Subcutaneous white adipose tissue independently regulates burn-induced hypermetabolism via immune-adipose crosstalk.

Severe burns induce a chronic hypermetabolic state that persists well past wound closure, indicating that additional internal mechanisms must be involved. Adipose tissue is suggested to be a central regulator in perpetuating hypermetabolism, although this has not been directly tested. Here, we show that thermogenic adipose tissues are activated in parallel to increases in hypermetabolism independent of cold stress. Using an adipose tissue transplantation model, we discover that burn-derived subcutaneous white adipose tissue alone is sufficient to invoke a hypermetabolic response in a healthy recipient mouse. Concomitantly, transplantation of healthy adipose tissue alleviates metabolic dysfunction in a burn recipient. We further show that the nicotinic acetylcholine receptor signaling pathway may mediate an immune-adipose crosstalk to regulate adipose tissue remodeling post-injury. Targeting this pathway could lead to innovative therapeutic interventions to counteract hypermetabolic pathologies.

Lactate shuttling drives the browning of white adipose tissue after burn.

High levels of plasma lactate are associated with increased mortality in critically injured patients, including those with severe burns. Although lactate has long been considered a waste product of glycolysis, it was recently revealed that it acts as a potent inducer of white adipose tissue (WAT) browning, a response implicated in mediating postburn cachexia, hepatic steatosis, and sustained hypermetabolism. Despite the clinical presentation of hyperlactatemia and browning in burns, whether these two pathological responses are linked is currently unknown. Here, we report that elevated lactate plays a causal signaling role in mediating adverse outcomes after burn trauma by directly promoting WAT browning. Using WAT obtained from human burn patients and mouse models of thermal injury, we show that the induction of postburn browning is positively correlated with a shift toward lactate import and metabolism. Furthermore, daily administration of l-lactate is sufficient to augment burn-induced mortality and weight loss in vivo. At the organ level, increased lactate transport amplified the thermogenic activation of WAT and its associated wasting, thereby driving postburn hepatic lipotoxicity and dysfunction. Mechanistically, the thermogenic effects of lactate appeared to result from increased import through MCT transporters, which in turn increased intracellular redox pressure, [NADH/NAD+], and expression of the batokine, FGF21. In fact, pharmacological inhibition of MCT-mediated lactate uptake attenuated browning and improved hepatic function in mice after injury. Collectively, our findings identify a signaling role for lactate that impacts multiple aspects of postburn hypermetabolism, necessitating further investigation of this multifaceted metabolite in trauma and critical illness.NEW & NOTEWORTHY To our knowledge, this study was the first to investigate the role of lactate signaling in mediating white adipose tissue browning after burn trauma. We show that the induction of browning in both human burn patients and mice is positively correlated with a shift toward lactate import and metabolism. Daily l-lactate administration augments burn-induced mortality, browning, and hepatic lipotoxicity in vivo, whereas pharmacologically targeting lactate transport alleviates burn-induced browning and improves liver dysfunction after injury.

Recent Developments in Islet Biology: A Review With Patient Perspectives.

Navigating the coronavirus disease-2019 (COVID-19, now COVID) pandemic has required resilience and creativity worldwide. Despite early challenges to productivity, more than 2,000 peer-reviewed articles on islet biology were published in 2021. Herein, we highlight noteworthy advances in islet research between January 2021 and April 2022, focussing on 5 areas. First, we discuss new insights into the role of glucokinase, mitogen-activated protein kinase-kinase/extracellular signal-regulated kinase and mitochondrial function on insulin secretion from the pancreatic ß cell, provided by new genetically modified mouse models and live imaging. We then discuss a new connection between lipid handling and improved insulin secretion in the context of glucotoxicity, focussing on fatty acid-binding protein 4 and fetuin-A. Advances in high-throughput "omic" analysis evolved to where one can generate more finely tuned genetic and molecular profiles within broad classifications of type 1 diabetes and type 2 diabetes. Next, we highlight breakthroughs in diabetes treatment using stem cell-derived ß cells and innovative strategies to improve islet survival posttransplantation. Last, we update our understanding of the impact of severe acute respiratory syndrome-coronavirus-2 infection on pancreatic islet function and discuss current evidence regarding proposed links between COVID and new-onset diabetes. We address these breakthroughs in 2 settings: one for a scientific audience and the other for the public, particularly those living with or affected by diabetes. Bridging biomedical research in diabetes to the community living with or affected by diabetes, our partners living with type 1 diabetes or type 2 diabetes also provide their perspectives on these latest advances in islet biology.

Beyond Genetics: The Role of Metabolism in Photoreceptor Survival, Development and Repair.

Vision commences in the retina with rod and cone photoreceptors that detect and convert light to electrical signals. The irreversible loss of photoreceptors due to neurodegenerative disease leads to visual impairment and blindness. Interventions now in development include transplanting photoreceptors, committed photoreceptor precursors, or retinal pigment epithelial (RPE) cells, with the latter protecting photoreceptors from dying. However, introducing exogenous human cells in a clinical setting faces both regulatory and supply chain hurdles. Recent work has shown that abnormalities in central cell metabolism pathways are an underlying feature of most neurodegenerative disorders, including those in the retina. Reversal of key metabolic alterations to drive retinal repair thus represents a novel strategy to treat vision loss based on cell regeneration. Here, we review the connection between photoreceptor degeneration and alterations in cell metabolism, along with new insights into how metabolic reprogramming drives both retinal development and repair following damage. The potential impact of metabolic reprogramming on retinal regeneration is also discussed, specifically in the context of how metabolic switches drive both retinal development and the activation of retinal glial cells known as Müller glia. Müller glia display latent regenerative properties in teleost fish, however, their capacity to regenerate new photoreceptors has been lost in mammals. Thus, re-activating the regenerative properties of Müller glia in mammals represents an exciting new area that integrates research into developmental cues, central metabolism, disease mechanisms, and glial cell biology. In addition, we discuss this work in relation to the latest insights gleaned from other tissues (brain, muscle) and regenerative species (zebrafish).

Islet Biology During COVID-19: Progress and Perspectives.

The coronavirus-2019 (COVID-19) pandemic has had significant impact on research directions and productivity in the past 2 years. Despite these challenges, since 2020, more than 2,500 peer-reviewed articles have been published on pancreatic islet biology. These include updates on the roles of isocitrate dehydrogenase, pyruvate kinase and incretin hormones in insulin secretion, as well as the discovery of inceptor and signalling by circulating RNAs. The year 2020 also brought advancements in in vivo and in vitro models, including a new transgenic mouse for assessing beta-cell proliferation, a "pancreas-on-a-chip" to study glucose-stimulated insulin secretion and successful genetic editing of primary human islet cells. Islet biologists evaluated the functionality of stem-cell-derived islet-like cells coated with semipermeable biomaterials to prevent autoimmune attack, revealing the importance of cell maturation after transplantation. Prompted by observations that COVID-19 symptoms can worsen for people with obesity or diabetes, researchers examined how islets are directly affected by severe acute respiratory syndrome coronavirus 2. Herein, we highlight novel functional insights, technologies and therapeutic approaches that emerged between March 2020 and July 2021, written for both scientific and lay audiences. We also include a response to these advancements from patient stakeholders, to help lend a broader perspective to developments and challenges in islet research.

Silencing the G-protein coupled receptor 3-salt inducible kinase 2 pathway promotes human ß cell proliferation.

Loss of pancreatic ß cells is the hallmark of type 1 diabetes, for which provision of insulin is the standard of care. While regenerative and stem cell therapies hold the promise of generating single-source or host-matched tissue to obviate immune-mediated complications, these will still require surgical intervention and immunosuppression. Here we report the development of a high-throughput RNAi screening approach to identify upstream pathways that regulate adult human ß cell quiescence and demonstrate in a screen of the GPCRome that silencing G-protein coupled receptor 3 (GPR3) leads to human pancreatic ß cell proliferation. Loss of GPR3 leads to activation of Salt Inducible Kinase 2 (SIK2), which is necessary and sufficient to drive cell cycle entry, increase ß cell mass, and enhance insulin secretion in mice. Taken together, our data show that targeting the GPR3-SIK2 pathway is a potential strategy to stimulate the regeneration of ß cells.

The AMPK-Related Kinases SIK1 and SIK3 Mediate Key Tumor-Suppressive Effects of LKB1 in NSCLC.

Mutations in the LKB1 (also known as STK11) tumor suppressor are the third most frequent genetic alteration in non-small cell lung cancer (NSCLC). LKB1 encodes a serine/threonine kinase that directly phosphorylates and activates 14 AMPK family kinases ("AMPKRs"). The function of many of the AMPKRs remains obscure, and which are most critical to the tumor-suppressive function of LKB1 remains unknown. Here, we combine CRISPR and genetic analysis of the AMPKR family in NSCLC cell lines and mouse models, revealing a surprising critical role for the SIK subfamily. Conditional genetic loss of Sik1 revealed increased tumor growth in mouse models of Kras-dependent lung cancer, which was further enhanced by loss of the related kinase Sik3. As most known substrates of the SIKs control transcription, gene-expression analysis was performed, revealing upregulation of AP1 and IL6 signaling in common between LKB1- and SIK1/3-deficient tumors. The SIK substrate CRTC2 was required for this effect, as well as for proliferation benefits from SIK loss. SIGNIFICANCE: The tumor suppressor LKB1/STK11 encodes a serine/threonine kinase frequently inactivated in NSCLC. LKB1 activates 14 downstream kinases in the AMPK family controlling growth and metabolism, although which kinases are critical for LKB1 tumor-suppressor function has remained an enigma. Here we unexpectedly found that two understudied kinases, SIK1 and SIK3, are critical targets in lung cancer.This article is highlighted in the In This Issue feature, p. 1469.

Differentiation of Normal and Radioresistant Prostate Cancer Xenografts Using Magnetization Transfer-Prepared MRI.

The ability of MRI to differentiate between normal and radioresistant cancer was investigated in prostate tumour xenografts in mice. Specifically, the process of magnetization exchange between water and other molecules was studied. It was found that magnetization transfer from semisolid macromolecules (MT) and chemical exchange saturation transfer (CEST) combined were significantly different between groups (p < 0.01). Further, the T2 relaxation of the semisolid macromolecular pool (T2,B), a parameter specific to MT, was found to be significantly different (p < 0.01). Also significantly different were the rNOE contributions associated with methine groups at -0.9 ppm with a saturation B1 of 0.5 µT (p < 0.01) and with other aliphatic groups at -3.3 ppm with 0.5 and 2 µT (both p < 0.05). Independently, using a live-cell metabolic assay, normal cells were found to have a greater metabolic rate than radioresistant ones. Thus, MRI provides a novel, in vivo method to quantify the metabolic rate of tumours and predict their radiosensitivity.

Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism.

G protein αs (GNAS) mediates receptor-stimulated cAMP signalling, which integrates diverse environmental cues with intracellular responses. GNAS is mutationally activated in multiple tumour types, although its oncogenic mechanisms remain elusive. We explored this question in pancreatic tumourigenesis where concurrent GNAS and KRAS mutations characterize pancreatic ductal adenocarcinomas (PDAs) arising from intraductal papillary mucinous neoplasms (IPMNs). By developing genetically engineered mouse models, we show that GnasR201C cooperates with KrasG12D to promote initiation of IPMN, which progress to invasive PDA following Tp53 loss. Mutant Gnas remains critical for tumour maintenance in vivo. This is driven by protein-kinase-A-mediated suppression of salt-inducible kinases (Sik1-3), associated with induction of lipid remodelling and fatty acid oxidation. Comparison of Kras-mutant pancreatic cancer cells with and without Gnas mutations reveals striking differences in the functions of this network. Thus, we uncover Gnas-driven oncogenic mechanisms, identify Siks as potent tumour suppressors, and demonstrate unanticipated metabolic heterogeneity among Kras-mutant pancreatic neoplasms.

AMPK and Friends: Central Regulators of ß Cell Biology.

If left unchecked, prediabetic hyperglycemia can progress to diabetes and often life-threatening attendant secondary complications. Central to the process of glucose homeostasis are pancreatic ß cells, which sense elevations in plasma glucose and additional dietary components and respond by releasing the appropriate quantity of insulin, ensuring the arrest of hepatic glucose output and glucose uptake in peripheral tissues. Given that ß cell failure is associated with the transition from prediabetes to diabetes, improved ß cell function ('compensation') has a central role in preventing type 2 diabetes mellitus (T2DM). Recent data have shown that both insulin secretion and ß cell mass dynamics are regulated by the liver kinase B1-AMP-activated kinase (LKB1-AMPK) pathway and related kinases of the AMPK family; thus, an improved understanding of the biological roles of AMPK in the ß cell is now of considerable interest.

Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors.

PDX1+/NKX6-1+ pancreatic progenitors (PPs) give rise to endocrine cells both in vitro and in vivo. This cell population can be successfully differentiated from human pluripotent stem cells (hPSCs) and hold the potential to generate an unlimited supply of ß cells for diabetes treatment. However, the efficiency of PP generation in vitro is highly variable, negatively impacting reproducibility and validation of in vitro and in vivo studies, and consequently, translation to the clinic. Here, we report the use of a proteomics approach to phenotypically characterize hPSC-derived PPs and distinguish these cells from non-PP populations during differentiation. Our analysis identifies the pancreatic secretory granule membrane major glycoprotein 2 (GP2) as a PP-specific cell surface marker. Remarkably, GP2 is co-expressed with NKX6-1 and PTF1A in human developing pancreata, indicating that it marks the multipotent pancreatic progenitors in vivo. Finally, we show that isolated hPSC-derived GP2+ cells generate ß-like cells (C-PEPTIDE+/NKX6-1+) more efficiently compared to GP2- and unsorted populations, underlining the potential therapeutic applications of GP2.Pancreatic progenitors (PPs) can be derived from human pluripotent stem cells in vitro but efficiency of differentiation varies, making it hard to sort for insulin-producing cells. Here, the authors use a proteomic approach to identify the secretory granule membrane glycoprotein 2 as a marker for PDX1+/NKX6-1+ PPs.

The Keap1-Nrf2 Stress Response Pathway Promotes Mitochondrial Hyperfusion Through Degradation of the Mitochondrial Fission Protein Drp1.

AIMS: Mitochondrial function is coupled to metabolic and survival pathways through both direct signaling cascades and dynamic changes in mitochondrial morphology. For example, a hyperfused mitochondrial reticulum is activated upon cellular stress and is protective against cell death. As part of a genome-wide small inhibitory ribonucleic acid screen, we identified the central redox regulator, Keap1, as a novel regulator of mitochondrial morphology. Here, we aimed to determine the mechanism through which redox signaling and Keap1 mediate changes in mitochondrial morphology. RESULTS: We found that the Nrf2 transcription factor is required for mitochondrial hyperfusion induced by knockdown of Keap1. Nrf2, which is negatively regulated by Keap1, mediates the cell's response to stress by controlling the expression of several hundred genes, including proteasome expression. We next showed that increased proteasome activity, a result of increased Nrf2 activity, is responsible for the degradation of the mitochondrial fission protein Drp1, which occurs in an ubiquitin-independent manner. INNOVATION: Our study described a novel pathway by which Nrf2 activation, known to occur in response to increased oxidative stress, decreases mitochondrial fission and contributes to a hyperfused mitochondrial network. CONCLUSION: This study has identified the Keap1-Nrf2 nexus and modulation of proteasomal activity as novel avenues to inhibit mitochondrial fission. These findings are important, because inhibiting mitochondrial fission is a promising therapeutic approach to restore the balance between fission and fusion, which is attractive for an increasing number of disorders linked to mitochondrial dysfunction. Antioxid. Redox Signal. 27, 1447-1459.

High-throughput Functional Genomics Identifies Regulators of Primary Human Beta Cell Proliferation.

The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing the proliferation of non-transformed human cells. Here, we report the development of a high-throughput RNAi screening strategy specifically for use in primary cells and demonstrate that silencing the cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21 as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens for dissecting therapeutically relevant state changes in primary human cells.

Identification of Kinase-substrate Pairs Using High Throughput Screening.

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Phenotypic Characterization of MIP-CreERT1Lphi Mice With Transgene-Driven Islet Expression of Human Growth Hormone.

There is growing concern over confounding artifacts associated with ß-cell-specific Cre-recombinase transgenic models, raising questions about their general usefulness in research. The inducible ß-cell-specific transgenic (MIP-CreERT(1Lphi)) mouse was designed to circumvent many of these issues, and we investigated whether this tool effectively addressed concerns of ectopic expression and disruption of glucose metabolism. Recombinase activity was absent from the central nervous system using a reporter line and high-resolution microscopy. Despite increased pancreatic insulin content, MIP-CreERT mice on a chow diet exhibited normal ambient glycemia, glucose tolerance and insulin sensitivity, and appropriate insulin secretion in response to glucose in vivo and in vitro. However, MIP-CreERT mice on different genetic backgrounds were protected from high-fat/ streptozotocin (STZ)-induced hyperglycemia that was accompanied by increased insulin content and islet density. Ectopic human growth hormone (hGH) was highly expressed in MIP-CreERT islets independent of tamoxifen administration. Circulating insulin levels remained similar to wild-type controls, whereas STZ-associated increases in α-cell number and serum glucagon were significantly blunted in MIP-CreERT(1Lphi) mice, possibly due to paracrine effects of hGH-induced serotonin expression. These studies reveal important new insight into the strengths and limitations of the MIP-CreERT mouse line for ß-cell research.

To Be(ta Cell) or Not to Be(ta cell): New Mouse Models for Studying Gene Function in the Pancreatic ß-Cell.

A challenge in the pancreatic ß-cell field has been to identify a promoter fragment that is active only in the ß-cell compartment and inactive in other regions, such as the hypothalamic region of the brain. The presence of Cre recombinase alone in some models may also affect glucoregulation, confounding interpretation of gene function in the ß-cell. A paper presented within describes the development and characterization of 2 new transgenic mice expressing Cre recombinase under the mouse insulin1 promoter that are useful for ß-cell-specific gene ablation: the first is constitutive and coexpresses DsRed (Ins1-Cre-DsRed); the second allows ß-cell-specific expression of the reverse tetracycline-controlled transactivator, which can be used for drug-dependent expression of a target gene of interest for overexpression studies. These novel models show robust specificity and efficiency and will be valuable tools for functional studies of gene action in ß-cells, potentially alleviating current issues associated with previously available mouse lines.

Homozygous mutations in MFN2 cause multiple symmetric lipomatosis associated with neuropathy.

Multiple symmetric lipomatosis (MSL) is a mitochondrial disorder with impaired brown fat metabolism that has been associated with MERRF mutations in some, but not all, patients. We studied a sibling pair and an unrelated indiviadual who presented with MSL and neuropathy to determine the genetic etiology of this disorder in patients who did not carry the MSL-associated MERRF mutation. Whole-exome sequencing was performed on the siblings, and a rare, shared homozygous mutation in MFN2 (c.2119C>T: p.R707W) was identified. The mutation was not present in their healthy siblings. In silico programs predict it to be pathogenic, and heterozygous carriers of the MFN2 p.R707W substitution are known to have Charcot-Marie-Tooth (CMT) disease. A third, unrelated patient with multiple symmetrical lipomatosis and neuropathy also harbored the same homozygous mutation and had been previously diagnosed with CMT. Functional studies in patient fibroblasts demonstrate that the p.R707W substitution impairs homotypic (MFN2-MFN2) protein interactions required for normal activity and renders mitochondria prone to perinuclear aggregation. These findings show that homozygous mutations at p.R707W in MFN2 are a novel cause of multiple symmetrical lipomatosis.