Glucose Uptake In Mice Research Articles

Rosmarinic acid against cognitive impairment via RACK1/HIF-1α regulated microglial polarization in sepsis-surviving mice

Microglial polarization modulation has been considered the potential therapeutic strategy for relieving cognitive impairment in sepsis survivors. Rosmarinic acid (RA), a water-soluble polyphenolic natural compound, processes a strong protective effect on various types of neurological disorders including Parkinson's disease, depression, and anxiety. However, its role and potential molecular mechanisms in sepsis-associated cognitive impairment remain unclear. To investigate the preventive and therapeutic effect of RA on sepsis-associated cognitive impairment and elucidate the potential mechanism of RA on regulating microglial polarization, we established a CLP-induced cognitive impairment model in mice and a lipopolysaccharide-induced microglia polarization cell model in BV-2. RACK1 siRNA was designed to identify the potential molecular mechanism of RACK1 on microglial polarization. The preventive and therapeutic effect of RA on cognitive impairment followed by PET-CT and behavioral tests including open-field test and tail suspension test. RACK1/HIF-1α pathway and microglial morphology in the hippocampus or BV-2 cells were measured. The results showed that RA significantly ameliorated the CLP-induced depressive and anxiety-like behaviors and promoted whole-brain glucose uptake in mice. Moreover, RA markedly improved CLP-induced hippocampal neuron loss and microglial activation by inhibiting microglial M1 polarization. Furthermore, experiments showed RACK1 was involved in the regulation of LPS-induced microglial M1 polarization via HIF-1α, and RA suppressed lipopolysaccharide or sepsis-associated microglial M1 polarization via RACK1/HIF-1α pathway (rescued the decrease of RACK1 and increase of HIF-1α). Taken together, RA could be a potential preventive and therapeutic medication in improving cognitive impairment through RACK1/HIF-1α pathway-regulated microglial polarization.

Different effects of CYP27A1 and CYP7B1 on cognitive function: Two mouse models in comparison

The oxysterol 27-hydroxycholesterol (27OHC) is produced by the enzyme sterol 27-hydroxylase (Cyp27A1) and is mainly catabolized to 7α-Hydroxy-3-oxo-4-cholestenoic acid (7-HOCA) by the enzyme cytochrome P-450 oxysterol 7α-hydroxylase (Cyp7B1). 27OHC is mostly produced in the liver and can reach the brain by crossing the blood-brain barrier. A large body of evidence shows that CYP27A1 overexpression and high levels of 27OHC have a detrimental effect on the brain, causing cognitive and synaptic dysfunction together with a decrease in glucose uptake in mice. In this work, we analyzed two mouse models with high levels of 27OHC: Cyp7B1 knock-out mice and CYP27A1 overexpressing mice. Despite the accumulation of 27OHC in both models, Cyp7B1 knock-out mice maintained intact learning and memory capacities, neuronal morphology, and brain glucose uptake over time. Neurons treated with the Cyp7B1 metabolite 7-HOCA did not show changes in synaptic genes and 27OHC-treated Cyp7B1 knock-out neurons could not counteract 27OHC detrimental effects. This suggests that 7-HOCA and Cyp7B1 deletion in neurons do not mediate the neuroprotective effects observed in Cyp7B1 knock-out animals. RNA-seq of neuronal nuclei sorted from Cyp7B1 knock-out brains revealed upregulation of genes likely to confer neuroprotection to these animals. Differently from Cyp7B1 knock-out mice, transcriptomic data from CYP27A1 overexpressing neurons showed significant downregulation of genes associated with synaptic function and several metabolic processes. Our results suggest that the differences observed in the two models may be mediated by the higher levels of Cyp7B1 substrates such as 25-hydroxycholesterol and 3β-Adiol in the knock-out mice and that CYP27A1 overexpressing mice may be a more suitable model for studying 27-OHC-specific signaling. We believe that future studies on Cyp7B1 and Cyp27A1 will contribute to a better understanding of the pathogenic mechanisms of neurodegenerative diseases like Alzheimer’s disease and may lead to potential new therapeutic approaches.

Overexpression of a Short Sulfonylurea Splice Variant Increases Cardiac Glucose Uptake and Uncouples Mitochondria by Regulating ROMK Activity.

The mitochondrial splice variant of the sulfonylurea receptor (SUR2A-55) is associated with protection from myocardial ischemia-reperfusion (IR) injury, increased mitochondrial ATP sensitive K+ channel activity (mitoKATP) and altered glucose metabolism. While mitoKATP channels composed of CCDC51 and ABCB8 exist, the mitochondrial K+ pore regulated by SUR2A-55 is unknown. We explored whether SUR2A-55 regulates ROMK to form an alternate mitoKATP. We assessed glucose uptake in mice overexpressing SUR2A-55 (TGSUR2A-55) compared with WT mice during IR injury. We then examined the expression level of ROMK and the effect of ROMK modulation on mitochondrial membrane potential (Δψm) in WT and TGSUR2A-55 mice. TGSUR2A-55 had increased glucose uptake compared to WT mice during IR injury. The expression of ROMK was similar in WT compared to TGSUR2A-55 mice. ROMK inhibition hyperpolarized resting cardiomyocyte Δψm from TGSUR2A-55 mice but not from WT mice. In addition, TGSUR2A-55 and ROMK inhibitor treated WT isolated cardiomyocytes had enhanced mitochondrial uncoupling. ROMK inhibition blocked diazoxide induced Δψm depolarization and prevented preservation of Δψm from FCCP perfusion in WT and to a lesser degree TGSUR2A-55 mice. In conclusion, cardio-protection from SUR2A-55 is associated with ROMK regulation, enhanced mitochondrial uncoupling and increased glucose uptake.

Selection for Growth Does Not Affect Apparent Energetic Efficiency of Jejunal Glucose Uptake in Mice

Five-wk-old male mice from high growth (M16) and randomly bred control (ICR) lines, plus their reciprocal crosses, ICR × M16 and M16 × ICR, were used to investigate whether whole-body O2 consumption, jejunal respiration, jejunal glucose absorption and the apparent energetic efficiency of jejunal active glucose uptake in mice are altered by genetic selection for growth as well as by heterosis and maternal effects. Whole-body O2 consumption was measured in 12 mice from each line or cross. The mice were later killed for measurement of jejunal O2, using tissue respiration chambers and jejunal glucose transport determined by 3H-3-O-methylglucose accumulation. No heterosis or maternal effects were detected in jejunal glucose active transport and active glucose uptake. Selection for growth (M16 vs. ICR) increased daily gain (1.54 vs. 1.09 g, P < 0.001), small intestinal length and weight, but did not enhance jejunal glucose transport. The apparent energetic efficiency of jejunal active glucose uptake among lines was not different (54.0, 50.4, 51.6 and 47.1 nmol ATP expended/nmol glucose uptake for M16, ICR, M16 × ICR and ICR × M16, respectively, P > 0.63). Selection for growth in mice did not result in more energetically efficient jejunal glucose absorption.

Selection for Body Composition Does Not Affect Energetic Efficiency of Jejunal Glucose Uptake in Mice

Five-wk-old male mice from three lines were used to examine whether the apparent energetic efficiency of active jejunal glucose uptake in mouse jejunum is altered by genetic selection for different body composition. The mice lines were selected as follows: HE, high percentage of body fat with no change in body weight as a constraint; LF, low percentage of body fat; and RS, randomly bred control. Body weight was similar in all lines. Total jejunal O2 consumption and ouabain-sensitive O2 consumption were used to estimate the energy expenditure associated with glucose absorption and Na+/K+-ATPase activity. Tritiated 3-O-methyl-D-glucose was used to determine glucose uptake by mouse jejunum. Line LF, when compared with line HE, had lower body fat as indicated by epididymal fat pad weight (143 vs. 362 mg/mouse, P < 0.001). There were no significant differences in small intestinal weight, length and density (mg/cm) between LF and HE lines. Jejunal villus width was greater in line LF compared with line HE (115 vs. 92 µm, P < 0.002). Jejunal glucose transport and O2 consumption were not different between LF and HE lines. Ouabain-sensitive O2 consumption was not significantly different among the three lines. No differences were noted in the apparent energetic efficiency of active glucose uptake among lines.

Measurement of skeletal muscle glucose uptake in mice in response to acute treadmill running.

Skeletal muscle contractions stimulate glucose uptake into the working muscles during exercise. Because this signaling pathway is independent of insulin, exercise constitutes an important alternative pathway to increase glucose uptake, also in insulin-resistant muscle. Therefore, much effort is being put into understanding the molecular regulation of exercise-stimulated glucose uptake by skeletal muscle. To delineate the causal molecular mechanisms whereby muscle contraction or exercise regulate glucose uptake, the investigation of genetically manipulated rodents is necessary. Presented here is a modified and optimized protocol assessing exercise-induced muscle glucose uptake in mice in response to acute treadmill running. Using this high-throughput protocol, running capacity can accurately and reproducibly be determined in mice, and basal- and exercise-stimulated skeletal muscle glucose uptake and intracellular signaling can precisely and dose-dependently be measured in awake mice in vivo without the need for catheterization and with minimal loss of blood.

Microglial activation states drive glucose uptake and FDG-PET alterations in neurodegenerative diseases.

2-Deoxy-2-[18F]fluoro-d-glucose positron emission tomography (FDG-PET) is widely used to study cerebral glucose metabolism. Here, we investigated whether the FDG-PET signal is directly influenced by microglial glucose uptake in mouse models and patients with neurodegenerative diseases. Using a recently developed approach for cell sorting after FDG injection, we found that, at cellular resolution, microglia displayed higher glucose uptake than neurons and astrocytes. Alterations in microglial glucose uptake were responsible for both the FDG-PET signal decrease in Trem2-deficient mice and the FDG-PET signal increase in mouse models for amyloidosis. Thus, opposite microglial activation states determine the differential FDG uptake. Consistently, 12 patients with Alzheimer’s disease and 21 patients with four-repeat tauopathies also exhibited a positive association between glucose uptake and microglial activity as determined by 18F-GE-180 18-kDa translocator protein PET (TSPO-PET) in preserved brain regions, indicating that the cerebral glucose uptake in humans is also strongly influenced by microglial activity. Our findings suggest that microglia activation states are responsible for FDG-PET signal alterations in patients with neurodegenerative diseases and mouse models for amyloidosis. Microglial activation states should therefore be considered when performing FDG-PET.

Atrophy of White Adipose Tissue Accompanied with Decreased Insulin-Stimulated Glucose Uptake in Mice Lacking the Small GTPase Rac1 Specifically in Adipocytes.

Insulin stimulates glucose uptake in adipose tissue and skeletal muscle by inducing plasma membrane translocation of the glucose transporter GLUT4. Although the small GTPase Rac1 is a key regulator downstream of phosphoinositide 3-kinase (PI3K) and the protein kinase Akt2 in skeletal muscle, it remains unclear whether Rac1 also regulates glucose uptake in white adipocytes. Herein, we investigated the physiological role of Rac1 in white adipocytes by employing adipocyte-specific rac1 knockout (adipo-rac1-KO) mice. Subcutaneous and epididymal white adipose tissues (WATs) in adipo-rac1-KO mice showed significant reductions in size and weight. Actually, white adipocytes lacking Rac1 were smaller than controls. Insulin-stimulated glucose uptake and GLUT4 translocation were abrogated in rac1-KO white adipocytes. On the other hand, GLUT4 translocation was augmented by constitutively activated PI3K or Akt2 in control, but not in rac1-KO, white adipocytes. Similarly, to skeletal muscle, the involvement of another small GTPase RalA downstream of Rac1 was demonstrated. In addition, mRNA levels of various lipogenic enzymes were down-regulated in rac1-KO white adipocytes. Collectively, these results suggest that Rac1 is implicated in insulin-dependent glucose uptake and lipogenesis in white adipocytes, and reduced insulin responsiveness due to the deficiency of Rac1 may be a likely explanation for atrophy of WATs.

Mouse Spexin: (III) Differential Regulation by Glucose and Insulin in Glandular Stomach and Functional Implication in Feeding Control.

Spexin (SPX), a neuropeptide with diverse functions, is a novel satiety factor in fish models and its role in feeding control has been recently confirmed in mammals. In mouse, food intake was shown to trigger SPX expression in glandular stomach with parallel rise in serum SPX and these SPX signals could inhibit feeding via central actions within the hypothalamus. However, the mechanisms for SPX regulation by food intake are still unclear. To examine the role of insulin signal caused by glucose uptake in SPX regulation, the mice were IP injected with glucose and insulin, respectively. In this case, serum SPX was elevated by glucose but not altered by insulin. Meanwhile, SPX transcript expression in the glandular stomach was up-regulated by glucose but the opposite was true for insulin treatment. Using in situ hybridization, the differential effects on SPX gene expression were located in the gastric mucosa of glandular stomach. Co-injection experiments also revealed that glucose stimulation on serum SPX and SPX mRNA expressed in glandular stomach could be blocked by insulin. In gastric mucosal cells prepared from glandular stomach, the opposite effects on SPX transcript expression by glucose and insulin could still be noted with similar blockade of the stimulatory effects of glucose by insulin. In this cell model, SPX gene expression induced by glucose was mediated by glucose uptake via GLUT, ATP synthesis by glycolysis/respiratory chain, and subsequent modulation of KATP channel activity, but the voltage-sensitive Ca2+ channels were not involved. The corresponding inhibition by insulin, however, was mediated by PI3K/Akt, MEK1/2/ERK1/2, and P38 MAPK cascades coupled to insulin receptor but not IGF-1 receptor. Apparently, glucose uptake in mice can induce SPX expression in the glandular stomach through ATP synthesis via glucose metabolism and subsequent modification of KATP channel activity, which may contribute to SPX release into circulation to act as the satiety signal after food intake. The insulin rise caused by glucose uptake, presumably originated from the pancreas, may serve as a negative feedback to inhibit the SPX response by activating MAPK and PI3K/Akt pathways in the stomach.

Characterization of Viral Insulin-Like Peptides Reveals Unique White Adipose Tissue Specific Characteristics

The members of the insulin superfamily are well conserved across the evolution tree. We recently showed that four viruses in the Iridoviridae family possess genes that share high similarity with human insulin and IGF-1. By chemically synthesizing single chain (sc, IGF-1 like) forms of these viral insulin/IGF-1 like peptides (VILPs), we previously showed that sc VILPs have insulin/IGF properties in vitro and in vivo. However, characteristics of double chain (dc, insulin-like) VILPs remain unknown. In this study, we characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1). We showed that GIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A, IR-B) and they bind to IGF-1R with a higher affinity than human insulin. These dcVILPs stimulate receptor phosphorylation and post-receptor signaling in vitro and in vivo. LCDV-1 dcVILP stimulated a weak response in in vitro signaling experiments, although we could not determine binding competition. Both GIV and SGIV dcVILPs stimulated glucose uptake in mice. In vivo infusion experiments in awake mice revealed that while insulin (2.5 mU/kg/min) and GIV dcVILP (125 mU/kg/min) stimulate a comparable glucose uptake in heart, skeletal muscle and brown adipose tissue, GIV dcVILP stimulates ~2 fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This is due to increased Akt phosphorylation and glucose transporter type 4 (GLUT4) expression compared to insulin specifically in WAT. Taken together, these results show that dc GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. This observation evokes questions about their potential roles in human disease including diabetes and cancer. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action and design new analogues that specifically target the tissues.

Characterization of viral insulins reveals white adipose tissue-specific effects in mice

ObjectiveMembers of the insulin/insulin-like growth factor (IGF) superfamily are well conserved across the evolutionary tree. We recently showed that four viruses in the Iridoviridae family possess genes that encode proteins highly homologous to human insulin/IGF-1. Using chemically synthesized single-chain (sc), i.e., IGF-1-like, forms of the viral insulin/IGF-1-like peptides (VILPs), we previously showed that they can stimulate human receptors. Because these peptides possess potential cleavage sites to form double chain (dc), i.e., more insulin-like, VILPs, in this study, we have characterized dc forms of VILPs for Grouper iridovirus (GIV), Singapore grouper iridovirus (SGIV) and Lymphocystis disease virus-1 (LCDV-1) for the first time. MethodsThe dcVILPs were chemically synthesized. Using murine fibroblast cell lines overexpressing insulin receptor (IR-A or IR-B) or IGF1R, we first determined the binding affinity of dcVILPs to the receptors and characterized post-receptor signaling. Further, we used C57BL/6J mice to study the effect of dcVILPs on lowering blood glucose. We designed a 3-h dcVILP in vivo infusion experiment to determine the glucose uptake in different tissues. ResultsGIV and SGIV dcVILPs bind to both isoforms of human insulin receptor (IR-A and IR-B) and to the IGF1R, and for the latter, show higher affinity than human insulin. These dcVILPs stimulate IR and IGF1R phosphorylation and post-receptor signaling in vitro and in vivo. Both GIV and SGIV dcVILPs stimulate glucose uptake in mice. In vivo infusion experiments revealed that while insulin (0.015 nmol/kg/min) and GIV dcVILP (0.75 nmol/kg/min) stimulated a comparable glucose uptake in heart and skeletal muscle and brown adipose tissue, GIV dcVILP stimulated 2-fold higher glucose uptake in white adipose tissue (WAT) compared to insulin. This was associated with increased Akt phosphorylation and glucose transporter type 4 (GLUT4) gene expression compared to insulin in WAT. ConclusionsOur results show that GIV and SGIV dcVILPs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help us to better understand insulin action, design new analogs that specifically target the tissues and provide new insights into their potential role in disease.

Hydrocarboxylic acid receptor 1 in BAT regulates glucose uptake in mice fed a high-fat diet.

Interscapular brown adipose tissue (BAT) has the capability to take up glucose from the circulation. Despite the important role of BAT in the control of glucose homeostasis, the metabolic fate and function of glucose in BAT remain elusive as there is clear dissociation between glucose uptake and BAT thermogenesis. Interestingly, intracellular glycolysis and lactate production appear to be required for glucose uptake by BAT. Here, we specifically examine whether activation of lactate receptors in BAT plays a key role in regulating glucose homeostasis in mice fed a high-fat diet (HFD). When C57BL/6J mice are given HFD for 5 weeks at 28°C, male, but not female, mice gain body weight and develop hyperglycemia. Importantly, high-fat feeding upregulates expression of the lactate receptor hydroxycarboxylic acid receptor 1 (HCAR1) in female C57BL/6J mice, whereas male C57BL/6J mice show reduced HCAR1 expression in BAT. Treatment with the HCAR1 agonist lowers systemic glucose levels in male DIO mice. This reduction is associated with increased glucose uptake in BAT. Therefore, our results suggest that HCAR1 in BAT may contribute to the development of hyperglycemia in male C57BL/6J DIO mice.

228-OR: Cerebral Glucose Transport and Metabolism in Obesity

Metabolic derangement triggered by a high fat diet has been associated with decreased brain glucose uptake in mice through downregulation of GLUT-1 expression at the blood-brain barrier. Likewise, a prior study by our group using 1H magnetic resonance spectroscopy (MRS) showed diminished change in brain glucose level during acute hyperglycemia in patients with obesity and diabetes. However, whether this reflected differences in absolute brain concentrations remains unknown. We utilized 13C MRS scanning at 4 Tesla to compare the rate of glucose transport (Tmax) and cerebral metabolic rate of glucose (CMRgl) in the occipital lobe during a 2-hour hyperglycemic clamp. Plasma glucose concentration of ∼180mg/dl was achieved using a variable 1-13C-glucose infusion. The study included 8 healthy lean (1M/7F, age 29 ± 5 years, BMI 21 ± 2 kg/m2, HgbA1c 5.4 ± 0.2%) and 6 obese participants (4M/2F, age 30 ± 3 years, BMI 33 ± 3 kg/m2, HgbA1c 5.5 ± 0.2%). Insulin and free fatty acids (FFA) levels were measured during the clamp. Despite nearly identical plasma glucose concentrations at steady state (lean 182.5 ± 9.0 mg/dL, obese 186.5 ± 13.5 mg/dL, P=0.55, averaged over time 60-120 minutes), the absolute brain glucose concentrations were 30% less in obese compared to lean participants (P=0.02). Moreover, using previously reported Michaelis-Menten Kt of 1.1 mM, the calculated Tmax/CMRgl at steady state were 37% less in obese participants (P=0.01), suggesting reduced glucose transport. A trend of higher insulin and FFA levels in obese participants during hyperglycemia was observed. In addition, FFA levels at steady state were negatively correlated with absolute brain glucose concentrations (r= -0.575, P=0.05). We conclude that obesity is associated with diminished absolute concentration of brain glucose during acute hyperglycemia, likely explained by reduced cerebral glucose transport capacity. These findings may have implications for understanding the impact of obesity on central regulation of feeding behavior as well as neurocognitive function. Disclosure F. Gunawan: None. L. Jiang: None. J. Leventhal: None. J.J. Pach: None. E. Sanchez Rangel: None. R. Belfort-DeAguiar: Research Support; Self; Silver Palate Kitchens, Inc. A. Coppoli: None. D.L. Rothman: None. R. Sherwin: Other Relationship; Self; ICON plc., IQVIA, MannKind Corporation. G.F. Mason: None. J.J. Hwang: None. Funding American Diabetes Association (1-17-ICTS-013 to J.J.H.)

CRISPR-Engineered Human Brown-Like Adipocytes Promotes Metabolic Homeostasis through Activation of Endogenous Brown Fat

Brown and brown-like beige adipocytes dissipate energy for thermogenesis and have been proposed to combat obesity and metabolic disorders. Uncoupling protein 1 (UCP1) is uniquely expressed in brown and beige adipocytes, and facilitates fuel utilization and energy expenditure. Ectopic UCP1 overexpression improves obesity and insulin resistance in mice. Recently, we utilized the CRISPR/Cas9 synergistic activation mediator and specific gRNAs to boost endogenous UCP1 expression in human white adipocytes. The CRISPR/Cas9 engineered human brown fat-like (referred to as HUMBLE, hereafter) cells had acquired human brown fat features when comparing bona fide human brown adipocytes derived from the same individual. Obese mice receiving a transplantation of HUMBLE cells showed improved glucose tolerance and insulin sensitivity, as well as increased energy expenditure. This was mediated, at least in part, via activation of endogenous brown fat, which showed an increase in heat production and glucose uptake in mice receiving HUMBLE cells. Co-culture with HUMBLE cells or treatment of conditional medium from HUMBLE cells promoted glucose uptake and thermogenesis in brown adipocytes. Metabolomic analysis identified an increased ratio of arginine and asymmetrical dimethylarginine (ADMA) in the sera of mice receiving HUMBLE cell transplantation. Arginine is known to promote nitric oxide biosynthesis, which can activate thermogenic function of brown fat. Consistently, the level of nitric oxide was increased in the endogenous brown fat of mice receiving HUMBLE cells. Inhibition of nitric oxide synthase abolished the HUMBLE cells-mediated activation of brown adipocytes in vitro and in vivo. In summary, CRISPR/Cas9-mediated activation of endogenous UCP1 expression in human white adipocytes creates brown fat-like features, and activates endogenous brown fat, potentially through arginine-related metabolites to improve the systemic glucose utilization. Disclosure C. Wang: None. M. Lundh: None. R. Kriszt: Employee; Self; QuintilesIMS. T. Huang: None. L.O. Leiria: None. F. Shamsi: None. M. Lynes: None. J. Darcy: None. B. Emanuelli: None. M. Kiebish: None. Y. Tseng: Other Relationship; Self; Chugai Pharmaceutical Co., Ltd.. Research Support; Self; MedImmune.

A Tbc1d1 Ser231Ala-knockin mutation partially impairs AICAR- but not exercise-induced muscle glucose uptake in mice.

TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab GTPase-activating protein (RabGAP) that has been implicated in regulating GLUT4 trafficking. TBC1D1 can be phosphorylated by the AMP-activated protein kinase (AMPK) on Ser231, which consequently interacts with 14-3-3 proteins. Given the key role for AMPK in regulating insulin-independent muscle glucose uptake, we hypothesised that TBC1D1-Ser231 phosphorylation and/or 14-3-3 binding may mediate AMPK-governed glucose homeostasis. Whole-body glucose homeostasis and muscle glucose uptake were assayed in mice bearing a Tbc1d1 Ser231Ala-knockin mutation or harbouring skeletal muscle-specific Ampkα1/α2 (also known as Prkaa1/2) double-knockout mutations in response to an AMPK-activating agent, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Exercise-induced muscle glucose uptake and exercise capacity were also determined in the Tbc1d1 Ser231Ala-knockin mice. Skeletal muscle-specific deletion of Ampkα1/a2 in mice prevented AICAR-induced hypoglycaemia and muscle glucose uptake. The Tbc1d1 Ser231Ala-knockin mutation also attenuated the glucose-lowering effect of AICAR in mice. Glucose uptake and cell surface GLUT4 content were significantly lower in muscle isolated from the Tbc1d1 Ser231Ala-knockin mice upon stimulation with a submaximal dose of AICAR. However, this Tbc1d1 Ser231Ala-knockin mutation neither impaired exercise-induced muscle glucose uptake nor affected exercise capacity in mice. TBC1D1-Ser231 phosphorylation and/or 14-3-3 binding partially mediates AMPK-governed glucose homeostasis and muscle glucose uptake in a context-dependent manner.

Impact of Image-Derived Input Function and Fit Time Intervals on Patlak Quantification of Myocardial Glucose Uptake in Mice.

Limited blood volume in mice precludes repeated sampling, rendering a reliable image-derived input function (IDIF) desirable for quantification of glucose uptake. We aimed to compare different IDIF volumes and to evaluate the effects of changing fit time interval on Patlak uptake kinetics in hearts of healthy mice. C57BL/6 mice (n=27) were studied under a range of metabolic conditions: no intervention (ctl), overnight fasting, insulin and glucose (6 mU/g, 1 mg/g) under isoflurane, and under ketamine-xylazine anesthesia to suppress glucose uptake. Dynamic PET imaging with 18F-FDG (7.7±0.9 MBq) was conducted. Images were analyzed using left ventricle cavity, left atrial cavity, or inferior vena cava as the IDIF. Patlak analysis was conducted using variable fit time intervals: automated fit, fit from 10 to 60 min (late), fit from 2 to 30 min (early), or fit from 2 to 10 min (very early). Both the ventricle and the atrial cavities displayed spill-in from the myocardium in late frames as compared with the vena cava (percentage injected dose per gram, ctl: 21.4±6.1 vs. 10.0±3.9 vs. 2.5±0.3, P<0.001). Higher and more rapid passage of peak activity was observed in the vena cava, but the area under the curve over 2 min was similar. The Patlak slope was significantly higher for the vena cava than atrial IDIF (mL/g/min, ctl: 0.11±0.02 vs. 0.07±0.01; fasting: 0.09±0.03 vs. 0.06±0.02; insulin: 0.52±0.09 vs. 0.23±0.12; ketamine-xylazine: 0.001±0.001 vs. 0.002±0.002; P<0.01). The rate of glucose uptake was similarly elevated depending on the IDIF (P<0.01). The various IDIF Patlak values were significantly correlated (r=0.867, P<0.001). Automated fit performed reliably in untreated, fasted, and ketamine-xylazine-treated mice, with no statistical difference compared with late, early, or very early fits. The Patlak composite rate constant (Ki) was markedly underestimated with automated and late fit after acute insulin treatment, reflecting the rapid early 18F-FDG uptake. The choice of IDIF has a profound effect on Patlak kinetics and calculated 18F-FDG uptake. Adjustment of the time interval for fit may be necessary for accurate calculations, particularly with acute insulin treatment. Even without partial-volume correction, the inferior vena cava provides a reliable and reproducible IDIF for Patlak analysis of myocardial glucose uptake in mice.

Glucose Transporter 4 (GLUT4) is Not the Only GLUT Responsible for Ca 2+ /Calmodulin‐Dependent Protein Kinase Kinase α (CaMKKα)‐Induced Glucose Uptake in Mouse Skeletal Muscle

Chronic activation of Ca2+/calmodulin-dependent protein kinase kinase α (CaMKKα) signaling for 2 weeks stimulates skeletal muscle glucose uptake in both insulin-sensitive and insulin-resistant mice; yet surprisingly the glucose transporter(s) that mediate this effect are currently unknown. The goal of this study was to determine if glucose transporter 4 (GLUT4) is the main glucose transporter responsible for CaMKKα-induced muscle glucose uptake. To stimulate CaMKKα signaling, muscles from 13-14 week old, male, wild-type/control, muscle-specific GLUT4 heterozygous and muscle-specific GLUT4 knockout mice were transfected with plasmid DNA containing constitutively active CaMKKα or empty vector by in vivo electroporation. After 2 weeks, active CaMKKα protein levels were robustly increased and were not different between groups. In vivo muscle [3H]-2-deoxyglucose uptake was examined in the absence of insulin stimulation. Constitutively active CaMKKα increased glucose uptake ~250% in wild-type/control mice, ~100...

A gut microbial metabolite of ginsenosides, compound K, induces intestinal glucose absorption and Na(+) /glucose cotransporter 1 gene expression through activation of cAMP response element binding protein.

The Na(+) /glucose cotransporter 1 (SGLT1) plays a crucial role in glucose uptake in intestinal epithelial cells (IECs), which has been shown essential in ameliorating intestinal inflammation. Ginseng has historically been used to treat inflammatory disorders. Understanding the regulatory mechanism of ginseng-mediated induction of SGLT1 gene expression in human intestinal cells is therefore important. We demonstrate that ginsenoside compound K (CK) enhances SGLT1-mediated glucose uptake in mice and human intestinal Caco-2 cells. Transient transfection analysis using SGLT1 promoter-luciferase reporters demonstrated that the presence of an essential cAMP response element (CRE) is required for CK-mediated induction of SGLT1 gene expression. The ChIP assays indicated that increased CRE-binding protein (CREB) and CREB-binding protein (CBP) binding to the SGLT1 promoter in CK-treated cells is associated with an activated chromatin state. Our result showed that the increased CREB phosphorylation is directly correlated with SGLT1 expression in IECs. Further studies indicated that the epidermal growth factor receptor (EGFR) signaling pathway is involved in the CK-mediated effect. These findings provide a novel mechanism for the CK-mediated upregulation of SGLT1 expression through EGFR-CREB signaling activation, which could contribute to reducing gut inflammation.

Exercise-induced muscle glucose uptake in mice with graded, muscle-specific GLUT-4 deletion

To investigate the importance of the glucose transporter GLUT-4 for muscle glucose uptake during exercise, transgenic mice with skeletal muscle GLUT-4 expression approximately 30–60% of normal (CON) and approximately 5–10% of normal (KO) were generated using the Cre/Lox system and compared with wild-type (WT) mice during approximately 40 min of treadmill running (KO: 37.7 ± 1.3 min; WT: 40 min; CON: 40 min, P = 0.18). In WT and CON animals, exercise resulted in an overall increase in muscle glucose uptake. More specifically, glucose uptake was increased in red gastrocnemius of WT mice and in the soleus and red gastrocnemius of CON mice. In contrast, the exercise-induced increase in muscle glucose uptake in all muscles was completely abolished in KO mice. Muscle glucose uptake increased during exercise in both red and white quadriceps of WT mice, while the small increases in CON mice were not statistically significant. In KO mice, there was no change at all in quadriceps muscle glucose uptake. No differences in muscle glycogen use during exercise were observed between any of the groups. However, there was a significant increase in plasma glucose levels after exercise in KO mice. The results of this study demonstrated that a reduction in skeletal muscle GLUT-4 expression to approximately 10% of normal levels completely abolished the exercise-induced increase in muscle glucose uptake.

Rac1 Is a Novel Regulator of Contraction-Stimulated Glucose Uptake in Skeletal Muscle

In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (∼60–100%) and humans (∼40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20–58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake.