Background: Mechanisms of insulin resistance in type 2 diabetes are not known. Results: A first dynamic mathematical model based on data from human adipocytes yields systems level understanding of insulin resistance. Conclusion: Attenuation of an mTORC1-derived feedback in diabetes explains reduced sensitivity and signal strength throughout the insulin-signaling network. Significance: Findings give a molecular basis for insulin resistance in the signaling network.

Insulin and other hormones control target cells through a network of signal-mediating molecules. Such networks are extremely complex due to multiple feedback loops in combination with redundancy, shared signal mediators, and cross-talk between signal pathways. We present a novel framework that integrates experimental work and mathematical modeling to quantitatively characterize the role and relation between coexisting submechanisms in complex signaling networks. The approach is independent of knowing or uniquely estimating model parameters because it only relies on (i) rejections and (ii) core predictions (uniquely identified properties in unidentifiable models). The power of our approach is demonstrated through numerous iterations between experiments, modelbased data analyses, and theoretical predictions to characterize the relative role of co-existing feedbacks governing insulin signaling. We examined phosphorylation of the insulin receptor and insulin receptor substrate-1 and endocytosis of the receptor in response to various different experimental perturbations in primary human adipocytes. The analysis revealed that receptor endocytosis is necessary for two identified feedback mechanisms involving mass and information transfer, respectively. Experimental findings indicate that interfering with the feedback may substantially increase overall signaling strength, suggesting novel therapeutic targets for insulin resistance and type 2 diabetes. Because the central observations are present in other signaling networks, our results may indicate a general mechanism in hormonal control.Hormonal control of target cells involves signal transduction from ligand-activated receptors to control of rate-limiting enzymes or proteins that affect key steps in metabolism or other processes within the cell. The signal transduction is carried out by a network of interacting signal mediators. A high degree of complexity is due to the presence of feedback and feed-forward loops, both negative and positive, and the fact that the importance of different interactions changes over time and according to intracellular location. This, in combination with redundancy, shared signal mediators, shared signal paths, and ample cross-talk between signals, leads to a complexity that poses new challenges to progress in dissecting and understanding cellular control. Many diseases, such as cancer and insulin resistance and type 2 diabetes, arise from malfunctioning in signaling networks.Insulin controls target cells through binding to its receptor at the cell surface (1), which activates the intracellular domains of the insulin receptor (IR) 4 to trans-autophosphorylate at specific tyrosine residues. The receptor can then transduce the insulin signal into the cell and to its various effectuating systems, such as glucose uptake and antilipolysis. Foremost of the directly downstream signal-mediating proteins are members of the insulin receptor substrate (IRS) family, in particular IRS1, which is rapidly phosphorylated at specific tyrosine residues by...

Type 2 diabetes (T2D) is strongly linked to obesity and an adipose tissue unresponsive to insulin. The insulin resistance is due to defective insulin signaling, but details remain largely unknown. We examined insulin signaling in adipocytes from T2D patients, and contrary to findings in animal studies, we observed attenuation of insulin activation of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). As a consequence, mTORC1 downstream effects were also affected in T2D: feedback signaling by insulin to signal-mediator insulin receptor substrate-1 (IRS1) was attenuated, mitochondria were impaired and autophagy was strongly upregulated. There was concomitant autophagic destruction of mitochondria and lipofuscin particles, and a dependence on autophagy for ATP production. Conversely, mitochondrial dysfunction attenuated insulin activation of mTORC1, enhanced autophagy and attenuated feedback to IRS1. The overactive autophagy was associated with large numbers of cytosolic lipid droplets, a subset with colocalization of perlipin and the autophagy protein LC3/atg8, which can contribute to excessive fatty acid release. Patients with diagnoses of T2D and overweight were consecutively recruited from elective surgery, whereas controls did not have T2D. Results were validated in a cohort of patients without diabetes who exhibited a wide range of insulin sensitivities. Because mitochondrial dysfunction, inflammation, endoplasmic-reticulum stress and hypoxia all inactivate mTORC1, our results may suggest a unifying mechanism for the pathogenesis of insulin resistance in T2D, although the underlying causes might differ. the dephosphorylation by phosphotyrosine protein phosphatases (13).TOR coordinates control of cell growth and metabolism in accordance with nutrient availability in unicellular organisms. During evolution of multicellular organisms this control was seized by insulin and other growth factors. However, the ancient ability of TOR to sense nutrient levels in cells independently of insulin is retained in multicellular organisms, including man, giving TOR a key role in cellular control of metabolism and cell growth, as well as tolerance to starvation through control of autophagy. In mammalian cells mTOR, in complex with the protein raptor (mTORC1), is activated by insulin and the insulin receptor substrate-1 (IRS1) via either or both of the two signaling branches of insulin that lead to activation of protein kinase B/Akt or the Map-kinase ERK1/2, respectively. By responding to amino acid and energy levels in the cell, mTORC1 thus integrates insulin signaling with nutrient availability to control cellular processes such as cell growth, protein synthesis, mitochondrial function and autophagy ( Figure 1A), reviewed in (14). In several studies, in particular on animals after high-fat feeding regimens, insulin resistance has been coupled with hyperactive mTORC1 (reviewed in [6]). However, because mTORC1 mediates the positive feedback signal to phosphorylation of IRS1 at serine 307, we wanted to further...

Key pointsr The molecular and cellular mechanisms involved in short-term regulation of white adipocyte adipokine release remain elusive.r Here we have examined effects of intracellular cAMP, Ca 2+ and ATP on exocytosis and adipokine secretion by a combination of membrane capacitance patch-clamp recordings and biochemical measurements of secreted adipokines.r Our findings show that white adipocyte exocytosis is stimulated by cAMP/Epac (exchange proteins activated by cAMP)-dependent but Ca 2+ -and PKA-independent mechanisms and can largely be correlated to release of adiponectin vesicles residing in a readily releasable vesicle pool.r A combination of Ca 2+ and ATP augments exocytosis/adiponectin secretion via a direct action on the release process and by recruitment of new releasable vesicles.r Our results elucidate several previously unknown cellular mechanisms involved in regulation of white adipocyte exocytosis/secretion. The well-established disturbances of adipokine secretion in obese individuals highlight the significance of understanding how white adipocyte adipokine release is controlled.Abstract We examined the effects of cAMP, Ca 2+ and ATP on exocytosis and adipokine release in white adipocytes by a combination of membrane capacitance patch-clamp recordings and biochemical measurements of adipokine secretion. 3T3-L1 adipocyte exocytosis proceeded even in the complete absence of intracellular Ca 2+ ([Ca 2+ ] i ; buffered with BAPTA) provided cAMP (0.1 mM) was included in the intracellular (pipette-filling) solution. Exocytosis typically plateaued within ß10 min, probably signifying depletion of a releasable vesicle pool. Inclusion of 3 mM ATP in combination with elevation of [Ca 2+ ] i to ࣙ700 nM augmented the rate of cAMP-evoked exocytosis ß2-fold and exocytosis proceeded for longer periods (ࣙ20 min) than with cAMP alone. Exocytosis was stimulated to a similar extent upon substitution of cAMP by the Epac of the membrane-permeable PKA inhibitor . The adipokines leptin, resistin and apelin were present in low amounts in the incubation medium (1-6% of measured adiponectin). Adipsin was secreted in substantial quantities (50% of adiponectin concentration) but release of this adipokine was unaffected by forsk-IBMX. We propose that white adipocyte exocytosis is stimulated by cAMP/Epac-dependent but Ca 2+ -and PKA-independent release of vesicles residing in a readily releasable pool and that the release is augmented by a combination of Ca 2+ and ATP. We further suggest that secreted vesicles chiefly contain adiponectin.

Type 2 diabetes is a metabolic disease that profoundly affects energy homeostasis. The disease involves failure at several levels and subsystems and is characterized by insulin resistance in target cells and tissues (i.e. by impaired intracellular insulin signaling). We have previously used an iterative experimental-theoretical approach to unravel the early insulin signaling events in primary human adipocytes. That study, like most insulin signaling studies, is based on in vitro experimental examination of cells, and the in vivo relevance of such studies for human beings has not been systematically examined. Herein, we develop a hierarchical model of the adipose tissue, which links intracellular insulin control of glucose transport in human primary adipocytes with whole-body glucose homeostasis. An iterative approach between experiments and minimal modeling allowed us to conclude that it is not possible to scale up the experimentally determined glucose uptake by the isolated adipocytes to match the glucose uptake profile of the adipose tissue in vivo. However, a model that additionally includes insulin effects on blood flow in the adipose tissue and GLUT4 translocation due to cell handling can explain all data, but neither of these additions is sufficient independently. We also extend the minimal model to include hierarchical dynamic links to more detailed models (both to our own models and to those by others), which act as submodules that can be turned on or off. The resulting multilevel hierarchical model can merge detailed results on different subsystems into a coherent understanding of whole-body glucose homeostasis. This hierarchical modeling can potentially create bridges between other experimental model systems and the in vivo human situation and offers a framework for systematic evaluation of the physiological relevance of in vitro obtained molecular/cellular experimental data.

In this article, we report a functionalised ZnO-nanorod-based selective electrochemical sensor for intracellular glucose. To adjust the sensor for intracellular glucose measurements, we grew hexagonal ZnO nanorods on the tip of a silver-covered borosilicate glass capillary (0.7 µm diameter) and coated them with the enzyme glucose oxidase. The enzyme-coated ZnO nanorods exhibited a glucose-dependent electrochemical potential difference versus an Ag/AgCl reference micro-electrode. The potential difference was linear over the concentration range of interest (0.5 µM -1000 µM). The measured glucose concentration in human adipocytes or frog oocytes using our ZnO nanorod sensor was consistent with values of glucose concentration reported in the literature; furthermore, the sensor was able to show that insulin increased the intracellular glucose concentration. This nanoelectrode device demonstrates a simple technique to measure intracellular glucose concentration.

Background: Molecular mechanisms of insulin resistance in diabetes are poorly understood. Results: Quantitative systems-wide data reveal that a single mechanism can explain insulin resistance throughout the signaling network in human adipocytes. Conclusion:The most important aspect of the insulin resistance mechanism is an attenuated feedback signal. Significance: The study demonstrates how insulin resistance originates and propagates throughout the signaling network in cells from patients with diabetes.

Abstract:In this study, a potentiometric intracellular glucose biosensor was fabricated by immobilization of glucose oxidase on nanoflake ZnO. Nanoflake ZnO with a wall thickness around 200 nm was grown on the tip of a borosilicate glass capillary and used as a selective intracellular glucose biosensor for the measurement of glucose concentrations in human adipocytes and frog oocytes. The results showed a fast response within 4 s and a logarithmic linear glucose-dependent electrochemical potential difference over a wide range of glucose concentration (500 nM-10 mM). Our measurements of intracellular glucose were consistent with the values of intracellular glucose concentrations reported in the literature. The monitoring capability of the sensor was demonstrated by following the increase in the intracellular glucose concentration induced by insulin in frog oocytes. In addition, the nanoflake ZnO material provided 1.8 times higher sensitivity than previously used ZnO nanorods under the same conditions. Moreover, the fabrication method in our experiment is simple and the resulting nanosensor showed good performance in sensitivity, stability, selectivity, reproducibility, and anti-interference. All these results demonstrate that the nanoflake ZnO can provide a promising material for reliable measurements of intracellular glucose concentrations within single living cells. Key Words:Glucose oxidase (GOD), Intracellular, Potentiometric biosensor, Nanoflake ZnO, Nafion membrane Among all glucose biosensors, enzyme-based electrochemical glucose biosensors have been in the main focus of biosensor research because of their simplicity, relatively low cost, and high sensitivity [14][15][16]. The intrinsic advantages of electrochemical biosensors are their robustness, easy miniaturization, excellent detection limits, also with small sample volumes, and ability to be used in 3 turbid biofluids. In enzyme-based electrochemical biosensors, enzyme immobilization is regarded to be one of the most important issues. Since proper immobilization of enzymes on a suitable matrix and their stability are important factors in the fabrication of biosensors, the search of support materials that provide large surface area for higher enzyme loading and a compatible microenvironment that helps enzyme bioactivity is thus of great importance.Recently, zinc oxide (ZnO) nanostructures have attracted considerable interest in the applications of biosensors due to many advantages, including non-toxicity, bio-safety, excellent biological compatibility, high electron-transfer rates, enhanced analytical performance, increased sensitivity, and easy preparation [17][18][19][20][21][22]. In addition, it is important to note that ZnO is relatively stable around biological pH-values, which makes ZnO compatible with biological fluids and species [23].Furthermore, the high isoelectric point (IEP) of ZnO (IEP 9.5) makes it a good matrix for immobilizing low IEP acidic proteins or DNA by electrostatic interactions with high binding stability [24][25][26]. This wi...