulin-immunoreactive β-like cells, lowered glycemia, and increased plasma insulin levels without apparent adverse effects.

Conclusion

These results provide proof of principle of gut cell conversion into β-like cells by a small molecule FoxO1 inhibitor, paving the way for clinical applications.

Objective

Ependymin-Related Protein 1 (EPDR1) was recently identified as a secreted human batokine regulating mitochondrial respiration linked to thermogenesis in brown fat. Despite that EPDR1 is expressed in human pancreatic β-cells and that glucose-stimulated mitochondrial metabolism is critical for stimulus-secretion coupling in β-cells, the role of EPDR1 in β-cell metabolism and function has not been investigated.

Methods

EPDR1 mRNA levels in human pancreatic islets from non-diabetic (ND) and type 2 diabetes (T2D) subjects were assessed. Human islets, EndoC-βH1 and INS1 832/13 cells were transfected with scramble (control) and EPDR1 siRNAs (EPDR1-KD) or treated with human EPDR1 protein, and glucose-stimulated insulin secretion (GSIS) assessed by ELISA. Mitochondrial metabolism was investigated by extracellular flux analyzer, confocal microscopy and mass spectrometry-based metabolomics analysis.

Results

EPDR1 mRNA expression was upregulated in human islets from T2D and obese donors and positively correlated to BMI of donors. In T2D donors, EPDR1 mRNA levels negatively correlated with HbA1c and positively correlated with GSIS. EPDR1silencing in human islets and β-cell lines reduced GSIS whereas treatment with human EPDR1 protein increased GSIS. Epdr1 silencing in INS1 832/13 cells reduced glucose- and pyruvate- but not K⁺-stimulated insulin secretion. Metabolomics analysis in Epdr1-KD INS1 832/13 cells suggests diversion of glucose-derived pyruvate to lactate production and decreased malate-aspartate shuttle and the tricarboxylic acid (TCA) cycle activity. The glucose-stimulated rise in mitochondrial respiration and ATP/ADP-ratio was impaired in Epdr1-deficient cells.

Conclusion

These results suggests that to maintain glucose homeostasis in obese people, upregulation of EPDR1 may improve β-cell function via channelling glycolysis-derived pyruvate to the mitochondrial TCA cycle.

Objectives

The Mitochondrial Unfolded Protein Response (UPR^mt) is a compartment-specific mitochondrial quality control (MQC) mechanism that uses the transcription factor ATF5 to induce the expression of protective enzymes to restore mitochondrial function. Acute exercise is a stressor that has the potential to temporarily disrupt organellar protein homeostasis, however, the roles of ATF5 and the UPR^mt in maintaining basal mitochondrial content, function and exercise-induced MQC mechanisms in skeletal muscle are not known.

Methods

ATF5 KO and WT mice were examined at rest or after a bout of acute endurance exercise. We measured protein content in whole muscle, nuclear, cytosolic and mitochondrial fractions, in addition to mRNA transcript levels in whole muscle. Using isolated mitochondria, we quantified rates of oxygen consumption and ROS emission to observe the effects of the absence of ATF5 on organelle function.

Results

ATF5 KO mice exhibited a larger and less functional muscle mitochondrial pool, most likely a culmination of enhanced biogenesis via increased PGC-1 expression, and attenuated mitophagy. The absence of ATF5 resulted in a reduction in antioxidant proteins and increases in mitochondrial ROS emission, cytosolic cytochrome c, and the expression of mitochondrial chaperones. KO muscle also displayed enhanced exercise-induced stress kinase signaling, but a blunted mitophagic and UPR^mt gene expression response, complemented by significant increases in the basal mRNA abundance and nuclear localization of ATF4. Instead of promoting its nuclear translocation, acute exercise caused the enrichment of ATF5 in mitochondrial fractions. We also identified PGC-1 as an additional regulator of the basal expression of UPR^mt genes.

Conclusion

The transcription factor ATF5 retains a critical role in the maintenance of mitochondrial homeostasis and the appropriate response of muscle to acute exercise for the optimization of mitochondrial quality control.

Objective

Internal clocks time behavior and physiology, including the gut microbiome, in a circadian (∼24 h) manner. Mismatch between internal and external time, e.g. during shift work, disrupts circadian system coordination promoting the development of obesity and type 2 diabetes (T2D). Conversely, body weight changes induce microbiota dysbiosis. The relationship between circadian disruption and microbiota dysbiosis in metabolic diseases, however, remains largely unknown.

Methods

Core and accessory clock gene expression in different gastrointestinal (GI) tissues were determined by qPCR in two different models of circadian disruption - mice with Bmal1 deficiency in the circadian pacemaker, the suprachiasmatic nucleus (Bmal1^SCNfl/-), and wild-type mice exposed to simulated shift work (SSW). Body composition and energy balance were evaluated by nuclear magnetic resonance (NMR), bomb calorimetry, food intake and running-wheel activity. Intestinal permeability was measured in an Ussing chamber. Microbiota composition and functionality were evaluated by 16S rRNA gene amplicon sequencing, PICRUST2.0 analysis and targeted metabolomics. Finally, microbiota transfer was conducted to evaluate the functional impact of SSW-associated microbiota on the host's physiology.

Results

Both chronodisruption models show desynchronization within and between peripheral clocks in GI tissues and reduced microbial rhythmicity, in particular in taxa involved in short-chain fatty acid (SCFA) fermentation and lipid metabolism. In Bmal1SCNfl/- mice, loss of rhythmicity in microbial functioning associates with previously shown increased body weight, dysfunctional glucose homeostasis and adiposity. Similarly, we observe an increase in body weight in SSW mice. Germ-free colonization experiments with SSW-associated microbiota mechanistically link body weight gain to microbial changes. Moreover, alterations in expression of peripheral clock genes as well as clock-controlled genes (CCGs) relevant for metabolic functioning of the host were observed in recipients, indicating a bidirectional relationship between microbiota rhythmicity and peripheral clock regulation.

Conclusions

Collectively, our data suggest that loss of rhythmicity in bacteria taxa and their products, which likely originates in desynchronization of intestinal clocks, promotes metabolic abnormalities during shift work.

Objective

Bone is a highly dynamic organ that undergoes constant bone formation and remodeling, and glucose as a major nutrient is necessary for bone formation and remodeling. Retinoblastoma (Rb1) is a critical regulator of mesenchymal stem cells (MSCs) fate, but how Rb1 regulates bone formation and remodeling is poorly understood.

Methods

We generated MSCs- and osteoprogenitors-specific Rb1 knockout mouse models and utilized these models to explore the function and mechanism of Rb1 in regulating bone formation and remodeling in vivo and in vitro primary cell culture.

Results

Rb1 deficiency in MSCs significantly increased bone mass and impaired osteoclastogenesis. Consistently, depletion of Rb1 in osteoprogenitors significantly promoted bone formation. Mechanistically, loss of Rb1 in MSCs elevated YAP nuclear translocation and transcriptional activity of YAP/TEAD1 complex, thereby increasing the transcriptional expression of Glut1 and OPG. Moreover Prx1-Cre; Rb1^f/f mice displayed hypoglycemia with increased systemic glucose tolerance instead of increased insulin level. In vitro data revealed that Rb1-mutant MSCs enhanced glucose uptake and lactate and ATP production. Increased osteogenesis caused by increased glucose metabolism and decreased osteoclastogenesis caused by increased expression of OPG eventually resulted in increased bone formation and remodeling.

Conclusions

Collectively, these findings demonstrated that Rb1 in MSCs inhibits YAP-medicated Glut1 and OPG expression to control glucose metabolism, osteogenesis and osteoclastogenesis during bone formation and remodeling, which provide new insights that controlling Rb1 signaling may be a potential strategy for osteopetrosis.

Objective

Rostral zona incerta (ZIR) evokes feeding by sending GABA transmission to paraventricular thalamus (PVT). Although central serotonin (5-HT) signaling is known to play critical roles in the regulation of food intake and eating disorders, it remains unknown whether raphe 5-HT neurons functionally innervate ZIR-PVT neural pathway for feeding control. Here, we sought to reveal how raphe 5-HT signaling regulates both ZIR and PVT for feeding control.

Methods

We used retrograde neural tracers to map 5-HT projections in Sert-Cre mice and slice electrophysiology to examine the mechanism by which 5-HT modulates ZIR GABA neurons. We also used optogenetics to test the effects of raphe-ZIR and raphe-PVT 5-HT projections on feeding motivation and food intake in mice regularly fed, 24 h fasted, and with intermittent high-fat high-sugar (HFHS) diet. In addition, we applied RNAscope in situ hybridization to identify 5-HT receptor subtype mRNA in ZIR.

Results

We show raphe 5-HT neurons sent projections to both ZIR and PVT with partial collateral axons. Photostimulation of 5-HT projections inhibited ZIR but excited PVT neurons to decrease motivated food consumption. However, both acute food deprivation and intermittent HFHS diet downregulated 5-HT inhibition on ZIR GABA neurons, abolishing the inhibitory regulation of raphe-ZIR 5-HT projections on feeding motivation and food intake. Furthermore, we found high-level 5-HT1a and 5-HT2c as well as low-level 5-HT7 mRNA expression in ZIR. Intermittent HFHS diet increased 5-HT7 but not 5-HT1a or 5-HT2c mRNA levels in the ZIR.

Conclusions

Our results reveal that raphe-ZIR 5-HT projections dynamically regulate ZIR GABA neurons for feeding control, supporting that a dynamic fluctuation of ZIR 5-HT inhibition authorizes daily food intake but a sustained change of ZIR 5-HT signaling leads to overeating induced by HFHS diet.

Objectives

Insulin treatment remains the sole effective intervention for Type 1 Diabetes. Here, we investigated the therapeutic potential of converting intestinal epithelial cells to insulin-producing, glucose-responsive β-like cells by targeted inhibition of FOXO1. We have previously shown that this can be achieved by genetic ablation in gut Neurogenin3 progenitors, adenoviral or shRNA-mediated inhibition in human gut organoids, and chemical inhibition in Akita mice, a model of insulin-deficient diabetes.

Methods

We profiled two novel FOXO1 inhibitors in reporter gene assays, and hepatocyte gene expression studies, and in vivo pyruvate tolerance test (PTT) for their activity and specificity. We evaluated their glucose-lowering effect in mice rendered insulin-deficient by administration of streptozotocin.

Results

We provide evidence that two novel FOXO1 inhibitors, FBT432 and FBT374 have glucose-lowering and gut β-like cell-inducing properties in mice. FBT432 is also highly effective in combination with a Notch inhibitor in this model.

Conclusion

The data add to a growing body of evidence suggesting that FOXO1 inhibition be pursued as an alternative treatment to insulin administration in diabetes.

Objective

Nonalcoholic fatty liver disease (NAFLD) ranges from steatosis to nonalcoholic steatohepatitis (NASH), which often progresses to hepatocellular carcinoma (HCC) through a largely undefined mechanism. NASH and HCC depend on inflammatory signaling, whose master regulator is the NFκB transcription factor family, activated by canonical and non-canonical pathways.

Methods

Here, we investigated non-canonical NFκB-inducing kinase (NIK/MAP3K14) in metabolic NASH, NASH to HCC transition, and DEN-induced HCC. To this end, we performed dietary and chemical interventions in mice that were analyzed via single nucleus sequencing, gene expression and histochemical methods. Ultimately, we verified our mouse results in human patient samples.

Results

We revealed that hepatocyte-specific NIK deficiency (NIKLKO) ameliorated metabolic NASH complications and reduced hepatocarcinogenesis, independent of its role in the NFκB pathway. Instead, hepatic NIK attenuated hepatoprotective JAK2/STAT5 signaling that is a prerequisite for NASH and NASH to HCC progression in mice and humans.

Conclusions

Our data suggest NIK-mediated inhibitory JAK2 phosphorylation at serine 633 that might be amenable for future therapeutic interventions in patients.

Objective

The glucagon gene (Gcg) encodes preproglucagon, which is cleaved to form glucagon-like peptide 1 (GLP1) and other mature signaling molecules implicated in metabolic functions. To date there are no transgenic rat models available for precise manipulation of GLP1-expressing cells in the brain and periphery.

Methods

To visualize and manipulate Gcg-expressing cells in rats, CRISPR/Cas9 was used to express iCre under control of the Gcg promoter. Gcg-Cre rats were bred with tdTomato reporter rats to tag Gcg-expressing cells. Cre-dependent AAVs and RNAscope in situ hybridization were used to evaluate the specificity of iCreexpression by GLP1 neurons in the caudal nucleus of the solitary tract (cNTS) and intermediate reticular nucleus (IRt), and by intestinal and pancreatic secretory cells. Food intake was assessed in heterozygous (Het) Gcg-Cre rats after chemogenetic stimulation of cNTS GLP1 neurons expressing an excitatory DREADD.

Results

While genotype has minimal effect on body weight or composition in chow-fed Gcg-Cre rats, homozygous (Homo) rats have lower plasma glucose levels. In neonatal and adult Gcg-Cre/tdTom rats, reporter-labeled cells are present in the cNTS and IRt, and in additional brain regions (e.g., basolateral amygdala, piriform cortex) that lack detectable Gcg mRNA in adults but display transient developmental or persistently low Gcg expression. Compared to wildtype (WT) rats, hindbrain GcgmRNA and GLP1 protein in brain and plasma are markedly reduced in Homo Gcg-Cre rats. Chemogenetic stimulation of cNTS GLP1 neurons reduced overnight chow intake in males but not females, the effect in males was blocked by antagonism of central GLP1 receptors, and hypophagia was enhanced when combined with a subthreshold dose of cholecystokinin-8 to stimulate gastrointestinal vagal afferents.

Conclusions

Gcg-Cre rats are a novel and valuable experimental tool for analyzing the development, anatomy, and function of Gcg-expressing cells in the brain and periphery. In addition, Homo Gcg-Cre rats are a unique model for assessing the role of Gcg-encoded proteins in glucose homeostasis and energy metabolism.

Objective

Obesity and its associated comorbidities represent a global health challenge with a need for well-tolerated, effective, and mechanistically diverse pharmaceutical interventions. Oxyntomodulin is a gut peptide that activates the glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) and reduces bodyweight by increasing energy expenditure and reducing energy intake in humans. Here we describe the pharmacological profile of the novel glucagon receptor (GCGR)/GLP-1 receptor (GLP-1R) dual agonist BI 456906.

Methods

BI 456906 was characterized using cell-based in vitro assays to determine functional agonism. In vivo pharmacological studies were performed using acute and subchronic dosing regimens to demonstrate target engagement for the GCGR and GLP-1R, and weight lowering efficacy.

Results

BI 456906 is a potent, acylated peptide containing a C18 fatty acid as a half-life extending principle to support once-weekly dosing in humans. Pharmacological doses of BI 456906 provided greater bodyweight reductions in mice compared with maximally effective doses of the GLP-1R agonist semaglutide. BI 456906’s superior efficacy is the consequence of increased energy expenditure and reduced food intake. Engagement of both receptors in vivo was demonstrated via glucose tolerance, food intake, and gastric emptying tests for the GLP-1R, and liver nicotinamide N-methyltransferase mRNA expression and circulating biomarkers (amino acids, fibroblast growth factor-21) for the GCGR. The dual activity of BI 456906 at the GLP-1R and GCGR was supported using GLP-1R knockout and transgenic reporter mice, and an ex vivo bioactivity assay.

Conclusions

BI 456906 is a potent GCGR/GLP-1R dual agonist with robust anti-obesity efficacy achieved by increasing energy expenditure and decreasing food intake.

Objective

Thyroid transcription factor-1 (TTF-1), a homeodomain-containing transcription factor, is predominantly expressed in discrete areas of the hypothalamus, which acts as the central unit for the regulation of whole-body energy homeostasis. Current study designed to identify the roles of TTF-1 on the responsiveness of the hypothalamic circuit activity to circulating leptin and the development of obesity linked to the insensitivity of leptin.

Methods

We generated conditional knock-out mice by crossing TTF-1^flox/flox mice with leptin receptor (ObRb)^Cre or proopiomelanocortin (POMC)^Cre transgenic mice to interrogate the contributions of TTF-1 in leptin signaling and activity. Changes of food intake, body weight and energy expenditure were evaluated in standard or high fat diet-treated transgenic mice by using an indirect calorimetry instrument. Molecular mechanism was elucidated with immunohistochemistry, immunoblotting, quantitative PCR, and promoter assays.

Results

The selective deletion of TTF-1 gene expression in cells expressing the ObRb or POMC enhanced the anorexigenic effects of leptin as well as the leptin-induced phosphorylation of STAT3. We further determined that TTF-1 inhibited the transcriptional activity of the ObRb gene. In line with these findings, the selective deletion of the TTF-1 gene in ObRb-positive cells led to protective effects against diet-induced obesity via the amelioration of leptin resistance.

Conclusions

Collectively, these results suggest that hypothalamic TTF-1 participates in the development of obesity as a molecular component involved in the regulation of cellular leptin signaling and activity. Thus, TTF-1 may represent a therapeutic target for the treatment, prevention, and control of obesity.

Objectives

Glucagon-like peptide-1 receptor (GLP-1R) agonists reduce the rates of major cardiovascular events, including myocardial infarction in people with type 2 diabetes, and decrease infarct size while preserving ventricular function in preclinical studies. Nevertheless, the precise cellular sites of GLP-1R expression that mediate the cardioprotective actions of GLP-1 in the setting of ischemic cardiac injury are uncertain.

Methods

Publicly available single cell RNA sequencing (scRNA-seq) datasets on mouse and human heart cells were analyzed for Glp1r/GLP1R expression. Fluorescent activated cell sorting was used to localize Glp1r expression in cell populations from the mouse heart. The importance of endothelial and hematopoietic cells for the cardioprotective response to liraglutide in the setting of acute myocardial infarction (MI) was determined by inactivating the Glp1r in Tie2+ cell populations. Cardiac gene expression profiles regulated by liraglutide were examined using RNA-seq to interrogate mouse atria and both infarcted and non-infarcted ventricular tissue after acute coronary artery ligation.

Results

In mice, cardiac Glp1r mRNA transcripts were exclusively detected in endocardial cells by scRNA-seq. In contrast, analysis of human heart by scRNA-seq localized GLP1R mRNA transcripts to populations of atrial and ventricular cardiomyocytes. Moreover, very low levels of GIPR, GCGR and GLP2R mRNA transcripts were detected in the human heart. Cell sorting and RNA analyses detected cardiac Glp1rexpression in endothelial cells (ECs) within the atria and ventricle in the ischemic and non-ischemic mouse heart. Transcriptional responses to liraglutide administration were not evident in wild type mouse ventricles following acute MI, however liraglutide differentially regulated genes important for inflammation, cardiac repair, cell proliferation, and angiogenesis in the left atrium, while reducing circulating levels of IL-6 and KC/GRO within hours of acute MI. Inactivation of the Glp1r within the Tie2+ cell expression domain encompassing ECs revealed normal cardiac structure and function, glucose homeostasis and body weight in Glp1r^Tie2−/−mice. Nevertheless, the cardioprotective actions of liraglutide to reduce infarct size, augment ejection fraction, and improve survival after experimental myocardial infarction (MI), were attenuated in Glp1r^Tie2−/− mice.

Conclusions

These findings identify the importance of the murine Tie2+ endothelial cell GLP-1R as a target for the cardioprotective actions of GLP-1R agonists and support the importance of the atrial and ventricular endocardial GLP-1R as key sites of GLP-1 action in the ischemic mouse heart. Hitherto unexplored species-specific differences in cardiac GLP-1R expression challenge the exclusive use of mouse models for understanding the mechanisms of GLP-1 action in the normal and ischemic human heart.

Objective

Treatment with glucagon receptor antagonists (GRAs) reduces blood glucose but causes dyslipidemia and accumulation of fat in the liver. We investigated the acute and chronic effects of glucagon on lipid metabolism in mice.

Methods

Chronic effects of glucagon receptor signaling on lipid metabolism were studied using oral lipid tolerance tests (OLTTs) in overnight fasted glucagon receptor knockout (Gcgr^−/−) mice, and in C57Bl/6JRj mice treated with a glucagon receptor antibody (GCGR Ab) or a long-acting glucagon analogue (GCGA) for eight weeks. Following treatment, liver tissue was harvested for RNA-sequencing and triglyceride measurements. Acute effects were studied in C57Bl/6JRj mice treated with a GRA or GCGA 1 h or immediately before OLTTs, respectively. Direct effects of glucagon on hepatic lipolysis were studied using isolated perfused mouse liver preparations. To investigate potential effects of GCGA and GRA on gastric emptying, paracetamol was, in separate experiments, administered immediately before OLTTs.

Results

Plasma triglyceride concentrations increased 2-fold in Gcgr^−/− mice compared to their wild-type littermates during the OLTT (P = 0.001). Chronic treatment with GCGR Ab increased, whereas GCGA treatment decreased, plasma triglyceride concentrations during OLTTs (P < 0.05). Genes involved in lipid metabolism were upregulated upon GCGR Ab treatment while GCGA treatment had opposite effects. Acute GRA and GCGA treatment, respectively, increased (P = 0.02) and decreased (P = 0.003) plasma triglyceride concentrations during OLTTs. Glucagon stimulated hepatic lipolysis, evident by an increase in free fatty acid concentrations in the effluent from perfused mouse livers. In line with this, GCGR Ab treatment increased, while GCGA treatment decreased, liver triglyceride concentrations. The effects of glucagon appeared independent of changes in gastric emptying of paracetamol.

Conclusions

Glucagon receptor signaling regulates triglyceride metabolism, both chronically and acutely, in mice. These data expand glucagon´s biological role and implicate that intact glucagon signaling is important for lipid metabolism. Glucagon agonism may have beneficial effects on hepatic and peripheral triglyceride metabolism.

Objective

The β-carotene oxygenase 1 (BCO1) is the enzyme responsible for the cleavage of β-carotene to retinal, the first intermediate in vitamin A formation. Preclinical studies suggest that BCO1 expression is required for dietary β-carotene to affect lipid metabolism. The goal of this study was to generate a gene therapy strategy that over-expresses BCO1 in the adipose tissue and utilizes the β-carotene stored in adipocytes to produce vitamin A and reduce obesity.

Methods

We generated a novel adipose-tissue-specific, adeno-associated vector to over-express BCO1 (AT-AAV-BCO1) in murine adipocytes. We tested this vector using a unique model to achieve β-carotene accumulation in the adipose tissue, in which Bco1^−/− mice were fed β-carotene. An AT-AAV over-expressing green fluorescent protein was utilized as control. We evaluated the adequate delivery route and optimized cellular and organ specificity, dosage, and exposure of our vectors. We also employed morphometric analyses to evaluate the effect of BCO1 expression in adiposity, as well as HPLC and mass spectrometry to quantify β-carotene and retinoids in tissues, including retinoic acid.

Results

AT-AAV-BCO1 infusions in the adipose tissue of the mice resulted in the production of retinoic acid, a vitamin A metabolite with strong effects on gene regulation. AT-AAV-BCO1 treatment also reduced adipose tissue size and adipocyte area by 35% and 30%, respectively. These effects were sex-specific, highlighting the complexity of vitamin A metabolism in mammals.

Conclusions

The over-expression of BCO1 through delivery of an AT-AAV-BCO1 leads to the conversion of β-carotene to vitamin A in adipocytes, which subsequently results in reduction of adiposity. These studies highlight for the first time the potential of adipose tissue β-carotene as a target for BCO1 over-expression in the reduction of obesity.

Objective

Pancreatic insulin was discovered a century ago, and this discovery led to the first lifesaving treatment for diabetes. While still controversial, nearly one hundred published reports suggest that insulin is also produced in the brain, with most focusing on hypothalamic or cortical insulin-producing cells. However, specific function for insulin produced within the brain remains poorly understood. Here we identify insulin expression in the hindbrain's dorsal vagal complex (DVC), and determine the role of this source of insulin in feeding and metabolism, as well as its response to diet-induced obesity in mice.

Methods

To determine the contribution of Ins2-producing neurons to feeding behavior in mice, we used the cross of transgenic RipHER-cre mouse and channelrhodopsin-2 expressing animals, which allowed us to optogenetically stimulate neurons expressing Ins2 in vivo. To confirm the presence of insulin expression in Rip-labeled DVC cells, in situ hybridization was used. To ascertain the specific role of insulin in effects discovered via optogenetic stimulation a selective, CNS applied, insulin receptor antagonist was used. To understand the physiological contribution of insulin made in the hindbrain a virogenetic knockdown strategy was used.

Results

Insulin gene expression and presence of insulin-promoter driven fluorescence in rat insulin promoter (Rip)-transgenic mice were detected in the hypothalamus, but also in the DVC. Insulin mRNA was present in nearly all fluorescently labeled cells in DVC. Diet-induced obesity in mice altered brain insulin gene expression, in a neuroanatomically divergent manner; while in the hypothalamus the expected obesity-induced reduction was found, in the DVC diet-induced obesity resulted in increased expression of the insulin gene. This led us to hypothesize a potentially divergent energy balance role of insulin in these two brain areas. To determine the acute impact of activating insulin-producing neurons in the DVC, optic stimulation of light-sensitive channelrhodopsin 2 in Rip-transgenic mice was utilized. Optogenetic photoactivation induced hyperphagia after acute activation of the DVC insulin neurons. This hyperphagia was blocked by central application of the insulin receptor antagonist S961, suggesting the feeding response was driven by insulin. To determine whether DVC insulin has a necessary contribution to feeding and metabolism, virogenetic insulin gene knockdown (KD) strategy, which allows for site-specific reduction of insulin gene expression in adult mice, was used. While chow-fed mice failed to reveal any changes of feeding or thermogenesis in response to the KD, mice challenged with a high-fat diet consumed less food. No changes in body weight were identified, possibly resulting from compensatory reduction in thermogenesis.

Conclusions

Together, our data suggest an important role for hindbrain insulin and insulin-producing cells in energy homeostasis.

Objective

The endocrine pancreatic β-cells play a pivotal role in maintaining whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate β-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homeostasis.

Methods

Mice lacking ROCK1 in pancreatic β-cells (RIP-Cre; ROCK1^loxP/loxP, β-ROCK1^−/−) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. An insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from β-ROCK1^−/− mice or β-cell lines with knockdown of ROCK1 was also evaluated. A proximity ligation assay was performed to determine the physical interactions between PK and ROCK1.

Results

Mice with a deficiency of ROCK1 in pancreatic β-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, β-ROCK1^−/− mice displayed a progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium and ATP levels and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown β-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that glucose stimulation in β-cells greatly enhanced ROCK1 binding to PK.

Conclusions

Our findings demonstrate that β-cell ROCK1 is essential for glucose-stimulated insulin secretion and for glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling.

Objective

The endocrine pancreatic β-cells play a pivotal role in maintaining whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate β-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homeostasis.

Methods

Mice lacking ROCK1 in pancreatic β-cells (RIP-Cre; ROCK1^loxP/loxP, β-ROCK1^−/−) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. An insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from β-ROCK1^−/− mice or β-cell lines with knockdown of ROCK1 was also evaluated. A proximity ligation assay was performed to determine the physical interactions between PK and ROCK1.

Results

Mice with a deficiency of ROCK1 in pancreatic β-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, β-ROCK1^−/− mice displayed a progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium and ATP levels and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown β-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that glucose stimulation in β-cells greatly enhanced ROCK1 binding to PK.

Conclusions

Our findings demonstrate that β-cell ROCK1 is essential for glucose-stimulated insulin secretion and for glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling.

Objective

The hypothalamus regulates feeding and glucose homeostasis through the balanced action of different neuropeptides, which are cleaved and activated by the proprotein convertases PC1/3 and PC2. However, the recent association of polymorphisms in the proprotein convertase FURIN with type 2 diabetes, metabolic syndrome, and obesity, prompted us to investigate the role of FURIN in hypothalamic neurons controlling glucose and feeding.

Methods

POMC-Cre^+/− mice were bred with Furin^fl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMCFurKO), and Furin^fl/fl mice were used as controls. POMCFurKO and controls were periodically monitored on both normal chow diet and high fat diet (HFD) for body weight and glucose tolerance by established in-vivo procedures. Food intake was measured in HFD-fed FurKO and controls. Hypothalamic Pomc mRNA was measured by RT-qPCR. ELISAs quantified POMC protein and resulting peptides in the hypothalamic extracts of POMCFurKO mice and controls. The in-vitroprocessing of POMC was studied by biochemical techniques in HEK293T and CHO cell lines lacking FURIN.

Results

In control mice, Furin mRNA levels were significantly upregulated on HFD feeding, suggesting an increased demand for FURIN activity in obesogenic conditions. Under these conditions, the POMCFurKO mice were hyperphagic and had increased body weight compared to Furin^fl/fl mice. Moreover, protein levels of POMC were elevated and ACTH concentrations markedly reduced. Also, the ratio of α-MSH/POMC was decreased in POMCFurKO mice compared to controls. This indicates that POMC processing was significantly reduced in the hypothalami of POMCFurKO mice, highlighting for the first time the involvement of FURIN in the cleavage of POMC. Importantly, we found that in vitro, the first stage in processing where POMC is cleaved into proACTH was achieved by FURIN but not by PC1/3 or the other proprotein convertases in cell lines lacking a regulated secretory pathway.

Conclusions

These results suggest that FURIN processes POMC into proACTH before sorting into the regulated secretory pathway, challenging the dogma that PC1/3 and PC2 are the only convertases responsible for POMC cleavage. Furthermore, its deletion affects feeding behaviors under obesogenic conditions.

Objective

Zinc transporter 8 (ZnT8) is a major humoral target in human type 1 diabetes (T1D). Polymorphic variants of Slc30A8, which encodes ZnT8, are also associated with protection from type 2 diabetes (T2D). The current study examined whether ZnT8 might play a role beyond simply being a target of autoimmunity in the pathophysiology of T1D.

Methods

The phenotypes of NOD mice with complete or partial global loss of ZnT8 were determined using a combination of disease incidence, histological, transcriptomic, and metabolic analyses.

Results

Unexpectedly, while complete loss of ZnT8 accelerated spontaneous T1D, heterozygosity was partially protective. In vivo and in vitro studies of ZnT8 deficient NOD.SCID mice suggested that the accelerated disease was due to more rampant autoimmunity. Conversely, beta cells in heterozygous animals uniquely displayed increased mitochondrial fitness under mild proinflammatory conditions.

Conclusions

In pancreatic beta cells and immune cell populations, Zn²⁺ plays a key role as a regulator of redox signaling and as an independent secondary messenger. Importantly, Zn²⁺ also plays a major role in maintaining mitochondrial homeostasis. Our results suggest that regulating mitochondrial fitness by altering intra-islet zinc homeostasis may provide a novel mechanism to modulate T1D pathophysiology.