The Role of TBC1D4 in Exercise- and Insulin-induced Glucose Metabolism in Human Skeletal Muscle
Condition: Exercise; Insulin Sensitivity; Skeletal Muscle
MeSH terms: Insulin Resistance
Study type: Interventional

Summary: 
      Recently a common Greenlandic nonsense p.Arg684erTer variant (in which arginine is replaced
      by a termination codon) in the gene TBC1D4 was discovered. The variant has an allele
      frequency of 17%. Homozygous carriers of this TBC1D4 variant have impaired glucose tolerance
      and a 10-fold enhanced risk of developing type 2 diabetes (T2D). The investigators propose to
      carry out comprehensive metabolic phenotyping of adult Inuits carrying zero or two alleles of
      the TBC1D4 variant. The investigators hypothesise that regulation of TBC1D4 in skeletal
      muscle is pivotal in regulating glucose uptake during exercise, during physiological insulin
      stimulation, and for the ability of an acute bout of exercise to improve insulin sensitivity
      to regulate glucose metabolism in humans.

      The overall aims in the present project are to:

        1. Determine whether the TBC1D4 p.Arg684Ter variant affects the regulation of glucose
           uptake in skeletal muscle during exercise and during physiological insulin stimulation.

        2. Determine the effect of the TBC1D4 p.Arg684Ter variant for the ability of acute exercise
           to insulin sensitize skeletal muscle to regulate glucose metabolism.

        3. Define the metabolic pathways affected by the p.Arg684Ter variant in order to identify
           causal factors responsible for the diabetic phenotype of Inuit carriers.

      The knowledge generated will contribute to additional explanatory clues to the increased
      frequency of T2D in the carriers.
    

Detailed description: 
      Recently a common Greenlandic nonsense p.Arg684erTer variant (in which arginine is replaced
      by a termination codon) in the gene TBC1D4 was discovered. The variant has an allele
      frequency of 17%. Homozygous carriers of this TBC1D4 variant have impaired glucose tolerance
      and a 10-fold enhanced risk of T2D. The investigators propose to carry out comprehensive
      metabolic phenotyping of adult Inuits carrying zero or two alleles of the TBC1D4 variant. The
      investigators hypothesise that regulation of TBC1D4 in skeletal muscle is pivotal in
      regulating glucose uptake during exercise, during physiological insulin stimulation, and for
      the ability of an acute bout of exercise to improve insulin sensitivity to regulate glucose
      metabolism in humans.

      Our overall aims in the present project are to:

        1. Determine whether the TBC1D4 p.Arg684Ter variant affects the regulation of glucose
           uptake in skeletal muscle during exercise and during physiological insulin stimulation.

        2. Determine the effect of the TBC1D4 p.Arg684Ter variant for the ability of acute exercise
           to insulin sensitize skeletal muscle to regulate glucose metabolism.

        3. Define the metabolic pathways affected by the p.Arg684Ter variant in order to identify
           causal factors responsible for the diabetic phenotype of Inuit carriers.

      The knowledge generated will contribute to additional explanatory clues to the increased
      frequency of T2D in the carriers.

      Study population:

      The Inuit Health in Transition (IHIT) study is a study of adults in West and East Greenland.
      The participants were selected through a stratified random sample and data were collected in
      1999-2010 using clinical procedures, sampling of biological material, and questionnaires. To
      accomplish the goal, the investigators will carry out comprehensive metabolic phenotyping of
      Inuits carrying zero (n=10) or two (n=10) alleles of the TBC1D4 p.Arg684Ter variant recruited
      from this cohort. Based on an effect of 1.2 SD as found for plasma glucose levels during an
      Oral Glucose Tolerance Test (OGTT) in the previous study (6) between homozygous carriers and
      non-carriers of the TBCD14 variant, the investigators achieve 89% power at p=0.05 when
      recruiting 10 homozygous carriers and 10 non-carriers.

      Methods:

      Genotyping of the IHIT cohort has already been performed. Thus, the investigators can re-call
      study participants for the present study based on their known genotype. The investigators
      intend to perform highly invasive physiological studies of subjects harboring the TBC1D4
      point mutation and in control subjects matched by age, gender and BMI. The participants will
      be nondiabetic (25-50 yrs) males recruited through the IHIT cohort register (n=4,200). The
      recruitment, screening and initial clinical investigations (oral glucose tolerance test and
      physical exercise tests, including customization to a one-legged cycling ergometer) will take
      place in Greenland. The most invasive part of the study, described below, will take place in
      Copenhagen, Denmark.

      During the stay in Copenhagen participants will receive an energy-matched, standardized diet
      to be consumed 3 days prior to the intervention study. On the study day the participants will
      arrive in the laboratory after an overnight fast. Catheters will be placed in both femoral
      veins (V) and in one femoral artery (A). A muscle biopsy will be obtained from vastus
      lateralis of one leg (resting leg). The participants will then perform one legged knee
      extensor exercise for one hour leaving the other leg as a rested control leg. Immediately
      after exercise a biopsy will be obtained from the exercising leg and the participants will
      rest in the fasted state for 3 hours before a third set of muscle biopsies (from both legs)
      and one abdominal subcutaneous fat biopsy are taken. Subsequently, insulin sensitivity will
      be evaluated during a 2-hour physiological hyperinsulinemic euglycemic clamp. By the end of
      the clamp a final set of biopsies (both legs) and one abdominal subcutaneous fat biopsy will
      be obtained. Throughout the study day sampling of artery and venous blood/plasma samples
      allow estimation of substrate extraction by the two legs. Further, measures of artery blood
      flow by Ultrasound Doppler technique will allow a final calculation of substrate
      uptake/release across the two legs before exercise, during exercise and in recovery from
      exercise. Importantly, the ability for insulin to stimulate theses process can be evaluated
      in both a rested and in the prior exercised leg.

      The investigators plan to carry out deep transcript profiling of muscle tissue samples and
      define correlations between age, sex, BMI, genetic variants, and the transcriptional profiles
      in order to understand the impact on gene regulation of identified metabolic genetic
      variants. In order to define key biochemical pathways affected by the mutated TBC1D4, the
      investigators will perform metabolomic analyses of the muscle biopsy samples. In order to
      identify putative targeted pathways affected by the mutation, the investigators will perform
      mass spectrometry-based interactome as well as proteomic / phosphoproteomic analyses. Based
      on these data and data obtained from the metabolic analyses, the investigators will be better
      able to identify the affected metabolic processes involved in the progression towards the
      diabetic phenotype of the Inuit carriers. Such pathways will in subsequent analyses be
      studied biochemically in more detail including additional (to phosphorylation) post
      translational modifications (e.g. glycosylation) as well as expression/activities of key
      enzymes in glucose and fat metabolism.
    

Intervention name(s): Acute exercise
Intervention type(s): Other
Intervention description(s): One hour of acute one-legged knee-extensor exercise followed by 3 hours recovery and 2 hours insulin (1.5 mU/min/kg) stimulation.
Other intervention name(s): Insulin stimulation
Intervention MeSH terms: Insulin
Allocation: Non-Randomized
Intervention model: Parallel Assignment
Intervention model description: A case-control study with two groups of participants with different genetic background on the TBC1D4 gene:
Homozygote carriers of a p.Arg684T TBC1D4 gene-variant
None carriers (matched controls) of the p.Arg684T TBC1D4 gene-variant
Masking: None (Open Label)
Primary purpose: Basic Science