Effects of Metformin on Hepatic Free Fatty Acid Metabolism in Patients Diagnosed With Type 2 Diabetes: A C11 PET Study
Phase: PHASE4
Conditions: Type 2 Diabetes, Dyslipidemia
Sponsor: Lars Christian Gormsen

Project	  description:	  
	  
Title:	  	  
	  
Effects	  of	  metformin	  on	  hepatic	  fatty	  acid	  metabolism	  in	  patients	  with	  type	  2	  diabetes	  
assessed	  by	  positron	  emmission	  tomography.	  
	  
	  
Investigator	  and	  sponsor:	  
Lars	  Christian	  Gormsen,	  consultant,	  PhD	  
Department	  of	  Nuclear	  Medicine	  &	  PET	  Center	  
Palle	  Juul-­‐Jensens	  Boulevard	  165	  
Dk-­‐8200	  Aarhus	  N	  
Tel:	  +45	  7845	  6260	  
Cell:	  +45	  2281	  1631	  
lars.christian.gormsen@clin.au.dk	  	  
	  
Version	  2.4.1	  -­‐	  date	  4/2	  2013	  
	  
Regional	  Scientific	  Committee	  Region	  Midtjylland:	  1-­‐10-­‐72-­‐522-­‐12	  
Clinical	  Trials	  Gov	  ID:	  NCT01729156	  
Data	  Protection	  Agency:	  1-­‐16-­‐02-­‐303-­‐12	  
	  
	  
	  

Introduction	  and	  purpose:	  	  
	  
Type	  2	  diabetes	  (T2D)	  is	  characterized	  by	  insulin	  resistance,	  impaired	  glycemic	  control	  and	  
elevated	  levels	  of	  potentially	  harmful	  circulating	  lipids.	  One	  of	  the	  most	  widely	  used	  drugs	  to	  
control	  blood	  sugar	  levels	  in	  patients	  with	  T2D	  is	  metformin,	  which	  has	  well	  known	  inhibitory	  
effects	  on	  hepatic	  gluconeogenesis,	  but	  also	  possesses	  poorly	  understood	  lipid	  lowering	  
effects.	  Thus,	  metformin	  supresses	  circulating	  triglycerides	  to	  an	  extent	  that	  is	  not	  fully	  
explained	  by	  decreased	  hepatic	  de	  novo	  lipogenesis.	  The	  pharmacological	  effects	  of	  
metformin	  are	  thought	  to	  principally	  occur	  in	  the	  liver,	  which	  has	  been	  a	  serious	  obstacle	  to	  
human	  studies.	  The	  liver	  is	  an	  inaccessible	  organ	  and	  any	  experimental	  procedures	  designed	  
to	  uncover	  intracellular	  reactions	  has	  previously	  involved	  surgical	  procedures	  with	  biopsies.	  
Advances	  in	  molecular	  imaging	  may	  change	  that.	  Positron	  emission	  tomography	  (PET)	  is	  a	  
new	  and	  advanced	  technology	  in	  which	  tracer	  amounts	  of	  positron	  emitting	  isotopes	  are	  
attached	  to	  the	  molecules	  under	  investigation,	  in	  this	  case	  fatty	  acids.	  This	  non-­‐invasive	  
technique	  allows	  quantification	  of	  the	  fate	  of	  fatty	  acids	  in	  the	  liver	  in	  vivo	  and	  can	  therefore	  
be	  used	  to	  assess	  how	  large	  a	  proportion	  of	  lipids	  that	  are	  oxidized,	  reesterified	  or	  resecreted	  
from	  the	  liver.	  It	  is	  therefore	  our	  aim	  to	  employ	  the	  PET	  scanning	  technique	  to	  study	  the	  effect	  
of	  metformin	  on	  hepatic	  fatty	  acid	  handling	  in	  the	  liver	  in	  patients	  with	  type	  2	  diabetes.	  	  
	  
Specific	  purposes:	  
• By	  [11C]palmitate	  PET	  scans	  to	  investigate	  hepatic	  fatty	  acid	  uptake,	  oxidation	  and	  
resecretion	  as	  very-­‐low-­‐density-­‐lipoprotein	  (VLDL)	  in	  a	  population	  of	  patients	  with	  
newly	  diagnosed	  T2D.	  Patients	  will	  be	  studied	  in	  a	  parallel	  group,	  placebo	  controlled,	  
double-­‐blinded	  study	  before	  initiation	  of	  metformin	  therapy	  and	  after	  3	  months	  
treatment.	  An	  age-­‐	  and	  BMI	  matched	  group	  treated	  with	  metformin	  for	  3	  months	  will	  
serve	  as	  healthy	  controls.	  
• By	  whole	  body	  [1-­‐14C]VLDL-­‐TG	  to	  quantitate	  hepatic	  production	  and	  secretion	  of	  
VLDL-­‐TG	  before	  and	  after	  metformin	  treatment.	  
• By	  14CO2-­‐breath	  collection	  to	  quantitate	  the	  effect	  of	  metformin	  on	  whole	  body	  
oxidation	  of	  VLDL	  derived	  fatty	  acids	  
• By	  [3-­‐3H]glucose	  infusion	  to	  quantitate	  the	  impact	  of	  metformin	  on	  whole	  body	  
glucose	  disposal	  as	  well	  as	  endogenous	  glucose	  production	  and	  to	  correlate	  any	  
changes	  with	  changes	  in	  lipid	  metabolism	  
• By	  dual	  X-­‐ray	  absorptiometry	  to	  determine	  the	  impact	  of	  metformin	  treatment	  on	  
body	  composition	  
	  
It	  is	  our	  hypothesis	  that	  T2D	  hypertriglyceridemia	  is	  primarily	  caused	  by	  defective	  hepatic	  
oxidation	  of	  fatty	  acids	  with	  subsequent	  increased	  resecretion	  as	  VLDL-­‐TG.	  We	  also	  
hypothesize	  that	  treatment	  for	  3	  months	  with	  metformin	  will	  normalize	  hepatic	  fatty	  acid	  
oxidation	  resulting	  in	  diminished	  reesterification	  and	  resecretion	  as	  triglycerides.	  
Background:	  
The	  prevalence	  of	  diabetes	  in	  general	  and	  T2D	  in	  particular	  has	  been	  growing	  
steadily	  during	  the	  past	  decades(1).	  T2D	  is	  most	  often	  associated	  with	  obesity	  and	  is	  
characterized	  by	  insulin	  resistance	  and	  elevated	  blood	  glucose	  but	  also	  by	  profound	  
dyslipidemia.	  This	  is	  the	  so-­‐called	  diabetic	  lipid	  triad	  of	  elevated	  low-­‐density-­‐lipoprotein	  
(LDL)	  cholesterol,	  decreased	  high-­‐density-­‐lipoprotein	  (HDL)	  cholesterol	  and	  
hypertriglyceridemia(2).	  Each	  component	  of	  the	  dyslipidemic	  triad	  is	  associated	  with	  

increased	  risk	  of	  cardiovascular	  disease	  and	  mortality	  and	  is	  therefore	  a	  potential	  risk	  factor	  
for	  diabetic	  patients.	  	  
Triglycerides	  are	  transported	  in	  the	  circulation	  packaged	  in	  lipoproteins	  that	  are	  
large	  particles	  consisting	  of	  scaffolding	  and	  surface	  proteins	  (among	  them	  ApoB’s)	  with	  a	  core	  
of	  various	  lipids.	  ApoB48	  containing	  chylomicrons	  dominate	  in	  the	  immediate	  postprandial	  
period,	  whereas	  ApoB100	  containing	  VLDLs	  are	  secreted	  continuously	  albeit	  at	  a	  slower	  rate	  
after	  feeding(3).	  VLDL	  associated	  TG	  (VLDL-­‐TG)	  is	  formed	  in	  the	  liver	  by	  a	  series	  of	  processes	  
involving	  reesterification	  of	  fatty	  acids	  released	  from	  subcutaneous	  and	  visceral	  adipose	  
tissue	  depots.	  VLDLs	  serve	  as	  transport	  molecules	  ensuring	  a	  constant	  exchange	  of	  lipids	  
between	  adipose	  tissue,	  muscle	  and	  the	  liver	  and	  also	  ”absorb”	  any	  surplus	  fatty	  acids	  not	  
oxidized	  by	  fuel	  consuming	  tissue	  and	  released	  due	  to	  bursts	  of	  exercise	  or	  catecholamine	  
stimulation.	  In	  healthy	  humans,	  the	  total	  pool	  of	  circulating	  VLDLs	  is	  surprisingly	  stable	  as	  is	  
the	  production,	  secretion	  and	  elimination	  of	  VLDL	  particles.	  In	  patients	  with	  diabetes,	  by	  
contrast,	  release	  of	  fatty	  acids	  from	  adipose	  tissue	  depots	  exceeds	  what	  is	  oxidized	  in	  the	  liver	  
and	  in	  muscle,	  and	  as	  a	  result,	  more	  fatty	  acids	  are	  reesterified	  and	  resecreted	  as	  VLDL-­‐TG.	  
This	  expands	  the	  VLDL-­‐TG	  pool	  leading	  to	  diabetic	  hypertriglyceridemia.	  What	  causes	  this	  
inbalance	  between	  released	  and	  oxidized	  fatty	  acids	  is	  still	  somewhat	  unclear.	  It	  has	  been	  
argued	  that	  the	  major	  culprit	  is	  adipose	  tissue	  resistance	  with	  increased	  release	  of	  fatty	  acids,	  
but	  also	  defects	  in	  insulin	  action	  in	  the	  liver	  and	  increased	  de	  novo	  lipogenesis	  due	  to	  elevated	  
blood	  sugar	  has	  been	  suggested.	  To	  date,	  the	  precise	  pathophysiology	  remains	  obscure.	  
Treatment	  of	  diabetic	  hypertriglyceridemia	  is	  aimed	  at	  preventing	  formation	  of	  
VLDL	  particles	  in	  the	  liver(4).	  Statins	  are	  a	  series	  of	  drugs	  originally	  designed	  to	  inhibit	  
cholesterol	  production	  that	  also	  prevent	  hepatic	  formation	  of	  mature	  VLDL	  particles	  due	  to	  
lack	  of	  cholesterol	  content.	  Inhibition	  of	  VLDL-­‐TG	  substrate	  delivery	  to	  the	  liver	  can	  be	  
achieved	  by	  blocking	  the	  hormone	  sensitive	  lipase	  with	  acipimox,	  a	  nicotinic	  acid	  analogue(5),	  
or	  by	  increasing	  fatty	  acid	  oxidation	  with	  fibrates(6).	  Somewhat	  surprisingly,	  the	  glucose	  
lowering	  biguanide,	  metformin,	  also	  reduces	  levels	  of	  circulating	  triglycerides.	  This	  has	  
previously	  been	  attributed	  to	  diminished	  conversion	  of	  glucose	  to	  lipids	  via	  hepatic	  de	  novo	  
lipogenis,	  but	  recent	  studies	  indicate	  that	  metformin	  may	  affect	  lipid	  metabolism	  more	  
directly(7),	  either	  in	  the	  liver	  or	  in	  the	  periphery.	  In	  vitro	  and	  animal	  studies	  have	  thus	  
demonstrated	  that	  metformin	  may	  increase	  hepatocyte	  fatty	  acid	  oxidation(8).	  Metformin	  has	  
also	  been	  shown	  to	  increase	  endothelial	  lipolysis	  of	  VLDL	  derived	  TGs(9)	  releasing	  more	  fatty	  
acids	  for	  oxidation	  in	  muscle.	  Whether	  the	  effect	  of	  metformin	  is	  primarily	  hepatic	  or	  
peripheral	  or	  a	  combination	  of	  both	  is	  still	  as	  unclear	  as	  whether	  diabetic	  dyslipidemia	  is	  
caused	  by	  hepatic	  or	  peripheral	  insulin	  resistance.	  The	  advent	  of	  the	  PET	  technique	  may	  very	  
well	  change	  this.	  
In	  short,	  the	  PET	  scan	  technique	  involves	  tracing	  the	  fate	  of	  certain	  metabolites	  
in	  vivo.	  This	  is	  done	  by	  incorporating	  a	  positron	  emitting	  isotope	  into	  any	  molecule	  being	  
investigated	  (the	  radiotracer),	  inject	  it	  as	  a	  bolus	  into	  the	  patient	  and	  subsequently	  monitor	  
time-­‐activity	  curves	  in	  various	  organs.	  Organ	  time-­‐activity	  curves	  reflect	  radiotracer	  exchange	  
between	  the	  circulation	  and	  intracellular	  pools	  e.g.	  lipid	  droplets	  in	  the	  liver	  or	  VLDL-­‐TG	  
particles	  ready	  for	  secretion.	  Using	  dynamic	  PET	  scans,	  Iozzo	  et.	  al	  have	  validated	  a	  kinetic	  
model	  that	  accurately	  describe	  the	  hepatic	  fate	  of	  [C11]palmitate(10,	  11)	  but	  [C11]palmitate	  	  
can	  also	  be	  used	  to	  estimate	  myocardial	  fatty	  acid	  uptake	  and	  oxidation(12).	  PET	  scans	  are	  
non-­‐invasive	  and	  are	  in	  theory	  only	  limited	  by	  the	  field	  of	  view	  of	  the	  scanner,	  typically	  ~20	  
centimeter.	  Estimating	  fatty	  acid	  metabolism	  in	  a	  single	  scan	  is	  thus	  feasible	  in	  neighboring	  
organs	  like	  the	  liver,	  visceral	  fat	  and	  myocardium	  (see	  figure	  1).	  

In	  addition	  to	  the	  fatty	  acid	  tracer,	  we	  have	  in	  our	  lab	  developed	  and	  validated	  a	  
whole	  body	  VLDL-­‐TG	  tracer,	  [1-­‐14C]VLDL,	  which	  enables	  measurements	  of	  VLDL-­‐TG	  
production,	  secretion	  and	  oxidation	  in	  tissues	  outside	  the	  PET	  scanner	  field	  of	  view(13,	  14).	  
Combining	  whole	  body	  [1-­‐14C]VLDL	  tracer	  kinetics	  with	  [C11]palmitate	  PET	  scans	  thus	  
yields	  simultaneous	  measurements	  of	  hepatic	  and	  peripheral	  lipid	  metabolism	  (see	  figure	  2).	  
It	  is	  therefore	  our	  aim	  to	  establish	  whether	  the	  lipid	  lowering	  effect	  of	  metformin	  
is	  a	  result	  of	  increased	  hepatic	  fatty	  acid	  oxidation	  or	  increased	  oxidation	  of	  VLDL	  derived	  TGs	  
in	  skeletal	  muscle.	  Insights	  into	  the	  mechanisms	  of	  metformin	  action	  will	  provide	  us	  with	  an	  
opportunity	  to	  tailor	  lipid	  lowering	  therapy	  in	  diabetes	  patients.	  
	  
	  
METHODS:	  
	  
Design:	   The	   study	   will	   be	   a	   randomized,	   double-­‐blinded,	   placebo-­‐controlled	   parallel	   study	  
with	  24	  newly	  diagnosed	  type	  2	  diabetes	  patients	  and	  12	  age,	  sex	  and	  BMI	  matched	  healthy	  
controls.	   Patients	   and	   controls	   will	   be	   studied	   before	   intervention	   and	   after	   3	   months	  
treatment	  with	  either	  metformin	  (2	  x	  1000	  mg)	  or	  placebo	  (T2D	  patients).	  Healthy	  controls	  
will	  be	  studied	  after	  3	  months	  metformin	  treatment	  (see	  figure	  3).	  	  
	  
	  
	  
Intervention	  T2D	  patients:	  Treatment	  with	  metformin	  or	  metformin	  placebo:	  500	  mg	  twice	  
daily	  for	  2	  to	  4	  weeks	  followed	  by	  1000	  mg	  daily	  for	  the	  remaining	  8-­‐12	  weeks.	  Dose	  will	  be	  
reduced	  in	  patients	  with	  moderate	  renal	  insufficiency	  (eGFR	  50-­‐80	  ml/min.	  =	  25-­‐50%,	  eGFR	  
30-­‐50	  ml/min.	  =	  50-­‐75%).	  Treatment	  will	  be	  initiated	  after	  day	  0	  for	  a	  total	  of	  90	  days.	  In	  this	  
patient	  cohort,	  the	  study	  is	  double-­‐blinded	  and	  randomized.	  
	  

Intervention	   healthy	   controls:	   Treatment	  with	  metformin	  or	  metformin	  placebo:	  500	  mg	  
twice	   daily	   for	   2	   to	   4	   weeks	   followed	   by	   1000	   mg	   daily	   for	   the	   remaining	   8-­‐12	  
weeks.	  	  Treatment	  will	  be	  initiated	  after	  day	  0	  for	  a	  total	  of	  90	  days.	  In	  this	  patient	  cohort,	  the	  
study	  is	  not	  blinded	  –subjects	  serve	  as	  their	  own	  controls	  (before	  and	  after	  treatment).	  
	  
Study	  subjects:	  24	  newly	  diagnosed	  T2D	  patients	  >50	  years	  will	  be	  recruited	  by	  physicians	  in	  
the	  endocrine	  outpatient	  unit	  (MEA,	  Aarhus	  University	  Hospital).	  12	  healthy	  controls	  will	  be	  
recruited	  by	  advertisements	  in	  local	  papers.	  
	  
Inclusion	  criteria	  patients:	  
• 
Newly diagnosed (>3 and <12 måneder) Type 2 diabetes  
• 
Age 50-70 year 
• 
BMI<40 
 
Inclusion	  criteria	  healthy	  controls:	  
• Age	  50-­‐70	  year	  
• Sex	  and	  BMI	  matched	  with	  included	  patients	  
	  
Exclusion	  crtiteria	  patients	  
• 
Metformin treatment >6 months 
• 
Liver disease in the form of NASH (non-alkoholic steatohepatitis)  
• 
Cancer 
• 
Anaemia 
• 
HbA1C>8.5 % 
• 
Other clinically relevant abnormal biochemical parameter 
• 
Chronic or acute pancreatitis 
• 
Known substance or alcohol abuse 
• 
Allergy towards metformin  
• 
Claustrophobia 
• 
Extreme obesity > 130 kg 
	  
Study	  power:	  We	  have	  previously	  studied	  T2D	  patients	  and	  found	  that	  their	  average	  VLDL-­‐
TG	   secretion	   was	   ~87	   µmol/min.	   Interventional	   studies	   using	   metformin	   to	   treat	   patients	  
with	  type	  2	  diabetes	  report	  TG	  concentration	  decreases	  of	  ~10	  %(15),	  10	  %(16),	  13	  %(17),	  
17	   %(18)	   and	   17	   %(19)	   and	   an	   extensive	   meta-­‐analysis	   of	   clinical	   studies	   have	   shown	   an	  
average	  decrease	  of	  ~10	  %(20).	  Total	  TG	  concentration	  reflects	  TG	  in	  all	  lipoprotein	  fractions	  
but	  there	  are	  strong	  indications	  that	  metformin	  primarily	  affects	  VLDL	  associated	  TG.	  Since	  
the	  proportion	  of	  TG	  in	  VLDL	  particles	  is	  60-­‐75	  %	  (21),	  VLDL-­‐TG	  concentration	  will	  decrease	  
more	  than	  10	  %,	  estimated	  14	  %.	  In	  addition,	  we	  have	  previously	  observed	  a	  50	  %	  greater	  
decrease	  in	  VLDL-­‐TG	  production	  than	  VLDL-­‐TG	  concentration	  during	  insulin	  stimulation	  (22).	  
In	   our	   view,	   metformin	   treatment	   in	   the	   dose	   proposed	   by	   us	   will	   therefore	   result	   in	   a	  
decrease	  in	  hepatic	  VLDL-­‐TG	  production	  ~20	  %	  or	  0.20	  x	  87	  µmol/min	  =	  17	  µmol/min.	  Based	  
on	  our	  previous	  studies	  in	  comparable	  patient	  populations(23),	  the	  SD	  of	  VLDL-­‐TG	  secretion	  

change	  is	  13	  µmol/min.	  α	  is	  set	  at	  0.05	  and	  power	  β	  set	  at	  0.8.	  We	  will	  then	  have	  to	  recruit	  
~11	  participants	  in	  each	  group.	  
	  
Scans:	   PET/CT	  scans:	  PET/CT	  scans	  will	  be	  preceded	  by	  at	  least	  6	  hours	  fasting.	  A	  catheter	  
will	  be	  placed	  in	  an	  antecubital	  vein	  for	  injection	  of	  the	  radiotracer.	  First,	  a	  low	  dose	  CT	  scan	  
will	   be	   performed	   in	   order	   to	   aid	   anatomical	   localization	   of	   the	   liver	   and	   for	   attenuation	  
correction	  of	  the	  subsequent	  PET	  scan.	  Second,	  380	  MBq	  [C11]palmitate	  is	  injected	  as	  a	  bolus	  
over	  10	  seconds.	  Dynamic	  imaging	  of	  [C11]palmitate	  will	  be	  done	  by	  list	  mode	  aquisition	  of	  
data	  in	  32	  frames	  of	  increasing	  length	  (from	  5	  s	  a	  frame	  in	  the	  first	  minute	  to	  600	  seconds	  a	  
frame	  in	  the	  end	  of	  the	  PET	  study).	  The	  PET	  scan	  will	  last	  a	  total	  of	  50	  minutes.	  Arterialized	  
blood	  samples	  will	  be	  obtained	  in	  order	  to	  measure	  radioactive	  palmitate	  metabolites	  (TGs,	  
phospholipids	  and	  CO2).	  The	  input	  function	  will	  be	  corrected	  for	  radioactive	  metabolites.	  	  
	  
PET	   data	   analysis:	   Kinetics:	  Hepatic	   metabolism	   of	   the	   radiotracer	   [C11]palmitate	   will	   be	  
calculated	  based	  on	  a	  3-­‐compartment	  model	  developed	  and	  validated	  by	  Iozzo	  et.	  al	  at	  Turkku	  
PET	  Center.	  In	  this	  model,	  the	  input	  function	  is	  based	  on	  image	  derived	  data	  from	  regions	  of	  
interest	  (ROIs)	  in	  the	  aorta,	  portal	  vein	  and	  hepatic	  right	  lobe.	  The	  dual	  input	  from	  the	  hepatic	  
artery	  and	  the	  portal	  vein	  will	  be	  weighted	  with	  80	  %	  to	  the	  portal	  vein	  and	  the	  remaining	  20	  
%	  from	  the	  artery.	  The	  analysis	  yields	  the	  parameters	  hepatic	  fatty	  acid	  uptake	  (K1),	  hepatic	  
fatty	  acid	  oxidation	  (k2),	  resterification	  (k3)	  and	  resecretion	  as	  VLDL-­‐TG	  (k4).	  	  
	  
[1-­‐14C]VLDL-­‐TG	   metabolism:	   Study	   subjects	   will	   be	   transferred	   to	   the	   Medical	   Research	  
Lab	  immediately	  after	  the	  PET	  scan	  and	  a	  bolus	  (~10	  mCi)	  ex-­‐vivo	  labeled	  [1-­‐14C]VLDL	  will	  
be	   injected.	   Blood	   samples	   will	   be	   taken	   every	   five	   minutes	   for	   30	   minutes	   and	   every	   30	  
minutes	   for	   the	   following	   2,5	   hours	   in	   order	   to	   estimate	   VLDL-­‐TG	   secretion.	   Oxidation	   of	  
VLDL-­‐TG	  will	  be	  assessed	  by	  collecting	  breath	  samples	  every	  30	  minutes	  and	  analyzing	  them	  
for	  14CO2	  content.	  	  
	  
[3-­‐3H]glucose	   metabolism:	   [3-­‐3H]glucose	   will	   be	   infused	   continously	   during	   the	   basal	  
period	  and	  subsequently	  for	  another	  2	  hours	  during	  a	  hyper-­‐insulinemic	  euglycemic	  clamp	  
(HE	  clamp).	  HE-­‐clamping	  is	  utilized	  to	  estimate	  whole	  body	  insulin	  sensitivity	  in	  subjects	  and	  
can	  also	  be	  employed	  to	  asses	  hepatic	  endogenous	  glucose	  production.	  	  	  
	  
Body	   composition:	   Both	   patients	   and	   healthy	   controls	   will	   have	   their	   body	   composition	  
assessed	  by	  dual	  X-­‐ray	  absorptiometry	  (DXA)	  (Hologic).	  In	  the	  DXA	  scanner,	  minute	  amounts	  
of	   X-­‐rays	   are	   used	   to	   determine	   various	   tissue	   attenuation	   patterns	   that	   reflect	   body	  
composition.	  	  
	  
Blood	  samples:	  	  Serum	  insulin,	  C-­‐peptide,	  cortisol	  and	  glucagone	  will	  be	  measured	  by	  Radio-­‐
Immuno-­‐Assay	   (RIA);	   interleukin-­‐6	   (Luminex);	   retinol	   binding	   protein	   4	   (Elisa);	   plasma	  

glucose	   (YSI	   STAT	   2300	   Glucose	   Analyzer),	   glycerol	   and	   lactate	   (Floruometric	   enzymatic	  
method),	  serum	  non-­‐esterified	  FFAs	  (radiochemical	  assay),	  plasma	  acyl	  og	  desacyl	  ghrelin.	  
	  
Patient	   data:	   Demographic	   data	   and	   other	   information	   regarding	   patients	   and	   disease	  
history	  are	  collected	  before	  the	  onset	  of	  the	  trial.	  All	  information	  are	  recorded	  in	  the	  Case	  
Report	  Form	  (CRF).	  	  
	  
	  
Data	   and	   permissions:	   All	   electronically	   collected	   data	   are	   archived	   on	   the	   PET	   Center	  
server	  (Tiger).	  Manual	  data	  are	  kept	  in	  a	  locked	  room	  at	  the	  PET	  Center.	  All	  participants	  are	  
registered	  under	  a	  study	  subject	  ID	  with	  initials	  provided	  by	  the	  blinding	  and	  randomization	  
authority	  –	  The	  Hospital	  Pharmacy.	  Participants	  are	  aware	  that	  the	  local	  Ethical	  Committee	  
and	  the	  Danish	  Data	  Agency	  must	  have	  access	  to	  all	  data.	  The	  study	  is	  approved	  by	  the	  Danish	  
Medicines	   Agency	   (EUDRA-­‐CT	   2012-­‐000808-­‐16),	   The	   Danish	   Data	   Agency	   and	   the	   local	  
Ethical	  Committe	  (1-­‐16-­‐02-­‐303-­‐12).	  	  
	  
Radioactivity	  dose:	  	  
PET	  scan	  with	  low	  dose	  CT	  to	  determine	  hepatic	  fatty	  acid	  metabolism	  ([11C]palmitate	  ):	  	  
	  
According	  to	  the	  lastest	  ICRP	  106,	  maximal	  realistic	  effective	  dose	  for	  C11-­‐labeled	  tracers	  is	  
0.011	  mSv/MBq.	  We	  plan	  to	  inject	  380	  MBq	  per	  study	  day	  and	  the	  total	  injected	  dose	  will	  then	  
be:	  
	  
0.011	  mSv/MBq	  x	  380	  MBq/day	  x	  2	  days	  =	  8,36	  mSv.	  
	  
Low	  dose	  CT	  of	  the	  abdominal	  region	  is	  1	  mSv.	  	  	  
	  
Total	  PET	  related	  radiation	  dose	  is	  then	  9,36	  mSv.	  The	  [14C]VLDL	  tracer	  yields	  an	  additional	  
0,4	  mSv	  per	  study	  day	  and	  the	  DXA	  scan	  0,15	  mSv.	  
	  
Accumulated	  radiation	  dose:	  9,91	  mSV.	  
	  
This	  dose	  is	  roughly	  equivalent	  to	  half	  a	  diagnostic	  CT	  of	  the	  thorax,	  abdomen	  and	  pelvis	  (18	  
mSv).	  It	  is	  also	  euquivalent	  to	  3	  times	  the	  normal	  background	  radiation	  (3	  mSv	  per	  year).	  
For	   persons	   aged	   60	   this	   corresponds	   to	   an	   additional	   risk	   of	   dying	   from	   cancer	   of	   about	  
0.0005	   (0.05	   %)	   which	   is	   comparable	   to	   smoking	   9	   packets	   of	   cigarettes	   or	   driving	   6000	  
kilometers	  in	  a	  car	  (http://www.xrayrisk.com/	  (24)).	  	  
	  
Ethics:	  
The	  study	  is	  performed	  according	  to	  the	  principles	  of	  the	  Helsinki	  declaration	  II	  and	  has	  been	  
approved	   by	   the	   local	   Ethical	   Committee,	   the	   Danish	   Medicines	   Agency	   and	   the	   Data	  

Protection	   Agency.	   All	   PET	   related	   procedures	   have	   been	   performed	   at	   the	   PET	   Center	  
numerous	   times	   both	   in	   an	   experimental	   and	   a	   clinical	   setting.	   The	   Clinical	   Research	  
laboratory	   has	   extensive	   experience	   in	   doing	   metabolic	   studies	   and	   has	   experienced	   no	  
serious	  adverse	  study	  related	  events	  for	  the	  past	  5	  years.	  	  
	  
	  
Rationale	  for	  the	  study:	  
Hypertriglyceridemia	  is	  almost	  universally	  present	  in	  patients	  with	  T2D	  and	  has	  	  
been	   convincingly	   demonstrated	   to	   be	   a	   serious	   risk	   factor	   for	   the	   development	   of	  
cardiovascular	   disease	   (CVD).	   Since	   CVD	   is	   associated	   with	   an	   increased	   morbidity	   and	  
mortality,	   many	   ongoing	   studies	   are	   designed	   to	   foster	   ideas	   of	   how	   to	   lower	   potentially	  
dangerous	  circulating	  lipids.	  In	  this	  context,	  it	  is	  of	  great	  interest	  to	  determine	  whether	  the	  
lipid	   lowering	   effect	   of	   metformin	   is	   independent	   of	   its	   beneficial	   effects	   on	   glucose	  
metabolism	  and	  whether	  the	  effect	  indeed	  hinges	  on	  improved	  or	  ”normalized”	  hepatic	  fatty	  
acid	  oxidation.	  If	  that	  is	  the	  case,	  future	  lipid	  lowering	  treatment	  strategies	  in	  patients	  with	  
T2D	  should	  include	  drugs	  with	  a	  different	  mode	  of	  action,	  e.g.	  statins	  or	  liraglutide	  rather	  than	  
fibrates.	   This	   study	   is	   therefore	   directly	   designed	   to	   modify	   treatment	   of	   a	   large	   group	   of	  
patients	  and	  the	  risk	  of	  using	  ionizing	  radiation	  (category	  2B)	  is	  in	  our	  opinion	  acceptable.	  To	  
further	  minimize	  radiation	  related	  risks,	  the	  study	  is	  restricted	  to	  patients	  >50	  years.	  	  
	  
Publication:	  
Lars	   Gormsen,	   Søren	   Nielsen	   and	   other	   associated	   researchers	   will	   cooperate	   in	   gathering	  
data,	  interpretating	  results	  and	  writing	  the	  papers.	  All	  results,	  both	  negative	  and	  positive,	  will	  
be	  reported.	  The	  Vancouver	  Criteria	  will	  be	  adhered	  to.	  
	  
	  
	  
	  
	  
	  
Reference	  list	  
	  
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Wild	  S,	  Roglic	  G,	  Green	  A,	  Sicree	  R,	  King	  H.	  Global	  prevalence	  of	  diabetes:	  estimates	  for	  
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Increased	  VLDL-­‐triglyceride	  secretion	  precedes	  impaired	  control	  of	  endogenous	  
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