Substrate Cycling in Energy Metabolism
Condition: Burns; Insulin Resistance
MeSH terms: Insulin Resistance
Study type: Interventional
Phase: Phase 2

Summary: 
      Insulin resistance and hyperglycemia contribute to negative outcomes in burned patients. We
      will assess insulin sensitivity in traditional terms of glucose metabolism, and with regard
      to the responsiveness of both muscle and liver protein metabolism, in severely burned
      patients. Plasma free fatty acid (FFA) and tissue TG levels will be manipulated via
      inhibition of peripheral lipolysis with nicotinic acid or activation of plasma lipoprotein
      lipase activity with heparin, stimulation of tissue fatty acid oxidation and thus reduction
      of tissue TG with the peroxisome proliferate-activated receptor (PPAR) alpha agonist
      fenofibrate. Methodological approaches will include stable isotope tracer techniques to
      quantify kinetic responses of protein, glucose and lipid metabolism in vivo, quantification
      of intracellular stores of TG and glycogen by means of magnetic resonance spectroscopy (MRS),
      as well as quantitative analysis of tissue levels of active products of fatty acids, key
      intermediates of the insulin signaling pathway, glycogen, the enzyme activities of citrate
      synthase and glycogen synthase and the activity of the muscle mitochondria. These studies
      will clarify the physiological and clinical significance of the alterations of tissue lipid
      metabolism that occur after burn injury, thereby forming the basis for new therapeutic
      approaches not only in this specific clinical condition but in other clinical circumstances
      in which hepatic and/or muscle TG is elevated.

      We will investigate the general hypothesis that the accumulation of intracellular TG in liver
      and muscle either directly causes insulin resistance in those tissues or serves as an
      indictor of the intracellular accumulation of active fatty acid products, such as fatty acyl
      CoA and diacylglycerol, which in turn disrupt insulin action.

      The following specific hypotheses will be investigated:

        1. Intracellular TG is elevated in both muscle and liver in severely burned patients. The
           reduction of the fat in the liver and the insulin resistance will improve clinical
           outcomes, glucose and protein metabolism.

        2. The insulin signaling pathway, as reflected by phosphoinositol-3-kinase (PI3K) and PKC
           activity, is impaired in tissues with elevated TG.

        3. Fatty acids, or their active intracellular products, are the direct inhibitors of
           insulin action, rather than the tissue TG itself.
    

Detailed description: 
      We will study patients with severe burns, defined as 2nd or 3rd degree burn covering >40% of
      total body surface area (TBSA). We propose to study burned children from Shriners Burns
      Hospital. The Shriners census is such that approximately 50 children with severe burns are
      treated every year. We will study patients immediately prior to their third surgical
      procedure, approximately 12-15 days after injury. One half of the patients will be given
      fenofibrate (5 mg/kg/day) daily delivered through feeding tube from the time of consent
      following admission until 12-14 days post-burn. This length of time after injury will ensure
      that untreated patients will have a large accumulation of hepatic TG. Because the "control"
      group of patients will have elevated liver TGs, the "experimental" group will have their
      hepatic TGs lowered by fenofibrate. By studying the patients the day before the operations,
      it will be possible to remove the staples used in skin grafting without risk of loss of
      adhesion of the graph, thereby ensuring safety in the MRS. Femoral line inserted for the
      surgery can also be utilized, and all patient generally receive a full transfusion during
      surgeries, minimizing any study related blood loss. In addition to the liver, we will study
      the muscle in burn patients. Patients will be studied during brief fasted state. The will be
      fasted four hours prior to the study and then through out the study. Their TPN will be
      immediately reconnected following the study. The surgical team at the Shrine places 3Fr, 8 cm
      polyethylene catheters (Cook, Inc., Bloomington, IN) into the femoral vein and femoral artery
      under local anesthesia the day before surgery. Both femoral catheters will be used for blood
      sampling, while the femoral arterial catheter will also used for indocyanine green infusion
      for the determination of leg blood flow. Systemic concentration of indocyanine green will be
      measured from a central vein, as standard procedure has a multi-lumen subclavian line in all
      patients. Patency of all catheters is maintained by saline infusion.

      Patients will be infused with stable (non-radioactive) isotope tracers of glucose,
      phenylalanine and palmitate for up to 8 hours. After 4 hours, without interruption of the
      tracer infusion, an infusion of insulin will be started and maintained at the rate of 1.5
      mU/kg•min for the final 4 h. Blood glucose concentrations will be measured throughout the
      insulin infusion and glucose infused as necessary to maintain the basal plasma glucose
      concentration.

      A biopsy of the quadriceps will be obtained with a Bergstrom needle at the beginning of the
      study, 4 h (immediately before the insulin infusion) and at the end of the 4 h insulin
      infusion. We will use the A-V balance technique to address the relation between tissue fatty
      acid and TG metabolism and the insulin responsiveness of glucose uptake and myofibrillar and
      mitochondrial protein synthesis and net protein balance.

      b. Subjects Patients are admitted to the burn unit within 48 h of injury. Fluid resuscitation
      is provided as previously described (94). Within 48 h of admission, the burn wound is excised
      and subsequently grafted by autograft or cadaveric allograft. Patients typically return to
      the operating room for reharvesting of donor sites every five to seven days. The experiments
      proposed here will be performed the day prior to the third surgery at day 12-15, as femoral
      catheters are normally inserted at the time for access during surgery. Enteral feeding with
      Vivonex TEN (Sandoz Nutrition Corp, Minneapolis, MN) is started within 24h of admission and
      continued until the patient is capable of food by mouth. All patients will be eligible for
      the study unless one of the exclusion criterion listed below apply.

      c. Procedures From day 1 to day 22 patients will be maintained on enteral feeding of a high
      carbohydrate/amino acid mixture (Vivonex, Novartis, Minneapolis, MN). Vivonex contains 300
      kcal/serving in the following caloric breakdown: 82.3% carbohydrate, 15% protein, 2.7% fat
      (linoleic acid). Patients will be given 25 kcal/kg of Vivonex plus an additional 45 kcal/kg
      for each percentage point of total body surface area burned. One half of the patients will be
      given fenofibrate (5 mg/kg/day - maximum daily dose) from the time of the first tracer study
      until the time of the second tracer study.

      The tracer study subjects can commence once catheters in the femoral artery and vein have
      been placed by the surgical team, if necessary, since the majority of patients wil have
      pre-existing lines placed for clinical reasons. The catheters will be used for sampling and
      in a peripheral vein for infusing, as in our previous studies (e.g., 4). Enteral
      administration of a mixture of carbohydrate and amino acids (Vivonex) will be stopped four
      hours prior to the study, and will be started immediately following the study.

      On the day after the tracer infusion the amount of liver and muscle TG and liver glycogen
      will be determined by MRS. After metal staples are removed, patients will be transported to
      the clinical MRS facilities at UTMB Dept. of Radiology, where measurements will be performed
      (see below for details), After obtaining baseline samples, tracer infusions will be started
      as described in Figure 2. Half the patients with high tissue TG will be given nicotinic acid
      (500 mg orally) at the start of period 2 to lower FFA levels acutely. In the group given
      fenofibrate (200 mg/d) or propranolol 0.5mg/kg every 6 hours to lower FFA, half will be
      infused with heparin (0.5 U/kg•min, 2.8 U/ kg prime IV) at a dose sufficient to activate
      lipoprotein lipase, thereby elevating plasma FFA, while not affecting blood coagulation.
      After baseline blood samples from the femoral artery, femoral vein, and peripheral vein are
      collected, an 8 hour continuous infusion of primed-constant infusions of 6,6-d2-glucose (0.08
      mg/kg•min, prime = 6.8 mg/kg) and d5-phenylalanine (0.20 µmol/kg•min, prime = 8.0 µmol/kg)
      will be given in order to quantify hepatic glucose production and protein synthetic rates,
      respectively. In addition, 2 hours into the protocol, U-13C16-palmitate (0.16 µmol/kg per
      minute) will be started with NaH13CO3 prime (150 µmol/kg) in order to quantify hepatic fatty
      acid uptake and oxidation. These tracer infusions will also be maintained throughout the 8
      hour tracer study. Blood samples (2- 12 ml) will be taken from the artery, femoral vein and
      peripheral vein simultaneously at 120, 180, 210, 225 and 240 minutes (see Appendix 2 for full
      timeline). Muscle tissue biopsies will be obtained at the start of period 1, and at 4 hours
      of period 1 to measure protein kinetics and also determine biochemical parameters. Then,
      period 2 will start. At the start of period 2, a primed, constant infusion of
      15N-phenylalanine will be started and maintained throughout period 2. The different tracer of
      phenylalanine will be used to quantify the plasma protein synthetic rates using the same
      tracer protocol as in period 1. We have previously shown that the two phenylalanine tracers
      yield the same results (70). The tracer technique will enable us to measure the primary
      endpoints of insulin responsiveness of the liver, i.e., endogenous glucose production and
      synthetic rates of albumin and fibrinogen. At 4 hours, hyperinsulinemia will be initiated by
      the infusion of insulin at the rate of 1.5 mu/kg•min, which will result in circulating levels
      of approximately 200 uU/ml (5). This rate of infusion was based on our previous experience
      with insulin infusion in burned patients (e.g., 1-5). We anticipate a considerable variation
      in the baseline insulin concentrations, such that if a low rate of infusion were to be used,
      the resulting "hyperinsulinemia" in some patients would likely be below the baseline
      concentration in others. Consequently, we have chosen a rate of infusion that will result in
      a clear-cut difference between the baseline and "hyperinsulinemic" values. Further, although
      during the insulin infusion we anticipate that insulin concentrations will also be variable,
      our endpoints will be assessed in terms of the magnitude of change from the baseline value in
      each subject. This statistical approach should minimize concern regarding subject
      variability. The dosage was selected because we have previously shown that protein metabolism
      is responsive to this rate of infusion (5), but that it is below the maximally-effective dose
      (4). Blood glucose concentration will be monitored throughout the second period, and glucose
      will be infused (if necessary) to maintain glucose concentrations at the baseline level.
      Since the baseline concentrations of glucose will vary, this means that during
      hyperinsulinemia the glucose concentrations will likely differ between subjects, but we have
      selected this approach because in this way only the insulin concentration will differ between
      periods 1 and 2, thereby simplifying interpretation of the changes in substrate and protein
      kinetics from period 1 to 2. The sampling schedule will be the same as in period 1, including
      the timing of the biopsy (i.e., at 4 h of period 2).

      Leg blood flow will be measured by indocyanine green infusion, ad described previously (14).
      Whole-body indirect calorimetry will be performed to quantify whole-body carbohydrate and fat
      oxidation.
    

Intervention name(s): fenofibrate
Intervention type(s): Drug
Intervention MeSH terms: Fenofibrate
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Double
Primary purpose: Treatment
Keywords: Glucose metabolism; Fat Metabolism; Insulin Resistance; Protein Metabolism; Tracer Methodology