Insulin-responsive Glucose Transporter Research Articles

Click Reaction Inspired Enzyme Inhibitors in Diabetes Care: An Update in the Field of Chronic Metabolic Disorder.

Diabetes is a chronic metabolic disorder that impacts all age groups and affects a large population worldwide. Humans receive glucose from almost every food source, and after absorption from the gut, it reaches the liver, which functions as the distribution center for it. The insulin-responsive glucose transporter type 4 (GLUT-4) is a major carrier of glucose to the various cells (majorly expressed in myocytes, adipocytes, and cardiomyocytes) in a well-fed state. In fasting periods, the glucose supply is maintained by glycogenolysis and gluconeogenesis. In diabetes, the distribution of glucose is hampered due to several reasons. Furthermore, to treat this disorder, several drugs have been synthesized, and click chemistry plays an important role. A more recent concept for producing pharmaceuticals with a click chemistry approach makes any reaction more practical and stereospecific, along with a higher yield of products and a smaller number of by-products. This approach comprises a compiled study of the activity of numerous compelling antidiabetic drugs containing 1, 2, 3-triazole derivatives supported by click chemistry. In this review, we discuss the synthetic antidiabetic drugs made via click chemistry and their commendable role in improving diabetes care.

The glucose transporter GLUT12, a new actor in obesity and cancer.

Obesity constitutes a global health epidemic which worsens the main leading death causes such as type 2 diabetes, cardiovascular diseases, and cancer. Changes in the metabolism in patients with obesity frequently lead to insulin resistance, along with hyperglycemia, dyslipidemia and low-grade inflammation, favoring a more aggressive tumor microenvironment. One of the hallmarks of cancer is the reprogramming of the energy metabolism, in which tumor cells change oxidative phosphorylation to aerobic glycolysis or "Warburg effect". Aerobic glycolysis is faster than oxidative phosphorylation, but less efficient in terms of ATP production. To obtain sufficient ATP, tumor cells increase glucose uptake by the glucose transporters of the GLUT/SLC2 family. The human glucose transporter GLUT12 was isolated from the breast cancer cell line MCF7. It is expressed in adipose tissue, skeletal muscle and small intestine, where insulin promotes its translocation to the plasma membrane. Moreover, GLUT12 over-expression in mice increases the whole-body insulin sensitivity. Thus, GLUT12 has been proposed as a second insulin-responsive glucose transporter. In obesity, GLUT12 is downregulated and does not respond to insulin. In contrast, GLUT12 is overexpressed in human solid tumors such as breast, prostate, gastric, liver and colon. High glucose concentration, insulin, and hypoxia upregulate GLUT12 both in adipocytes and tumor cells. Inhibition of GLUT12 mediated Warburg effect suppresses proliferation, migration, and invasion of cancer cells and xenografted tumors. This review summarizes the up-to-date information about GLUT12 physiological role and its implication in obesity and cancer, opening new perspectives to consider this transporter as a therapeutic target.

Adipocyte traits limiting cellular insulin responsiveness and glucose transport.

Adipocyte dysfunction is a hallmark of systemic insulin resistance. Insulin-responsive glucose transporter 4 (GLUT4) is downregulated in the insulin resistant state, and cellular insulin responsiveness varies depending on fat depot origin and degree of adipose expansion. Here, we have resolved factors limiting cellular insulin responsiveness, by examining adipocyte function and traits related to glucose transport at the cellular level. Subcutaneous (inguinal) and visceral (epididymal) adipocytes were isolated from C57BL/6J mice fed either chow or high-fat diet. Cell size was determined using coulter counter method, glucose uptake and cytosolic volume were assessed using glucose tracer assays. Total and GLUT4 protein content expressions were determined by Western blot. We found that basal glucose uptake per cell was preserved independent of diet or fat depot origin. Insulin-stimulated glucose uptake per cell was sustained in visceral adipocytes but decreased with adipose expansion in subcutaneous adipocytes. In parallel, the cytosolic space and total protein increased proportionally to total cellular volumetric expansion in visceral, but not in subcutaneous, adipocytes, whereas GLUT4 content decreased exclusively in expanding subcutaneous adipocytes. Together, these data support the existence of distinct phenotypic adipocyte traits that could limit cellular insulin responsiveness. Potentially, these characteristics account for fat depot-specific differences related to glucose transport capacity.NEW & NOTEWORTHY This work illustrates that adipocyte characteristics related to fat depot origin rather than adipocyte size per se limit cellular insulin responsiveness and glucose uptake in male C57BL/6J mice. These findings contribute to the overall understanding of factors limiting adipocyte function and how adipose progression affects insulin response and glucose transport capacity differently in diverse fat depots. Future studies examining whether the proposed characteristics hold true in adipocytes derived from female mice or human origin are needed.

Studying the role of random translocation of GLUT4 in cardiomyocytes on calcium oscillations

Defective excitation-contraction coupling (ECC) is one of the causes of contractile dysfunction linked to diabetic cardiomyopathy (DCM). This defect in ECC is mainly caused by the dysregulation of the calcium homeostasis in normal cardiomyocytes due to prolonged elevated blood glucose levels. Different types of channels inside the cardiomyocytes help maintain calcium homeostasis in the cell. Free cytosolic calcium concentration changes are one of several variables that affect complicated physiological cardiac processes and significantly impact proper heart functioning. Glucose concentration in the cardiac cell also plays a vital role in altering the calcium homeostasis in the DCM condition. In the current study, we develop a mathematical model that explicitly represents many of the known signalling components mediating translocation of the insulin-responsive glucose transporter type 4 (GLUT4) to gain insight into the complexities of metabolic insulin signalling pathways in cardiac cells. We examined the effect of the random movement of GLUT4 responsible for the entry of glucose in the cardiomyocytes. In non-paced cardiac cells calcium remains in the steady state in the absence of external pacing and avoid oscillation for healthy cardiomyocyte functioning. Intracellular calcium transients were observed during action potential. We investigated the circumstances under which the system transits from a stable state to an oscillatory state. We studied the effect of random translocation of GLUT4 on calcium oscillation. By altering system parameters, we induced insulin resistance in cardiomyocytes to mimic diabetic conditions and proposed some potential restoration strategies to restore normal calcium dynamics. Early bifurcation was observed with the introduction of randomness in the system.

Non-monotonic dose-response of di-(2-ethylhexyl) phthalate isolated from Penicillium citrinum XT6 on adipogenesis and expression of PPARγ and GLUT4 in 3T3-L1 adipocytes.

Adipogenesis is the fat cell formation process regulated by peroxisome proliferator-activated receptors (PPARγ). The insulin-responsive glucose transporter 4 (GLUT4) has a major role in glucose uptake and metabolism in insulin target tissues (i.e.,adipose and muscle cells). The interplay between PPARγ and GLUT4 is essential for proper glucose homeostasis. This study aimed to isolate, elucidate, and investigate the effect of an isolated compound from Penicillium citrinum XT6 on adipogenesis, PPARγ, and GLUT4 expression in 3T3-L1 adipocytes. The isolated compound was determined by analyzing spectroscopic data (LC-MS, FT-IR, Spectrophotometry UV-Vis, and NMR). The adipogenesis activity of the isolated compound in 3T3-L1 cells was determined by the Oil Red O staining method. RT-PCR was used to analyze the gene expression of PPARγ and GLUT4. Di-(2-ethylhexyl)-phthalate (DEHP) was the isolated compound from P.citrinum XT6. The results revealed adipogenesis stimulation and inhibition, as well as PPARγ and GLUT4 expressions. DEHP showed a non-monotonic dose-response (NMDR) effect on adipogenesis and PPARγ and GLUT4 expression. It is the first study that reveals DEHP's NMDR effects on lipid and glucose metabolism in adipocytes.

Oral administration of wild plant-derived minerals and red ginseng ameliorates insulin resistance in fish through different pathways.

Many kinds of fish are characterized by a limited efficiency to use carbohydrates. For this reason, raw fish and mixed feed containing a lot of fish meal have been used as feed for fish farming. However, continuing to use high-protein diets not only increases the cost of fish farming, but may also fuel animal protein shortages. Furthermore, carbohydrates are added to improve the texture of the feed and act as a binding agent and are usually contained at 20% in the feed. It makes sense, therefore, to find ways to make good use of carbohydrates rather than wasting them. The physiological mechanisms of glucose intolerance in fish are not yet well understood. Therefore, we investigated the glucose utilization of fish, omnivorous goldfish Carassius auratus and carnivorous rainbow trout Oncorhynchus mykiss. Furthermore, the effects of oral administration of wild plant-derived minerals and red ginseng on the glucose utilization in these fish muscle cells were investigated. As a result, we found the following. (1) An extremely high insulin resistance in fish muscle and the symptom was more pronounced in carnivorous rainbow trout. (2) Administration of wild plant-derived minerals promotes the translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface of white muscle via activation of the PI3 kinase axis, whereas administration of red ginseng not only promotes GLUT4 transfer and translocation to the cell surface of white muscle via AMPK activation as well as promoting glucose uptake into muscle cells via a pathway separate from the insulin signaling system. (3) In fish, at least goldfish and rainbow trout, both PI3K/Akt and AMPK signaling cascades exist to promote glucose uptake into muscle cells, as in mammals.

Tectorigenin targets PKACα to promote GLUT4 expression in skeletal muscle and improve insulin resistance in vitro and in vivo.

The decreased expression and dysfunction of glucose transporter 4 (GLUT4), the insulin-responsive glucose transporter, are closely related to the occurrence of insulin resistance (IR). To improve the expression of GLUT4 may represent a promising strategy to prevent and treat IR and type 2 diabetes (T2DM). Here, we demonstrate that the natural compound tectorigenin (TG) enhances GLUT4 expression, glucose uptake and insulin responsiveness via activating AMP-activated protein kinase (AMPK)/myocyte enhancer factor 2 (MEF2) signaling in both normal and IR skeletal muscle cells and tissues. Accordingly, prophylactic and therapeutic uses of TG can significantly ameliorate IR and hyperglycemia in T2DM mice. Mechanistically, we identify protein kinase A catalytic subunit α (PKACα) as the target of TG to increase GLUT4 expression and TG-PKACα binding promotes the dissociation of PKACα from the regulatory subunits, leading to the activation of PKA/AMPK signaling. PKACα knockdown in local quadriceps muscles almost completely abolished the therapeutic effects of TG on IR and T2DM, as well as the enhancement on AMPK signaling and GLUT4 expression in skeletal muscle. This study supports TG as a new drug candidate to treat IR and its related diseases, but also enriches our knowledge of PKA signaling in glucose metabolism in skeletal muscle.

Exocytosis Proteins: Typical and Atypical Mechanisms of Action in Skeletal Muscle.

Insulin-stimulated glucose uptake in skeletal muscle is of fundamental importance to prevent postprandial hyperglycemia, and long-term deficits in insulin-stimulated glucose uptake underlie insulin resistance and type 2 diabetes. Skeletal muscle is responsible for ~80% of the peripheral glucose uptake from circulation via the insulin-responsive glucose transporter GLUT4. GLUT4 is mainly sequestered in intracellular GLUT4 storage vesicles in the basal state. In response to insulin, the GLUT4 storage vesicles rapidly translocate to the plasma membrane, where they undergo vesicle docking, priming, and fusion via the high-affinity interactions among the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) exocytosis proteins and their regulators. Numerous studies have elucidated that GLUT4 translocation is defective in insulin resistance and type 2 diabetes. Emerging evidence also links defects in several SNAREs and SNARE regulatory proteins to insulin resistance and type 2 diabetes in rodents and humans. Therefore, we highlight the latest research on the role of SNAREs and their regulatory proteins in insulin-stimulated GLUT4 translocation in skeletal muscle. Subsequently, we discuss the novel emerging role of SNARE proteins as interaction partners in pathways not typically thought to involve SNAREs and how these atypical functions reveal novel therapeutic targets for combating peripheral insulin resistance and diabetes.

Enhanced recombinant expression and purification of human IRAP for biochemical and crystallography studies

Insulin-regulated aminopeptidase (IRAP) in humans is a membrane bound enzyme that has multiple functions. It was first described as a companion protein of the insulin-responsive glucose transporter, Glut4, in specialized vesicles. The protein has subsequently been shown to be identical to the oxytocinase/aminopeptidase or the angiotensin IV (Ang IV) receptor (AT4 receptor). Some AT4 ligand peptides, such as Ang IV and LVV-hemorphin-7, have been shown to act as IRAP inhibitors that exert memory-enhancing properties. As such IRAP has been a target for developing cognitive enhancers. To facilitate detailed mechanistic studies of IRAP catalysis and inhibition, and to pave the way for biophysical and structural studies of IRAP in complex with peptide inhibitors, we report here an optimized expression and purification system using High Five insect cells. We also report biochemical characterizations of the purified recombinant IRAP with a standard aminopeptidase substrate and an optimized IRAP peptide inhibitor with a Ki of 98 nM.

Histone deacetylase 5 regulates interleukin 6 secretion and insulin action in skeletal muscle

ObjectivePhysical exercise training is associated with increased glucose uptake in skeletal muscle and improved glycemic control. HDAC5, a class IIa histone deacetylase, has been shown to regulate transcription of the insulin-responsive glucose transporter GLUT4 in cultured muscle cells. In this study, we analyzed the contribution of HDAC5 to the transcriptional network in muscle and the beneficial effect of muscle contraction and regular exercise on glucose metabolism. MethodsHDAC5 knockout mice (KO) and wild-type (WT) littermates were trained for 8 weeks on treadmills, metabolically phenotyped, and compared to sedentary controls. Hdac5-deficient skeletal muscle and cultured Hdac5-knockdown (KD) C2C12 myotubes were utilized for studies of gene expression and glucose metabolism. Chromatin immunoprecipitation (ChIP) studies were conducted to analyze Il6 promoter activity using H3K9ac and HDAC5 antibodies. ResultsGlobal transcriptome analysis of Hdac5 KO gastrocnemius muscle demonstrated activation of the IL-6 signaling pathway. Accordingly, knockdown of Hdac5 in C2C12 myotubes led to higher expression and secretion of IL-6 with enhanced insulin-stimulated activation of AKT that was reversed by Il6 knockdown. Moreover, Hdac5-deficient myotubes exhibited enhanced glucose uptake, glycogen synthesis, and elevated expression levels of the glucose transporter GLUT4. Transcription of Il6 was further enhanced by electrical pulse stimulation in Hdac5-deficient C2C12 myotubes. ChIP identified a ∼1 kb fragment of the Il6 promoter that interacts with HDAC5 and demonstrated increased activation-associated histone marker AcH3K9 in Hdac5-deficient muscle cells. Exercise intervention of HDAC5 KO mice resulted in improved systemic glucose tolerance as compared to WT controls. ConclusionsWe identified HDAC5 as a negative epigenetic regulator of IL-6 synthesis and release in skeletal muscle. HDAC5 may exert beneficial effects through two different mechanisms, transcriptional control of genes required for glucose disposal and utilization, and HDAC5-dependent IL-6 signaling cross-talk to improve glucose uptake in muscle in response to exercise.

A Pea (Pisum sativum L.) Seed Vicilins Hydrolysate Exhibits PPARγ Ligand Activity and Modulates Adipocyte Differentiation in a 3T3-L1 Cell Culture Model.

Legume consumption has been reported to induce beneficial effects on obesity-associated metabolic disorders, but the underlying mechanisms have not been fully clarified. In the current work, pea (Pisum sativum L.) seed meal proteins (albumins, legumins and vicilins) were isolated, submitted to a simulated gastrointestinal digestion, and the effects of their hydrolysates (pea albumins hydrolysates (PAH), pea legumins hydrolysates (PLH) and pea vicilin hydrolysates (PVH), respectively) on 3T3-L1 murine pre-adipocytes were investigated. The pea vicilin hydrolysate (PVH), but not native pea vicilins, increased lipid accumulation during adipocyte differentiation. PVH also increased the mRNA expression levels of the adipocyte fatty acid-binding protein (aP2) and decreased that of pre-adipocyte factor-1 (Pref-1) (a pre-adipocyte marker gene), suggesting that PVH promotes adipocyte differentiation. Moreover, PVH induced adiponectin and insulin-responsive glucose transporter 4 (GLUT4) and stimulated glucose uptake. The expression levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, were up-regulated in 3T3-L1 cells treated with PVH during adipocyte differentiation. Finally, PVH exhibited PPARγ ligand activity. Lactalbumin or other pea hydrolysates (PAH, PLH) did not exhibit such effects. These findings show that PVH stimulates adipocyte differentiation via, at least in part, the up-regulation of PPARγ expression levels and ligand activity. These effects of PVH might be relevant in the context of the beneficial health effects of legume consumption in obesity-associated metabolic disorders.

Role of LXR alpha in regulating expression of glucose transporter 4 in adipocytes — Investigation on improvement of health of diabetic patients

BackgroundThe insulin-responsive glucose transporter 4 (GLUT4) plays prominent role in insulin-mediated facilitated glucose uptake into most of the cell types, majorly muscle, liver and adipose tissue. Impaired expression of GLUT4 has been linked to insulin resistance and diabetes. In adipocytes, excess lipids that are generated from liver and by de novo lipogenesis utilizing blood sugar, are stored. There are various nuclear factors, co-factors and signaling mechanisms that directly and indirectly regulate glucose transporter activity in adipocytes. Molecular mechanism behind the regulation of GLUT4 in adipocytes has not elucidated well. ObjectiveTherefore, the present study focuses to explore the role of Liver X receptor- alpha (LXRα) on GLUT4 expression and the possible co-factors involved during in vitro adipogenesis and is assessed by modulating the activity of LXRα with specific agonist and antagonist ligands in 3T3L1 differentiated cells. ResultsThe results demonstrate that SR 9238 (300nM), a strong inhibitor of LXRα, decreased the rate of adipogenesis through reduced lipid droplet formation in adipocytes without affecting the cell morphology. The FMOC-l-Leucine (FLL), a known partial ligand of PPARγ, enhanced the expression of LXRα. Thus, it can be concluded that LXRα has a significant role in adipocyte differentiation and FLL interaction promoted the transcription of LXRα, thereby promotes GLUT4 expression. The results in this study identified the role of LXRα in regulating the expression of GLUT4 through SRC1. ConclusionThe study is of much relevance in treatment of diabetes and also opens the possibility of identifying new drug molecules that target LXRα.

Insulin acutely increases glucose transporter 1 on plasma membranes and glucose uptake in an AKT-dependent manner in chicken adipocytes

Avian glucose transporters (GLUT) responsible for insulin-responsive glucose uptake into adipocytes remain poorly characterized. We aimed to identify the insulin-responsive GLUT using primary culture of chicken adipocytes. Acute stimulation with 1 μM insulin for 20 min increased 2-deoxyglucose uptake, AKT protein phosphorylation, and GLUT1 protein levels on the plasma membrane of the chicken adipocytes, whereas pretreatment with 10 μM triciribine, an AKT inhibitor, canceled these effects. Furthermore, the insulin stimulation did not affect GLUT12 protein levels on the plasma membrane of the chicken adipocytes. Our results suggest that GLUT1 is an insulin-responsive GLUT in chicken adipocytes.

Genetic diversity of CHC22 clathrin impacts its function in glucose metabolism.

CHC22 clathrin plays a key role in intracellular membrane traffic of the insulin-responsive glucose transporter GLUT4 in humans. We performed population genetic and phylogenetic analyses of the CHC22-encoding CLTCL1 gene, revealing independent gene loss in at least two vertebrate lineages, after arising from gene duplication. All vertebrates retained the paralogous CLTC gene encoding CHC17 clathrin, which mediates endocytosis. For vertebrates retaining CLTCL1, strong evidence for purifying selection supports CHC22 functionality. All human populations maintained two high frequency CLTCL1 allelic variants, encoding either methionine or valine at position 1316. Functional studies indicated that CHC22-V1316, which is more frequent in farming populations than in hunter-gatherers, has different cellular dynamics than M1316-CHC22 and is less effective at controlling GLUT4 membrane traffic, altering its insulin-regulated response. These analyses suggest that ancestral human dietary change influenced selection of allotypes that affect CHC22's role in metabolism and have potential to differentially influence the human insulin response.

1177-P: Discovery of New Therapeutic Antibodies to Two Metabolically Relevant Targets—GPCR CB1 and Transporter GLUT4—Using Virus-Like Particles

Integral membrane proteins are important drug targets, and monoclonal antibodies (mAbs) against them are highly sought for therapeutic and diagnostic purposes. However, the complex structure of multipass membrane proteins makes discovery of these mAbs particularly challenging. By employing strategies such as sequential immunization with DNA (prime) and virus-like particles (boost), and using divergent species (chickens) as immunization hosts, we routinely isolate diverse panels of mAbs against structurally complex and highly conserved membrane protein targets. Here, we report the isolation and characterization of mAbs recognizing two distinct metabolically relevant targets: the 12-transmembrane insulin-responsive glucose transporter GLUT4 and the 7-transmembrane GPCR cannabinoid receptor-1 (CB1). In both cases, we isolated diverse panels of mAbs against native conformational epitopes. Affinities ranged from 2.6 nM (anti-CB1 IM-102) to 1 pM (anti-GLUT4 LM048). Two anti-GLUT4 mAbs discriminated between inward-open and outward-open conformational states, including one mAb (LM048) that inhibited transporter function. Similarly, we isolated several anti-CB1 mAbs that antagonized CB1-mediated calcium flux. Lead mAbs were confirmed to be specific for their target proteins, based on reactivity profiling in the Membrane Proteome Array of >5,600 human membrane proteins. Identified mAbs represent novel and valuable molecular tools for understanding and potentially treating common metabolic diseases, such as type 2 diabetes mellitus and nonalcoholic steatohepatitis/fatty liver disease (NASH/NAFLD). Furthermore, the technologies underpinning their discovery can be rapidly and routinely employed to facilitate discovery of mAbs against other therapeutically relevant membrane protein targets. Disclosure D. Tucker: None. T. Charpentier: None. N. Molino: None. E. Rosenberg: None. B. Screnci: None. L.J. Stafford: None. C. Sulli: None. R. Chambers: None. B.J. Doranz: None. J.B. Rucker: None. Funding National Institutes of Health

A novel mutation in Slc2a4 as a mouse model of fatigue.

Chronic fatigue is a debilitating disorder with widespread consequences, but effective treatment strategies are lacking. Novel genetic mouse models of fatigue may prove invaluable for studying its underlying physiological mechanisms and for testing treatments and interventions. In a screen of voluntary wheel-running behavior in N-ethyl-N-nitrosourea mutagenized C57BL/6J mice, we discovered two lines with low body weights and aberrant wheel-running patterns suggestive of a fatigue phenotype. Affected progeny from these lines had lower daily activity levels and exhibited low amplitude circadian rhythm alterations. Their aberrant behavior was characterized by frequent interruptions and periods of inactivity throughout the dark phase of the light-dark cycle and increased levels of activity during the rest or light phase. Expression of the behavioral phenotypes in offspring of strategic crosses was consistent with a recessive inheritance pattern. Mapping of phenotypic abnormalities showed linkage with a single locus on chromosome 1, and whole exome sequencing identified a single point mutation in the Slc2a4 gene encoding the GLUT4 insulin-responsive glucose transporter. The single nucleotide change (A-T, which we named "twiggy") was in the distal end of exon 10 and resulted in a premature stop (Y440*). Additional metabolic phenotyping confirmed that these mice recapitulate phenotypes found in GLUT4 knockout mice. However, to the best of our knowledge, this is the first time a mutation in this gene has been shown to result in extensive changes in general behavioral patterns. These findings suggest that GLUT4 may be involved in circadian behavioral abnormalities and could provide insights into fatigue in humans.

PID1 regulates insulin-dependent glucose uptake by controlling intracellular sorting of GLUT4-storage vesicles

The phosphotyrosine interacting domain-containing protein 1 (PID1) serves as a cytosolic adaptor protein of the LDL receptor-related protein 1 (LRP1). By regulating its intracellular trafficking, PID1 controls the hepatic, LRP1-dependent clearance of pro-atherogenic lipoproteins. In adipose and muscle tissues, LRP1 is present in endosomal storage vesicles containing the insulin-responsive glucose transporter 4 (GLUT4). This prompted us to investigate whether PID1 modulates GLUT4 translocation and function via its interaction with the LRP1 cytosolic domain. We initially evaluated this in primary brown adipocytes as we observed an inverse correlation between brown adipose tissue glucose uptake and expression of LRP1 and PID1. Insulin stimulation in wild type brown adipocytes induced LRP1 and GLUT4 translocation from endosomal storage vesicles to the cell surface. Loss of PID1 expression in brown adipocytes prompted LRP1 and GLUT4 sorting to the plasma membrane independent of insulin signaling. When placed on a diabetogenic high fat diet, systemic and adipocyte-specific PID1-deficient mice presented with improved hyperglycemia and glucose tolerance as well as reduced basal plasma insulin levels compared to wild type control mice. Moreover, the improvements in glucose parameters associated with increased glucose uptake in adipose and muscle tissues from PID1-deficient mice. The data provide evidence that PID1 serves as an insulin-regulated retention adaptor protein controlling translocation of LRP1 in conjunction with GLUT4 to the plasma membrane of adipocytes. Notably, loss of PID1 corrects for insulin resistance-associated hyperglycemia emphasizing its pivotal role and therapeutic potential in the regulation of glucose homeostasis.

Molecular and metabolic adaption of glucose metabolism in the red and white muscle of the omnivorous GIFT tilapia Oreochromis niloticus to a glucose load

In this experiment, Genetically improved farmed Nile tilapia Oreochromis niloticus were intraperitoneally injected with 1 g glucose/kg of body weight or saline. Red and white muscle tissues were collected at 0, 1, 2, 4, 6 and 12 h after the glucose tolerance test (GTT) or saline injection, and the time course of changes in molecular and metabolic adaption of glucose metabolism of these two tissues were evaluated. The results showed that the expression of insulin-responsive glucose transporter 4 (glut4) was up-regulated at 4 h after the GTT in the red muscle, implying an increase of glucose uptake. However, the expression of glut4 in the white muscle did not change with glucose load. The glycolysis of red muscle in tilapia was stimulated during 2–4 h after the GTT, as the expression of hexokinase 1b (hk1b), hk2, phosphofructokinase muscle type a (pfkma) and pfkmb and the activity of HK and PFK increased. By contrast, only the expression of hk1b was up-regulated at 6 h after the GTT in the white muscle. The mRNA level of glycogen synthase 1 (gys1) and glycogen content increased at 2 and 6 h, respectively after the GTT in the red muscle, suggesting that glucose storage was provoked. However, glycogen content in the white muscle was not impacted by GTT. Lipogenesis was stimulated in the red muscle as reflected by up-regulated expression of acetyl-CoA carboxylase α (accα) (during 2–4 h) and accβ (during 4–12 h) with GTT. In the white muscle, however, the expression of accα was not changed, and mRNA level of accβ was not up-regulated until 6 h after the GTT. Taken together, it was concluded that the glycolytic and glycogen synthesis mechanisms in the red muscle were highly regulated by an acute glucose load while those in the white muscle were less responsive to this stimulus.

RAPID COMMUNICATION: Residual feed intake in beef cattle is associated with differences in protein turnover and nutrient transporters in ruminal epithelium.

Residual feed intake (RFI) is a widely used measure of feed efficiency in cattle. Although the precise biologic mechanisms associated with improved feed efficiency are not well-known, most-efficient steers (i.e., with low RFI coefficient) downregulate abundance of proteins controlling protein degradation in skeletal muscle. Whether cellular mechanisms controlling protein turnover in ruminal tissue differ by RFI classification is unknown. The aim was to investigate associations between RFI and signaling through the mechanistic target of rapamycin (MTOR) and ubiquitin-proteasome pathways in ruminal epithelium. One hundred and forty-nine Red Angus cattle were allocated to 3 contemporary groups according to sex and herd origin. Animals were offered a finishing diet for 70 d to calculate the RFI coefficient for each. Within each group, the 2 most-efficient (n = 6) and least-efficient animals (n = 6) were selected. Compared with least-efficient animals, the most-efficient animals consumed less feed (P < 0.05; 18.36 vs. 23.39 kg/d DMI). At day 70, plasma samples were collected for insulin concentration analysis. Ruminal epithelium was collected immediately after slaughter to determine abundance and phosphorylation status of 29 proteins associated with MTOR, ubiquitin-proteasome, insulin signaling, and glucose and amino acid transport. Among the proteins involved in cellular protein synthesis, most-efficient animals had lower (P ≤ 0.05) abundance of MTOR, p-MTOR, RPS6KB1, EIF2A, EEF2K, AKT1, and RPS6KB1, whereas MAPK3 tended (P = 0.07) to be lower. In contrast, abundance of p-EEF2K, p-EEF2K:EEF2K, and p-EIF2A:EIF2A in most-efficient animals was greater (P ≤ 0.05). Among proteins catalyzing steps required for protein degradation, the abundance of UBA1, NEDD4, and STUB1 was lower (P ≤ 0.05) and MDM2 tended (P = 0.06) to be lower in most-efficient cattle. Plasma insulin and ruminal epithelium insulin signaling proteins did not differ (P > 0.05) between RFI groups. However, abundance of the insulin-responsive glucose transporter SLC2A4 and the amino acid transporters SLC1A3 and SLC1A5 also was lower (P ≤ 0.05) in most-efficient cattle. Overall, the data indicate that differences in signaling mechanisms controlling protein turnover and nutrient transport in ruminal epithelium are components of feed efficiency in beef cattle.

Novel dual-color drug screening model for GLUT4 translocation in adipocytes

Insulin-responsive glucose transporter type 4 (GLUT4) translocation plays a major role in controlling glucose uptake in adipose tissue and muscle, maintaining homeostasis and preventing hyperglycemia. Screening for chemicals enhancing GLUT4 translocation is an approach for identifying hits of drug development for type 2 diabetes. Here we developed a novel functional dual-color probe, pHluorin-GLUT4-mOrange2, and constructed 3T3-L1 adipocytes based screening system to simply and efficiently screen new compounds stimulating GLUT4 translocation. Based on this system, we successfully identified a few hits facilitating GLUT4 translocation. In conclusion, we developed an easy-to-apply dual color GLUT4 probe to monitor GLUT4 translocation in insulin-responsive cells, which could be alternatively employed to high-throughput screen compounds regulating GLUT4 translocation and glucose uptake, even to dissect GLTU4 approaching, docking and fusion with the plasma membrane (PM), and to reveal relevant molecular mechanisms involved in these steps as expected.