From the Division of Endocrinology and Metabolism, Department of Internal Medicine III, University of Vienna Medical School, Vienna, Austria.
Address correspondence and reprint requests to Michael Roden, MD, Division of Endocrinology and Metabolism, Department of Internal Medicine III, University of Vienna Medical School, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: michael.roden{at}akh-wien.ac.at .
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ABSTRACT |
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INTRODUCTION |
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Insufficient insulin replacement, decreased portal insulin-to-glucagon
ratios and/or hyperglycemia-induced insulin resistance may account for the
abnormalities of hepatic glucose metabolism in type 1 diabetes. However,
hyperinsulinemic clamp tests and intensive insulin treatment completely
normalized endogenous glucose production in some
(6,8,16)
but not in all studies (10).
Even intraportal hyperinsulinemia does not reverse all of the alterations in
hepatic carbon fluxes (17). In
addition, chronic near-normoglycemia alone was not sufficient to reduce
endogenous glucose production, which was suppressed only by plasma insulin
concentrations as high as 6 nmol/l
(18). It has been suggested
that only a near-physiological mode of insulin delivery by intermittent
infusion will reactivate liver metabolism and reduce both HbA1c
levels and the frequency of hypoglycemic events in patients with brittle
diabetes (19). Nevertheless,
it is unknown at present whether or not improvement of both insulinemia and
glycemia reverses the defects in hepatic glycogen metabolism in type 1
diabetes under physiological conditions of mixed meal ingestion.
Therefore, this study was designed with the following intentions: 1) to determine rates of net hepatic glycogen synthesis after ingestion of a standardized mixed meal dinner, 2) to determine rates of net hepatic glycogen breakdown during nighttime in type 1 diabetic and nondiabetic subjects, and 3) to test the hypothesis that hepatic glycogen deposition can be completely restored to normal levels by short-term improvement of insulin treatment and glycemia for 24 h using variable intravenous insulin infusion. We applied 13C nuclear magnetic resonance (NMR) spectroscopy to noninvasively follow the time course of hepatic glycogen concentrations in vivo (20). This method enables quantification of both net glycogen synthesis and breakdown during mixed meal ingestion (1,7,21) and avoids limitations of other experimental approaches such as liver biopsies (22), hepatic venous cannulation (23), or isotopic dilution methodology (6,24).
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RESEARCH DESIGN AND METHODS |
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Experimental protocol. All subjects were advised to ingest a carbohydraterich weight-maintaining diet and to refrain from strenuous physical exercise for at least 3 days before the study. Diabetic patients were instructed to omit NPH or Zn insulin and to correct plasma glucose concentrations with regular insulin (Actrapid; Novo Nordisk, Copenhagen, Denmark) for 24 h before admission. Nondiabetic subjects were studied once, whereas type 1 diabetic patients were studied twice: once during current metabolic control with correction of plasma glucose by subcutaneous insulin injection (protocol 1) and once during improved glycemic control by variable intravenous insulin infusion (protocol 2).
On the first study day, nondiabetic and diabetic subjects in protocol 1
were admitted to the clinical research facility at 7:00 A.M. Teflon
catheters were inserted in antecubital veins of the left arm for blood
sampling. Three standard mixed meals (60% carbohydrate, 20% protein, and 20%
fat) were served at 8:00 A.M. (720 kcal solid), 12:30 P.M. (710 kcal solid),
and 5:00 P.M. (800 kcal liquid meal). During protocol 1, diabetic patients
received subcutaneous insulin injections before the meals to avoid excessive
increases of plasma glucose concentrations >22.2 mmol/l. Plasma glucose
concentrations were measured every 15 min between 7:30 A.M. and midnight and,
when appropriate, during nighttime.
During protocol 2, diabetic patients were admitted to the clinical research
facility at 12:00 A.M. on the day before the study. Teflon catheters were
inserted in antecubital veins of the right and left arm for blood sampling and
for insulin infusion, respectively. Insulin was administered as a variable
intravenous infusion (0.2 to 1.5 mU · kg-1 ·
min-1) to slowly lower the plasma glucose level and keep it between
5.56-6.67 mmol/l. On the next day, three standard mixed meals were served as
described above. To achieve plasma glucose concentrations obtained in control
subjects, insulin infusion rates were frequently adjusted based on the actual
plasma glucose concentration and increased accordingly before ingestion of
each meal. Plasma glucose concentrations were measured every 15 min during
protocol 2.
In all studies, blood samples were taken every 2 h to allow determination of plasma concentrations of free insulin, C-peptide, glucagon, and free fatty acids (FFAs).
Analytical methods. Plasma glucose concentrations were measured
using the glucose oxidase method (Glucose Analyzer II; Beckman Instruments,
Fullerton, CA). HbA1c was quantified by using high-performance
liquid chromatography (Bio-Rad, Richmond, CA). Plasma concentrations of FFAs
were determined by using a colorimetric assay (intra- and interassay CVs 4.3
and 5.7%) (Wako, Neuss, Germany). Free insulin (CVs 8%)
(Pharmacia-Upjohn, Uppsala, Sweden), C-peptide (CVs
9%) (CIS,
Gif-Sur-Yvette, France), and glucagon (CVs
8%) (Biochem, Freiburg,
Germany) were determined by radioimmunoassay.
13C NMR spectroscopy. After dinner, liver glycogen
concentrations were determined in all subjects from 7:30 P.M. until the
plateau was reached (10:30 P.M.) and from 7:00 A.M. to 8:00 A.M. on the
morning of the next day. These time periods were chosen because the difference
in net hepatic glycogen accumulation between nondiabetic and diabetic subjects
becomes maximal after dinner
(7). In three subjects, the
measurements were performed except for short breaks from 7:30 P.M. until 8:00
A.M. of the next morning to confirm linear decrease of liver glycogen
concentrations between 10:30 P.M. and 8:00 A.M.
(5).
In vivo 13C NMR spectroscopy was performed on the 3 T Medspec 30/80-DBX system (Bruker Medical, Ettlingen, Germany) installed at the General Hospital of Vienna, Austria. A double-tuned 1H (125.6 MHz) and 13C (31.5 MHz) 10-cm circular coil was used for data collection. Subjects were lying in the supine position in the magnet with the coil positioned rigidly over the lateral aspect of the liver. The liver borders were determined by percussion, and the correct position of the coil was confirmed with a multislice gradient echoimage. Magnetic field homogeneity was optimized on the water signal to a line width of 60-80 Hz. Spectra were acquired using a modified 1D-ISIS sequence (20) without proton decoupling (pulse length 150 µs/135° in the coil plane, time of resonance 150 ms, acquisition time 25.6 ms, number of scans 5,000, total scan time 13 min). Spectra were zero filled to 4k, gaussian and exponentially filtered, and phase-corrected manually. Hepatic glycogen was quantified by integration of the C1 glycogen doublet at 100.5 ppm using the same frequency bandwidth for all spectra (± 300 Hz). Absolute quantification of the hepatic glycogen concentration was obtained by comparing the peak integral with that of a glycogen standard solution obtained under identical conditions. Corrections for loading and sensitive volume of the coil were performed.
Because hepatic glycogen synthase and phosphorylase are simultaneously active in humans, 13C NMR spectroscopy does not measure total changes but does measure net changes in liver glycogen concentrations so that rates of net glycogen synthesis and net glycogenolysis can be assessed. Individual rates of net glycogen synthesis and net glycogen breakdown were calculated from linear regression of the net glycogen concentration-time curves between 7:30 and 10:30 P.M. and from 10:30 P.M. to 8:00 A.M., respectively.
Determination of liver volume. In all subjects, liver volumes were measured using magnetic resonance imaging in a 1.5 T Vision imager (Siemens, Munich, Germany) using a body array coil and inphase and postphase multislice FLASH imaging sequences. Slice number and position were chosen to cover the whole organ. Liver tissue was manually segmented, and the area of each region of interest was determined in each slice. Areas were added and multiplied by the sum of slice thickness (0.8 mm) and interslice distance (8 mm). Liver volumes were determined 2 h after dinner on the first study day.
Calculations and statistics. Data are means ± SE. One-way analysis of variance with Bartlett's test for equal variances and post hoc testing by the Newman-Keuls test was used for statistical comparisons between and within the different groups. In addition, data of type 1 diabetic patients before and during short-term intensified insulin treatment were compared using the paired Student's t test. Statistical significance was considered at P < 0.05. All calculations were performed using the Sigma Stat software package (Jandel, San Rafael, CA).
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RESULTS |
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Plasma C-peptide, insulin, glucagon, and FFAs. Fasting plasma
C-peptide concentrations were markedly lower in diabetic subjects (0.32
± 0.06 ng/ml in poorly controlled diabetic subjects vs. 0.26 ±
0.01 ng/ml in insulin-infused diabetic subjects, NS) than in nondiabetic
subjects (1.61 ± 0.18 ng/ml; P < 0.001 vs. poorly
controlled diabetic subjects and insulin-infused diabetic subjects). Whereas
plasma C-peptide concentrations were below the detection limit (0.25 ng/ml) in
five diabetic patients, two patients exhibited detectable plasma C-peptide
levels (0.57 and 0.54 ng/ml). Plasma C-peptide did not change from fasting
levels in the diabetic subjects, but increased (P < 0.0001) in the
control subjects. Following correction of glycemia by subcutaneous or
intravenous insulin before 8:00 A.M., mean fasting plasma insulin was
slightly, but not significantly, higher in diabetic subjects (134 ± 62
pmol/l in poorly controlled diabetic subjects and 180 ± 84 pmol/l in
insulin-infused diabetic subjects) than in control subjects (40 ± 6
pmol/l) (Fig. 2A).
During the study day, mean plasma insulin concentration was higher in diabetic
patients during intensive insulin treatment (396 ± 87 pmol/l in
insulin-infused diabetic subjects) than during poor metabolic control (205
± 22 pmol/l in poorly controlled diabetic subjects, P <
0.05) and increased compared with nondiabetic control subjects (109 ±
20 pmol/l, P < 0.01). Plasma glucagon concentrations were not
different in the fasting state (160 ± 24, 156 ± 21, and 181
± 28 pg/ml in poorly controlled diabetic patients, insulin-infused
diabetic patients, and control subjects, respectively) and throughout the
studies (165 ± 7, 177 ± 5, and 187 ± 7 pg/ml in poorly
controlled diabetic patients, insulin-infused diabetic patients, and control
subjects, respectively) (Fig.
2B). Fasting plasma FFA concentrations were slightly
higher in diabetic patients during poor metabolic control (524 ± 97
µmol/l in the poorly controlled diabetic subjects) than during intensified
insulin treatment (319 ± 38 µmol/l in the insulin-infused diabetic
subjects, NS) and control studies (353 ± 56 µmol/l, NS)
(Fig. 2C). After 10:00
P.M., plasma FFA increased further only in diabetic patients during protocol 1
(698 ± 64 µmol/l in poorly controlled diabetic patients),
1.8-fold higher than during protocol 2 (396 ± 66 µmol/l in
insulin-infused diabetic patients; P < 0.025) and during control
studies (380 ± 56 µmol/l; P < 0.01) performed at 3:30
A.M.
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Hepatic glycogen synthesis and breakdown. Liver glycogen
concentrations linearly increased from 8:00 A.M. to 11:00 P.M.
Figure 3 illustrates the time
course of liver glycogen concentrations and determination of rates of net
glycogen synthesis by a line-fitting procedure in one nondiabetic subject.
After dinner, hepatic glycogen concentrations were 29% lower (P
< 0.001) in poorly controlled diabetic subjects than in nondiabetic
subjects (Table 1). Rates of net
glycogen synthesis were
74% lower (P < 0.001) in diabetic
patients (Fig. 4A).
Short-term improvement of metabolic control resulted in an increase of
23% (P < 0.01) of maximal liver glycogen contents
(Table 1), which was still
reduced (P < 0.05) compared with control studies. Similarly, net
glycogen synthesis doubled (P = 0.017 vs. poorly controlled diabetic
subjects), but was
52% lower (P < 0.001) than in nondiabetic
subjects.
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During overnight fasting, liver glycogen linearly decreased from 11:00 P.M.
to 8:00 A.M. (Fig. 3). The
maximum decrement was lower in poorly controlled diabetic subjects than in
nondiabetic subjects (-58 ± 9 mmol/l liver vs. -113 ± 13 mmol/l
liver; P < 0.001) and gradually increased during tight metabolic
control (-82 ± 8 mmol/l liver in the insulin-infused diabetic subjects;
P < 0.05 vs. poorly controlled diabetic subjects; P <
0.01 vs. control subjects). Rates of net glycogen breakdown were reduced by
47% in poorly controlled diabetic patients (P < 0.001 vs.
control subjects), but improved during protocol 2 (P = 0.011 vs.
insulin-infused and P < 0.05 vs. control subjects)
(Fig. 4B). The
following morning, hepatic glycogen had decreased (P < 0.001) in
all protocols, with no differences among them
(Table 1).
Mean liver volume was not different between diabetic and nondiabetic subjects (1,627 ± 102 cm3 in the poorly controlled diabetic subjects vs. 1,502 ± 43 cm3 in the control subjects, unpaired Student's t test: NS).
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DISCUSSION |
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The glycemic profiles of poorly controlled type 1 diabetic patients
exhibited a prolonged inappropriate increase of plasma glucose, particularly
after breakfast. Most likely, this results from peripheral insulin resistance
due to the diurnal secretion pattern of growth hormone
(7,27)
and is further supported by the observed nocturnal rise of plasma FFA, which
strongly correlates with peripheral insulin resistance
(28,29)
and per se reduces skeletal muscle glucose uptake and glycogen synthesis
(30,31).
Although mean peripheral plasma insulin and glucagon concentrations were
comparable with that of nondiabetic humans, portal hypoinsulinemia and a
decreased insulin-to-glucagon ratio may account for the defect in postprandial
glycogen accumulation. Net hepatic glycogen synthesis, as measured in this
study, is the result of simultaneously active fluxes through glycogen synthase
and phosphorylase (32), which
are differentially regulated by glucose, insulin, and glucagon under in vivo
conditions
(33,34).
Rates of net glycogen synthesis of nondiabetic subjects were in agreement with
previous reports using either liver biopsies
(35) or 13C NMR
spectroscopy
(1,7,21).
In the poorly controlled diabetic subjects, the prevailing hyperglycemia will
primarily inhibit glycogen phosphorylase flux, while hypoinsulinemia and
relative hyperglucagonemia in the portal vein may stimulate glycogen cycling
(33,34).
Furthermore, even short periods of hyperglycemia are sufficient to reduce
nonoxidative glucose disposal
(36). In addition to the
effects of acute endocrine and metabolic changes, chronic alterations in the
activities of hepatic glucoregulatory enzymes, such as glucokinase
(37), may contribute to
reduced glycogen synthesis. Such a defect was recently suggested in type 2
diabetic subjects, because both UDP-glucose flux and percent contribution of
extracellular glucose to hepatic glycogen synthesis were reduced
(38). The finding that
glucokinase-deficient type 2 maturity-onset diabetes of young (MODY-2)
patients exhibit only 12% lower peak liver glycogen concentrations after
dinner as compared with healthy subjects
(21) also indicates that other
enzymes, such as glycogen synthase
(39,40),
might add to defective glycogen accumulation in poorly controlled type 1
diabetes.
In addition, this study followed the time course of liver glycogen
breakdown during overnight fasting. The decrease of intrahepatic glycogen
concentrations was linear and allowed us to calculate rates of net glycogen
breakdown
(1,5,20).
Corrections for changes in liver volume were not performed, because previous
studies found no (5) or only
minor loss (20) of liver
volumes in subjects fasted for 64 h. Such decreases in liver size will
result in changes of glycogen concentrations that are below the detection
limit of the applied NMR technique. To the extent that simultaneous glycogen
synthesis occurs, net glycogen breakdown will underestimate the flux through
glycogen phosphorylase. Rates of glycogen breakdown in nondiabetic subjects
(
0.19 mmol/l liver/min) were identical to previous reports
(1,41),
whereas no data were available for type 1 diabetes. The observed decrease of
rates of net glycogen breakdown in poorly controlled type 1 diabetic patients
may result from hyperglycemia that inhibits glycogen phosphorylase
(34) and/or relative portal
vein hyperglucagonemia (33).
In addition, as individual net glycogenolytic rates are correlated with the
initial hepatic glycogen concentration
(5), the lower maximal
postprandial liver glycogen concentrations may have contributed to reduced
glycogen breakdown. Finally, the defects in liver glycogen metabolism could
partly be because of the action of epinephrine, cortisol, and growth hormone,
which stimulate splanchnic glucose output after glucose ingestion in humans
(42,43)
and worsen hyperglycemia in insulin-deficient dogs
(44) and in human type 1
diabetes (45).
During hyperglycemic-hyperinsulinemic clamp tests, hepatic glycogen
synthesis was normalized in 90%-pancreatectomized rats
(46), as well as in five
poorly controlled type 1 diabetic subjects
(17). However,
supraphysiological plasma concentrations of glucose (17 and 9 mmol/l) and
insulin (
3,300 and 400 pmol/l) were required to achieve this effect.
Moreover, administration of somatostatin without concomitant glucagon infusion
will have caused portal hypoglucagonemia in those type 1 diabetic patients
(17), which favors net hepatic
glycogen synthesis (33). In
the present study, improvement of insulin replacement by variable intravenous
insulin infusion resulted in glycemic profiles close to that of nondiabetic
subjects. Under these conditions, more likely reflecting the aim of improved
metabolic control in clinical practice, net glycogen accumulation of type 1
diabetic patients doubled, but was still half of that in control subjects. The
failure to normalize glycogen synthesis could result, in part, from the
decrease of
57% of plasma glucose during short-term improvement of
metabolic control. It is conceivable that hyperglycemia per se may compensate
for defective glycogen synthesis in the poorly controlled diabetic patients.
This could be because of glucose-induced glycogen synthesis following a portal
glucose load (47) and/or
because of reduction of simultaneous ongoing glycogenolysis
(34). Alternatively,
hyperglycemia may increase glycogen repletion by the indirect (gluconeogenic)
pathway in diabetic rats (46)
and humans who also exhibit reduced hepatic pyruvate oxidation
(17).
One might speculate that infusion of insulin into a peripheral vein instead
of physiological insulin delivery might be responsible for the difference
between nondiabetic and type 1 diabetic subjects, despite their acutely
improved metabolic control. During insulin infusion, mean plasma insulin
concentration in the peripheral vein doubled and was 190 pmol/l higher in
the diabetic patients. Thus, portal vein insulin concentrations were likely to
be similar in both groups
(48). The observed increase in
systemic insulin will decrease hepatic and renal gluconeogenesis, as well as
lipolysis, which is mirrored by the observed decrease of plasma FFA
concentrations. Consequently, endogenous glucose production should fall, net
hepatic glucose output should approach zero
(49), and glycogen synthesis
should be maximal. It is of note that the efficacy of insulin to inhibit
hepatic glucose production also depends on its pulsatile secretion pattern
(50). However, in the present
study, frequent adjustments of the insulin infusion at intervals as low as 5
min and peak rates of up to 12 mU · kg-1 ·
min-1 should have provided for near-physiological conditions
(19,51).
On the other hand, given comparable glycemic profiles in the absence of
glucosuria, whole-body glucose disposal has to be similar in well- controlled
diabetic and nondiabetic subjects. Therefore, in the well-controlled diabetic
patients, the higher peripheral insulin concentration could have increased
muscular glucose uptake, leaving less glucose available for hepatic glycogen
formation.
Nevertheless, insufficient duration of normalized glycemia and insulinemia
may account for the failure to completely restore glycogen synthesis and
breakdown in our type 1 diabetic patients. Near-normalization of glycemia by
intensive subcutaneous insulin therapy for 2 weeks was required to
decrease tracer-determined glucose production to the normal range in the
fasted
(6,16,18)
and in the postprandial state
(6). This time period might be
necessary to overcome hepatic insulin resistance, which is a common feature of
insulin-dependent diabetes, despite physiological daily insulin requirements
(8,52),
as observed in our patients, whose nocturnal suppression of plasma FFA albeit
indicates normal insulin sensitivity.
In conclusion, poorly controlled type 1 diabetic subjects present with 1) markedly impaired hepatic glycogen synthesis after a mixed meal dinner, 2) reduced glycogen breakdown during overnight fasting, and 3) improved, but not normalized, glycogen synthesis and breakdown during refined insulinemia and glycemia. Postprandial enlargement of hepatic glycogen storage can provide more glucose for the immediate increase in endogenous glucose production during hypoglycemia counterregulation. Therefore, despite potentially increased frequency of hypoglycemia (14,15), intensive insulin therapy might rather serve to protect against severe hypoglycemic episodes.
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ACKNOWLEDGMENTS |
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We gratefully acknowledge the excellent technical assistance of A. Hofer, H. Lentner, P. Nowotny, and the laboratory staff of the Division of Endocrinology and Metabolism, as well as the radiological technical assistants of the Department of Radiology. We also thank Prof. H. Imhof, MD, MR Unit, and Prof. E. Moser, PhD, Institute of Medical Physics, University of Vienna, for cooperation and support.
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FOOTNOTES |
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Received for publication May 1, 2000 and accepted in revised form October 16, 2000
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REFERENCES |
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