The ability of metabolically active tissues to increase glucose uptake in response to insulin is critical to whole-body glucose homeostasis. This report describes the Dual Tracer Test, a robust method involving sequential retro-orbital injection of [14C]2-deoxyglucose ([14C]2DG) alone, followed 40 min later by injection of [3H]2DG with a maximal dose of insulin to quantify both basal and insulin-stimulated 2DG uptake in the same mouse. The collection of both basal and insulin-stimulated measures from a single animal is imperative for generating high-quality data since differences in insulin action may be misinterpreted mechanistically if basal glucose uptake is not accounted for. The approach was validated in a classic diet-induced model of insulin resistance and a novel transgenic mouse with reduced GLUT4 expression that, despite ubiquitous peripheral insulin resistance, did not exhibit fasting hyperinsulinemia. This suggests that reduced insulin-stimulated glucose disposal is not a primary contributor to chronic hyperinsulinemia. The Dual Tracer Test offers a technically simple assay that enables the study of insulin action in many tissues simultaneously. By administering two tracers and accounting for both basal and insulin-stimulated glucose transport, this assay halves the required sample size for studies in inbred mice and demonstrates increased statistical power to detect insulin resistance, relative to other established approaches, using a single tracer. The Dual Tracer Test is a valuable addition to the metabolic phenotyping toolbox.

Despite the fact that genes and the environment are known to play a central role in islet function, our knowledge of how these parameters interact to modulate insulin secretory function remains relatively poor. Presently, we performed ex vivo glucose-stimulated insulin secretion and insulin content assays in islets of 213 mice from 13 inbred mouse strains on chow, Western diet (WD), and a high-fat, carbohydrate-free (KETO) diet. Strikingly, among these 13 strains, islets from the commonly used C57BL/6J mouse strain were the least glucose responsive. Using matched metabolic phenotyping data, we performed correlation analyses of isolated islet parameters and found a positive correlation between basal and glucose-stimulated insulin secretion, but no relationship between insulin secretion and insulin content. Using in vivo metabolic measures, we found that glucose tolerance determines the relationship between ex vivo islet insulin secretion and plasma insulin levels. Finally, we showed that islet glucose-stimulated insulin secretion decreased with KETO in almost all strains, concomitant with broader phenotypic changes, such as increased adiposity and glucose intolerance. This is an important finding as it should caution against the application of KETO diet for beta-cell health. Together these data offer key insights into the intersection of diet and genetic background on islet function and whole body glucose metabolism.

Adipocyte lipolysis is tightly regulated to provide fatty acids when needed and this is suppressed postprandially by insulin. This regulation remains incompletely understood. We have identified the abhydrolase ABHD15 as a key player in this process together with the phosphodiesterase PDE3B. In the absence of either ABHD15 or PDE3B, insulin is not able to suppress lipolysis. ABHD15 and PDE3B form a complex and in response to insulin they are both phosphorylated by Akt and bind to the adaptor protein 14-3-3. Insertion of a constitutive 14-3-3 binding site into either ABHD15 or PDE3B results in complete suppression of lipolysis even under pro-lipolytic conditions. Notably, we have discovered that the kinase Akt interacts with ABHD15 and insulin and/or 14-3-3 binding to ABHD15 abolishes the interaction. In summary we propose a model where ABHD15 binds to and suppresses the phosphodiesterase activity of PDE3B in the absence of insulin thereby facilitating unrestrained lipolysis and supply of fatty acids during starvation or crisis. Akt directly interacts with the ABHD15/PDE3B complex leading to phosphorylation and 14-3-3 binding upon insulin stimulation, which activates a pool of PDE3B thus repressing PKA and the lipolytic drive. Disclosure J. Stoeckli: Research Support; Pfizer Inc. A. Zadoorian: None. G. Yang: None. D. E. James: Research Support; Eli Lilly and Company, Novo Nordisk, Pfizer Inc. Funding Pfizer Inc.

Abstract Genetic and environmental factors play a major role in metabolic health. However, they do not act in isolation, as a change in an environmental factor such as diet may exert different effects based on an individual’s genotype. Here, we sought to understand how such gene–diet interactions influenced nutrient storage and utilization, a major determinant of metabolic disease. We subjected 178 inbred strains from the Drosophila genetic reference panel (DGRP) to diets varying in sugar, fat, and protein. We assessed starvation resistance, a holistic phenotype of nutrient storage and utilization that can be robustly measured. Diet influenced the starvation resistance of most strains, but the effect varied markedly between strains such that some displayed better survival on a high carbohydrate diet (HCD) compared to a high-fat diet while others had opposing responses, illustrating a considerable gene × diet interaction. This demonstrates that genetics plays a major role in diet responses. Furthermore, heritability analysis revealed that the greatest genetic variability arose from diets either high in sugar or high in protein. To uncover the genetic variants that contribute to the heterogeneity in starvation resistance, we mapped 566 diet-responsive SNPs in 293 genes, 174 of which have human orthologs. Using whole-body knockdown, we identified two genes that were required for glucose tolerance, storage, and utilization. Strikingly, flies in which the expression of one of these genes, CG4607 a putative homolog of a mammalian glucose transporter, was reduced at the whole-body level, displayed lethality on a HCD. This study provides evidence that there is a strong interplay between diet and genetics in governing survival in response to starvation, a surrogate measure of nutrient storage efficiency and obesity. It is likely that a similar principle applies to higher organisms thus supporting the case for nutrigenomics as an important health strategy.

Lipolysis is a tightly regulated catabolic process essential for maintaining whole‐body energy homeostasis. It exists in a dynamic balance between activation by β‐adrenergic stimulation during starvation or prolonged exercise, and inhibition by insulin following nutrient intake. Dysregulation of lipolysis can lead to ectopic fat accumulation in the blood and insulin‐sensitive tissues, encouraging the development of metabolic disorders such as type 2 diabetes and non‐alcoholic fatty liver disease. Despite this, the mechanism by which insulin regulates lipolysis remains controversial and incomplete. The prevailing model hinges on the enzymatic function of the Akt substrate, phosphodiesterase 3B (PDE3B) in explaining insulin’s antilipolytic action. However, several studies have challenged this and suggested that whilst PDE3B is essential, its function alone cannot explain this regulation. Instead, we implicate another protein, α/β‐hydrolase domain‐containing protein 15 (ABHD15) as a novel player in insulin‐mediated lipolysis inhibition. Through stable knockdown or knockout of ABHD15 in 3T3‐L1, brown or fat explant adipocytes, we demonstrate a significant defect in insulin’s inhibition of lipolysis that is rescued upon ABHD15 re‐expression. Moreover, through the characterisation of ABHD15 and PDE3B mutants lacking novel insulin‐regulated phosphorylation sites previously identified by our lab, we have explored a putative mechanism by which these proteins facilitate insulin’s inhibition of lipolysis. In summary, we have identified a novel regulator of lipolysis that will likely shed light on the mechanism of this important process.Support or Funding InformationAustralian Government Research Training Program Scholarship, The Chen Family Research Scholarship.

Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry-based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and in situ muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross-species phosphosite responses, as well as unique model-specific regulation. We identified > 22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK-mediated phosphorylation of STIM1 negatively regulates store-operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross-species resource of exercise-regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.

Insulin and exercise stimulate glucose uptake into skeletal muscle via different pathways. Both stimuli converge on the translocation of the glucose transporter GLUT4 from intracellular vesicles to the cell surface. Two Rab guanosine triphosphatases-activating proteins (GAPs) have been implicated in this process: AS160 for insulin stimulation and its homolog, TBC1D1, are suggested to regulate exercise-mediated glucose uptake into muscle. TBC1D1 has also been implicated in obesity in humans and mice. We investigated the role of TBC1D1 in glucose metabolism by generating TBC1D1−/− mice and analyzing body weight, insulin action, and exercise. TBC1D1−/− mice showed normal glucose and insulin tolerance, with no difference in body weight compared with wild-type littermates. GLUT4 protein levels were reduced by ∼40% in white TBC1D1−/− muscle, and TBC1D1−/− mice showed impaired exercise endurance together with impaired exercise-mediated 2-deoxyglucose uptake into white but not red muscles. These findings indicate that the RabGAP TBC1D1 plays a key role in regulating GLUT4 protein levels and in exercise-mediated glucose uptake in nonoxidative muscle fibers.

Aside from glucose metabolism, insulin regulates a variety of pathways in peripheral tissues. Under insulin-resistant conditions, it is well known that insulin-stimulated glucose uptake is impaired, and many studies attribute this to a defect in Akt signaling. Here we make use of several insulin resistance models, including insulin-resistant 3T3-L1 adipocytes and fat explants prepared from high fat-fed C57BL/6J and ob/ob mice, to comprehensively distinguish defective from unaffected aspects of insulin signaling and its downstream consequences in adipocytes. Defective regulation of glucose uptake was observed in all models of insulin resistance, whereas other major actions of insulin such as protein synthesis and anti-lipolysis were normal. This defect corresponded to a reduction in the maximum response to insulin. The pattern of change observed for phosphorylation in the Akt pathway was inconsistent with a simple defect at the level of Akt. The only Akt substrate that showed consistently reduced phosphorylation was the RabGAP AS160 that regulates GLUT4 translocation. We conclude that insulin resistance in adipose tissue is highly selective for glucose metabolism and likely involves a defect in one of the components regulating GLUT4 translocation to the cell surface in response to insulin.

Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPK-dependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry.

Background: We searched for novel regulators of insulin-stimulated glucose transport in adipocytes. Results: Tumor suppressor candidate 5 (TUSC5) colocalized with GLUT4, and manipulation of TUSC5 expression levels affected insulin-regulated glucose transport. Conclusion: TUSC5 is a novel regulator of insulin-stimulated glucose transport. Significance: TUSC5 contributes to insulin-sensitizing effects of PPARγ agonists in adipocytes. Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

The vascular endothelial growth factor (VEGF) family of cytokines are important regulators of angiogenesis that have emerged as important targets for the treatment of obesity. While serum VEGF levels rise during obesity, recent studies using genetic models provide conflicting evidence as to whether VEGF prevents or accelerates metabolic dysfunction during obesity. In the current study, we sought to identify the effects of VEGF-A neutralization on parameters of glucose metabolism and insulin action in a dietary mouse model of obesity. Within only 72 h of administration of the VEGF-A–neutralizing monoclonal antibody B.20-4.1, we observed almost complete reversal of high-fat diet–induced insulin resistance principally due to improved insulin sensitivity in the liver and in adipose tissue. These effects were independent of changes in whole-body adiposity or insulin signaling. These findings show an important and unexpected role for VEGF in liver insulin resistance, opening up a potentially novel therapeutic avenue for obesity-related metabolic disease.

are associated with adverse pregnancy outcomes including foetal overgrowth and increased risk of metabolic disease in the offspring. Our aim was to investigate the effect of a low glycemic index (GI) versus a conventional high fibre (HF) diet on pregnancy outcomes and neonatal body composition in women at high risk of developing GDM. 139 women (mean± SD age: 34.7± 0.4 y; prepregnancy BMI: 25.3± 0.5 kg/m2) at high risk of GDM (age≥ 35 yr, family history of type 2 diabetes, ethnicity, previous GDM or BMI≥ 30 kg/m2) were randomised to follow a low GI (LGI, n = 72; target GI≈ 50) or a high fibre, moderate GI diet (HF, n = 67; target GI≈ 60). Women were enrolled at 14—20 weeks gestation (mean 18 weeks). Dietary intake was assessed by 3-day food records at study entry. Neonatal body composition was measured by air displacement plethysmography using PeaPod®. Pregnancy outcomes were collected from medical records. The dietary intervention did not result in any differences in birth weight (LGI 3270± 50 vs HF 3260± 60 g, p = 0.906), ponderal index (LGI 2.71± 0.03 vs HF 2.69± 0.03 kg/m3, p = 0.958), birth weight centile (LGI 46.2± 3.2 vs HF 42.8± 3.3, p = 0.888), %fat (LGI 10± 1 vs HF 10± 1%, p = 0.347), or adverse pregnancy outcomes. However, increased maternal BMI (p = 0.02), mode of delivery (p = 0.01) and mother’s dietary GI at baseline (p = 0.035) were predictors of a higher birth weight z-score. In intensively monitored women at high risk of GDM, a low GI diet and a conventional high fibre diet produced similar pregnancy outcomes. However, maternal BMI and a high GI diet in early pregnancy appear to increase birth weight. These findings suggest that interventions to improve pregnancy outcomes need to begin prior to conception.

A major challenge of the post-genomics era is to define the connectivity of protein phosphorylation networks. Here, we quantitatively delineate the insulin signaling network in adipocytes by high-resolution mass spectrometry-based proteomics. These data reveal the complexity of intracellular protein phosphorylation. We identified 37,248 phosphorylation sites on 5,705 proteins in this single-cell type, with approximately 15% responding to insulin. We integrated these large-scale phosphoproteomics data using a machine learning approach to predict physiological substrates of several diverse insulin-regulated kinases. This led to the identification of an Akt substrate, SIN1, a core component of the mTORC2 complex. The phosphorylation of SIN1 by Akt was found to regulate mTORC2 activity in response to growth factors, revealing topological insights into the Akt/mTOR signaling network. The dynamic phosphoproteome described here contains numerous phosphorylation sites on proteins involved in diverse molecular functions and should serve as a useful functional resource for cell biologists.

The APPL1 and APPL2 proteins (APPL (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein)) are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction through their ability to act as Rab effectors. (Rabs are a family of Ras GTPases involved in membrane trafficking.) Both APPL1 and APPL2 comprise an N-terminal membrane-curving BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain. The structure and interactions of APPL1 are well characterized, but little is known about APPL2. Here, we report the crystal structure and low resolution solution structure of the BARPH domains of APPL2. We identify a previously undetected hinge site for rotation between the two domains and speculate that this motion may regulate APPL2 functions. We also identified Rab binding partners of APPL2 and show that these differ from those of APPL1, suggesting that APPL-Rab interaction partners have co-evolved over time. Isothermal titration calorimetry data reveal the interaction between APPL2 and Rab31 has a K(d) of 140 nM. Together with other biophysical data, we conclude the stoichiometry of the complex is 2:2.

The Rab GTPase-activating protein TBC1D4/AS160 regulates GLUT4 trafficking in adipocytes. Nonphosphorylated AS160 binds to GLUT4 vesicles and inhibits GLUT4 translocation, and AS160 phosphorylation overcomes this inhibitory effect. In the present study we detected several new functional features of AS160. The second phosphotyrosine-binding domain in AS160 encodes a phospholipid-binding domain that facilitates plasma membrane (PM) targeting of AS160, and this function is conserved in other related RabGAP/Tre-2/Bub2/Cdc16 (TBC) proteins and an AS160 ortholog in Drosophila. This region also contains a nonoverlapping intracellular GLUT4-containing storage vesicle (GSV) cargo-binding site. The interaction of AS160 with GSVs and not with the PM confers the inhibitory effect of AS160 on insulin-dependent GLUT4 translocation. Constitutive targeting of AS160 to the PM increased the surface GLUT4 levels, and this was attributed to both enhanced AS160 phosphorylation and 14-3-3 binding and inhibition of AS160 GAP activity. We propose a model wherein AS160 acts as a regulatory switch in the docking and/or fusion of GSVs with the PM.

Insulin stimulates glucose transport in adipocytes by triggering translocation of GLUT4 glucose transporters to the plasma membrane (PM) and several Rabs including Rab10 have been implicated in this process. To delineate the molecular regulation of this pathway, we conducted a TBC/RabGAP overexpression screen in adipocytes. This identified TBC1D13 as a potent inhibitor of insulin‐stimulated GLUT4 translocation without affecting other trafficking pathways. To determine the potential Rab substrate for TBC1D13 we conducted a yeast two‐hybrid screen and found that the GTP bound forms of Rabs 1 and 10 specifically interacted with TBC1D13 but not with eight other TBC proteins. Surprisingly, a comprehensive in vitro screen for TBC1D13 GAP activity revealed Rab35 but not Rab10 as a specific substrate. TBC1D13 also displayed in vivo GAP activity towards Rab35. Overexpression of constitutively active Rab35 but not constitutively active Rab10 reversed the block in insulin‐stimulated GLUT4 translocation observed with TBC1D13 overexpression. These studies implicate an important role for Rab35 in insulin‐stimulated GLUT4 translocation in adipocytes.