Homeostatic coordination of cellular phosphate uptake and efflux requires an organelle-based receptor for the inositol pyrophosphate IP8.

Phosphate (Pi) serves countless metabolic pathways and is involved in macromolecule synthesis, energy storage, cellular signaling, and bone maintenance. Herein, we describe the coordination of Pi uptake and efflux pathways to maintain mammalian cell Pi homeostasis. We discover that XPR1, the presumed Pi efflux transporter, separately supervises rates of Pi uptake. This direct, regulatory interplay arises from XPR1 being a binding partner for the Pi uptake transporter PiT1, involving a predicted transmembrane helix/extramembrane loop in XPR1, and its hitherto unknown localization in a subset of intracellular LAMP1-positive puncta (named "XLPVs"). A pharmacological mimic of Pi homeostatic challenge is sensed by the inositol pyrophosphate IP8, which functionalizes XPR1 to respond in a temporally hierarchal manner, initially adjusting the rate of Pi efflux, followed subsequently by independent modulation of PiT1 turnover to reset the rate of Pi uptake. These observations generate a unifying model of mammalian cellular Pi homeostasis, expanding opportunities for therapeutic intervention.

IP8: A quantitatively minor inositol pyrophosphate signaling molecule that punches above its weight.

The inositol pyrophosphates (PP-IPs) are specialized members of the wider inositol phosphate signaling family that possess functionally significant diphosphate groups. The PP-IPs exhibit remarkable functionally versatility throughout the eukaryotic kingdoms. However, a quantitatively minor PP-IP - 1,5 bisdiphosphoinositol tetrakisphosphate (1,5-IP8) - has received considerably less attention from the cell signalling community. The main purpose of this review is to summarize recently-published data which have now brought 1,5-IP8 into the spotlight, by expanding insight into the molecular mechanisms by which this polyphosphate regulates many fundamental biological processes.

Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues.

Technical challenges have to date prevented a complete profiling of the levels of myo-inositol phosphates (InsPs) and pyrophosphates (PP-InsPs) in mammalian tissues. Here, we have deployed capillary electrophoresis mass spectrometry to identify and record the levels of InsPs and PP-InsPs in several tissues obtained from wild type mice and a newly created PPIP5K2 knockout strain. We observe that the mouse colon harbours unusually high levels of InsPs and PP-InsPs. Additionally, the PP-InsP profile is considerably more complex than previously reported for animal cells: using chemically synthesized internal stable isotope references and high-resolution mass spectra, we characterize two new PP-InsP isomers as 4/6-PP-InsP5 and 2-PP-InsP5. The latter has not previously been described in nature. The analysis of feces and the commercial mouse diet suggests that the latter is one potential source of noncanonical isomers in the colon. However, we also identify both molecules in the heart, indicating unknown synthesis pathways in mammals. We also demonstrate that the CE-MS method is sensitive enough to measure PP-InsPs from patient samples such as colon biopsies and peripheral blood mononuclear cells (PBMCs). Strikingly, PBMCs also contain 4/6-PP-InsP5 and 2-PP-InsP5. In summary, our study substantially expands PP-InsP biology in mammals.

Nucleolar Architecture Is Modulated by a Small Molecule, the Inositol Pyrophosphate 5-InsP7.

Inositol pyrophosphates (PP-InsPs); are a functionally diverse family of eukaryotic molecules that deploy a highly-specialized array of phosphate groups as a combinatorial cell-signaling code. One reductive strategy to derive a molecular-level understanding of the many actions of PP-InsPs is to individually characterize the proteins that bind them. Here, we describe an alternate approach that seeks a single, collective rationalization for PP-InsP binding to an entire group of proteins, i.e., the multiple nucleolar proteins previously reported to bind 5-InsP7 (5-diphospho-inositol-1,2,3,4,6-pentakisphosphate). Quantitative confocal imaging of the outer nucleolar granular region revealed its expansion when cellular 5-InsP7 levels were elevated by either (a) reducing the 5-InsP7 metabolism by a CRISPR-based knockout (KO) of either NUDT3 or PPIP5Ks; or (b), the heterologous expression of wild-type inositol hexakisphosphate kinase, i.e., IP6K2; separate expression of a kinase-dead IP6K2 mutant did not affect granular volume. Conversely, the nucleolar granular region in PPIP5K KO cells shrank back to the wild-type volume upon attenuating 5-InsP7 synthesis using either a pan-IP6K inhibitor or the siRNA-induced knockdown of IP6K1+IP6K2. Significantly, the inner fibrillar volume of the nucleolus was unaffected by 5-InsP7. We posit that 5-InsP7 acts as an 'electrostatic glue' that binds together positively charged surfaces on separate proteins, overcoming mutual protein-protein electrostatic repulsion the latter phenomenon is a known requirement for the assembly of a non-membranous biomolecular condensate.

Development of Novel IP6K Inhibitors for the Treatment of Obesity and Obesity-Induced Metabolic Dysfunctions.

Obesity and obesity-induced metabolic dysfunctions are significant risk factors for nonalcoholic fatty liver disease and cardiovascular diseases. Thus, obesity is an economic and social burden in developed countries. Blocking the synthesis of inositol pyrophosphates by inositol hexakisphosphate kinase (IP6K) has been identified as a potential therapeutic strategy for obesity and related diseases. We have developed a novel and potent IP6K inhibitor 20 (UNC7467) (IC50 values: IP6K1 8.9 nM; IP6K2 4.9 nM; IP6K3 1320 nM). Inositol phosphate profiling of the HCT116 colon cancer cell line demonstrates that 20 reduced levels of inositol pyrophosphates by 66-81%, without significantly perturbing levels of other inositol phosphates. Furthermore, intraperitoneal injection of 20 in diet-induced obese mice improved glycemic profiles, ameliorated hepatic steatosis, and reduced weight gain without altering food intake. Thus, inhibitor 20 can be used as an in vivo probe for IP6K-related research. Moreover, it may have therapeutic relevance in treating obesity and related diseases.

Metabolic supervision by PPIP5K, an inositol pyrophosphate kinase/phosphatase, controls proliferation of the HCT116 tumor cell line.

Identification of common patterns of cancer metabolic reprogramming could assist the development of new therapeutic strategies. Recent attention in this field has focused on identifying and targeting signal transduction pathways that interface directly with major metabolic control processes. In the current study we demonstrate the importance of signaling by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks) to the metabolism and proliferation of the HCT116 colonic tumor cell line. We observed reciprocal cross talk between PPIP5K catalytic activity and glucose metabolism, and we show that CRISPR-mediated PPIP5K deletion suppresses HCT116 cell proliferation in glucose-limited culture conditions that mimic the tumor cell microenvironment. We conducted detailed, global metabolomic analyses of wild-type and PPIP5K knockout (KO) cells by measuring both steady-state metabolite levels and by performing isotope tracing experiments. We attribute the growth-impaired phenotype to a specific reduction in the supply of precursor material for de novo nucleotide biosynthesis from the one carbon serine/glycine pathway and the pentose phosphate pathway. We identify two enzymatic control points that are inhibited in the PPIP5K KO cells: serine hydroxymethyltransferase and phosphoribosyl pyrophosphate synthetase, a known downstream target of AMP-regulated protein kinase, which we show is noncanonically activated independently of adenine nucleotide status. Finally, we show the proliferative defect in PPIP5K KO cells can be significantly rescued either by addition of inosine monophosphate or a nucleoside mixture or by stable expression of PPIP5K activity. Overall, our data describe multiple, far-reaching metabolic consequences for metabolic supervision by PPIP5Ks in a tumor cell line.

Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry.

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling.

Clinical value of bedside abdominal sonography performed by certified sonographer in emergency evaluation of blunt abdominal trauma.

PURPOSE: To investigate the accuracy and efficiency of bedside ultrasonography application performed by certified sonographer in emergency patients with blunt abdominal trauma. METHODS: The study was carried out from 2017 to 2019. Findings in operations or on computed tomography (CT) were used as references to evaluate the accuracy of bedside abdominal ultrasonography. The time needed for bedside abdominal ultrasonography or CT examination was collected separately to evaluate the efficiency of bedside abdominal ultrasonography application. RESULTS: Bedside abdominal ultrasonography was performed in 106 patients with blunt abdominal trauma, of which 71 critical patients received surgery. The overall diagnostic accordance rate was 88.68%. The diagnostic accordance rate for liver injury, spleen injury, kidney injury, gut perforation, retroperitoneal hematoma and multiple abdominal organ injury were 100%, 94.73%, 94.12%, 20.00%, 100% and 81.48%, respectively. Among the 71 critical patients, the diagnostic accordance rate was 94.37%, in which the diagnostic accordance rate for liver injury, spleen injury, kidney injury, gut perforation and multiple abdominal organ injury were 100%, 100%, 100%, 20.00% and 100%. The mean time for imaging examination of bedside abdominal ultrasonography was longer than that for CT scan (4.45 ± 1.63 vs. 2.38 ± 1.19) min; however, the mean waiting time before examination (7.37 ± 2.01 vs. 16.42 ± 6.37) min, the time to make a diagnostic report (6.42 ± 3.35 vs. 36.26 ± 13.33) min, and the overall time (17.24 ± 2.33 vs. 55.06 ± 6.96) min were shorter for bedside abdominal ultrasonography than for CT scan. CONCLUSION: Bedside ultrasonography application provides both efficiency and reliability for the assessment of blunt abdominal trauma. Especially for patients with free peritoneal effusion and critical patients, bedside ultrasonography has been proved obvious advantageous. However, for negative bedside ultrasonography patients with blunt abdominal trauma, we recommend further abdominal CT scan or serial ultrasonography scans subsequently.

InsP7 is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics.

Regulation of enzymatic 5' decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5' decapping promotes accumulation of mRNAs into processing (P) bodies-membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7 (5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7 inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout of PPIP5Ks (diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e., PPIP5K KO), which elevates cellular 5-InsP7 levels by two- to threefold (i.e., within the physiological rheostatic range). The PPIP5K KO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7 synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7 analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7 levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

Control of XPR1-dependent cellular phosphate efflux by InsP8 is an exemplar for functionally-exclusive inositol pyrophosphate signaling.

Homeostasis of cellular fluxes of inorganic phosphate (Pi) supervises its structural roles in bones and teeth, its pervasive regulation of cellular metabolism, and its functionalization of numerous organic compounds. Cellular Pi efflux is heavily reliant on Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1), regulation of which is largely unknown. We demonstrate specificity of XPR1 regulation by a comparatively uncharacterized member of the inositol pyrophosphate (PP-InsP) signaling family: 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8). XPR1-mediated Pi efflux was inhibited by reducing cellular InsP8 synthesis, either genetically (knockout [KO] of diphosphoinositol pentakisphosphate kinases [PPIP5Ks] that synthesize InsP8) or pharmacologically [cell treatment with 2.5 µM dietary flavonoid or 10 µM N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine], to inhibit inositol hexakisphosphate kinases upstream of PPIP5Ks. Attenuated Pi efflux from PPIP5K KO cells was quantitatively phenocopied by KO of XPR1 itself. Moreover, Pi efflux from PPIP5K KO cells was rescued by restoration of InsP8 levels through transfection of wild-type PPIP5K1; transfection of kinase-dead PPIP5K1 was ineffective. Pi efflux was also rescued in a dose-dependent manner by liposomal delivery of a metabolically resistant methylene bisphosphonate (PCP) analog of InsP8; PCP analogs of other PP-InsP signaling molecules were ineffective. High-affinity binding of InsP8 to the XPR1 N-terminus (Kd = 180 nM) was demonstrated by isothermal titration calorimetry. To derive a cellular biology perspective, we studied biomineralization in the Soas-2 osteosarcoma cell line. KO of PPIP5Ks or XPR1 strongly reduced Pi efflux and accelerated differentiation to the mineralization end point. We propose that catalytically compromising PPIP5K mutations might extend an epistatic repertoire for XPR1 dysregulation, with pathological consequences for bone maintenance and ectopic calcification.

A two-way switch for inositol pyrophosphate signaling: Evolutionary history and biological significance of a unique, bifunctional kinase/phosphatase.

The inositol pyrophosphates (PP-InsPs) are a unique subgroup of intracellular signals with diverse functions, many of which can be viewed as reflecting an overarching role in metabolic homeostasis. Thus, considerable attention is paid to the enzymes that synthesize and metabolize the PP-InsPs. One of these enzyme families - the diphosphoinositol pentakisphosphate kinases (PPIP5Ks) - provides an extremely rare example of separate kinase and phosphatase activities being present within the same protein. Herein, we review the current state of structure/function insight into the PPIP5Ks, the separate specialized activities of the two metazoan PPIP5K genes, and we describe a phylogenetic analysis that places PPIP5K evolutionary origin within the Excavata, the very earliest of eukaryotes. These different aspects of PPIP5K biology are placed in the context of a single, overriding question. Why are they bifunctional: i.e., what is the particular significance of the ability to turn PP-InsP signaling on or off from two separate 'switches' in a single protein?

PPIP5K2 and PCSK1 are Candidate Genetic Contributors to Familial Keratoconus.

Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future.

Correction: Bacillus anthracis Spore Surface Protein BclA Mediates Complement Factor H Binding to Spores and Promotes Spore Persistence.

[This corrects the article DOI: 10.1371/journal.ppat.1005678.].

Inhibition of Inositol Polyphosphate Kinases by Quercetin and Related Flavonoids: A Structure-Activity Analysis.

Dietary flavonoids inhibit certain protein kinases and phospholipid kinases by competing for their ATP-binding sites. These nucleotide pockets have structural elements that are well-conserved in two human small-molecule kinases, inositol hexakisphosphate kinase (IP6K) and inositol polyphosphate multikinase (IPMK), which synthesize multifunctional inositol phosphate cell signals. Herein, we demonstrate that both kinases are inhibited by quercetin and 16 related flavonoids; IP6K is the preferred target. Relative inhibitory activities were rationalized by X-ray analysis of kinase/flavonoid crystal structures; this detailed structure-activity analysis revealed hydrophobic and polar ligand/protein interactions, the degree of flexibility of key amino acid side chains, and the importance of water molecules. The seven most potent IP6K inhibitors were incubated with intact HCT116 cells at concentrations of 2.5 µM; diosmetin was the most selective and effective IP6K inhibitor (>70% reduction in activity). Our data can instruct on pharmacophore properties to assist the future development of inositol phosphate kinase inhibitors. Finally, we propose that dietary flavonoids may inhibit IP6K activity in cells that line the gastrointestinal tract.

IL-17R-EGFR axis links wound healing to tumorigenesis in Lrig1+ stem cells.

Lrig1 marks a distinct population of stem cells restricted to the upper pilosebaceous unit in normal epidermis. Here we report that IL-17A-mediated activation of EGFR plays a critical role in the expansion and migration of Lrig1+ stem cells and their progenies in response to wounding, thereby promoting wound healing and skin tumorigenesis. Lrig1-specific deletion of the IL-17R adaptor Act1 or EGFR in mice impairs wound healing and reduces tumor formation. Mechanistically, IL-17R recruits EGFR for IL-17A-mediated signaling in Lrig1+ stem cells. While TRAF4, enriched in Lrig1+ stem cells, tethers IL-17RA and EGFR, Act1 recruits c-Src for IL-17A-induced EGFR transactivation and downstream activation of ERK5, which promotes the expansion and migration of Lrig1+ stem cells. This study demonstrates that IL-17A activates the IL-17R-EGFR axis in Lrig1+ stem cells linking wound healing to tumorigenesis.

Act1 is a negative regulator in T and B cells via direct inhibition of STAT3.

Although Act1 (adaptor for IL-17 receptors) is necessary for IL-17-mediated inflammatory responses, Act1- (but not Il17ra-, Il17rc-, or Il17rb-) deficient mice develop spontaneous SLE- and Sjögren's-like diseases. Here, we show that Act1 functions as a negative regulator in T and B cells via direct inhibition of STAT3. Mass spectrometry analysis detected an Act1-STAT3 complex, deficiency of Act1 (but not Il17ra-, Il17rc-, or Il17rb) results in hyper IL-23- and IL-21-induced STAT3 activation in T and B cells, respectively. IL-23R deletion or blockade of IL-21 ameliorates SLE- and Sjögren's-like diseases in Act1-/- mice. Act1 deficiency results in hyperactivated follicular Th17 cells with elevated IL-21 expression, which promotes T-B cell interaction for B cell expansion and antibody production. Moreover, anti-IL-21 ameliorates the SLE- and Sjögren's-like diseases in Act1-deficient mice. Thus, IL-21 blocking antibody might be an effective therapy for treating SLE- and Sjögren's-like syndrome in patients containing Act1 mutation.

Mutations in Diphosphoinositol-Pentakisphosphate Kinase PPIP5K2 are associated with hearing loss in human and mouse.

Autosomal recessive nonsyndromic hearing loss is a genetically heterogeneous disorder. Here, we report a severe-to-profound sensorineural hearing loss locus, DFNB100 on chromosome 5q13.2-q23.2. Exome enrichment followed by massive parallel sequencing revealed a c.2510G>A transition variant in PPIP5K2 that segregated with DFNB100-associated hearing loss in two large apparently unrelated Pakistani families. PPIP5Ks enzymes interconvert 5-IP7 and IP8, two key members of the inositol pyrophosphate (PP-IP) cell-signaling family. Their actions at the interface of cell signaling and bioenergetic homeostasis can impact many biological processes. The c.2510G>A transition variant is predicted to substitute a highly invariant arginine residue with histidine (p.Arg837His) in the phosphatase domain of PPIP5K2. Biochemical studies revealed that the p.Arg837His variant reduces the phosphatase activity of PPIP5K2 and elevates its kinase activity. We found that in mouse inner ear, PPIP5K2 is expressed in the cochlear and vestibular sensory hair cells, supporting cells and spiral ganglion neurons. Mice homozygous for a targeted deletion of the Ppip5k2 phosphatase domain exhibit degeneration of cochlear outer hair cells and elevated hearing thresholds. Our demonstration that PPIP5K2 has a role in hearing in humans indicates that PP-IP signaling is important to hair cell maintenance and function within inner ear.

Structural and biochemical characterization of Siw14: A protein-tyrosine phosphatase fold that metabolizes inositol pyrophosphates.

Inositol pyrophosphates (PP-InsPs) are "energetic" intracellular signals that are ubiquitous in animals, plants, and fungi; structural and biochemical characterization of PP-InsP metabolic enzymes provides insight into their evolution, reaction mechanisms, and regulation. Here, we describe the 2.35-Å-resolution structure of the catalytic core of Siw14, a 5-PP-InsP phosphatase from Saccharomyces cerevisiae and a member of the protein tyrosine-phosphatase (PTP) superfamily. Conclusions that we derive from structural data are supported by extensive site-directed mutagenesis and kinetic analyses, thereby attributing new functional significance to several key residues. We demonstrate the high activity and exquisite specificity of Siw14 for the 5-diphosphate group of PP-InsPs. The three structural elements that demarcate a 9.2-Å-deep substrate-binding pocket each have spatial equivalents in PTPs, but we identify how these are specialized for Siw14 to bind and hydrolyze the intensely negatively charged PP-InsPs. (a) The catalytic P-loop with the CX5R(S/T) PTP motif contains additional, positively charged residues. (b) A loop between the α5 and α6 helices, corresponding to the Q-loop in PTPs, contains a lysine and an arginine that extend into the catalytic pocket due to displacement of the α5 helix orientation through intramolecular crowding caused by three bulky, hydrophobic residues. (c) The general-acid loop in PTPs is replaced in Siw14 with a flexible loop that does not use an aspartate or glutamate as a general acid. We propose that an acidic residue is not required for phosphoanhydride hydrolysis.

Inositol hexakisphosphate kinase 1 is a metabolic sensor in pancreatic ß-cells.

Diphosphoinositol pentakisphosphate (IP7) is critical for the exocytotic capacity of the pancreatic ß-cell, but its regulation by the primary instigator of ß-cell exocytosis, glucose, is unknown. The high Km for ATP of the IP7-generating enzymes, the inositol hexakisphosphate kinases (IP6K1 and 2) suggests that these enzymes might serve as metabolic sensors in insulin secreting ß-cells and act as translators of disrupted metabolism in diabetes. We investigated this hypothesis and now show that glucose stimulation, which increases the ATP/ADP ratio, leads to an early rise in IP7 concentration in ß-cells. RNAi mediated knock down of the IP6K1 isoform inhibits both glucose-mediated increase in IP7 and first phase insulin secretion, demonstrating that IP6K1 integrates glucose metabolism and insulin exocytosis. In diabetic mouse islets the deranged ATP/ADP levels under both basal and glucose-stimulated conditions are mirrored in both disrupted IP7 generation and insulin release. Thus the unique metabolic sensing properties of IP6K1 guarantees appropriate concentrations of IP7 and thereby both correct basal insulin secretion and intact first phase insulin release. In addition, our data suggest that a specific cell signaling defect, namely, inappropriate IP7 generation may be an essential convergence point integrating multiple metabolic defects into the commonly observed phenotype in diabetes.