Complex physiology and clinical implications of time-restricted eating.

Time-restricted eating (TRE) is a dietary intervention that limits food consumption to a specific time window each day. The effect of TRE on body weight and physiological functions has been extensively studied in rodent models, which have shown considerable therapeutic effects of TRE and important interactions among time of eating, circadian biology, and metabolic homeostasis. In contrast, it is difficult to make firm conclusions regarding the effect of TRE in people because of the heterogeneity in results, TRE regimens, and study populations. In this review, we 1) provide a background of the history of meal consumption in people and the normal physiology of eating and fasting; 2) discuss the interaction between circadian molecular metabolism and TRE; 3) integrate the results of preclinical and clinical studies that evaluated the effects of TRE on body weight and physiological functions; 4) summarize other time-related dietary interventions that have been studied in people; and 4) identify current gaps in knowledge and provide a framework for future research directions.

Mechanisms of Insulin Action and Insulin Resistance.

The 1921 discovery of insulin was a Big Bang from which a vast and expanding universe of research into insulin action and resistance has issued. In the intervening century, some discoveries have matured, coalescing into solid and fertile ground for clinical application; others remain incompletely investigated and scientifically controversial. Here, we attempt to synthesize this work to guide further mechanistic investigation and to inform the development of novel therapies for type 2 diabetes (T2D). The rational development of such therapies necessitates detailed knowledge of one of the key pathophysiological processes involved in T2D: insulin resistance. Understanding insulin resistance, in turn, requires knowledge of normal insulin action. In this review, both the physiology of insulin action and the pathophysiology of insulin resistance are described, focusing on three key insulin target tissues: skeletal muscle, liver, and white adipose tissue. We aim to develop an integrated physiological perspective, placing the intricate signaling effectors that carry out the cell-autonomous response to insulin in the context of the tissue-specific functions that generate the coordinated organismal response. First, in section II, the effectors and effects of direct, cell-autonomous insulin action in muscle, liver, and white adipose tissue are reviewed, beginning at the insulin receptor and working downstream. Section III considers the critical and underappreciated role of tissue crosstalk in whole body insulin action, especially the essential interaction between adipose lipolysis and hepatic gluconeogenesis. The pathophysiology of insulin resistance is then described in section IV. Special attention is given to which signaling pathways and functions become insulin resistant in the setting of chronic overnutrition, and an alternative explanation for the phenomenon of ?selective hepatic insulin resistanceË® is presented. Sections V, VI, and VII critically examine the evidence for and against several putative mediators of insulin resistance. Section V reviews work linking the bioactive lipids diacylglycerol, ceramide, and acylcarnitine to insulin resistance; section VI considers the impact of nutrient stresses in the endoplasmic reticulum and mitochondria on insulin resistance; and section VII discusses non-cell autonomous factors proposed to induce insulin resistance, including inflammatory mediators, branched-chain amino acids, adipokines, and hepatokines. Finally, in section VIII, we propose an integrated model of insulin resistance that links these mediators to final common pathways of metabolite-driven gluconeogenesis and ectopic lipid accumulation.

Decreased sarcoplasmic reticulum phospholipids in human skeletal muscle are associated with metabolic syndrome.

Metabolic syndrome affects more than one in three adults and is associated with increased risk of diabetes, cardiovascular disease, and all-cause mortality. Muscle insulin resistance is a major contributor to the development of the metabolic syndrome. Studies in mice have linked skeletal muscle sarcoplasmic reticulum (SR) phospholipid composition to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase activity and insulin sensitivity. To determine if the presence of metabolic syndrome alters specific phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species in human SR, we compared SR phospholipid composition in skeletal muscle from sedentary subjects with metabolic syndrome and sedentary control subjects without metabolic syndrome. Both total PC and total PE were significantly decreased in skeletal muscle SR of sedentary metabolic syndrome patients compared with sedentary controls, particularly in female participants, but there was no difference in the PC:PE ratio between groups. Total SR PC levels, but not total SR PE levels or PC:PE ratio, were significantly negatively correlated with BMI, waist circumference, total fat, visceral adipose tissue, triglycerides, fasting insulin, and homeostatic model assessment for insulin resistance. These findings are consistent with the existence of a relationship between skeletal muscle SR PC content and insulin resistance in humans.

Adaptor protein Abelson interactor 1 in homeostasis and disease.

Dysregulation of Abelson interactor 1 (ABI1) is associated with various states of disease including developmental defects, pathogen infections, and cancer. ABI1 is an adaptor protein predominantly known to regulate actin cytoskeleton organization processes such as those involved in cell adhesion, migration, and shape determination. Linked to cytoskeleton via vasodilator-stimulated phosphoprotein (VASP), Wiskott-Aldrich syndrome protein family (WAVE), and neural-Wiskott-Aldrich syndrome protein (N-WASP)-associated protein complexes, ABI1 coordinates regulation of various cytoplasmic protein signaling complexes dysregulated in disease states. The roles of ABI1 beyond actin cytoskeleton regulation are much less understood. This comprehensive, protein-centric review describes molecular roles of ABI1 as an adaptor molecule in the context of its dysregulation and associated disease outcomes to better understand disease state-specific protein signaling and affected interconnected biological processes.

Transcriptomics of acute myeloid leukaemia core bone marrow biopsies reveals distinct therapy response-specific osteo-mesenchymal profiles.

While the bone marrow (BM) microenvironment is significantly remodelled in acute myeloid leukaemia (AML), molecular insight into AML-specific alterations in the microenvironment has been historically limited by the analysis of liquid marrow aspirates rather than core biopsies that contain solid-phase BM stroma. We assessed the effect of anthracycline- and cytarabine-based induction chemotherapy on both haematopoietic and non-haematopoietic cells directly in core BM biopsies using RNA-seq and histological analysis. We compared matched human core BM biopsies at diagnosis and 2 weeks after cytarabine- and anthracycline-based induction therapy in responders (<5% blasts present after treatment) and non-responders (≥5% blasts present after treatment). Our data indicated enrichment in vimentin (VIM), platelet-derived growth factor receptor beta (PDGFRB) and Snail family transcriptional repressor 2 (SNAI2) transcripts in responders, consistent with the reactivation of the mesenchymal population in the BM stroma. Enrichment of osteoblast maturation-related transcripts of biglycan (BGN), osteopontin (SPP1) and osteonectin (SPARC) was observed in non-responders. To the best of our knowledge, this is the first report demonstrating distinct osteogenic and mesenchymal transcriptome profiles specific to AML response to induction chemotherapy assessed directly in core BM biopsies. Detailing treatment response-specific alterations in the BM stroma may inform optimised therapeutic strategies for AML.

Metabolic control analysis of hepatic glycogen synthesis in vivo.

Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK)-mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC)-fed male rats (control), 2) high fat diet (HFD)-fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.

Mass spectrometry-based proteomic platforms for better understanding of SARS-CoV-2 induced pathogenesis and potential diagnostic approaches.

While protein-protein interaction is the first step of the SARS-CoV-2 infection, recent comparative proteomic profiling enabled the identification of over 11,000 protein dynamics, thus providing a comprehensive reflection of the molecular mechanisms underlying the cellular system in response to viral infection. Here we summarize and rationalize the results obtained by various mass spectrometry (MS)-based proteomic approaches applied to the functional characterization of proteins and pathways associated with SARS-CoV-2-mediated infections in humans. Comparative analysis of cell-lines versus tissue samples indicates that our knowledge in proteome profile alternation in response to SARS-CoV-2 infection is still incomplete and the tissue-specific response to SARS-CoV-2 infection can probably not be recapitulated efficiently by in vitro experiments. However, regardless of the viral infection period, sample types, and experimental strategies, a thorough cross-comparison of the recently published proteome, phosphoproteome, and interactome datasets led to the identification of a common set of proteins and kinases associated with PI3K-Akt, EGFR, MAPK, Rap1, and AMPK signaling pathways. Ephrin receptor A2 (EPHA2) was identified by 11 studies including all proteomic platforms, suggesting it as a potential future target for SARS-CoV-2 infection mechanisms and the development of new therapeutic strategies. We further discuss the potentials of future proteomics strategies for identifying prognostic SARS-CoV-2 responsive age-, gender-dependent, tissue-specific protein targets.

PKCε contributes to lipid-induced insulin resistance through cross talk with p70S6K and through previously unknown regulators of insulin signaling.

Insulin resistance drives the development of type 2 diabetes (T2D). In liver, diacylglycerol (DAG) is a key mediator of lipid-induced insulin resistance. DAG activates protein kinase C ε (PKCε), which phosphorylates and inhibits the insulin receptor. In rats, a 3-day high-fat diet produces hepatic insulin resistance through this mechanism, and knockdown of hepatic PKCε protects against high-fat diet-induced hepatic insulin resistance. Here, we employed a systems-level approach to uncover additional signaling pathways involved in high-fat diet-induced hepatic insulin resistance. We used quantitative phosphoproteomics to map global in vivo changes in hepatic protein phosphorylation in chow-fed, high-fat-fed, and high-fat-fed with PKCε knockdown rats to distinguish the impact of lipid- and PKCε-induced protein phosphorylation. This was followed by a functional siRNA-based screen to determine which dynamically regulated phosphoproteins may be involved in canonical insulin signaling. Direct PKCε substrates were identified by motif analysis of phosphoproteomics data and validated using a large-scale in vitro kinase assay. These substrates included the p70S6K substrates RPS6 and IRS1, which suggested cross talk between PKCε and p70S6K in high-fat diet-induced hepatic insulin resistance. These results identify an expanded set of proteins through which PKCε may drive high-fat diet-induced hepatic insulin resistance that may direct new therapeutic approaches for T2D.

Bone marrow-specific loss of ABI1 induces myeloproliferative neoplasm with features resembling human myelofibrosis.

Although the pathogenesis of primary myelofibrosis (PMF) and other myeloproliferative neoplasms (MPNs) is linked to constitutive activation of the JAK-STAT pathway, JAK inhibitors have neither curative nor MPN-stem cell-eradicating potential, indicating that other targetable mechanisms are contributing to the pathophysiology of MPNs. We previously demonstrated that Abelson interactor 1 (Abi-1), a negative regulator of Abelson kinase 1, functions as a tumor suppressor. Here we present data showing that bone marrow-specific deletion of Abi1 in a novel mouse model leads to development of an MPN-like phenotype resembling human PMF. Abi1 loss resulted in a significant increase in the activity of the Src family kinases (SFKs), STAT3, and NF-κB signaling. We also observed impairment of hematopoietic stem cell self-renewal and fitness, as evidenced in noncompetitive and competitive bone marrow transplant experiments. CD34+ hematopoietic progenitors and granulocytes from patients with PMF showed decreased levels of ABI1 transcript as well as increased activity of SFKs, STAT3, and NF-κB. In aggregate, our data link the loss of Abi-1 function to hyperactive SFKs/STAT3/NF-κB signaling and suggest that this signaling axis may represent a regulatory module involved in the molecular pathophysiology of PMF.

Biological nitrogen fixation in maize: optimizing nitrogenase expression in a root-associated diazotroph.

Plants depend upon beneficial interactions between roots and root-associated microorganisms for growth promotion, disease suppression, and nutrient availability. This includes the ability of free-living diazotrophic bacteria to supply nitrogen, an ecological role that has been long underappreciated in modern agriculture for efficient crop production systems. Long-term ecological studies in legume-rhizobia interactions have shown that elevated nitrogen inputs can lead to the evolution of less cooperative nitrogen-fixing mutualists. Here we describe how reprogramming the genetic regulation of nitrogen fixation and assimilation in a novel root-associated diazotroph can restore ammonium production in the presence of exogenous nitrogen inputs. We isolated a strain of the plant-associated proteobacterium Kosakonia sacchari from corn roots, characterized its nitrogen regulatory network, and targeted key nodes for gene editing to optimize nitrogen fixation in corn. While the wild-type strain exhibits repression of nitrogen fixation in conditions replete with bioavailable nitrogen, such as fertilized greenhouse and field experiments, remodeled strains show elevated levels in the rhizosphere of corn in the greenhouse and field even in the presence of exogenous nitrogen. Such strains could be used in commercial applications to supply fixed nitrogen to cereal crops.

Usp25m protease regulates ubiquitin-like processing of TUG proteins to control GLUT4 glucose transporter translocation in adipocytes.

Insulin stimulates the exocytic translocation of specialized vesicles in adipocytes, which inserts GLUT4 glucose transporters into the plasma membrane to enhance glucose uptake. Previous results support a model in which TUG (Tether containing a UBX domain for GLUT4) proteins trap these GLUT4 storage vesicles at the Golgi matrix and in which insulin triggers endoproteolytic cleavage of TUG to translocate GLUT4. Here, we identify the muscle splice form of Usp25 (Usp25m) as a protease required for insulin-stimulated TUG cleavage and GLUT4 translocation in adipocytes. Usp25m is expressed in adipocytes, binds TUG and GLUT4, dissociates from TUG-bound vesicles after insulin addition, and colocalizes with TUG and insulin-responsive cargoes in unstimulated cells. Previous results show that TUG proteolysis generates the ubiquitin-like protein, TUGUL (for TUGubiquitin-like). We now show that TUGUL modifies the kinesin motor protein, KIF5B, and that TUG proteolysis is required to load GLUT4 onto these motors. Insulin stimulates TUG proteolytic processing independently of phosphatidylinositol 3-kinase. In nonadipocytes, TUG cleavage can be reconstituted by transfection of Usp25m, but not the related Usp25a isoform, together with other proteins present on GLUT4 vesicles. In rodents with diet-induced insulin resistance, TUG proteolysis and Usp25m protein abundance are reduced in adipose tissue. These effects occur soon after dietary manipulation, prior to the attenuation of insulin signaling to Akt. Together with previous data, these results support a model whereby insulin acts through Usp25m to mediate TUG cleavage, which liberates GLUT4 storage vesicles from the Golgi matrix and activates their microtubule-based movement to the plasma membrane. This TUG proteolytic pathway for insulin action is independent of Akt and is impaired by nutritional excess.

Anti-myostatin antibody increases muscle mass and strength and improves insulin sensitivity in old mice.

Sarcopenia, or skeletal muscle atrophy, is a debilitating comorbidity of many physiological and pathophysiological processes, including normal aging. There are no approved therapies for sarcopenia, but the antihypertrophic myokine myostatin is a potential therapeutic target. Here, we show that treatment of young and old mice with an anti-myostatin antibody (ATA 842) for 4 wk increased muscle mass and muscle strength in both groups. Furthermore, ATA 842 treatment also increased insulin-stimulated whole body glucose metabolism in old mice, which could be attributed to increased insulin-stimulated skeletal muscle glucose uptake as measured by a hyperinsulinemic-euglycemic clamp. Taken together, these studies provide support for pharmacological inhibition of myostatin as a potential therapeutic approach for age-related sarcopenia and metabolic disease.

Insulin-independent regulation of hepatic triglyceride synthesis by fatty acids.

A central paradox in type 2 diabetes is the apparent selective nature of hepatic insulin resistance--wherein insulin fails to suppress hepatic glucose production yet continues to stimulate lipogenesis, resulting in hyperglycemia, hyperlipidemia, and hepatic steatosis. Although efforts to explain this have focused on finding a branch point in insulin signaling where hepatic glucose and lipid metabolism diverge, we hypothesized that hepatic triglyceride synthesis could be driven by substrate, independent of changes in hepatic insulin signaling. We tested this hypothesis in rats by infusing [U-(13)C] palmitate to measure rates of fatty acid esterification into hepatic triglyceride while varying plasma fatty acid and insulin concentrations independently. These experiments were performed in normal rats, high fat-fed insulin-resistant rats, and insulin receptor 2'-O-methoxyethyl chimeric antisense oligonucleotide-treated rats. Rates of fatty acid esterification into hepatic triglyceride were found to be dependent on plasma fatty acid infusion rates, independent of changes in plasma insulin concentrations and independent of hepatocellular insulin signaling. Taken together, these results obviate a paradox of selective insulin resistance, because the major source of hepatic lipid synthesis, esterification of preformed fatty acids, is primarily dependent on substrate delivery and largely independent of hepatic insulin action.

Genetic activation of pyruvate dehydrogenase alters oxidative substrate selection to induce skeletal muscle insulin resistance.

The pyruvate dehydrogenase complex (PDH) has been hypothesized to link lipid exposure to skeletal muscle insulin resistance through a glucose-fatty acid cycle in which increased fatty acid oxidation increases acetyl-CoA concentrations, thereby inactivating PDH and decreasing glucose oxidation. However, whether fatty acids induce insulin resistance by decreasing PDH flux remains unknown. To genetically examine this hypothesis we assessed relative rates of pyruvate dehydrogenase flux/mitochondrial oxidative flux and insulin-stimulated rates of muscle glucose metabolism in awake mice lacking pyruvate dehydrogenase kinase 2 and 4 [double knockout (DKO)], which results in constitutively activated PDH. Surprisingly, increased glucose oxidation in DKO muscle was accompanied by reduced insulin-stimulated muscle glucose uptake. Preferential myocellular glucose utilization in DKO mice decreased fatty acid oxidation, resulting in increased reesterification of acyl-CoAs into diacylglycerol and triacylglycerol, with subsequent activation of PKC-θ and inhibition of insulin signaling in muscle. In contrast, other putative mediators of muscle insulin resistance, including muscle acylcarnitines, ceramides, reactive oxygen species production, and oxidative stress markers, were not increased. These findings demonstrate that modulation of oxidative substrate selection to increase muscle glucose utilization surprisingly results in muscle insulin resistance, offering genetic evidence against the glucose-fatty acid cycle hypothesis of muscle insulin resistance.

Imeglimin lowers glucose primarily by amplifying glucose-stimulated insulin secretion in high-fat-fed rodents.

Imeglimin is a promising new oral antihyperglycemic agent that has been studied in clinical trials as a possible monotherapy or add-on therapy to lower fasting plasma glucose and improve hemoglobin A1c (1-3, 9). Imeglimin was shown to improve both fasting and postprandial glycemia and to increase insulin secretion in response to glucose during a hyperglycemic clamp after 1-wk of treatment in type 2 diabetic patients. However, whether the ß-cell stimulatory effect of imeglimin is solely or partially responsible for its effects on glycemia remains to be fully confirmed. Here, we show that imeglimin directly activates ß-cell insulin secretion in awake rodents without affecting hepatic insulin sensitivity, body composition, or energy expenditure. These data identify a primary amplification rather than trigger the ß-cell mechanism that explains the acute, antidiabetic activity of imeglimin.

Reduced intestinal lipid absorption and body weight-independent improvements in insulin sensitivity in high-fat diet-fed Park2 knockout mice.

Mitochondrial dysfunction is associated with many human diseases and results from mismatch of damage and repair over the life of the organelle. PARK2 is a ubiquitin E3 ligase that regulates mitophagy, a repair mechanism that selectively degrades damaged mitochondria. Deletion of PARK2 in multiple in vivo models results in susceptibility to stress-induced mitochondrial and cellular dysfunction. Surprisingly, Park2 knockout (KO) mice are protected from nutritional stress and do not develop obesity, hepatic steatosis or insulin resistance when fed a high-fat diet (HFD). However, these phenomena are casually related and the physiological basis for this phenotype is unknown. We therefore undertook a series of acute HFD studies to more completely understand the physiology of Park2 KO during nutritional stress. We find that intestinal lipid absorption is impaired in Park2 KO mice as evidenced by increased fecal lipids and reduced plasma triglycerides after intragastric fat challenge. Park2 KO mice developed hepatic steatosis in response to intravenous lipid infusion as well as during incubation of primary hepatocytes with fatty acids, suggesting that hepatic protection from nutritional stress was secondary to changes in energy balance due to altered intestinal triglyceride absorption. Park2 KO mice showed reduced adiposity after 1-wk HFD, as well as improved hepatic and peripheral insulin sensitivity. These studies suggest that changes in intestinal lipid absorption may play a primary role in protection from nutritional stress in Park2 KO mice by preventing HFD-induced weight gain and highlight the need for tissue-specific models to address the role of PARK2 during metabolic stress.

ApoA5 knockdown improves whole-body insulin sensitivity in high-fat-fed mice by reducing ectopic lipid content.

ApoA5 has a critical role in the regulation of plasma TG concentrations. In order to determine whether ApoA5 also impacts ectopic lipid deposition in liver and skeletal muscle, as well as tissue insulin sensitivity, we treated mice with an antisense oligonucleotide (ASO) to decrease hepatic expression of ApoA5. ASO treatment reduced ApoA5 protein expression in liver by 60-70%. ApoA5 ASO-treated mice displayed approximately 3-fold higher plasma TG concentrations, which were associated with decreased plasma TG clearance. Furthermore, ApoA5 ASO-treated mice fed a high-fat diet (HFD) exhibited reduced liver and skeletal muscle TG uptake and reduced liver and muscle TG and diacylglycerol (DAG) content. HFD-fed ApoA5 ASO-treated mice were protected from HFD-induced insulin resistance, as assessed by hyperinsulinemic-euglycemic clamps. This protection could be attributed to increases in both hepatic and peripheral insulin responsiveness associated with decreased DAG activation of protein kinase C (PKC)-ε and PKCθ in liver and muscle, respectively, and increased insulin-stimulated AKT2 pho-sphory-lation in these tissues. In summary, these studies demonstrate a novel role for ApoA5 as a modulator of susceptibility to diet-induced liver and muscle insulin resistance through regulation of ectopic lipid accumulation in liver and skeletal muscle.

Targeting steroid receptor coactivator 1 with antisense oligonucleotides increases insulin-stimulated skeletal muscle glucose uptake in chow-fed and high-fat-fed male rats.

The steroid receptor coactivator 1 (SRC1) regulates key metabolic pathways, including glucose homeostasis. SRC1(-/-) mice have decreased hepatic expression of gluconeogenic enzymes and a reduction in the rate of endogenous glucose production (EGP). We sought to determine whether decreasing hepatic and adipose SRC1 expression in normal adult rats would alter glucose homeostasis and insulin action. Regular chow-fed and high-fat-fed male Sprage-Dawley rats were treated with an antisense oligonucleotide (ASO) against SRC1 or a control ASO for 4 wk, followed by metabolic assessments. SRC1 ASO did not alter basal EGP or expression of gluconeogenic enzymes. Instead, SRC1 ASO increased insulin-stimulated whole body glucose disposal by ~30%, which was attributable largely to an increase in insulin-stimulated muscle glucose uptake. This was associated with an approximately sevenfold increase in adipose expression of lipocalin-type prostaglandin D2 synthase, a previously reported regulator of insulin sensitivity, and an approximately 70% increase in plasma PGD2 concentration. Muscle insulin signaling, AMPK activation, and tissue perfusion were unchanged. Although GLUT4 content was unchanged, SRC1 ASO increased the cleavage of tether-containing UBX domain for GLUT4, a regulator of GLUT4 translocation. These studies point to a novel role of adipose SRC1 as a regulator of insulin-stimulated muscle glucose uptake.