The P300 acetyltransferase inhibitor C646 promotes membrane translocation of insulin receptor protein substrate and interaction with the insulin receptor.

Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRß) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol(4,5)P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRß's tyrosine kinase activity and directly promotes IRß interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.

Insulin receptor substrate 1, but not IRS2, plays a dominant role in regulating pancreatic alpha cell function in mice.

Dysregulated glucagon secretion deteriorates glycemic control in type 1 and type 2 diabetes. Although insulin is known to regulate glucagon secretion via its cognate receptor (insulin receptor, INSR) in pancreatic alpha cells, the role of downstream proteins and signaling pathways underlying insulin's activities are not fully defined. Using in vivo (knockout) and in vitro (knockdown) studies targeting insulin receptor substrate (IRS) proteins, we compared the relative roles of IRS1 and IRS2 in regulating alpha cell function. Alpha cell-specific IRS1-knockout mice exhibited glucose intolerance and inappropriate glucagon suppression during glucose tolerance tests. In contrast, alpha cell-specific IRS2-knockout animals manifested normal glucose tolerance and suppression of glucagon secretion after glucose administration. Alpha cell lines with stable IRS1 knockdown could not repress glucagon mRNA expression and exhibited a reduction in phosphorylation of AKT Ser/Thr kinase (AKT, at Ser-473 and Thr-308). AlphaIRS1KD cells also displayed suppressed global protein translation, including reduced glucagon expression, impaired cytoplasmic Ca2+ response, and mitochondrial dysfunction. This was supported by the identification of novel IRS1-specific downstream target genes, Trpc3 and Cartpt, that are associated with glucagon regulation in alpha cells. These results provide evidence that IRS1, rather than IRS2, is a dominant regulator of pancreatic alpha cell function.

From population to neuron: exploring common mediators for metabolic problems and mental illnesses.

Major mental illnesses such as schizophrenia (SZ) and bipolar disorder (BP) frequently accompany metabolic conditions, but their relationship is still unclear, in particular at the mechanistic level. We implemented an approach of "from population to neuron", combining population-based epidemiological analysis with neurobiological experiments using cell and animal models based on a hypothesis built from the epidemiological study. We characterized high-quality population data, olfactory neuronal cells biopsied from patients with SZ or BP, and healthy subjects, as well as mice genetically modified for insulin signaling. We accessed the Danish Registry and observed (1) a higher incidence of diabetes in people with SZ or BP and (2) higher incidence of major mental illnesses in people with diabetes in the same large cohort. These epidemiological data suggest the existence of common pathophysiological mediators in both diabetes and major mental illnesses. We hypothesized that molecules associated with insulin resistance might be such common mediators, and then validated the hypothesis by using two independent sets of olfactory neuronal cells biopsied from patients and healthy controls. In the first set, we confirmed an enrichment of insulin signaling-associated molecules among the genes that were significantly different between SZ patients and controls in unbiased expression profiling data. In the second set, olfactory neuronal cells from SZ and BP patients who were not pre-diabetic or diabetic showed reduced IRS2 tyrosine phosphorylation upon insulin stimulation, indicative of insulin resistance. These cells also displayed an upregulation of IRS1 protein phosphorylation at serine-312 at baseline (without insulin stimulation), further supporting the concept of insulin resistance in olfactory neuronal cells from SZ patients. Finally, Irs2 knockout mice showed an aberrant response to amphetamine, which is also observed in some patients with major mental illnesses. The bi-directional relationships between major mental illnesses and diabetes suggest that there may be common pathophysiological mediators associated with insulin resistance underlying these mental and physical conditions.

Irs2 deficiency alters hippocampus-associated behaviors during young adulthood.

Type 2 diabetes mellitus (T2DM), characterized by hyperglycemia and insulin resistance, has been recognized as a risk factor for cognitive impairment and dementia, including Alzheimer's disease (AD). Insulin receptor substrate2 (IRS2) is a major component of the insulin/insulin-like growth factor-1 signaling pathway. Irs2 deletion leads to life-threatening T2DM, promoting premature death in male mice regardless of their genetic background. Here, we showed for the first time that young adult male mice lacking Irs2 on a C57BL/6J genetic background (Irs2-/-/6J) survived in different experimental environments and displayed hippocampus-associated behavioral alterations. Young adult male Irs2-/-/6J mice also exhibit aberrant alterations in energy and nutrient sensors, such as AMP-activated protein kinase (AMPK) and glucose transporter3 (GLUT3), and reduced core body temperature accompanied by abnormal change in the temperature sensor in the brain. These results suggest that Irs2 deficiency-induced impairments of brain energy metabolism and thermoregulation contribute to hippocampus-associated behavioral changes in young adult male mice.

Ablation of insulin receptor substrates 1 and 2 suppresses Kras-driven lung tumorigenesis.

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer death worldwide, with 25% of cases harboring oncogenic Kirsten rat sarcoma (KRAS). Although KRAS direct binding to and activation of PI3K is required for KRAS-driven lung tumorigenesis, the contribution of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) in the context of mutant KRAS remains controversial. Here, we provide genetic evidence that lung-specific dual ablation of insulin receptor substrates 1/2 (Irs1/Irs2), which mediate insulin and IGF1 signaling, strongly suppresses tumor initiation and dramatically extends the survival of a mouse model of lung cancer with Kras activation and p53 loss. Mice with Irs1/Irs2 loss eventually succumb to tumor burden, with tumor cells displaying suppressed Akt activation and strikingly diminished intracellular levels of essential amino acids. Acute loss of IRS1/IRS2 or inhibition of IR/IGF1R in KRAS-mutant human NSCLC cells decreases the uptake and lowers the intracellular levels of amino acids, while enhancing basal autophagy and sensitivity to autophagy and proteasome inhibitors. These findings demonstrate that insulin/IGF1 signaling is required for KRAS-mutant lung cancer initiation, and identify decreased amino acid levels as a metabolic vulnerability in tumor cells with IR/IGF1R inhibition. Consequently, combinatorial targeting of IR/IGF1R with autophagy or proteasome inhibitors may represent an effective therapeutic strategy in KRAS-mutant NSCLC.

Hyperglycemia induces vascular smooth muscle cell dedifferentiation by suppressing insulin receptor substrate-1-mediated p53/KLF4 complex stabilization.

Hyperglycemia and insulin resistance accelerate atherosclerosis by an unclear mechanism. The two factors down-regulate insulin receptor substrate-1 (IRS-1), an intermediary of the insulin/IGF-I signaling system. We previously reported that IRS-1 down-regulation leads to vascular smooth muscle cell (VSMC) dedifferentiation and that IRS-1 deletion from VSMCs in normoglycemic mice replicates this response. However, we did not determine IRS-1's role in mediating differentiation. Here, we sought to define the mechanism by which IRS-1 maintains VSMC differentiation. High glucose or IRS-1 knockdown decreased p53 levels by enhancing MDM2 proto-oncogene (MDM2)-mediated ubiquitination, resulting in decreased binding of p53 to Krüppel-like factor 4 (KLF4). Exposure to nutlin-3, which dissociates MDM2/p53, decreased p53 ubiquitination and enhanced the p53/KLF4 association and differentiation marker protein expression. IRS-1 overexpression in high glucose inhibited the MDM2/p53 association, leading to increased p53 and p53/KLF4 levels, thereby increasing differentiation. Nutlin-3 treatment of diabetic or Irs1-/- mice enhanced p53/KLF4 and the expression of p21, smooth muscle protein 22 (SM22), and myocardin and inhibited aortic VSMC proliferation. Injecting normoglycemic mice with a peptide disrupting the IRS-1/p53 association reduced p53, p53/KLF4, and differentiation. Analyzing atherosclerotic lesions in hypercholesterolemic, diabetic pigs, we found that p53, IRS-1, SM22, myocardin, and KLF4/p53 levels are significantly decreased compared with their expression in nondiabetic pigs. We conclude that IRS-1 is critical for maintaining VSMC differentiation. Hyperglycemia- or insulin resistance-induced IRS-1 down-regulation decreases the p53/KLF4 association and enhances dedifferentiation and proliferation. Our results suggest that enhancing IRS-1-dependent p53 stabilization could attenuate the progression of atherosclerotic lesions in hyperglycemia and insulin-resistance states.

Insulin receptor substrate-1 deficiency drives a proinflammatory phenotype in KRAS mutant lung adenocarcinoma.

Insulin receptor substrate-1 (IRS-1) is a signaling adaptor protein that interfaces with many pathways activated in lung cancer. It has been assumed that IRS-1 promotes tumor growth through its ability to activate PI3K signaling downstream of the insulin-like growth factor receptor. Surprisingly, tumors with reduced IRS-1 staining in a human lung adenocarcinoma tissue microarray displayed a significant survival disadvantage, especially within the Kirsten rat sarcoma viral oncogene homolog (KRAS) mutant subgroup. Accordingly, adenoviral Cre recombinase (AdCre)-treated LSL-Kras/Irs-1(fl/fl) (Kras/Irs-1(-/-)) mice displayed increased tumor burden and mortality compared with controls. Mechanistically, IRS-1 deficiency promotes Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling via the IL-22 receptor, resulting in enhanced tumor-promoting inflammation. Treatment of Kras/Irs-1(+/+) and Kras/Irs-1(-/-) mice with JAK inhibitors significantly reduced tumor burden, most notably in the IRS-1-deficient group.

Down-regulation of Insulin Receptor Substrate 1 during Hyperglycemia Induces Vascular Smooth Muscle Cell Dedifferentiation.

Diabetes is a major risk factor for the development of atherosclerosis, but the mechanism by which hyperglycemia accelerates lesion development is not well defined. Insulin and insulin-like growth factor I (IGF-I) signal through the scaffold protein insulin receptor substrate 1 (IRS-1). In diabetes, IRS-1 is down-regulated, and cells become resistant to insulin. Under these conditions, the IGF-I receptor signals through an alternate scaffold protein, SHPS-1, resulting in pathophysiologic stimulation of vascular smooth muscle cell (VSMC) migration and proliferation. These studies were undertaken to determine whether IRS-1 is functioning constitutively to maintain VSMCs in their differentiated state and, thereby, inhibit aberrant signaling. Here we show that deletion of IRS-1 expression in VSMCs in non-diabetic mice results in dedifferentiation, SHPS-1 activation, and aberrant signaling and that these changes parallel those that occur in response to hyperglycemia. The mice showed enhanced sensitivity to IGF-I stimulation of VSMC proliferation and a hyperproliferative response to vascular injury. KLF4, a transcription factor that induces VSMC dedifferentiation, was up-regulated in IRS-1-/- mice, and the differentiation inducer myocardin was undetectable. Importantly, these changes were replicated in wild-type mice during hyperglycemia. These findings illuminate a new function of IRS-1: that of maintaining cells in their normal, differentiated state. Because IRS-1 is down-regulated in states of insulin resistance that occur in response to metabolic stresses such as obesity and cytokine stimulation, the findings provide a mechanism for understanding how patients with metabolic stress and/or diabetes are predisposed to developing vascular complications.

G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser318, Ser346, Ser612, and Ser789, and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R.

Serine 302 Phosphorylation of Mouse Insulin Receptor Substrate 1 (IRS1) Is Dispensable for Normal Insulin Signaling and Feedback Regulation by Hepatic S6 Kinase.

Constitutive activation of the mammalian target of rapamycin complex 1 and S6 kinase (mTORC1→ S6K) attenuates insulin-stimulated Akt activity in certain tumors in part through "feedback" phosphorylation of the upstream insulin receptor substrate 1 (IRS1). However, the significance of this mechanism for regulating insulin sensitivity in normal tissue remains unclear. We investigated the function of Ser-302 in mouse IRS1, the major site of its phosphorylation by S6K in vitro, through genetic knock-in of a serine-to-alanine mutation (A302). Although insulin rapidly stimulated feedback phosphorylation of Ser-302 in mouse liver and muscle, homozygous A302 mice (A/A) and their knock-in controls (S/S) exhibited similar glucose homeostasis and muscle insulin signaling. Furthermore, both A302 and control primary hepatocytes from which Irs2 was deleted showed marked inhibition of insulin-stimulated IRS1 tyrosine phosphorylation and PI3K binding after emetine treatment to raise intracellular amino acids and activate mTORC1 → S6K signaling. To specifically activate mTORC1 in mouse tissue, we deleted hepatic Tsc1 using Cre adenovirus. Although it moderately decreased IRS1/PI3K association and Akt phosphorylation in liver, Tsc1 deletion failed to cause glucose intolerance or promote hyperinsulinemia in mixed background A/A or S/S mice. Moreover, Tsc1 deletion failed to stimulate phospho-Ser-302 or other putative S6K sites within IRS1, whereas ribosomal S6 protein was constitutively phosphorylated. Following acute Tsc1 deletion from hepatocytes, Akt phosphorylation, but not IRS1/PI3K association, was rapidly restored by treatment with the mTORC1 inhibitor rapamycin. Thus, within the hepatic compartment, mTORC1 → S6K signaling regulates Akt largely through IRS-independent means with little effect upon physiologic insulin sensitivity.

IRS proteins and diabetic complications.

IRS proteins are cellular adaptor molecules that mediate many of the key metabolic actions of insulin. When tyrosine is phosphorylated by the activated insulin receptor, IRS proteins recruit downstream effectors, such as phosphoinositide 3-kinase and mitogen-activated protein kinase, in order to elicit cellular responses such as glucose uptake, lipid metabolism and cell proliferation. There are two main IRS proteins in humans (IRS1 and IRS2), both of which are widely expressed. Given their central role in the insulin signalling pathway, it is not surprising that male mice lacking Irs1 or Irs2 present with elevated blood glucose or type 2 diabetes, respectively. For reasons yet to be identified, female Irs2 (-/-) mice do not develop type 2 diabetes. A number of organs are affected by complications of diabetes; macrovascular complications include stroke and coronary artery disease, while nephropathy, neuropathy and retinopathy fall into the category of microvascular complications. Given the serious consequences of these complications on patient morbidity and mortality, it is essential to identify the molecular pathogenesis underlying diabetic complications, with a view to improving therapeutic intervention and patient outcomes. A number of recently published papers have converged on the hypothesis that the loss of insulin signalling and IRS proteins is instrumental to the development and/or progression of diabetic complications. This review will summarise some highlights from the published work in which this hypothesis is discussed.

Insulin and metabolic stress stimulate multisite serine/threonine phosphorylation of insulin receptor substrate 1 and inhibit tyrosine phosphorylation.

IRS1 and IRS2 are key substrates of the insulin receptor tyrosine kinase. Mass spectrometry reveals more than 50 phosphorylated IRS1 serine and threonine residues (Ser(P)/Thr(P) residues) in IRS1 from insulin-stimulated cells or human tissues. We investigated a subset of IRS1 Ser(P)/Thr(P) residues using a newly developed panel of 25 phospho-specific monoclonal antibodies (αpS/TmAb(Irs1)). CHO cells overexpressing the human insulin receptor and rat IRS1 were stimulated with insulin in the absence or presence of inhibitors of the PI3K → Akt → mechanistic target of rapamycin (mTOR) → S6 kinase or MEK pathways. Nearly all IRS1 Ser(P)/Thr(P) residues were stimulated by insulin and significantly suppressed by PI3K inhibition; fewer were suppressed by Akt or mTOR inhibition, and none were suppressed by MEK inhibition. Insulin-stimulated Irs1 tyrosine phosphorylation (Tyr(P)(Irs1)) was enhanced by inhibition of the PI3K → Akt → mTOR pathway and correlated with decreased Ser(P)-302(Irs1), Ser(P)-307(Irs1), Ser(P)-318(Irs1), Ser(P)-325(Irs1), and Ser(P)-346(Irs1). Metabolic stress modeled by anisomycin, thapsigargin, or tunicamycin increased many of the same Ser(P)/Thr(P) residues as insulin, some of which (Ser(P)-302(Irs1), Ser(P)-307(Irs1), and four others) correlated significantly with impaired insulin-stimulated Tyr(P)(Irs1). Thus, IRS1 Ser(P)/Thr(P) is an integrated response to insulin stimulation and metabolic stress, which associates with reduced Tyr(P)(Irs1) in CHO(IR)/IRS1 cells.

Nerve growth factor receptor TrkA, a new receptor in insulin signaling pathway in PC12 cells.

TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.

IRS1Ser³°7 phosphorylation does not mediate mTORC1-induced insulin resistance.

Increased mammalian target of rapamycin complex 1 (mTORC1) activity has been suggested to play important roles in development of insulin resistance in obesity. mTORC1 hyperactivity also increases endoplasmic reticulum (ER) stress, which in turn contributes to development of insulin resistance and glucose intolerance. Increased IRS1 phosphorylation at Ser307 in vitro is correlated with mTORC1- and ER stress-induced insulin resistance. This phosphorylation site correlates strongly with impaired insulin receptor signaling in diabetic mice and humans. In contrast, evidence from knock-in mice suggests that phosphorylation of IRS1 at Ser307 is actually required to maintain insulin sensitivity. To study the involvement of IRS1(Ser307) phosphorylation in mTORC1-mediated glucose intolerance and insulin sensitivity in vivo, we investigated the effects of liver specific TSC1 depletion in IRS1(Ser307Ala) mice and controls. Our results demonstrate that blockade of IRS1(Ser307) phosphorylation in vivo does not prevent mTORC1-mediated glucose intolerance and insulin resistance.

Inhibition of insulin signaling in endothelial cells by protein kinase C-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase (PI3K).

The regulation of endothelial function by insulin is consistently abnormal in insulin-resistant states and diabetes. Protein kinase C (PKC) activation has been reported to inhibit insulin signaling selectively in endothelial cells via the insulin receptor substrate/PI3K/Akt pathway to reduce the activation of endothelial nitric-oxide synthase (eNOS). In this study, it was observed that PKC activation differentially inhibited insulin receptor substrate 1/2 (IRS1/2) signaling of insulin's activation of PI3K/eNOS by decreasing only tyrosine phosphorylation of IRS2. In addition, PKC activation, by general activator and specifically by angiotensin II, increased the phosphorylation of p85/PI3K, which decreases its association with IRS1 and activation. Thr-86 of p85/PI3K was identified to be phosphorylated by PKC activation and confirmed to affect IRS1-mediated activation of Akt/eNOS by insulin and VEGF using a deletion mutant of the Thr-86 region of p85/PI3K. Thus, PKC and angiotensin-induced phosphorylation of Thr-86 of p85/PI3K may partially inhibit the activation of PI3K/eNOS by multiple cytokines and contribute to endothelial dysfunction in metabolic disorders.

IRS-2 Deficiency impairs NMDA receptor-dependent long-term potentiation.

The beneficial effects of insulin and insulin-like growth factor I on cognition have been documented in humans and animal models. Conversely, obesity, hyperinsulinemia, and diabetes increase the risk for neurodegenerative disorders including Alzheimer's disease (AD). However, the mechanisms by which insulin regulates synaptic plasticity are not well understood. Here, we report that complete disruption of insulin receptor substrate 2 (Irs2) in mice impairs long-term potentiation (LTP) of synaptic transmission in the hippocampus. Basal synaptic transmission and paired-pulse facilitation were similar between the 2 groups of mice. Induction of LTP by high-frequency conditioning tetanus did not activate postsynaptic N-methyl-D-aspartate (NMDA) receptors in hippocampus slices from Irs2(-/-) mice, although the expression of NR2A, NR2B, and PSD95 was equivalent to wild-type controls. Activation of Fyn, AKT, and MAPK in response to tetanus stimulation was defective in Irs2(-/-) mice. Interestingly, IRS2 was phosphorylated during induction of LTP in control mice, revealing a potential new component of the signaling machinery which modulates synaptic plasticity. Given that IRS2 expression is diminished in Type 2 diabetics as well as in AD patients, these data may reveal an explanation for the prevalence of cognitive decline in humans with metabolic disorders by providing a mechanistic link between insulin resistance and impaired synaptic transmission.

The role of hepatokines in NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is not only a consequence of insulin resistance, but it is also an important cause of insulin resistance and major non-communicable diseases (NCDs). The close relationship of NAFLD with visceral obesity obscures the role of fatty liver from visceral adiposity as the main pathomechanism of insulin resistance and NCDs. To overcome this limitation, in analogy to the concept of adipokines, in 2008 we introduced the term hepatokines to describe the role of fetuin-A in metabolism. Since then, several other hepatokines were tested for their effects on metabolism. Here we address the dysregulation of hepatokines in people with NAFLD. Then, we discuss pathophysiological mechanisms of cardiometabolic diseases specifically related to NAFLD by focusing on hepatokine-related organ crosstalk. Finally, we propose how the determination of major hepatokines and adipokines can be used for pathomechanism-based clustering of insulin resistance in NAFLD and visceral obesity to better implement precision medicine in clinical practice.

Hepatic follistatin increases basal metabolic rate and attenuates diet-induced obesity during hepatic insulin resistance.

OBJECTIVE: Body weight change and obesity follow the variance of excess energy input balanced against tightly controlled EE (energy expenditure). Since insulin resistance can reduce energy storage, we investigated whether genetic disruption of hepatic insulin signaling reduced adipose mass with increased EE. METHODS: Insulin signaling was disrupted by genetic inactivation of Irs1 (Insulin receptor substrate 1) and Irs2 in hepatocytes of LDKO mice (Irs1L/L·Irs2L/L·CreAlb), creating a state of complete hepatic insulin resistance. We inactivated FoxO1 or the FoxO1-regulated hepatokine Fst (Follistatin) in the liver of LDKO mice by intercrossing LDKO mice with FoxO1L/L or FstL/L mice. We used DEXA (dual-energy X-ray absorptiometry) to assess total lean mass, fat mass and fat percentage, and metabolic cages to measure EE (energy expenditure) and estimate basal metabolic rate (BMR). High-fat diet was used to induce obesity. RESULTS: Hepatic disruption of Irs1 and Irs2 (LDKO mice) attenuated HFD (high-fat diet)-induced obesity and increased whole-body EE in a FoxO1-dependent manner. Hepatic disruption of the FoxO1-regulated hepatokine Fst normalized EE in LDKO mice and restored adipose mass during HFD consumption; moreover, hepatic Fst disruption alone increased fat mass accumulation, whereas hepatic overexpression of Fst reduced HFD-induced obesity. Excess circulating Fst in overexpressing mice neutralized Mstn (Myostatin), activating mTORC1-promoted pathways of nutrient uptake and EE in skeletal muscle. Similar to Fst overexpression, direct activation of muscle mTORC1 also reduced adipose mass. CONCLUSIONS: Thus, complete hepatic insulin resistance in LDKO mice fed a HFD revealed Fst-mediated communication between the liver and muscle, which might go unnoticed during ordinary hepatic insulin resistance as a mechanism to increase muscle EE and constrain obesity.

BRD7 improves glucose homeostasis independent of IRS proteins.

Bromodomain-containing protein 7 (BRD7) has emerged as a player in the regulation of glucose homeostasis. Hepatic BRD7 levels are decreased in obese mice, and the reinstatement of hepatic BRD7 in obese mice has been shown to establish euglycemia and improve glucose homeostasis. Of note, the upregulation of hepatic BRD7 levels activates the AKT cascade in response to insulin without enhancing the sensitivity of the insulin receptor (InsR)-insulin receptor substrate (IRS) axis. In this report, we provide evidence for the existence of an alternative insulin signaling pathway that operates independently of IRS proteins and demonstrate the involvement of BRD7 in this pathway. To investigate the involvement of BRD7 as a downstream component of InsR, we utilized liver-specific InsR knockout mice. Additionally, we employed liver-specific IRS1/2 knockout mice to examine the requirement of IRS1/2 for the action of BRD7. Our investigation of glucose metabolism parameters and insulin signaling unveiled the significance of InsR activation in mediating BRD7's effect on glucose homeostasis in the liver. Moreover, we identified an interaction between BRD7 and InsR. Notably, our findings indicate that IRS1/2 is not necessary for BRD7's regulation of glucose metabolism, particularly in the context of obesity. The upregulation of hepatic BRD7 significantly reduces blood glucose levels and restores glucose homeostasis in high-fat diet-challenged liver-specific IRS1/2 knockout mice. These findings highlight the presence of an alternative insulin signaling pathway that operates independently of IRS1/2 and offer novel insights into the mechanisms of a previously unknown insulin signaling in obesity.