C. Ronald Kahn

RNA-independent cis-autoregulatory circuits within bidirectional gene pairs control metabolism

Brown fat-specific RNA labelling reveals a network of inter-organ communication by secreted microRNAs

Protein Kinase C δ: a critical hub regulating macrophage immunomodulatory functions during Mycobacterium tuberculosis infection

A host-modulating candidate gene involved in putative pathogen-killing pathways, with potential novel therapeutic intervention, Protein Kinase C – δ (PKCδ) has been recognized as a critical marker of inflammation with clinical and experimental evidence in recent years. Pulmonary microenvironment during Mtb infection is largely governed by lung resident macrophages, initiating innate and subsequent adaptive immune responses. We investigated the role of PKCδ in macrophages using a macrophage-specific PKCδ knockout mice model (LysM cre PKCδ flox/flox ). PKCδ deficiency in macrophages triggers an early lymphocytic immune response, increases neutrophil recruitment, and reduces inflammatory macrophages in the lungs, leading to higher Mtb burden and exacerbated pathology. Experimental and omics analysis further revealed that dysregulation of antimicrobial effector functions is detrimental to macrophage’s ability to restrict bacterial growth in vitro . Importantly this defect was mitigated by exogenous GM-CSF supplementation and/or overexpressing PKCδ in macrophages. Thus, PKCδ plays a crucial role in immune modulation during Mtb infection with GM-CSF amongst several downstream pathways through which PKCδ exerts its regulatory effects. Teaser PKCδ is crucial for immune modulation during Mtb infection revealing macrophages as a potential axis of signaling.

Loss of insulin signaling in microglia impairs cellular uptake of Aβ and neuroinflammatory response exacerbating AD-like neuropathology

Insulin receptors are present on cells throughout the body, including the brain. Dysregulation of insulin signaling in neurons and astrocytes has been implicated in altered mood, cognition, and the pathogenesis of Alzheimer's disease (AD). To define the role of insulin signaling in microglia, the primary phagocytes in the brain critical for maintenance and damage repair, we created mice with an inducible microglia-specific insulin receptor knockout (MG-IRKO). RiboTag profiling of microglial mRNAs revealed that loss of insulin signaling results in alterations of gene expression in pathways related to innate immunity and cellular metabolism. In vitro, loss of insulin signaling in microglia results in metabolic reprogramming with an increase in glycolysis and impaired uptake of Aβ. In vivo, MG-IRKO mice exhibit alterations in mood and social behavior, and when crossed with the 5xFAD mouse model of AD, the resultant mice exhibit increased levels of Aβ plaque and elevated neuroinflammation. Thus, insulin signaling in microglia plays a key role in microglial cellular metabolism and the ability of the cells to take up Aβ, such that reduced insulin signaling in microglia alters mood and social behavior and accelerates AD pathogenesis. Together, these data indicate key roles of insulin action in microglia and the potential of targeting insulin signaling in microglia in treatment of AD.

Cell-intrinsic insulin signaling defects in human iPS cell–derived hepatocytes in type 2 diabetes

Hepatic insulin resistance is central to type 2 diabetes (T2D) and metabolic syndrome, but defining the molecular basis of this defect in humans is challenging because of limited tissue access. Utilizing inducible pluripotent stem cells differentiated into hepatocytes from control individuals and patients with T2D and liquid chromatography with tandem mass spectrometry-based (LC-MS/MS-based) phosphoproteomics analysis, we identified a large network of cell-intrinsic alterations in signaling in T2D. Over 300 phosphosites showed impaired or reduced insulin signaling, including losses in the classical insulin-stimulated PI3K/AKT cascade and their downstream targets. In addition, we identified over 500 phosphosites of emergent, i.e., new or enhanced, signaling. These occurred on proteins involved in the Rho-GTPase pathway, RNA metabolism, vesicle trafficking, and chromatin modification. Kinome analysis indicated that the impaired phosphorylation sites represented reduced actions of AKT2/3, PKCθ, CHK2, PHKG2, and/or STK32C kinases. By contrast, the emergent phosphorylation sites were predicted to be mediated by increased action of the Rho-associated kinases 1 and 2 (ROCK1/2), mammalian STE20-like protein kinase 4 (MST4), and/or branched-chain α-ketoacid dehydrogenase kinase (BCKDK). Inhibiting ROCK1/2 activity in T2D induced pluripotent stem cell-derived hepatocytes restored some of the alterations in insulin action. Thus, insulin resistance in the liver in T2D did not simply involve a loss of canonical insulin signaling but the also appearance of new phosphorylations representing a change in the balance of multiple kinases. Together, these led to altered insulin action in the liver and identified important targets for the therapy of hepatic insulin resistance.

Weight loss in obesity and T2D is linked to ~65% lower plasma sEVs, restoring insulin signaling.

Hepatic SerpinA1 improves energy and glucose metabolism through regulation of preadipocyte proliferation and UCP1 expression

Lipodystrophy and obesity are associated with insulin resistance and metabolic syndrome accompanied by fat tissue dysregulation. Here, we show that serine protease inhibitor A1 (SerpinA1) expression in the liver is increased during recovery from lipodystrophy caused by the adipocyte-specific loss of insulin signaling in mice. SerpinA1 induces the proliferation of white and brown preadipocytes and increases the expression of uncoupling protein 1 (UCP1) to promote mitochondrial activation in mature white and brown adipocytes. Liver-specific SerpinA1 transgenic mice exhibit increased browning of adipose tissues, leading to increased energy expenditure, reduced adiposity and improved glucose tolerance. Conversely, SerpinA1 knockout mice exhibit decreased adipocyte mitochondrial function, impaired thermogenesis, obesity, and systemic insulin resistance. SerpinA1 forms a complex with the Eph receptor B2 and regulates its downstream signaling in adipocytes. These results demonstrate that SerpinA1 is an important hepatokine that improves obesity, energy expenditure and glucose metabolism by promoting preadipocyte proliferation and activating mitochondrial UCP1 expression in adipocytes.

7449 A Genome-wide CRISPR Screen Identifies Drivers of Impaired Glucose Uptake in Human Insulin Resistance

Disclosure: N. Haider: None. Y. Lee: None. P. Yi: None. C.R. Kahn: None. Insulin resistance is a major risk factor in the development of type 2 diabetes (T2D), and metabolic syndrome. Although 25% of people within the general non-diabetic population are insulin resistant, the primary underlying cause of insulin resistance remains elusive. In this study, we have used induced pluripotent stem cells (iPSC) derived from non-diabetic humans at both ends of the insulin sensitivity spectrum, i.e., the top 20% of insulin resistance vs. the top 20% of insulin sensitive, as well as individuals with T2D to model human insulin resistance. We find that iPSCs differentiated into myoblasts (iMyos) from T2D and non-diabetic individuals in the highest quintile of insulin resistance (I-Res) show impaired insulin signaling and defective insulin-stimulated glucose uptake, as compared to iMyos from the insulin sensitive individuals (I-Sen), indicating that these cells in vitro mirror the alterations seen in vivo in a cell autonomous manner. To uncover the primary upstream drivers that contribute to the impaired glucose uptake associated with insulin resistance, we conducted a genome-wide loss-of-function screen using a CRISPR-based approach to identify genes that positively or negatively regulated glucose uptake in I-Res and T2D iMyos. Knockdown of ∼525 genes show effect to rescue the impaired glucose uptake in both I-Res and T2D iMyos, i.e., were negative regulators of glucose uptake. We hypothesized that important negative regulators of glucose uptake might be over-expressed at the protein level in I-Res and T2D iMyos and crossing the iMyos proteomics data with the CRISPR screen revealed 8 top candidates that are increased at the protein level and whose knockdown rescued glucose uptake in I-Res and T2D iMyos. These included candidates associated with cytoskeleton changes, protein kinases, transmembrane transport, mitochondrial and nuclear proteins. In summary, iPSC derived myoblasts from patients with insulin resistance and T2D mirror defects in glucose uptake observed in vivo. Combining CRISPR screening and global proteomics, we have identified important upstream regulators of insulin resistance in humans. Presentation: 6/2/2024

Loss of Insulin Signaling in Microglia Impairs Cellular Uptake of Aβ and Neuroinflammatory Response Exacerbating Alzheimer-like Neuropathology

Insulin receptors are present on cells throughout the body, including the brain. Dysregulation of insulin signaling in neurons and astrocytes has been implicated in altered mood, cognition, and the pathogenesis of Alzheimers disease (AD). To define the role of insulin signaling in microglia, the primary phagocytes in brain critical for maintenance and damage repair, we created mice with an inducible microglia-specific insulin receptor knockout (MG-IRKO). RiboTag profiling of microglial mRNAs revealed that loss of insulin signaling results in alterations of gene expression in pathways related to innate immunity and cellular metabolism. In vitro, loss of insulin signaling in microglia results in metabolic reprograming with an increase in glycolysis and impaired uptake of Aβ. In vivo, MG-IRKO mice exhibit alterations in mood and social behavior, and when crossed with the 5xFAD mouse model of AD, the resultant mice exhibit increased levels of Aβ; plaque and elevated neuroinflammation. Thus, insulin signaling in microglia plays a key role in microglial cellular metabolism, neuroinflammation and the ability of the cells to take up Aβ; such that reduced insulin signaling in microglia alters mood and social behavior and accelerates AD pathogenesis. Together these data indicate key roles of insulin action in microglia and the potential of targeting insulin signaling in microglia in treatment of AD.

Multi-step regulation of microRNA expression and secretion into small extracellular vesicles by insulin

Tissues release microRNAs (miRNAs) in small extracellular vesicles (sEVs) including exosomes, which can regulate gene expression in distal cells, thus acting as modulators of local and systemic metabolism. Here, we show that insulin regulates miRNA secretion into sEVs from 3T3-L1 adipocytes and that this process is differentially regulated from cellular expression. Thus, of the 53 miRNAs upregulated and 66 miRNAs downregulated by insulin in 3T3-L1 sEVs, only 12 were regulated in parallel in cells. Insulin regulated this process in part by phosphorylating hnRNPA1, causing it to bind to AU-rich motifs in miRNAs, mediating their secretion into sEVs. Importantly, 43% of insulin-regulated sEV-miRNAs are implicated in obesity and insulin resistance. These include let-7 and miR-103, which we show regulate insulin signaling in AML12 hepatocytes. Together, these findings demonstrate an important layer to insulin's regulation of adipose biology and provide a mechanism of tissue crosstalk in obesity and other hyperinsulinemic states.

219-OR: Loss of Insulin Signaling in Microglia Impairs Cellular Uptake of Aß and Neuroinflammatory Response Exacerbating Alzheimer-Like Neuropathology

Insulin receptors are present on cells throughout the body. Dysregulation of insulin signaling in the brain has been implicated in mood, cognitive disorders, and pathogenesis of Alzheimer’s disease (AD). However, the brain is composed of multiple cells types, and little is known as to how brain insulin resistance in particular in different brain cell types contributes to the comorbidity of type 2 diabetes (T2D) and AD. In this regard, microglia are particularly interesting, since microglia are involved in the uptake and clearance of amyloid-β (Aβ) and formation of neuroinflammation. To investigate the role of insulin signaling in microglial function and development of AD, we have created an inducible microglia-specific insulin receptor knockout (MG-IRKO) and found that loss of microglial insulin signaling in these mice results in alterations in mood, social behavior and impairment in systemic metabolism. At a molecular level, utilizing RiboTag to isolate and profile microglial-specific ribosome-bounded mRNA, we found that loss of insulin receptor resulted in alterations in pathways associated with innate immunity and cellular metabolism in microglia. In vitro, loss of insulin signaling in microglial resulted in metabolic reprograming with a switch to more glycolytic metabolism, and this was associated with impaired ability of microglia in Aβ uptake. When the MG-IRKO mice were crossed with an AD mouse model (5xFAD mice) to create MGIRKO/5xFAD mice, the resultant mice exhibit increased levels of Aβ plaque formation and elevated neuroinflammation marked by increased IFN-γ. Thus, insulin signaling in microglia plays a key role in microglial cellular metabolism, neuroinflammation and the ability of the cells to uptake Aβ, such that loss of insulin signaling in microglia accelerates AD pathogenesis. Together, these data indicate the potential importance of targeting insulin signaling in microglia in treatment of AD. Disclosure W. Chen: None. V.R. Muñoz: None. C. Kahn: Consultant; Cellarity. Other Relationship; 1825 Therapeutics. Advisory Panel; TIXiMED, Senseion, ERX.

Retraction: Insulin-like Growth Factor-1 Induces Adhesion and Migration in Human Multiple Myeloma Cells via Activation of β1-Integrin and Phosphatidylinositol 3′-Kinase/AKT Signaling

There are concerns about the reliability of certain data arising from errors

Reciprocal communication between FAPs and muscle cells via distinct extracellular vesicle miRNAs in muscle regeneration

Muscle regeneration is a complex process relying on precise teamwork between multiple cell types, including muscle stem cells (MuSCs) and fibroadipogenic progenitors (FAPs). FAPs are also the main source of intramuscular adipose tissue (IMAT). Muscles without FAPs exhibit decreased IMAT infiltration but also deficient muscle regeneration, indicating the importance of FAPs in the repair process. Here, we demonstrate the presence of bidirectional crosstalk between FAPs and MuSCs via their secretion of extracellular vesicles (EVs) containing distinct clusters of miRNAs that is crucial for normal muscle regeneration. Thus, after acute muscle injury, there is activation of FAPs leading to a transient rise in IMAT. These FAPs also release EVs enriched with a selected group of miRNAs, a number of which come from an imprinted region on chromosome 12. The most abundant of these is miR-127-3p, which targets the sphingosine-1-phosphate receptor S1pr3 and activates myogenesis. Indeed, intramuscular injection of EVs from immortalized FAPs speeds regeneration of injured muscle. In late stages of muscle repair, in a feedback loop, MuSCs and their derived myoblasts/myotubes secrete EVs enriched in miR-206-3p and miR-27a/b-3p. The miRNAs repress FAP adipogenesis, allowing full muscle regeneration. Together, the reciprocal communication between FAPs and muscle cells via miRNAs in their secreted EVs plays a critical role in limiting IMAT infiltration while stimulating muscle regeneration, hence providing an important mechanism for skeletal muscle repair and homeostasis.

Interactions among insulin resistance, epigenetics, and donor sex in gene expression regulation of iPSC-derived myoblasts

FoxK1 associated gene regulatory network in hepatic insulin action and its relationship to FoxO1 and insulin receptor mediated transcriptional regulation

OBJECTIVE: Insulin acts on the liver via changes in gene expression to maintain glucose and lipid homeostasis. This study aimed to the Forkhead box protein K1 (FOXK1) associated gene regulatory network as a transcriptional regulator of hepatic insulin action and to determine its role versus FoxO1 and possible actions of the insulin receptor at the DNA level. METHODS: Genome-wide analysis of FoxK1 binding were studied by chromatin immunoprecipitation sequencing and compared to those for IR and FoxO1. These were validated by knockdown experiments and gene expression analysis. RESULTS: Chromatin immunoprecipitation (ChIP) sequencing shows that FoxK1 binds to the proximal promoters and enhancers of over 4000 genes, and insulin enhances this interaction for about 75% of them. These include genes involved in cell cycle, senescence, steroid biosynthesis, autophagy, and metabolic regulation, including glucose metabolism and mitochondrial function and are enriched in a TGTTTAC consensus motif. Some of these genes are also bound by FoxO1. Comparing this FoxK1 ChIP-seq data to that of the insulin receptor (IR) reveals that FoxK1 may act as the transcription factor partner for some of the previously reported roles of IR in gene regulation, including for LARS1 and TIMM22, which are involved in rRNA processing and cell cycle. CONCLUSION: These data demonstrate that FoxK1 is an important regulator of gene expression in response to insulin in liver and may act in concert with FoxO1 and IR in regulation of genes in metabolism and other important biological pathways.

Loss of insulin signaling in astrocytes exacerbates Alzheimer-like phenotypes in a 5xFAD mouse model

Brain insulin signaling controls peripheral energy metabolism and plays a key role in the regulation of mood and cognition. Epidemiological studies have indicated a strong connection between type 2 diabetes (T2D) and neurodegenerative disorders, especially Alzheimer's disease (AD), linked via dysregulation of insulin signaling, i.e., insulin resistance. While most studies have focused on neurons, here, we aim to understand the role of insulin signaling in astrocytes, a glial cell type highly implicated in AD pathology and AD progression. To this end, we created a mouse model by crossing 5xFAD transgenic mice, a well-recognized AD mouse model that expresses five familial AD mutations, with mice carrying a selective, inducible insulin receptor (IR) knockout in astrocytes (iGIRKO). We show that by age 6 mo, iGIRKO/5xFAD mice exhibited greater alterations in nesting, Y-maze performance, and fear response than those of mice with the 5xFAD transgenes alone. This was associated with increased Tau (T231) phosphorylation, increased Aβ plaque size, and increased association of astrocytes with plaques in the cerebral cortex as assessed using tissue CLARITY of the brain in the iGIRKO/5xFAD mice. Mechanistically, in vitro knockout of IR in primary astrocytes resulted in loss of insulin signaling, reduced ATP production and glycolic capacity, and impaired Aβ uptake both in the basal and insulin-stimulated states. Thus, insulin signaling in astrocytes plays an important role in the control of Aβ uptake, thereby contributing to AD pathology, and highlighting the potential importance of targeting insulin signaling in astrocytes as a site for therapeutics for patients with T2D and AD.

Fructose Induced KHK-C Increases ER Stress and Modulates Hepatic Transcriptome to Drive Liver Disease in Diet-Induced and Genetic Models of NAFLD

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform increases endoplasmic reticulum (ER) stress in a dose dependent fashion, so when fructose is coupled with a HFD intake it leads to unresolved ER stress. Conversely, a liver-specific knockdown of KHK in C57BL/6J male mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in genetically obesity ob/ob, db/db and lipodystrophic FIRKO male mice, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

Unique ligand and kinase-independent roles of the insulin receptor in regulation of cell cycle, senescence and apoptosis

Insulin acts through the insulin receptor (IR) tyrosine kinase to exert its classical metabolic and mitogenic actions. Here, using receptors with either short or long deletion of the β-subunit or mutation of the kinase active site (K1030R), we have uncovered a second, previously unrecognized IR signaling pathway that is intracellular domain-dependent, but ligand and tyrosine kinase-independent (LYK-I). These LYK-I actions of the IR are linked to changes in phosphorylation of a network of proteins involved in the regulation of extracellular matrix organization, cell cycle, ATM signaling and cellular senescence; and result in upregulation of expression of multiple extracellular matrix-related genes and proteins, down-regulation of immune/interferon-related genes and proteins, and increased sensitivity to apoptosis. Thus, in addition to classical ligand and tyrosine kinase-dependent (LYK-D) signaling, the IR regulates a second, ligand and tyrosine kinase-independent (LYK-I) pathway, which regulates the cellular machinery involved in senescence, matrix interaction and response to extrinsic challenges.

Leucine-973 is a crucial residue differentiating insulin and IGF-1 receptor signaling

Insulin and IGF-1 receptors (IR and IGF1R) are highly homologous and share similar signaling systems, but each has a unique physiological role, with IR primarily regulating metabolic homeostasis and IGF1R regulating mitogenic control and growth. Here, we show that replacement of a single amino acid at position 973, just distal to the NPEY motif in the intracellular juxtamembrane region, from leucine, which is highly conserved in IRs, to phenylalanine, the highly conserved homologous residue in IGF1Rs, resulted in decreased IRS-1/PI3K/Akt/mTORC1 signaling and increased Shc/Gab1/MAPK cell cycle signaling. As a result, cells expressing L973F-IR exhibited decreased insulin-induced glucose uptake, increased cell growth, and impaired receptor internalization. Mice with knockin of the L973F-IR showed similar alterations in signaling in vivo, and this led to decreased insulin sensitivity, a modest increase in growth, and decreased weight gain when mice were challenged with a high-fat diet. Thus, leucine-973 in the juxtamembrane region of the IR acts as a crucial residue differentiating IR signaling from IGF1R signaling.

Interaction of a viral insulin-like peptide with the IGF-1 receptor produces a natural antagonist

Lymphocystis disease virus-1 (LCDV-1) and several other Iridoviridae encode viral insulin/IGF-1 like peptides (VILPs) with high homology to human insulin and IGFs. Here we show that while single-chain (sc) and double-chain (dc) LCDV1-VILPs have very low affinity for the insulin receptor, scLCDV1-VILP has high affinity for IGF1R where it can antagonize human IGF-1 signaling, without altering insulin signaling. Consequently, scLCDV1-VILP inhibits IGF-1 induced cell proliferation and growth hormone/IGF-1 induced growth of mice in vivo. Cryo-electron microscopy reveals that scLCDV1-VILP engages IGF1R in a unique manner, inducing changes in IGF1R conformation that led to separation, rather than juxtaposition, of the transmembrane segments and hence inactivation of the receptor. Thus, scLCDV1-VILP is a natural peptide with specific antagonist properties on IGF1R signaling and may provide a new tool to guide development of hormonal analogues to treat cancers or metabolic disorders sensitive to IGF-1 without affecting glucose metabolism.