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Feb 2018

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Insulin Tolerance Test under Anaesthesia to Measure Tissue-specific Insulin-stimulated Glucose Disposal
麻醉下测量组织特异性胰岛素刺激的葡萄糖代谢的胰岛素耐量试验   

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Abstract

Insulin resistance is a pathophysiological state defined by impaired responses to insulin and is a risk factor for several metabolic diseases, most notably type 2 diabetes. Insulin resistance occurs in insulin target tissues including liver, adipose and skeletal muscle. Methods such as insulin tolerance tests and hyperinsulinaemic-euglycaemic clamps permit assessment of insulin responses in specific tissues and allow the study of the progression and causes of insulin resistance. Here we detail a protocol for assessing insulin action in adipose and muscle tissues in anesthetized mice administered with insulin intravenously.

Keywords: Insulin (胰岛素), Adipose tissue (脂肪组织), Muscle (肌肉), Glucose transport (葡萄糖转运), Glucose uptake (葡萄糖摄取), Insulin tolerance test (胰岛素耐量试验)

Background

Surrogate measures of whole-body insulin sensitivity such as homeostatic model assessment for insulin resistance (HOMA-IR) often do not reflect insulin-stimulated glucose uptake in peripheral tissues of the mouse (Lee et al., 2008; Mather, 2009). Ex vivo adipose tissue explants or isolated skeletal muscles preparations (Burchfield et al., 2018; Fazakerley et al., 2018b) can allow direct measurement of insulin responses in specific tissues if they are amenable to these procedures, but do not preserve the organism environment where finely articulated delivery of glucose and insulin via the vasculature, uptake into cells and intracellular metabolism may all contribute to the overall rate of tissue glucose uptake (Wasserman et al., 2011). Commonly employed methods to assess whole-body insulin action in mice include intraperitoneal (IP) or intravenous insulin tolerance tests (ITTs) and, the gold standard, hyperinsulinaemic-euglycaemic clamp (Ayala et al., 2010; Brandon et al., 2016). These techniques can assess whole-body insulin action by measuring either changes in blood glucose (ITT) or changes in the amount of glucose required to maintain glycemia (glucose infusion rate; hyperinsulinaemic-euglycaemic clamp) and can be adapted to measure tissue-specific insulin responses. For example, glucose tracers such as radiolabeled 2-deoxyglucose (2-DOG), which is often used as a surrogate for glucose in assessing glucose uptake as it is not metabolized through glycolysis and is ‘trapped’ in the cells following uptake, can be introduced to permit assessment of glucose uptake into specific tissues during these tests. Note that 2-DOG is only trapped in tissues that do not possess significant glucose-6-phosphatase activity, and so 2-DOG tracer is therefore not useful for assessing glucose disposal in the liver.

Both ITTs and hyperinsulinaemic-euglycaemic clamps can be performed in conscious or anesthetized mice. Certain anesthetics affect whole-body glucose metabolism and should therefore be avoided when assessing insulin action or glucose homeostasis [e.g., isoflurane (Pomplun et al., 2004; Tanaka et al., 2009; Windelov et al., 2016; Hoyer et al., 2018)]. However, anesthetics that minimally affect glucose metabolism, such as pentobarbitone (Guarino et al., 2013), may actually be advantageous for studying insulin-stimulated glucose uptake. This is because insulin-stimulated 2-DOG/glucose uptake in conscious mice may be affected by movement (e.g., due to mechanical- and/or exercise- stimulated glucose uptake) and/or stress responses [e.g., catecholamines (Cooney et al., 1985; Furler et al., 1991) and glucocorticoids (Pasieka and Rafacho, 2016)].

Here, we describe a protocol for a terminal intravenous ITT performed under pentobarbitone-induced anesthesia to assess insulin-stimulated glucose uptake into tissues of interest. The protocol can be performed with minimal delays between mice, making it amenable to assessing insulin action in large cohorts of animals. Intravenous administration of a bolus of insulin/tracer rapidly delivers insulin to tissues, minimizing the time between the start of the assay and initiation of insulin responses within tissues. Also, rapid equilibration of the 2-DOG tracer with the total blood glucose pool ensures that the tracer is immediately available for uptake into tissues. Alternative methods of insulin/tracer delivery (i.e., intraperitoneal injection, oral gavage) may result in delays or inconsistent insulin/tracer uptake into the central circulation. For example, in the case of IP injection, there may be a considerable delay before insulin reaches the circulation. These time lags may differ between mice or between injections, increasing experimental variability. Rapid and consistent tissue uptake is also particularly important when performing time-series experiments in live animals. Indeed, hepatic portal-vein administration of insulin has enabled the assessment of the time-resolved effects of insulin on the liver phosphoproteome (Humphrey et al., 2015). For oral gavage, 2-DOG may exhibit markedly different kinetics of appearance in the circulation compared to glucose since the sodium-dependent glucose transporters, which play a key role in oral glucose absorption, exhibit a strong preference for glucose (Bissonnette et al., 1996), and this may limit tracer availability to tissues during the assay.

In this protocol, mice are anesthetized with pentobarbitone and saline or insulin and radiolabeled 2-DOG tracer administered via the hepatic portal vein. Insulin action in muscle and adipose tissues of interest can be assessed via Western blotting for insulin signaling intermediates or through measuring radiolabeled 2-DOG tracer accumulation as an index of glucose uptake.

Materials and Reagents

  1. Pipette tips (e.g., Axygen, catalog numbers: T-1000-C, T-200-C)
  2. Cotton bud (e.g., McFarlane Medical Equipment, catalog number: 18049DE)
  3. 1.5 ml microfuge tubes (Axygen, catalog number: MCT-150-C)
  4. 2.0 ml microfuge tubes (Eppendorf, catalog number: 0030120.094)
  5. 5 ml polystyrene tubes (SARSTEDT, catalog number: 60.9921.531)
  6. 0.5 ml Ultra-Fine Insulin Syringes (Becton Dickinson, catalog number: 230-45094)
  7. Masking tape (e.g., Lyreco, catalog number: 737.665)
  8. 96-well plates (Corning, catalog number: CLS2595)
  9. Carbon Steel, size 22 scalpel blades (Livingstone, catalog number: SBLDCL22)
  10. Chromatography columns (Bio-Rad, catalog number: 731-1550)
  11. 6 ml pony vial scintillation tubes (PerkinElmer, catalog number: 6000292)
  12. Adult mice (> 8 weeks old)
    Note: Ensure experiments using mice are carried with the approval of local ethics committee. Mice may be subjected to different dietary/exercise regimes or be knockout/transgenic lines for genes of interest. For knockout/transgenic lines we recommend using littermate controls. 
  13. Double-distilled water (ddH2O)
  14. Bicinchoninic acid assay (BCA) protein assay kit (Thermo Fisher Scientific, catalog number: 23225)
  15. Antibodies
    1. Anti-phospho-Thr308 Akt antibody (Cell Signaling Technology, catalog number: 9275)
    2. Anti-phospho-Ser473 Akt antibody (Cell Signaling Technology, catalog number: 4051)
    3. Anti-Akt antibody (Cell Signaling Technology, catalog number: 4685)
  16. Pentobarbitone (Lethabarb, Virbac, Australia)
    Note: Pentobarbitone may be a regulated substance. Ensure that use follows local regulations. 
  17. Human insulin (Actrapid, Novo Nordisk)
  18. 0.9% Sodium Chloride Injection BP (Pfizer, catalog number: 158594)
  19. [3H]2-DOG (PerkinElmer, catalog number: NET328001MC)
  20. Accu-Chek performa Glucose Strips (Accu-Check II; Roche Diagnostics, catalog number: 06454038)
  21. Liquid nitrogen
  22. AG1-X8 resin (Bio-Rad, catalog number: 140-1443)
  23. Ulitma Gold XR Scintillation fluid (PerkinElmer, catalog number: 6013119)
  24. Barium hydroxide (Sigma, catalog number: 433373)
  25. Dry ice
  26. 2.75% zinc sulfate solution (see Recipes)
    Zinc sulfate (ZnSO4) (Sigma, catalog number: 307491)
  27. Phosphorylated [3H]2-DOG elution buffer (see Recipes)
    1. Trifluoroacetic acid (Sigma, catalog number: 302031)
    2. NaCl (Sigma, catalog number: S7653)
  28. Western blot lysis buffer (see Recipes)
    1. HEPES (Sigma, catalog number: 54457)
    2. Sucrose (Sigma, catalog number: S8501)
    3. EDTA (Sigma, catalog number: 03620)
    4. SDS (Sigma, catalog number: 71729)
    5. Protease inhibitors (Roche, cOmpleteTM, catalog number: 11697498001) 
    6. Sodium pyrophosphate (Sigma, catalog number: 71501)
    7. Sodium orthovanadate (Sigma, catalog number: S6508)
    8. Sodium fluoride (Sigma, catalog number: S7920)

Equipment

  1. Scalpel or surgical scissors (e.g., World Precision Instruments, catalog number: 501259-G)
  2. Pipettes (P20, P200, P1000) (e.g., Finnpipette F1 Pipettes, Thermo Fisher Scientific, catalog numbers: 4641050N, 4641080N, 4641100N)
  3. Echo-magnetic resonance imaging (MRI) scanner (Echo Medical Systems LLC, model: EchoMRI-4 in 1TM for Live Animals,) to assess mouse lean mass for insulin and pentobarbitone dosing
  4. Heat pads (Able Scientific, catalog number: ASCHP-RP)
  5. Blood glucometer (Roche Diagnostics, model: Accu-Check II)
  6. Mortar and pestle for tissue preparation (e.g., Maxwell & Williams, catalog number: AA1891)
  7. Ultrasonic tip-probe sonicator/homogenizer (Bendelin, model: Sonopuls)
  8. Refrigerated centrifuge (Thermo Fisher Scientific, model: Heraeus Fresco 21)
  9. Vortex (Ratek, model: VM1)
  10. Scintillation Counter (Beckman Coulter, model:LS6500)

Software

  1. Microsoft Excel or GraphPad Prism

Procedure

  1. Hepatic portal vein cannulation
    1. Determine mouse lean mass by ECHO-MRI according to the manufacturer’s instructions 24-48 h prior to procedure. 
    2. Remove food at least 2 h prior to starting experiment, depending on study requirements.
    3. Prepare a 96-well plate containing 75 μl 2.75% ZnSO4 per well for blood collection during the procedure. Prepare enough wells for collection of blood at 6 time points per mouse. Keep plate on ice throughout the procedure. 
    4. Inject mice with 80 mg/kg lean mass (determined by ECHO-MRI) pentobarbitone intraperitoneally. Mice with increased adiposity may require more pentobarbitone, up to 80 mg/kg body weight. See Videos 1 and 2.

      Video 1. Intraperitoneal injection of pentobarbitone (This video was made at The University of Sydney. Procedures were approved by The University of Sydney Animal Ethics Committee under project # 2017/1274)

      Video 2. Intraperitoneal injection of pentobarbitone (close up) (This video was made at The University of Sydney. Procedures were approved by The University of Sydney Animal Ethics Committee under project # 2017/1274)

    5. Assess the quality of anesthesia using the toe-pinch reflex once the animal appears to be fully unconscious. When the mouse does not respond (approximately 15-20 min after injection), place animal on back (dorsal recumbency) on a heat pad (~30 °C) and secure the limbs to the surface using tape.
      Note: In the event that a mouse still has a toe-pinch reflex 20 min post-anesthetic, administer up to 20% more pentobarbitone and test for toe-pinch reflex again after 10 min. Euthanize mouse if still responsive.
    6. Cut the tip (1 mm) off the tail with a scalpel blade. Take a baseline blood glucose measurement from a drop of blood taken from the tail using a glucometer. Take 5 μl blood from the tail for scintillation counting to act as a background sample. Add this 5 μl blood to one well of a 96-well plate containing 75 μl 2.75% ZnSO4
    7. Make a 3 cm incision through the skin and peritoneum using a scalpel or surgical scissors across the midline and perpendicular up towards the rib cage to open the abdominal cavity. Avoid puncturing the diaphragm. See Video 3. 
    8. Using a blunt sterile implement (e.g., cotton bud moistened with saline), carefully move the intestines to the right and the liver right and left medial lobes upward toward the ribcage to expose the hepatic portal vein and inferior vena cava (Figure 1A). See Video 3.

      Video 3. Surgery to access the hepatic portal vein (This video was made at The University of Sydney. Procedures were approved by The University of Sydney Animal Ethics Committee under project # 2017/1274)

    9. Using a 25-29 G insulin syringe, inject a bolus of saline (for non-insulin-stimulated glucose uptake) or insulin (for insulin-stimulated glucose uptake; 1 U/kg lean body mass) containing 5-10 μCi [3H]2-DOG tracer into the hepatic portal vein (Figures 1A and 1B) or inferior vena cava if preferred. See Video 4 and Figure 1.
      Note: Ensure that use of radioactive material is performed following local guidelines and regulations, including appropriate use of protective equipment, safe work practices, cleaning of workspaces after experiments and waste disposal.

      Video 4. Administration of saline to the hepatic portal vein. (This video was made at The University of Sydney. Procedures were approved by The University of Sydney Animal Ethics Committee under project # 2017/1274)

    10. The needle can either:
      1. Be rested to the side and left in the vein (Figure 1B).
      2. Be removed and a clamp applied to minimize bleeding.


        Figure 1. Hepatic portal vein cannulation in anesthetized mice. A. Location of the hepatic portal vein or inferior vena cava in mice. B. Picture of a Balb/c mouse during experiment where saline/insulin and tracer was injected via the hepatic portal vein and needle left in for the duration of the study.

    11. Measure blood glucose (from tail) and collect 5 μl blood (and add to ZnSO4 as in Step A6) per mouse to determine blood radioactivity after 2, 5, 10, 15, 20, and 30 min.
    12. After 30 min, terminate the mouse by cervical dislocation and excise tissues of interest (e.g., quadriceps muscle, tibialis anterior muscle, gastrocnemius muscle, epididymal adipose tissue, inguinal adipose tissue, heart). Tissues should be snapped frozen in liquid nitrogen and stored at -80 °C until further analysis. Tissues can be weighed before or after freezing.
      Note: Mice should be culled at a time where blood tracer counts are still decreasing and have not plateaued. We typically perform 30 min ITTs. 

  2. Western blot preparation
    1. Weigh out 50 mg epididymal white adipose tissue or muscle on dry ice, keeping the tissue frozen. 
    2. Add 250 μl of Western Blot Lysis Buffer to frozen tissue and immediately homogenize by sonication (90% power, 3 x 10 s, allowing sample to cool between pulses).
    3. Centrifuge at 13,000 x g for 10 min at 12 °C. 
    4. Remove the supernatant for muscle tissue, or carefully remove the infranatant for adipose tissue, being careful not to disturb the lipid layer. 
    5. Determine protein concentration using BCA or similar protein assay and prepare samples for Western blot as per standard protocol using phospho-specific antibodies to signaling intermediates of interest (e.g., phospho-and total Akt) (Figure 2).
      Note: Care should be taken to ensure that waste from SDS-PAGE is disposed of according to local regulations since samples contain radioactivity.


      Figure 2. Assessment of insulin signaling in adipose tissue. Adipose tissue was excised from mice administered with saline or insulin. Phosphorylation of Akt at activating sites (Thr308 and Ser473) was assessed by Western blotting with phospho-specific antibodies. Insulin increased Akt phosphorylation at both sites.

  3. Tracer disappearance and uptake
    1. To measure tracer disappearance from the blood, add 25 μl of a saturated Ba(OH)2 solution (in ddH2O) to wells containing blood samples in ZnSO4 (Step A5 above, final volume 105 μl). This deproteinizes the samples. Centrifuge the 96-well plate at 1,000 x g for 5 min to pellet precipitated protein and transfer 50 μl of the cleared sample to a scintillation vial and add 3 ml of scintillant. Count 3H DPM in each sample using a liquid scintillation counter. 
    2. To measure tracer uptake into a tissue of interest, powder tissue in liquid nitrogen using a mortar and pestle and then weigh an aliquot of powder for analysis. Smaller tissues such as the soleus muscle can be homogenized according to Step C3 and do not require powdering. 
    3. Homogenize ~40 mg tissue (as little as 10 mg soleus and EDL muscle can be used) in 1 ml ddH2O in a 1.5 ml tube by sonication (90% power, 3 x 10 s, allowing sample to cool between pulses) and centrifuge at 13,000 x g for 15 min. Collect supernatant (~800 μl) and transfer to a new 2.0 ml tube. Bring up volume to 2 ml with ddH2O.
    4. Prepare the phospho-2-DOG affinity elution columns by adding 1 ml AG1-X8 resin diluted in ddH2O (70% volume resin) to a 0.8 x 4 cm chromatography column using a wide-bore pipette tip (e.g., P1000 tip cut 5 mm from the tip using a scalpel blade to increase the aperture). 
    5. Place 5 ml tubes below the columns and add 1 ml of the supernatant to the columns followed by three 1 ml washes with ddH2O.
    6. Place new scintillation vials below the columns and add 1 ml of the elution buffer (see Recipes below) to columns followed by another 1 ml of elution buffer. 
    7. Add 3 ml scintillation fluid (PerkinElmer) to the scintillation vials, vortex thoroughly, and measure 3H DPM in each sample using a liquid scintillation counter to quantify [3H]2-DOG-6-P.

Data analysis

The aim of data analysis is to normalize for the amount of tracer delivered to each mouse and available for uptake into tissues, and the amount of tissue analyzed. Methods for normalizing such data have been discussed extensively elsewhere (Sokoloff et al., 1977; Goodner et al., 1980; Hom et al., 1984; Cooney et al., 1985; Kraegen et al., 1985).
  We describe two methods below: 1) the data are normalized to tracer availability in the blood to calculate the amount of tracer taken into the tissue of interest as a proportion of the amount of tracer available to the tissue, and 2) approximate an index of glucose uptake into tissues by using DPM and blood glucose to calculate a specific activity (DPM/mol glucose). In each case, data can be expressed per unit weight and/or protein/DNA content of the analyzed tissue.

Note: The methods described below use an area under the curve (AUC) calculation to normalize for tracer availability to tissues. This method works best if mice are culled when blood tracer counts are decreasing and have not plateaued. We typically perform 30 min ITTs.

  1. To calculate tracer 2-DOG clearance into specific tissues as a proportion of total 2-DOG available to the tissue:
    This calculation assumes that the 2-DOG tracer measured by a tail bleed is indicative of tracer available in the interstitial space for uptake into the tissue.
      First, extrapolate DPM per 5 μl to 1 ml to yield DPM/ml. Since the tracer will disappear from the blood by an exponential decay, calculate the AUC by fitting the DPM/ml at measured time points to a single exponential function, and integrate this function over the experimental period. This estimates the change in blood DPM/ml throughout the experiment (DPM/ml•min). This AUC value provides a normalization factor that takes into account differences in tracer availability between mice.
      Tissue DPMs can be normalized using this AUC value to calculate the proportion of available tracer taken up into the tissue. This can be further normalized to the weight of tissue analyzed (g). The final units are: 2-DOG clearance (ml/min/g). Data normalized using this method are presented in Figures 3A and 3B. These data show insulin-stimulated 2-DOG clearance into muscle and adipose tissues, but not into brain (Figure 3A) and that feeding mice a diet high in fat and sucrose leads to impaired insulin-stimulated 2-DOG clearance into epididymal adipose tissue and quadriceps muscle (Figure 3B).
  2. To obtain a tissue-specific index of glucose uptake:
    The blood glucose concentration during this assay is non-steady-state. This calculation aims to take into account differences in blood glucose over the course of the experiment and approximate glucose uptake into tissue. This calculation assumes that there is no discrimination between 2-DOG and glucose at the glucose transporter and therefore the rate of [3H]2-DOG accumulation in tissue is equivalent to the rate of glucose uptake into tissue (Ferre et al., 1985). Since the kinetics of 2-DOG and glucose uptake may differ, we advise referring to 2-DOG accumulation as a “glucose uptake index”.
      First calculate the AUC for blood DPM (as above by exponential curve fitting; DPM/ml•min) and blood glucose during the ITT (by the trapezoidal method; μmol/ml•min). The average specific activity of 2-DOG in the blood can be calculated by dividing the blood DPM AUC by blood glucose AUC (DPM/μmol). Tissue DPM can then be converted to an index for the rate of glucose uptake by dividing by the average specific activity, and further normalized to the weight of tissue analyzed (g) and expressed per min or h. The final units are: Glucose uptake index (μmol/g/h). Data normalized using this method are presented in Figure 3B. These data show that feeding mice a high fat high sucrose diet for 14 d lowers the insulin-stimulated glucose uptake index in epididymal adipose tissue and quadriceps muscle (Figure 3C).


    Figure 3. 2-DOG uptake into tissues. A. 2-DOG clearance into indicated tissues was assessed in C57Bl/6J mice following saline or insulin administration (Epi; Epididymal, Subcut; subcutaneous/inguinal, EDL; Extensor digitorum longus). Adipose and muscle tissues exhibited insulin-responsive 2-DOG clearance. B and C. Insulin-stimulated 2-DOG clearance (B) or glucose uptake index (C) for epididymal white adipose tissue (WAT) (left inset) and quadriceps muscle (right inset) in C57Bl/6J mice fed a chow diet or high fat high sucrose diet (HFHSD). *P < 0.05 versus mice fed a chow diet, Student’s t-test, n = 6-7. Data in B and C were recalculated from Fazakerley et al. (2018a).

Recipes

  1. 2.75% zinc sulfate solution
    100 ml of 2.75% zinc sulfate contains 4.44 g zinc sulfate in ddH2O
  2. Phosphorylated [3H]2-DOG elution buffer
    Low pH, high salt solution (e.g., 1% Trifluoroacetic acid, 2 M NaCl)
    100 ml of elution buffer contains 1 ml 100% Trifluoracetic acid and 11.69 g NaCl in ddH2O
  3. Western blot lysis buffer
    10 mM HEPES, pH 7.4
    250 mM sucrose
    1 mM EDTA
    2% SDS
    Protease and phosphatase inhibitors (1 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride)
    We advise making 10x stocks of HEPES pH 7.4, sucrose, EDTA and SDS.
    10 ml of lysis buffer contains, 23.8 mg HEPES, 855.6 mg sucrose, 2.9 mg EDTA, 576.8 mg SDS, 1x Roche cOmpleteTM protease inhibitors, 1 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride. Adjust the solution pH to 7.4

Acknowledgments

This protocol is a modified version of the protocol reported in Fazakerley et al., 2018a. This work was supported by National Health and Medical Research Council of Australia (NHMRC) Project Grants 1061122 and 1086850 (to D. E. J.). D.E.J. is a National Health and Medical Research Council of Australia Senior Principal Research Fellow. The contents of the published material are solely the responsibility of the individual authors and do not reflect the views of NHMRC. We thank Dr. James Krycer and Dr. Lake-Ee Quek for helpful discussion.

Competing interests

The authors have no competing interests to declare.

Ethics

All experiments were carried out with the approval of the University of Sydney Animal Ethics Committee, following guidelines issued by the National Health and Medical Research Council of Australia.

References

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  23. Windelov, J. A., Pedersen, J., and Holst, J. J. (2016). Use of anesthesia dramatically alters the oral glucose tolerance and insulin secretion in C57Bl/6 mice. Physiol Rep 4(11): e1282.

简介

胰岛素抵抗是由对胰岛素的反应受损所定义的病理生理状态,并且是几种代谢疾病的风险因素,最明显的是2型糖尿病。 胰岛素抵抗发生在胰岛素靶组织中,包括肝脏,脂肪和骨骼肌。 诸如胰岛素耐量试验和高胰岛素 - 正 - 葡萄糖钳夹的方法允许评估特定组织中的胰岛素反应,并允许研究胰岛素抗性的进展和原因。 在这里,我们详细介绍了评估静脉注射胰岛素的麻醉小鼠脂肪和肌肉组织中胰岛素作用的方案。
【背景】 全身胰岛素敏感性的替代指标,如胰岛素抵抗的稳态模型评估(HOMA-IR),往往不能反映胰岛素刺激的小鼠外周组织葡萄糖摄取(Lee et al。,2008 ;马瑟,2009)。 离体脂肪组织外植体或分离的骨骼肌制剂(Burchfield et al。,2018; Fazakerley et al。,2018b)可以直接测量特殊组织中的胰岛素反应,如果它们适合这些程序,但不保留生物体环境,其中通过脉管系统精细关节递送葡萄糖和胰岛素,摄入细胞和细胞内代谢可能都有助于组织葡萄糖摄取的总体速率(Wasserman et al。,2011)。常用的评估小鼠全身胰岛素作用的方法包括腹膜内(IP)或静脉注射胰岛素耐量试验(ITTs),以及金标准,高胰岛素血症 - 正常血糖钳(Ayala et al。,2010; Brandon et al。,2016)。这些技术可以通过测量血糖(ITT)的变化或维持血糖所需的葡萄糖量的变化(葡萄糖输注速率;高胰岛素 - 正葡萄糖钳夹)来评估全身胰岛素作用,并且可以适应于测量组织特异性胰岛素响应。例如,葡萄糖示踪剂,例如放射性标记的2-脱氧葡萄糖(2-DOG),其通常在评估葡萄糖摄取时用作葡萄糖的替代物,因为它不通过糖酵解代谢并且在摄取后被“捕获”在细胞中,可以是引入以允许在这些测试期间评估葡萄糖摄入特定组织。注意,2-DOG仅被捕获在不具有显着的葡萄糖-6-磷酸酶活性的组织中,因此2-DOG示踪剂因此不能用于评估肝脏中的葡萄糖处理。

ITT和高胰岛素 - 正 - 葡萄糖钳夹均可在有意识或麻醉的小鼠中进行。某些麻醉剂会影响全身葡萄糖代谢,因此在评估胰岛素作用或葡萄糖稳态时应予以避免[例如,异氟醚(Pomplun et al。,2004; Tanaka et al。,2009; Windelov et al。,2016; Hoyer et al。,2018)]。然而,最低限度影响葡萄糖代谢的麻醉剂,例如戊巴比妥(Guarino 等人,2013),实际上可能有利于研究胰岛素刺激的葡萄糖摄取。这是因为有意识的小鼠中胰岛素刺激的2-DOG /葡萄糖摄取可能受到运动的影响(例如,由于机械和/或运动刺激的葡萄糖摄取)和/或应激反应[例如,儿茶酚胺(Cooney et al。,1985; Furler et al。,1991)和糖皮质激素(Pasieka和Rafacho,2016)]。

在这里,我们描述了在戊巴比妥诱导的麻醉下进行的终末静脉内ITT的方案,以评估胰岛素刺激的葡萄糖摄取到感兴趣的组织中。该方案可以在小鼠之间以最小的延迟进行,使其适合于评估大群动物中的胰岛素作用。静脉内施用大剂量胰岛素/示踪剂可快速将胰岛素递送至组织,从而最小化测定开始与组织内胰岛素反应开始之间的时间。此外,2-DOG示踪剂与总血糖库的快速平衡确保示踪剂可立即用于摄取到组织中。胰岛素/示踪剂递送的替代方法(即,腹膜内注射,口服强饲法)可导致胰岛素/示踪剂摄入中枢循环的延迟或不一致。例如,在IP注射的情况下,在胰岛素到达循环之前可能存在相当大的延迟。这些时间滞后可能在小鼠之间或注射之间不同,增加了实验变异性。当在活体动物中进行时间序列实验时,快速且一致的组织摄取也是特别重要的。实际上,肝门静脉注射胰岛素可以评估胰岛素对肝脏磷酸化蛋白质组的时间分辨效应(Humphrey et al。,2015)。对于口服强饲法,与葡萄糖相比,2-DOG在循环中可能表现出明显不同的外观动力学,因为钠依赖性葡萄糖转运蛋白在口服葡萄糖吸收中起关键作用,表现出对葡萄糖的强烈偏好(Bissonnette et al。,1996),这可能会限制测定期间组织对示踪剂的可用性。

在该方案中,用戊巴比妥和盐水或胰岛素麻醉小鼠,并通过肝门静脉给予放射性标记的2-DOG示踪剂。可以通过用于胰岛素信号传导中间体的蛋白质印迹或通过测量放射性标记的2-DOG示踪剂积累作为葡萄糖摄取的指标来评估感兴趣的肌肉和脂肪组织中的胰岛素作用。

关键字:胰岛素, 脂肪组织, 肌肉, 葡萄糖转运, 葡萄糖摄取, 胰岛素耐量试验

材料和试剂

  1. 移液器吸头(例如,Axygen,目录号:T-1000-C,T-200-C)
  2. 棉花芽(例如,McFarlane Medical Equipment,目录号:18049DE)
  3. 1.5 ml微量离心管(Axygen,目录号:MCT-150-C)
  4. 2.0毫升微量离心管(Eppendorf,目录号:0030120.094)
  5. 5毫升聚苯乙烯管(SARSTEDT,目录号:60.9921.531)
  6. 0.5毫升超细胰岛素注射器(Becton Dickinson,目录号:230-45094)
  7. 遮蔽胶带(例如,Lyreco,目录号:737.665)
  8. 96孔板(Corning,目录号:CLS2595)
  9. 碳钢,22号手术刀片(Livingstone,目录号:SBLDCL22)
  10. 色谱柱(Bio-Rad,目录号:731-1550)
  11. 6毫升小马瓶闪烁管(PerkinElmer,目录号:6000292)
  12. 成年小鼠(> 8周龄)
    注意:确保使用小鼠进行实验,并获得当地伦理委员会的批准。小鼠可以经历不同的饮食/运动方案,或者是针对感兴趣的基因的敲除/转基因系。对于敲除/转基因品系,我们建议使用同窝对照。&nbsp;
  13. 双蒸水(ddH 2 O)
  14. Bicinchoninic酸测定(BCA)蛋白质测定试剂盒(Thermo Fisher Scientific,目录号:23225)
  15. 抗体
    1. 抗磷酸化Thr308 Akt抗体(Cell Signaling Technology,目录编号:9275)
    2. 抗磷酸化Ser473 Akt抗体(Cell Signaling Technology,目录编号:4051)
    3. 抗Akt抗体(Cell Signaling Technology,目录编号:4685)
  16. 戊巴比妥(Lethabarb,Virbac,澳大利亚)
    注意:戊巴比妥可能是一种受管制的物质。确保使用遵循当地法规。&nbsp;
  17. 人胰岛素(Actrapid,Novo Nordisk)
  18. 0.9%氯化钠注射液BP(辉瑞,目录号:158594)
  19. [ 3 H] 2-DOG(PerkinElmer,目录号:NET328001MC)
  20. Accu-Chek performa葡萄糖条(Accu-Check II;罗氏诊断,目录号:06454038)
  21. 液氮
  22. AG1-X8树脂(Bio-Rad,目录号:140-1443)
  23. Ulitma Gold XR闪烁液(PerkinElmer,目录号:6013119)
  24. 氢氧化钡(Sigma,目录号:433373)
  25. 干冰
  26. 2.75%硫酸锌溶液(见食谱)
    硫酸锌(ZnSO 4 )(Sigma,目录号:307491)
  27. 磷酸化[ 3 H] 2-DOG洗脱缓冲液(见食谱)
    1. 三氟乙酸(Sigma,目录号:302031)
    2. NaCl(Sigma,目录号:S7653)
  28. Western blot裂解缓冲液(见食谱)
    1. HEPES(Sigma,目录号:54457)
    2. 蔗糖(西格玛,目录号:S8501)
    3. EDTA(Sigma,目录号:03620)
    4. SDS(西格玛,目录号:71729)
    5. 蛋白酶抑制剂(Roche,cOmplete TM ,目录号:11697498001)&nbsp;
    6. 焦磷酸钠(西格玛,目录号:71501)
    7. 原钒酸钠(Sigma,目录号:S6508)
    8. 氟化钠(Sigma,目录号:S7920)

设备

  1. 手术刀或手术剪(例如,World Precision Instruments,目录号:501259-G)
  2. 移液器(P20,P200,P1000)(例如,Finnpipette F1移液器,Thermo Fisher Scientific,目录号:4641050N,4641080N,4641100N)
  3. 回声磁共振成像(MRI)扫描仪(Echo Medical Systems LLC,型号:EchoMRI-4 in 1 TM for Live Animals,)评估胰岛素和戊巴比妥剂量的小鼠瘦体重
  4. 热垫(Able Scientific,目录号:ASCHP-RP)
  5. 血液血糖仪(罗氏诊断,型号:Accu-Check II)
  6. 用于组织准备的研钵和研杵(例如,Maxwell&amp; Williams,目录号:AA1891)
  7. 超声波探头超声波探头/均质器(Bendelin,型号:Sonopuls)
  8. 冷冻离心机(Thermo Fisher Scientific,型号:Heraeus Fresco 21)
  9. Vortex(Ratek,型号:VM1)
  10. 闪烁计数器(Beckman Coulter,型号:LS6500)

软件

  1. Microsoft Excel或GraphPad Prism

程序

  1. 肝门静脉插管
    1. 在手术前24-48小时,根据制造商的说明,通过ECHO-MRI确定小鼠瘦体重。&nbsp;
    2. 根据研究要求,在开始实验前至少2小时取出食物。
    3. 制备96孔板,每孔含有75μl2.75%ZnSO 4 ,用于在手术期间采集血液。准备足够的水井,每只小鼠6个时间点收集血液。在整个过程中将盘子放在冰上。&nbsp;
    4. 通过腹膜内注射80mg / kg瘦体重(通过ECHO-MRI测定)戊巴比妥注射小鼠。肥胖增加的小鼠可能需要更多戊巴比妥,最高可达80 mg / kg体重。见视频1和2.
      视频1.腹腔注射戊巴比妥(本视频由悉尼大学制作。程序经悉尼大学动物伦理委员会批准,项目编号为2017/1274)
      视频2.腹腔注射戊巴比妥(特写)(该视频由悉尼大学制作。程序经悉尼大学动物伦理委员会批准,项目编号为2017/1274)

    5. 一旦动物完全失去知觉,使用脚趾反射评估麻醉质量。当小鼠没有反应时(注射后大约15-20分钟),将动物放在背垫(背侧卧位)上的热垫(~30°C)并用胶带将四肢固定在表面上。
      注意:如果鼠标在麻醉后20分钟仍有趾捏反射,则给予多达20%的戊巴比妥,并在10分钟后再次测试趾捏反射。如果仍然有反应,则安乐死鼠标。
    6. 用手术刀刀片切掉尾部(1毫米)。使用血糖仪从尾巴采集的一滴血液中进行基线血糖测量。从尾部取5μl血液进行闪烁计数,作为背景样本。将该5μl血液加入到含有75μl2.75%ZnSO 4 的96孔板的一个孔中。&nbsp;
    7. 使用手术刀或手术剪刀穿过皮肤和腹膜切开3厘米的切口,穿过中线,垂直朝向胸腔,打开腹腔。避免刺破隔膜。见视频3.&nbsp;
    8. 使用钝的无菌器具(例如,用生理盐水润湿的棉花芽),小心地将肠道向右移动,肝脏左右内侧叶片朝向胸腔向上移动以暴露肝门静脉和下腔静脉卡瓦酒(图1A)。见视频3.
      视频3.手术进入肝门静脉(该视频由悉尼大学制作。程序由悉尼大学动物伦理委员会批准,项目编号为2017/1274)

    9. 使用25-29G胰岛素注射器,注射含有5-10μCi的一团生理盐水(用于非胰岛素刺激的葡萄糖摄取)或胰岛素(用于胰岛素刺激的葡萄糖摄取; 1U / kg瘦体重)如果愿意,[3 H] 2-DOG示踪剂进入肝门静脉(图1A和1B)或下腔静脉。见视频4和图1.
      注意:确保按照当地的指导方针和规定执行放射性物质的使用,包括适当使用防护设备,安全工作实践,实验后清洁工作空间和废物处理。
      视频4.给肝门静脉注射盐水。 (该视频是在悉尼大学制作的。程序由悉尼大学动物伦理委员会根据项目#2017/1274批准)

    10. 针可以:
      1. 休息一侧,静脉留下(图1B)。
      2. 取下并使用夹子以减少出血。


        图1.麻醉小鼠的肝门静脉插管。 A.小鼠肝门静脉或下腔静脉的位置。 B.实验期间Balb / c小鼠的图片,其中通过肝门静脉注射盐水/胰岛素和示踪剂并在研究期间留下针头。

    11. 测量血糖(从尾部)并每只小鼠收集5μl血液(并按步骤A6加入ZnSO 4 )以确定2,5,10,15,20和30分钟后的血液放射性。
    12. 30分钟后,通过颈椎脱位终止小鼠并切除感兴趣的组织(例如,股四头肌,胫骨前肌,腓肠肌,附睾脂肪组织,腹股沟脂肪组织,心脏)。应将组织在液氮中快速冷冻并储存在-80℃直至进一步分析。可以在冷冻之前或之后称重组织。
      注意:小鼠应该在血液示踪剂计数仍在下降并且没有停顿的情况下进行剔除。我们通常执行30分钟的ITT。&nbsp;

  2. 蛋白质印迹制备
    1. 在干冰上称出50 mg附睾白色脂肪组织或肌肉,保持组织冷冻。&nbsp;
    2. 向冷冻组织中加入250μlWestern印迹裂解缓冲液,并立即通过超声处理均质化(90%功率,3×10秒,使样品在脉冲之间冷却)。
    3. 在12℃下以13,000 x g 离心10分钟。&nbsp;
    4. 去除肌肉组织的上清液,或小心地去除脂肪组织的infranatant,小心不要打扰脂质层。&nbsp;
    5. 使用BCA或类似的蛋白质测定确定蛋白质浓度,并使用针对感兴趣的中间体(例如,磷酸 - 和总Akt)的磷酸特异性抗体,按照标准方案制备用于蛋白质印迹的样品(图2)。
      注意:应注意确保SDS-PAGE的废物按照当地法规处理,因为样品中含有放射性物质。


      图2.脂肪组织中胰岛素信号传导的评估从给予盐水或胰岛素的小鼠中切除脂肪组织。通过磷酸特异性抗体的Western印迹评估活化位点(Thr308和Ser473)的Akt的磷酸化。胰岛素增加两个位点的Akt磷酸化。

  3. 示踪剂消失和摄取
    1. 为了测量血液中的示踪剂消失,将25μl饱和的Ba(OH) 2 溶液(在ddH 2 O中)加入到含有ZnSO 中血液样品的孔中4 (上述步骤A5,最终体积105μl)。这使样品脱蛋白质。将离心的96孔板在1000μM离心5分钟以沉淀沉淀的蛋白质,并将50μl澄清的样品转移到闪烁瓶中,并加入3ml闪烁剂。使用液体闪烁计数器计算每个样品中的 3 H DPM。&nbsp;
    2. 使用研钵和研杵测量示踪剂对感兴趣组织的吸收,在液氮中的粉末组织,然后称量等分试样的粉末用于分析。较小的组织如比目鱼肌可根据步骤C3均质化,不需要粉化。&nbsp;
    3. 通过超声处理(150%功率,3×10 s,允许)在1.5 ml管中的1 ml ddH 2 O中均质化约40 mg组织(少至10 mg比目鱼肌和EDL肌肉)样品在脉冲之间冷却)并在13,000 xg 下离心15分钟。收集上清液(约800μl)并转移至新的2.0ml管中。用ddH 2 O将体积调至2ml。
    4. 制备磷酸-2-DOG亲和洗脱柱,使用大口径移液管将1d稀释于ddH 2 O(70%体积树脂)的AG1-X8树脂加入到0.8 x 4 cm色谱柱中尖端(例如,P1000尖端使用手术刀刀片从尖端切割5 mm以增加孔径。)&nbsp;
    5. 将5ml管置于柱下方,向柱中加入1ml上清液,然后用ddH 2 洗涤3次,每次1ml。
    6. 将新的闪烁瓶放在柱子下面,加入1ml洗脱缓冲液(见下面的配方)到柱子中,再加入1ml洗脱缓冲液。&nbsp;
    7. 将3 ml闪烁液(PerkinElmer)加入闪烁瓶中,充分涡旋,并使用液体闪烁计数器测量每个样品中的 3 H DPM,以定量[ 3 H] 2 -DOG-6-P。

数据分析

数据分析的目的是使递送至每只小鼠的示踪剂的量标准化并且可用于摄取到组织中,以及分析的组织量。在其他地方已经广泛讨论了对这些数据进行标准化的方法(Sokoloff et al。,1977; Goodner et al。,1980; Hom et al。 ,1984; Cooney et al。,1985; Kraegen et al。,1985)。
&NBSP;我们描述了以下两种方法:1)将数据标准化为血液中的示踪剂可用性,以计算进入感兴趣组织的示踪剂量,作为组织可用示踪剂量的比例,和2)近似指数通过使用DPM和血糖来计算比活性(DPM / mol葡萄糖),从而将葡萄糖摄取到组织中。在每种情况下,数据可以表示每单位重量和/或分析组织的蛋白质/ DNA含量。

注意:下面描述的方法使用曲线下面积(AUC)计算来标准化组织的示踪剂可用性。如果在血液示踪剂计数降低且未达到平稳期时剔除小鼠,则此方法效果最佳。我们通常执行30分钟的ITT。

  1. 计算示踪剂2-DOG清除到特定组织中的比例,作为组织可用的总2-DOG的比例:
    该计算假设通过尾部流量测量的2-DOG示踪剂指示在间隙空间中可用于摄取到组织中的示踪剂。
    &NBSP;首先,将每5μl的DPM外推至1ml,得到DPM / ml。由于示踪剂将通过指数衰减从血液中消失,通过将测量时间点的DPM / ml拟合为单指数函数来计算AUC,并在实验期间整合该函数。这估计了整个实验中血液DPM / ml的变化(DPM / ml•min)。该AUC值提供归一化因子,其考虑了小鼠之间的示踪物可用性的差异。
    &NBSP;可以使用该AUC值对组织DPM进行标准化,以计算吸收到组织中的可用示踪剂的比例。这可以进一步标准化为分析的组织重量(g)。最终单位为:2-DOG清除率(ml / min / g)。使用该方法归一化的数据显示在图3A和3B中。这些数据显示胰岛素刺激的2-DOG清除进入肌肉和脂肪组织,但不进入大脑(图3A),给小鼠喂食高脂肪和蔗糖的饮食导致胰岛素刺激的2-DOG清除进入附睾脂肪组织和股四头肌(图3B)。
  2. 获得组织特异性葡萄糖摄取指数:
    该测定期间的血糖浓度是非稳态的。该计算旨在考虑实验过程中血糖的差异以及对组织的近似葡萄糖摄取。该计算假设在葡萄糖转运蛋白的2-DOG和葡萄糖之间没有区别,因此组织中[ 3 H] 2-DOG积累的速率等于葡萄糖摄入组织的速率。 (Ferre et al。,1985)。由于2-DOG和葡萄糖摄取的动力学可能不同,我们建议将2-DOG积累称为“葡萄糖摄取指数”。
    &NBSP;首先计算血液DPM的AUC(如上指数曲线拟合; DPM / ml•min)和ITT期间的血糖(通过梯形法;μmol/ ml•min)。可以通过将血液DPM AUC除以血糖AUC(DPM /μmol)来计算血液中2-DOG的平均比活性。然后可以通过除以平均比活性将组织DPM转化为葡萄糖摄取速率的指数,并进一步标准化为分析的组织重量(g)并以每分钟或每小时表达。最终单位是:葡萄糖摄取指数(μmol/ g / h)。使用该方法归一化的数据显示在图3B中。这些数据表明,给小鼠喂食高脂肪高蔗糖饮食14天会降低附睾脂肪组织和股四头肌中胰岛素刺激的葡萄糖摄取指数(图3C)。


    图3. 2-DOG摄入组织。 :一种。在盐水或胰岛素给药后,在C57Bl / 6J小鼠中评估指示组织中的2-DOG清除(Epi;附睾,Subcut;皮下/腹股沟,EDL; Extensor digitorum longus)。脂肪和肌肉组织表现出胰岛素响应性2-DOG清除率。 B和C.胰岛素刺激的2-DOG清除率(B)或葡萄糖摄取指数(C)用于喂食饲料或C57Bl / 6J小鼠的附睾白色脂肪组织(WAT)(左侧插图)和股四头肌(右侧插入)或高脂肪高蔗糖饮食(HFHSD)。 * P &lt;与饲喂饲料的小鼠相比,0.05,学生 t - 测试,n = 6-7。从Fazakerley 等(2018a)重新计算B和C中的数据。

食谱

  1. 1. 2.75%硫酸锌溶液
    100ml 2.75%硫酸锌在ddH 2 O中含有4.44g硫酸锌
  2. 磷酸化[ 3 H] 2-DOG洗脱缓冲液
    低pH,高盐溶液(例如,1%三氟乙酸,2 M NaCl)
    100 ml洗脱缓冲液含有1 ml 100%三氟乙酸和11.69 g NaCl,ddH 2 O
  3. Western blot裂解缓冲液
    10 mM HEPES,pH 7.4
    250毫克蔗糖
    1 mM EDTA
    2%SDS
    蛋白酶和磷酸酶抑制剂(1 mM焦磷酸钠,2 mM原钒酸钠,10 mM氟化钠)
    我们建议制备10倍HEPES pH 7.4,蔗糖,EDTA和SDS。
    10 ml裂解缓冲液含有23.8 mg HEPES,855.6 mg蔗糖,2.9 mg EDTA,576.8 mg SDS,1x Roche cOmplete TM 蛋白酶抑制剂,1mM焦磷酸钠,2mM原钒酸钠,10mM氟化钠。将溶液pH调节至7.4

致谢

该协议是Fazakerley et al。,2018a中报道的协议的修改版本。这项工作得到了澳大利亚国家健康与医学研究委员会(NHMRC)项目拨款1061122和1086850(致D. E. J.)的支持。 D.E.J.是澳大利亚国家健康与医学研究委员会高级首席研究员。出版材料的内容完全由作者个人负责,并不反映NHMRC的观点。我们感谢James Krycer博士和Lake-Ee Quek博士的有益讨论。

利益争夺

作者没有相互竞争的利益来宣布。

伦理

所有实验均经悉尼大学动物伦理委员会批准,遵循澳大利亚国家健康与医学研究委员会发布的指南。

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Fazakerley, D. J., Fritzen, A. M., Nelson, M. E., Thorius, I. H., Cooke, K. C., Humphrey, S. J., Cooney, G. J. and James, D. E. (2019). Insulin Tolerance Test under Anaesthesia to Measure Tissue-specific Insulin-stimulated Glucose Disposal. Bio-protocol 9(2): e3146. DOI: 10.21769/BioProtoc.3146.
  2. Fazakerley, D. J., Chaudhuri, R., Yang, P., Maghzal, G. J., Thomas, K. C., Krycer, J. R., Humphrey, S. J., Parker, B. L., Fisher-Wellman, K. H., Meoli, C. C., Hoffman, N. J., Diskin, C., Burchfield, J. G., Cowley, M. J., Kaplan, W., Modrusan, Z., Kolumam, G., Yang, J. Y., Chen, D. L., Samocha-Bonet, D., Greenfield, J. R., Hoehn, K. L., Stocker, R. and James, D. E. (2018a). Mitochondrial CoQ deficiency is a common driver of mitochondrial oxidants and insulin resistance. Elife 7: e32111.
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