© 1997 American Heart Association, Inc.
Articles |
Metabolic Basis of Hypotriglyceridemic Effects of Insulin in Normal Men
From the Department of Medicine, University of Helsinki, Helsinki, Finland (R.M., S.R., H.Y-J., M-R.T.); and the Department of Pathological Biochemistry, Royal Infirmary, Glasgow, Scotland, United Kingdom (C.J.P., T.D.G.W., M.C., D.B., P.S., J.S.).
Correspondence to Marja-Riitta Taskinen, MD, Department of Medicine, Division of Endocrinology and Diabetology, Helsinki University Central Hospital, Haartmaninkatu 4, FIN-00290 Helsinki, Finland.
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Abstract The mechanism by which acute insulin administration alters VLDL apolipoprotein (apo) B subclass metabolism and thus plasma triglyceride concentration was evaluated in 7 normolipidemic healthy men on two occasions, during a saline infusion and during an 8.5-hour euglycemic hyperinsulinemic clamp (serum insulin, 490±30 pmol/L). During the insulin infusion, plasma triglycerides decreased by 22% (P<.05), and serum free fatty acid decreased by 85% (P<.05). The plasma concentration of VLDL1 apo B fell 32% during the insulin infusion, while that of VLDL2 apo B remained constant. A bolus injection of [3-2H]leucine was given on both occasions to trace apo B kinetics in the VLDL1 and VLDL2 subclasses (Svedberg flotation rate, 60-400 and 20-60, respectively), and the kinetic basis for the change in VLDL levels caused by insulin was examined using a non-steady-state multicompartmental model. The mean rate of VLDL1 apo B synthesis decreased significantly by 35% (P<.05) after 0.5 hour of the insulin infusion (523±87 mg/d) compared with the saline infusion (808±91 mg/d). This parameter was allowed to vary with time to explain the fall in VLDL1 concentration. After 8.5 hours of hyperinsulinemia, the rate of VLDL1 apo B synthesis was 51% lower (321±105 mg/d) than during the saline infusion (651±81 mg/d, P<.05). VLDL2 apo B production was similar during the saline (269±35 mg/d) and insulin (265±37 mg/d) infusions. No significant changes were observed in the fractional catabolic rates of either VLDL1 or VLDL2 apo B. We conclude that acute hyperinsulinemia lowers plasma triglyceride and VLDL levels principally by suppressing VLDL1 apo B production but has no effect on VLDL2 apo B production. These findings indicate that the rates of VLDL1 and VLDL2 apo B production in the liver are independently regulated.
Key Words: VLDL • apolipoprotein B • stable isotopes • kinetics • euglycemic clamping
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Introduction |
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The cluster of hypertriglyceridemia, a low HDL cholesterol concentration, and the preponderance of small, dense LDL particles is a common lipid abnormality in NIDDM and the insulin resistance syndrome1 2 and increases the risk of coronary heart disease.3 4 5 To determine the mechanisms underlying hypertriglyceridemia in NIDDM, interest has recently been focused on the regulation of VLDL triglyceride and apo B production in the liver and the consequences of altered operation of the VLDL–LDL delipidation cascade. Key to a more detailed understanding of lipoprotein metabolism is the recognition of lipoprotein structural heterogeneity and the possibility that different VLDL and LDL subclasses may have various metabolic fates.6 The major subclasses of VLDL particles are large, triglyceride-rich VLDL1 particles with a Svedberg flotation rate of 60 to 400 and small, dense VLDL2 particles with a Svedberg flotation rate of 20 to 60. Insulin resistance has been associated with the predominance of large, triglyceride-rich VLDL1 particles.7 Recent findings have raised the possibility that secretion of large VLDL1 and small VLDL2 particles from the liver may be independently regulated,8 9 but so far no in vivo study has shown that during an acute experiment the production rates can change independently of one another. The plasma triglyceride concentration (ie, VLDL concentration) is the major regulator of LDL and HDL subclass distribution, and even moderate changes in the VLDL concentration influence the structure and metabolic properties of LDL10 and HDL.11 These relationships are of obvious importance in NIDDM, in which the primary defect is hypertriglyceridemia and small, dense LDL are commonly found.2
The role of insulin in the regulation of VLDL triglyceride and apo B metabolism is highly controversial. Reaven1 postulated that hypertriglyceridemia is caused by an increase in hepatic synthesis and secretion of VLDL triglyceride secondary to elevations of ambient plasma insulin and/or FFA concentrations. The basis of this concept is that chronic hyperinsulinemia is associated with overproduction of VLDL triglycerides.12 13 Studies using the perfused rat liver preparation have also provided evidence for an acute stimulatory effect of insulin on triglyceride synthesis.14 In contrast, studies using cultured rat15 16 and human17 18 hepatocytes have suggested that insulin actually inhibits secretion and release of apo B. The reason for the divergent observations is unclear. The effects of acute hyperinsulinemia on VLDL triglyceride19 20 21 22 and VLDL apo B20 21 22 23 production have recently been studied in vivo. Although some of the results were only semiquantitative, the data suggested that acute hyperinsulinemia suppressed VLDL apo B and triglyceride production. However, the precise mechanisms by which insulin regulates apo B production and its assembly with triglycerides are unknown.
Recent advances in the use of stable isotopes for lipoprotein kinetic studies have made possible repeated acute measurements of VLDL triglyceride and apo B kinetics.24 25 The present investigation was designed to define the site of acute insulin action on VLDL apo B subclass metabolism in healthy normolipidemic men. In pilot studies, different insulin concentrations were used to establish the dose dependency of inhibition of VLDL apo B production by insulin. Apo B turnovers using stable isotope [3-2H]leucine were then performed in a group of normolipidemic men to discover the effect of a euglycemic hyperinsulinemic clamp (insulin infusion rate, 1.0 mU · kg-1 · min-1) on VLDL1 and VLDL2 apo B production. We tested the hypothesis that VLDL1 and VLDL2 apo B syntheses are subject to independent regulation and that the hypotriglyceridemic effect of insulin is principally mediated via regulation of VLDL1 apo B production.
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Methods |
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Subjects
Seven healthy men volunteered for the study. A history was taken for all subjects, and all subjects underwent a physical examination and laboratory tests for exclusion of hepatic, renal, thyroid, and hematologic abnormalities. All subjects had normal oral glucose tolerance (75 g) according to World Health Organization criteria.26 Physical and biochemical characteristics of the subjects are shown in Table 1
. None of the subjects
was taking any medication known to affect lipid or glucose
metabolism. The subjects were instructed to ingest an
isocaloric, weight-maintaining diet for 1 month before and during the
study period. The subjects were asked to record their dietary
intake for 24 hours before the first admission and to replicate this
diet before the other admissions. The clinical and laboratory work was
done in Helsinki, Finland, and the GC-MS and kinetic analyses
were performed in Glasgow, Scotland, United Kingdom.
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The purpose, nature, and potential risks of the studies were explained to the subjects before their informed consent was obtained. The study protocol was approved by the Ethical Committee of Helsinki University Hospital.
Study Design
Each subject completed two turnover studies separated by a 1- to
2-month interval. The study design is outlined in Fig 1.
Apo B turnover studies were performed during a saline infusion and
during a euglycemic hyperinsulinemic clamp
(insulin infusion rate, 1.0
mU · kg-1 · min-1)
in random order. Two subjects participated in additional turnover
studies during a euglycemic
hyperinsulinemic clamp in which lower insulin doses
(0.5 mU · kg-1 [subjects 1 and 2] and
0.25 mU
· kg-1 · min-1
[subject 1]) were used. A heparin test for the measurement of
postheparin lipases was performed on a separate
day.27
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Apo B Turnover Protocol
All subjects were admitted to the metabolic ward of
Helsinki University Hospital before both turnover studies, after an
overnight (12-hour) fast. An indwelling cannula was inserted in an
antecubital vein for infusions. A second cannula was inserted
retrogradely into a heated hand vein to obtain arterialized
venous blood for blood sampling. At 0 minutes, infusion of saline or
insulin was started. A bolus injection of [3-2H]leucine
(C/D/N Isotopes, Inc, Vaudreuil, Quebec, Canada; 7 mg/kg body weight)
in saline was given at 30 minutes. Blood samples for the determination
of plasma [3-2H]leucine concentration were taken
immediately before the bolus injection and at 32, 34, 36, 38, 40, 42,
45, 50, 60, and 75 minutes and 1.5, 2.5, 3.5, 4.5, 6.5, and 8.5 hours.
To measure VLDL1 and VLDL2 apo B [3-2H]leucine
enrichment, blood samples were taken before the bolus and at 45, 60,
75, 90, 105, 120, 150, and 180 minutes and 3.5, 4.5, 5.5, 6.5, 7.5,
8.5, and 24 hours. The concentration of plasma apo B mass, composition
of the VLDL1 and VLDL2 fractions, and concentration of plasma
lipoprotein lipase were determined three times during the study period
(at 30 minutes and 4.5 and 8.5 hours). During the control study, saline
was infused at a rate of 200 mL/h, which was approximately equal to the
volume of insulin and glucose infusions during the clamp study. The
subjects continued to fast until 5 PM, when they were
served a hospital meal. They were served a light evening snack at 8
PM, then fasted until 8:30 AM the next morning,
when the last blood sample was drawn.
Euglycemic Hyperinsulinemic Clamp
Studies
The euglycemic hyperinsulinemic
clamp study was performed as previously described.28
Insulin (Actrapid Human, Novo Nordisk, Copenhagen, Denmark) was infused
in a primed continuous manner at a rate of 1
mU · kg-1 · min-1
for 8.5 hours. Normoglycemia was maintained by adjusting the rate of a
20% glucose infusion based on plasma glucose measurements, which were
performed at 5-minute intervals. Whole-body glucose uptake was
calculated from the mean values of the second-hour glucose infusion
rate after correcting for changes in the glucose pool
size.28 These measurements assume that
endogenous glucose production has been entirely
suppressed. This assumption is valid in normal volunteers even under
isotopic steady-state conditions using an insulin infusion rate of 1.0
mU · kg-1 · min-1.29
Isolation of Lipoproteins
VLDL1 and VLDL2 were isolated from plasma by cumulative
flotation gradient ultracentrifugation as detailed
previously.27 30 31 Briefly, 8.4 mL of plasma was mixed
with 0.6 mL of 1.019-g/mL-density NaCl solution and used to prepare
total VLDL by ultracentrifugation (100 000 rpm; 2
hours, 45 minutes; at 23°C) in a tabletop centrifuge
(TLA-100,3 Fixed Angle Rotor, Beckman Instruments, Palo Alto, Calif).
Total VLDL was aspirated from the top 2 mL, and its density was
increased to 1.118 g/mL by the addition of solid NaCl (170 mg/mL of
VLDL solution). A 2-mL aliquot of this preparation was layered over a
0.5-mL cushion of d 1.182 g/mL NaBr solution and a six-step
discontinuous salt gradient of 1.0988 to 1.0588 g/mL was constructed
above it as described by Lindgren et al.30 The Ti40 SW
rotor was subjected to centrifugation (Optima L-60,
Beckman Instruments, Palo Alto, Calif) at 39 000 rpm for 1 hour, 21
minutes at 23°C and decelerated without braking. VLDL1 was removed in
the top 1.0 mL of solution, which was replaced with 1.0 mL of
d 1.0588 g/mL solution before the separation was continued.
VLDL2 was then isolated from the top 0.5 mL of the gradient after
centrifugation at 18 500 rpm for 15 hours, 29 minutes
at 23°C. The apo B pool size samples were prepared in a similar
manner starting with 6 mL of plasma. Pool sizes for apo B were
calculated as the product of plasma volume (assumed to be 4.5% of
body weight) and the plasma concentration of apo B in VLDL1 and VLDL2.
The leucine content of the apo B pool was calculated from apo B amino
acid composition.32
Preparation and Analysis of Leucine in Apo B
Apo B in isolated lipoprotein fractions was precipitated by
isopropanol.33 Equal volumes of the lipoprotein fraction
and isopropanol were mixed and stored at 4°C overnight. The following
morning, samples were centrifuged at 3000 rpm for 30 minutes at
4°C, and the infranatant containing isopropanol-soluble proteins
under the thin apo B layer was removed. The apo B pellicle was then
delipidated with ethanol-diethylether, dried, and subsequently
hydrolyzed with 6 M HCl at 110°C for 22 to 24 hours.
To determine plasma leucine enrichments, proteins were precipitated from 1 mL of plasma by adding 1 mL of trichloroacetic acid (10%) and amino acids prepared from the supernatant by cation exchange chromatography using 2-mL columns filled with Dowex AG-50W-X8 resin (H+ form, 50 to 100 mesh; Biorad, Richmond, Calif). The amino acids that were bound to the resin were desorbed by 4 M NH4OH, which was subsequently removed by evaporation in a vacuum concentrator (Gyrovap, VA Howe, Banbury, United Kingdom), transferred into microvials, and dried again for derivation.
Determination of Leucine Enrichment by GC-MS
Amino acids were converted into tert-butyl-dimethyl-silyl
derivatives by incubation with a suitable volume of freshly prepared
1:1 mixtures of
N-methyl-N-(tert-buthyl-dimethyl-silyl)-trifluoro-acetamide
(Fluka, Buchs, Switzerland) and acetonitrile (silylation grade, Pierce
& Warriner, Ltd, United Kingdom) in crimped microvials at 70°C for 1
hour.34 Enrichments were determined immediately by GC-MS
using a quadrupole GC-MS instrument (MD 800, Fisons, Manchester, United
Kingdom). The method used for the analysis of
[3-2H]leucine enrichment in protein hydroxylates and
plasma amino acids has been described in detail
elsewhere.34 The GC conditions were as follows: 1 to 2
µL of sample was injected automatically by a fitted autosampler into
a 30-mx0.25-mm inner diameter, 0.25-µm film thickness capillary
column (J & W, Folsom, Calif) in which initial temperature was 110°C.
The temperature was increased to 210°C at a rate of 10°C per minute
and then to 310°C at 20°C per minute and maintained at 310°C for
1 minute. The injector temperature was 300°C. The split ratio was
1:50, and the carrier gas was helium with a head pressure of
approximately 70 kPa. The mass spectrometer was operated under electron
ionization conditions, and ion mass fragments at
m/z 277, m/z 276, and
m/z 274 were monitored by selective ion
recording. Peak areas were quantified in arbitrary units by the
GC-MS data management system (LabBase, Fisons, Manchester, United
Kingdom). A standard curve obtained from different mixtures of
[3-2H]leucine and natural leucine at a total leucine
concentration of 50 ng/µL demonstrated a linear relationship between
m/z 277 and m/z 276. The
theoretical values for specific isotopic enrichment ranged from 0.0 to
10.0%. Detailed analysis of standards with low isotopic
enrichment (0.00 to 1.00%) revealed that a level of 0.25% could
reliably be distinguished from 0.00% (m/z 277:
m/z 276=0.1697±0.0023 versus 0.1909±0.0024,
P<.00001). The ratios of m/z 277:
m/z 276 were multiplied by an average value for
the constant ratio of m/z 276:
m/z 274, and the resulting
m/z 277: m/z 274 values
were used to calculate the specific isotopic enrichment, E,
with the following formula35 :
E=(R–RN)/[(1+R)x(1+RN)]
where R is the m/z 277: m/z 274 peak area ratio for the enriched sample and RN is the equivalent ratio of naturally occurring leucine. A typical value for RN was 0.01697±0.00023 (n=10) as indicated above. Monitoring of the m+3 and m+2 peaks permitted greater loading of GC-MS and hence an enhanced ability to detect low enrichments with good precision.34 Using specific enrichment data, tracer/tracee ratios, Z, were derived with the following formula35 :
Z=E/(EI–E)
where EI is the isotopic abundance of the infused tracer, which was determined to be 0.998.
Kinetic Analysis of VLDL1 and VLDL2 Apo B
Tracer/tracee ratios and apo B masses determined as above were
used to derive kinetic rate constants describing the production
and catabolism of VLDL1 and VLDL2 apo B. The data were analyzed
with the CONSAM program36 using the non–steady-state
multicompartmental model shown in Fig 2. The model had
the following features. First, plasma leucine kinetics were described
by a four-compartment subsystem. The present experimental design
did not allow determination of all the rate constants for plasma
leucine. Some were fixed (Fig 2), having been determined in previous
studies on long-term (14-day) data in a large group of
subjects.34 Others were allowed to vary to adjust the
plasma leucine curve between individuals. Input of leucine into VLDL1
and VLDL2 apo B occurred from compartment 2 via a delay component
(compartment 5). The delay was set at 0.5 hours initially, but this
value was adjusted if required by the data (Table 2).
VLDL1 was modeled as a short delipidation chain in line with previously
published systems,34 and a remnant compartment was
included for subjects who required it to obtain a good fit. The default
was to set k8,6 to zero. Similarly, in most
patients k0,7 was not required, and this was set
to zero for baseline and clamp studies. If k0,7
was needed to generate a satisfactory fit in either turnover, it was
also fixed to the same value in the other study. Input of apo B
occurred at compartment 6 in VLDL1 and at compartment 9 in VLDL2. VLDL2
was modeled as a short delipidation chain with a single output.
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During the clamp test, the concentration of VLDL1 apo B in many
subjects fell markedly over the course of the infusion. Thus, the model
had to take account of this non–steady-state situation.
k6,5 was permitted to decrease with time
according to the equation shown in Fig 2
. An excellent fit was obtained
with this approach for both the tracer data and the change in mass in
VLDL1. The same model was applied to all turnovers, including those in
patients who showed a decrease in VLDL1 apo B concentration during
saline infusion. VLDL2 apo B mass did not change during the infusions,
and permitting VLDL2 production (k9,5)
to vary with time did not result in an improved fit to the observed
data. This parameter and k9,7,
k0,10, k1,2, and
k0,1 were allowed to vary freely between
turnovers but were not made functions of time.
The experimental tracer/tracee ratios were weighted within CONSAM by
applying a standard deviation of approximately
4x10-5 to the data. This
represented a CV of about 1% for peak ratios in VLDL1 and
VLDL2 apo B leucine. A CV of 5% was applied to the apo B pool sizes.
The mean tracer masses in VLDL1 and VLDL2 apo B are presented
in Fig 3. Individual rate constants are given in Table 7.
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Analytical Methods
Plasma glucose concentrations were measured in duplicate using
the glucose oxidation method (Beckman Glucose Analyzer II,
Beckman Instruments, Fullerton, Calif).37 Serum free
insulin concentrations were determined by double antibody
radioimmunoassay (Pharmacia Insulin RIA Kit, Pharmacia,
Uppsala, Sweden) after precipitation with polyethylene glycol.
Concentrations of cholesterol and triglyceride
(Hoffman-La Roche, kits 0736805 and 0736642, respectively),
phospholipids (Wako Chemicals, Neuss, Germany), and free
(nonesterified) cholesterol (Boehringer Mannheim,
Mannheim, Germany) were measured enzymatically in an automated Cobas
Mira analyzer (Basel, Switzerland). The concentration of
cholesteryl ester was calculated as the difference between total and
free cholesterol. Serum apo B was determined using an
immunochemical assay (Orion, Espoo, Finland), and serum apo A-I and apo
A-II were determined by a turbidimetric assay (Boehringer
Mannheim). Serum FFAs were quantified by the fluorometric method of
Miles et al.38 Serum glycerol was determined by an
enzymatic spectrophotometric method.39 The concentrations
of proteins in isolated lipoprotein fractions were determined according
to Kashyap et al.40 Plasma LPL during turnover studies and
postheparin LPL and hepatic lipase from heparin tests were
analyzed as previously described.27 41 Apo E
phenotyping was done from serum by isoelectric
focusing.42
Other Measurements
The percentage of body fat was determined using a
single-frequency bioelectrical impedance device (Bio-Electrical
Impedance Analyzer System, model BIA-101A, Mf.Clemens,
Mich.).43
Statistical Analysis
All data are expressed as the mean±SEM. Statistical comparisons
between two study occasions were made with the
nonparametric Wilcoxon signed rank test.
Repeated-measures analysis of variance was used to evaluate
whether there was any significant change in plasma
triglycerides, cholesterol, and lipoprotein
lipase during insulin or saline infusion. Variables with skewed
distributions (plasma triglycerides) were logarithmically
transformed before statistical comparisons. P
values < .05 were considered to be statistically
significant. Data analysis was performed using the SYSTAT
statistical package (SYSTAT Inc, Evanston, Ill).
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Insulin and Glucose Concentrations and Whole-Body Glucose Disposal Rates
The plasma glucose concentration was maintained at the baseline level and averaged 5.4±0.1 mmol/L during the insulin infusion. The CV of plasma glucose concentrations during hyperinsulinemia was 5.7±0.4%. During the saline infusion, the glucose concentration averaged 5.5±0.2 mmol/L. Serum free insulin averaged 490±30 pmol/L during the insulin infusion. The rate of whole-body glucose disposal during the insulin infusion was 37.5±5.6 µmol · kg-1.
Plasma Lipids, Apolipoproteins, and Lipase Activities
Fasting triglyceride, cholesterol, FFA,
and glycerol concentrations were comparable during both studies. Plasma
triglycerides (Fig 4) decreased during
insulin infusion (from 1.49±0.20 to 1.16±0.18 mmol/L
P<.05) but did not change during saline infusion
(1.35±0.15 versus 1.39±0.10 mmol/L, NS). Plasma
cholesterol concentrations decreased less during saline
infusion (from 5.25±0.21 to 4.93±0.22 mmol/L, NS) than during
hyperinsulinemia (from 5.21±0.23 to
4.57±0.19 mmol/L, P<.05). Serum FFAs (Fig 4) declined
rapidly after initiation of the insulin infusion and remained low
during the entire period (463±62 to 71±14 µmol/L,
P<.05). No significant change in FFAs occurred during
infusion of saline (494±69 versus 594±43 µmol/L, NS). Serum
glycerol (Fig 4) decreased during insulin infusion (from 79±9 to
54±11 µmol/L, P<.05) but remained unchanged during
saline infusion (93±12 versus 86±9 µmol/L, NS).
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Plasma LPL activity increased during both the saline (from 3.2±0.5 to
4.5±0.4 mU/mL, P<.05) and insulin (from 2.4±0.5 to
3.3±0.5 mU/mL, P<.05) infusions. Individual values of
plasma apolipoproteins and postheparin lipase activities
measured on a separate day are given in Table 3.
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VLDL1 and VLDL2 Composition
Table 4 shows the plasma concentrations of
different components of VLDL1 and VLDL2 during saline and insulin
infusions. The concentrations of triglycerides, free
cholesterol, phospholipids, and proteins in VLDL1 decreased
significantly with insulin infusion. These parameters
remained unchanged during saline infusion. The concentrations of VLDL2
components did not change significantly during either saline or insulin
infusion.
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Table 5 shows that a small, nonsignificant decrease in
plasma VLDL1 apo B pool size was observed during infusion of saline.
Plasma VLDL1 apo B pool size (Table 5) decreased by 32% during the
insulin infusion (P<.05). Only 1 subject on saline infusion
had levels of VLDL1 apo B pool size that fell at 4.5 hours and again at
8.5 hours, whereas on insulin either the starting level was lower than
on saline or there was a consistent decrease from 0.5 to 4.5 to
8.5 hours in 6 of the 7 subjects. VLDL2 apo B pool size (Table 6) remained unchanged during both the insulin and saline
infusions.
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The VLDL1 triglyceride/VLDL1 apo B ratio, an index of particle size, decreased during insulin infusion by 27% (from 30.2±2.5 to 22.1±1.2, P<.05) but did not change significantly during saline infusion (21.0±1.2 to 24.9±2.8, NS). The VLDL2 triglyceride/apo B ratio did not change significantly during either insulin or saline infusion (5.1±0.5 to 4.6±0.4 and 6.2±0.6 to 6.6±0.4, respectively, NS).
VLDL1 and VLDL2 Apo B Production
A pilot study was conducted to evaluate the dose dependency
of VLDL apo B production on insulin. VLDL1 apo B
production rates during saline and insulin infusions using
three (subject 1) or two (subject 2) insulin doses are shown in Fig 5. In subject 1, the decreases in VLDL1 apo B
production rates at different doses of insulin (0.25, 0.5, and
1.0
mU · kg-1 · min-1)
were comparable. In subject 2, the suppression of VLDL1 apo B was more
rapid using a higher (1.0
mU · kg-1 · min-1)
rather than lower (0.5
mU · kg-1 · min-1)
insulin dose. Therefore, we decided to use the insulin dose 1.0
mU · kg-1 · min-1
in further studies to observe efficient suppression of VLDL1 apo B
production.
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The individual synthetic rates of VLDL1 apo B during saline and insulin
(1.0 mU · kg-1) infusions are shown in Fig 6. VLDL1 apo B production decreased during
saline infusion in 4 of 7 subjects. In all but 1 subject (subject 7),
the rate of VLDL1 apo B production was at 30 minutes of insulin
infusion already lower than that seen during the saline infusion.
Subjects 6, 2, and 3 had lower (83%, 47%, and 3%, respectively)
VLDL1 apo B production after 30 minutes of insulin infusion
compared with 30 minutes of saline infusion; thereafter, the
production rate remained constant over the whole period of
insulin infusion, while in others it fell with time. VLDL1 apo B
synthesis decreased by 2.9% per hour (range, 0% to 11.4% per hour)
during the saline infusion and by 8.8% per hour (range, 0% to 23.9%
per hour) during the insulin infusion. In subjects in whom there was no
decline during the insulin infusion, a lower VLDL1 apo B
production rate was already established at 30 minutes. Thus,
overall, during saline infusion the synthesis of VLDL1 apo B decreased
from 808±91 mg/d at 30 minutes to 719±80 mg/d at 4.5 hours and to
651±81 mg/d at 8.5 hours. After 30 minutes of the insulin infusion,
VLDL1 apo B synthesis averaged 523±87 mg/d; it decreased to 385±90
mg/d at 4.5 hours and to 321±105 mg/d at 8.5 hours (P<.05
for insulin versus saline infusion at each time point). Table 5 shows
the mean values of VLDL1 apo B synthesis calculated from the
parameters after 4.5 hours of infusions.
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VLDL2 apo B de novo synthesis and input from VLDL1 during the saline
and insulin infusions (1.0
mU · kg-1 · min-1)
for all subjects are shown in Table 6. Insulin had no effect on de novo
VLDL2 apo B synthesis, but the delipidation rate of VLDL1 to VLDL2
decreased significantly (P<.05). In our pilot study, VLDL2
apo B de novo synthesis in subject 1 was 291, 582, 423, and 284 mg/d,
and input from VLDL1 was 738, 399, 447, and 348 mg/d during the saline
infusion and insulin infusions of 0.25, 0.5, and 1.0
mU · kg-1 · min-1.
In subject 2, VLDL2 apo B de novo synthesis was 346, 303, and 293 mg/d,
and input from VLDL1 was 854, 531, and 478 mg/d, respectively.
Fractional Catabolic Rates of VLDL1 and VLDL2 Apo B
The individual catabolic rates for VLDL1 apo B and VLDL2 apo B for
different pathways are presented in Tables 5 and 6. VLDL1 apo B
direct catabolism and transfer to VLDL2 were approximately similar
during the insulin and saline infusions. Mean fractional catabolic
rates of both VLDL1 apo B and VLDL2 apo B were comparable during the
saline and insulin infusions, although there was considerable
interindividual variation in the response to insulin.
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Discussion |
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The novel observation of this study is that insulin acutely suppresses VLDL1 apo B production but has no effect on de novo VLDL2 apo B production from the liver. Our data provide direct evidence for the concept that VLDL1 and VLDL2 apo B production are independently regulated as proposed by Gaw et al,9 who studied VLDL subclass metabolism in patients with moderate hypercholesterolemia. The present study also extends the findings of Lewis et al,20 21 22 who showed that insulin suppresses VLDL apo B and triglyceride production in healthy subjects; it focuses the action of the hormone on the triglyceride-rich VLDL subspecies. The acute inhibitory effect of insulin on VLDL triglyceride production was observed in earlier studies.19 44 Thus, the data from these in vivo studies are consistent with in vitro work demonstrating that acute hyperinsulinemia suppresses VLDL secretion from liver.15 16 17 18 However, these previous studies provided no data on the effect of acute insulin administration on VLDL subclass metabolism.
Previous in vivo studies exploring the effect of insulin on VLDL apo B metabolism have used exogenous labeling of lipoproteins with radioactive tracers.20 21 22 Endogenous labeling of apo B with stable isotopes represents a new approach that provides many advantages over previous techniques using radioactive tracers. Endogenous labeling of apolipoproteins allows direct measurement of the rate of VLDL apo B synthesis. Possible modification of the apo B protein during its isolation and exogenous labeling is avoided. Stable isotopes allow repeated studies without a risk of radiation exposure to the study subjects.
The regulation of the assembly and secretion of VLDL particles in the liver is a complex procedure.45 46 The two key components of VLDL are apo B and triglycerides. Apo B is synthesized in hepatocytes, and its secretion is primarily regulated posttranslationally.46 Pretranslational regulation may also exist. Apo B mRNA appears to increase when HepG2 cells are incubated with VLDL.47 Recently, an MTP was suggested to play a critical role in VLDL assembly.48 It has been postulated that a small amount of lipid, probably triglyceride, is required to promote the movement of apo B into the lumen of rough endoplasmic reticulum to initiate the lipoprotein assembly.49 MTP could be a mediator of this early step. Interestingly, insulin decreases MTP gene expression without altering MTP activity.50 However, in the present study, the duration of the insulin infusion was probably too short to have its effect mediated by the MTP gene.50 If initiation of the assembly process fails, prolonged association of apo B with the endoplasmic reticulum membrane51 predisposes the nascent polypeptide to rapid degradation.52 In vitro studies indicate that insulin may regulate this early step by enhancing hepatic degradation of apo B.53
The availability of triglyceride is crucial in the assembly and secretion of VLDL particles. Insulin regulates the flux of substrates used for triglyceride synthesis in the liver by suppressing the release of FFA from adipose tissue. Data on hepatic lipogenesis, measured using stable isotopes, indicate that de novo VLDL fatty acid synthesis represents a quantitatively minor pathway.54 In vitro studies indicate that about 70% of the secreted VLDL triglycerides are derived via lipolysis and re-esterification of intracellular FFA.55 The proportion of triglycerides derived from lipolysis versus de novo synthesis remained unchanged when hepatocytes were exposed to insulin for 24 hours.55 FFA, when added to the hepatoma cell culture medium, stimulates production of VLDL triglyceride and apo B in vitro.18 Thus, the major determinant for triglyceride synthesis seems to be the influx of FFA into the liver and the subsequent re-esterification rate of FFAs into triglycerides.
The question of whether insulin inhibits VLDL production independent of FFA availability was recently explored by Lewis et al.22 Elevation of plasma FFAs during hyperinsulinemia by heparin and intralipid infusions attenuated but did not completely abolish the suppressive effect of insulin on the production of VLDL triglycerides. VLDL apo B production remained unchanged, although there was a considerable variation in apo B production. The authors concluded that the suppression of FFA by insulin explains only part of the decrease of VLDL production during insulin infusion, which thus reinforces the concept that insulin has a direct inhibitory effect on VLDL production in the liver. In our study, the early effect of insulin on VLDL1 apo B suppression paralleled the decrease of plasma FFA concentrations. However, although the suppression of plasma FFA concentrations by insulin was similar in all subjects, the rate of suppression of VLDL1 apo B production varied markedly between the subjects, which may be explained by variation in the direct insulin action in the liver.
What is the mechanism underlying the specific suppression of VLDL1 apo B production by insulin? Because there is only one apo B molecule in each VLDL particle, the amount of apo B determines the number of VLDL particles secreted. We observed a decrease in the production of VLDL1 apo B, while VLDL2 apo B secretion remained unchanged. This resulted in a smaller number of total secreted VLDL particles. This finding suggests increased apo B degradation in the liver, which in turn could be mediated via decreased availability of triglycerides, by direct stimulation of apo B degradation in the liver, or both. Triglyceride availability and the extent to which newly formed particles are loaded with lipids determine the density of the newly secreted lipoproteins.9 In the present study, only VLDL1 particle size (triglyceride/apo B ratio) decreased, whereas there was no significant change in VLDL2 particle size. Thus, the shortage of triglycerides for VLDL assembly results in a smaller amount of and more dense VLDL1 particles. We speculate that the availability of intrahepatic cholesterol is an important regulator of VLDL2 apo B production. This idea is supported by the demonstration that the major subclass produced in excess in hypercholesterolemic patients is VLDL2.9 The actual site where the differentiation of VLDL particles to distinct subclasses occurs remains to be established.
Finally, insulin also regulates the removal of VLDL via its action on LPL, the rate-limiting enzyme of triglyceride hydrolysis. We observed no change in fractional catabolic rates of either VLDL1 or VLDL2 apo B. The decrease in particle size could be partly explained by the effect of insulin on LPL. However, we found no difference between plasma LPL values during saline or insulin infusion, although the activity of the enzyme did rise on both occasions for reasons that are not clear.
In conclusion, we propose that insulin directs the trafficking of apo B between its degradation and secretion according to physiological demands (ie, need of triglyceride-rich particles for energy supply). This mechanism is mediated mainly by VLDL1 particles, which represent the liver's chylomicrons delivering FFAs for an energy source in the fasting state. Postprandially, insulin would prevent the unnecessary secretion of VLDL1 particles in the presence of exogenous chylomicrons, thereby also relieving postprandial competition between chylomicrons and VLDL via the same lipolytic pathway.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The skillful technical assistance of Anne Salo and Helinä Perttunen-Nio is greatly appreciated. We thank Soile Aarnio for drawing the figures. This study was supported by grants from the Sigrid Juselius Foundation, Helsinki, Finland, and by British Heart Foundation Award BHF 190/1242. T.D.G.W. was the recipient of a fellowship from the Wellcome Trust.
Received May 7, 1996; accepted October 8, 1996.
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