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Division of Diabetes, Nutrition, and Metabolic Disorders, Department of Medicine, C.H.U. Sart- Tilman, B-4000 Liège, Belgium; and Clinical Physiology Unit, School of Medicine, University of Granada, Granada, Spain
Address correspondence and requests for reprints to: Prof. A. J. Scheen, Division of Diabetes, Nutrition, and Metabolic Disorders, Department of Medicine, C.H.U. Sart Tilman, B-4000 Liège 1, Belgium.
Abstract
Inhibition of tumor necrosis factor (TNF)-
results in a marked
increase in insulin sensitivity in obese rodents. We investigated the
influence of a TNF antagonist [Ro 45-2081, a recombinant fusion
protein that consists of the soluble TNF-receptor (p55) linked to the
Fc portion of human IgG1] on insulin sensitivity of patients with
android obesity. Seven patients (five women and two men; mean ±
SD age, 41 ± 4 yr; body mass index, 36.1 ± 4.7
kg/m2; waist to hip ratio, 0.99 ± 0.11) were studied
(three patients with normal glucose tolerance and four patients with
impaired glucose tolerance or mild diabetes; all were
hyperinsulinemic). Each patient underwent two consecutive euglycemic
hyperinsulinemic glucose-clamp tests: 48 h after injection of
placebo and 48 h after a single iv injection of 50 mg Ro 45-2081.
In both tests, steady-state plasma glucose and insulin levels were
similar. Insulin-mediated glucose disposal (2.23 ± 0.74
vs. 2.38 ± 0.99
mg/kg-1·min-1) and glucose metabolic
clearance rate (2.28 ± 0.85 vs. 2.48 ± 1.03
mL/kg-1·min-1) were similar after placebo
and after the drug. Indirect calorimetry showed no difference in
substrate oxidation rates between the two experimental conditions. In
conclusion, under the conditions of this study, no improvement in
insulin sensitivity was observed in obese insulin-resistant patients
following a single iv administration of a recombinant TNF receptor: Fc
fusion protein.
INSULIN resistance plays a key role
in numerous clinical conditions, such as type 2 diabetes mellitus,
obesity, or hypertension (1). Reduction of cellular insulin sensitivity
may be caused by several factors, and the primary cause of insulin
resistance in human obesity and type 2 diabetes remains largely unknown
(2). Genetically obese mice (ob/ob mice) and rats (fa/fa Zucker) have
increased expression of tumor necrosis factor (TNF)- in their
adipose tissue, and TNF- may be a mediator of the insulin resistance
observed in these animal models (3, 4). Recent evidences suggest that
TNF- overexpression is also present in both adipose tissue (5) and
skeletal muscle (6) of insulin-resistant obese subjects. TNF-
cellular production and plasma levels are markedly increased in human
obesity (7) and type 2 diabetes (8, 9). After weight loss in obese
insulin-resistant patients, TNF- expression and secretion fall, in
association with a decrease in serum TNF- concentration and a
restoration of insulin sensitivity (7). Infusion of TNF- reduced
insulin-mediated glucose disposal in animals (10), and the
administration of TNF- to healthy volunteers induced a state of
hyperinsulinemia without hyperglycemia, suggesting decreased insulin
sensitivity (11).
In animal models of obesity/insulin resistance, neutralizing TNF-
with injection of a soluble TNF-receptor-IgG fusion (chimeric) protein
(12) resulted in a 2- to 3-fold increase in insulin sensitivity, as
assessed by the glucose disposal rate during a euglycemic
hyperinsulinemic clamp (3). Moreover, obese mice TNF- deficient by a
targeted null mutation in the gene encoding TNF- were protected from
obesity-induced insulin resistance (13). However, the single study that
investigated the effects of neutralizing TNF- using an engineered
human anti-TNF- antibody failed to demonstrate any significant
effects on insulin sensitivity (assessed by a short insulin sensitivity
test) and glycemic control in patients with overt type 2 diabetes
(14).
In the present study, we investigated the effect of neutralizing
TNF- on insulin sensitivity in insulin-resistant patients presenting
android obesity but no overt diabetes. As in the study performed in
rodents (3), we used a recombinant fusion protein combining soluble
TNF- receptor and sequences of human IgG1 (Ro
45-2081, known to produce long-lasting neutralization of peripheral
TNF-) (12). The euglycemic hyperinsulinemic clamp technique was used
as a method to assess insulin sensitivity (15).
Materials and Methods
Patients
Seven obese subjects (five women and two men) volunteered to
participate in the study after giving full informed written consent.
The study was approved by the University of Liège Ethics
Committee for Human Investigations. Individual relevant anthropometric
and clinical data are presented in Table 1
. In addition to a full clinical
assessment, hematology, and blood chemistry assessments, eligibility
screening included an oral glucose tolerance test (OGTT) (75g glucose).
Three patients had normal glucose tolerance, but hyperinsulinism (peak
level, >575 pmol/L) was present during the OGTT. Four subjects had
marked impaired glucose tolerance or mild Type 2 noninsulin-dependent
diabetes mellitus, despite an important hyperinsulinemic response
during the OGTT. Treatments known to affect insulin sensitivity or
glucose metabolism were discontinued during, at least, the preceding 2
weeks.
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This was a single center, single-blind, sequential treatment (placebo, followed by active drug) study performed in hospitalized patients. On study day 1, and following an overnight fast, each patient received a single iv injection of placebo, followed 48 h later (day 3) by a euglycemic hyperinsulinemic clamp. After the clamp had been completed, each patient received a single iv dose of 50 mg Ro 45-2081, followed 48 h later (day 5) by a second euglycemic hyperinsulinemic clamp. Blood samples for pharmacokinetics assessment of Ro 45-2081 were collected. A second OGTT was performed on day 6.
Euglycemic hyperinsulinemic clamp
With the patient lying in supine position and after a 30-min period of baseline measurement, a primed-continuous (1 mU/kg-1·min-1) infusion of crystalline insulin (Actrapid HM; Novo-Nordisk, Copenhagen, Denmark) was started and continued for the next 120 min (15). Plasma glucose levels, measured every 5 min or less using a Beckman Coulter, Inc. Glucose Analyzer (Beckman Coulter, Inc. Instruments, Fullerton, CA), were maintained constant at basal levels by means of a variable infusion of 20% dextrose. Potassium chloride was simultaneously infused at a rate of 4 mmol/h to prevent insulin-induced lowering of plasma potassium concentration. Plasma insulin (16), C-peptide (17), glucagon (18), and free fatty acid (FFA) (19) concentrations were measured in blood samples regularly collected during the clamp. The indices of insulin sensitivity used were the glucose infusion rate (GIR) and the metabolic clearance rate of glucose (MCRG = GIR divided by the steady-state plasma glucose concentration) calculated for the last 30 min of the glucose clamp (15). To determine substrate oxidation rates and nonoxidative glucose disposal, an indirect calorimetry (Deltatrac; Datex Instruments, Helsinki, Finland) was coupled to the glucose clamp, as described previously (20).
Pharmacokinetics of Ro 45-2081
Blood samples were collected before, immediately after injection of placebo and test drug, and then 1, 4, and 48 h afterward. Serum concentrations of Ro 45-2081 were measured by means of an enzyme-linked immunological and biological binding assay specific for the sTNFR-55 part of the recombinant protein.
Statistical analysis
The Student t test for paired data has been used. Results are expressed as mean ± SD.
Results
All seven patients completed the study. Following the
administration of Ro 45-2081, there were no major adverse events or
laboratory abnormalities of clinical significance. However, in the week
following drug injection, five patients complained of occasional mild
gastric discomfort with nausea (vomiting in two patients). Two patients
complained of episodic asthenia and headache, and one patient reported
a feeling of anxiety. Such side effects might be related to the drug,
but all were mild, transient, and did not require any specific
treatment. In one 41-yr-old male patient (patient 1 in Table 1
), an
unexplained bradycardia presented 5 days after administration of the
drug, but all cardiovascular investigations were normal.
The pharmacokinetics study showed a similar disposition/elimination pattern in all patients, with an initial rapid decline phase, followed by a slow terminal elimination phase (terminal elimination half-life = 98 ± 15 h). Cmax, observed in the sample taken immediately after injection of 50 mg Ro 45-2081, was 15.2 ± 2.2 µg/mL. The circulating levels at the time of the glucose clamp test were 3.13 ± 1.13 µg/mL (range, 2.57–3.65).
In basal conditions and immediately before both clamp tests, plasma
glucose and insulin levels were similar, as well as plasma FFA
concentrations (Fig. 1). In both
occasions, plasma glucose levels were appropriately clamped around 5.5
mmol/L and plasma insulin increased from around 140 pmol/L at baseline
to steady-state levels that plateaued at about 700 pmol/L (Fig. 1),
with no differences between the two clamp conditions. Plasma glucagon
levels were unmodified during the euglycemic hyperinsulinemic clamp
performed after placebo injection (0 min, 38.0 ± 12.4
vs. 120 min, 39.1 ± 24.0 ng/L) and after Ro 45-2081
injection (0 min, 32.3 ± 31.4 vs. 120 min, 28.7
± 30.2 ng/L). Plasma C-peptide levels decreased from 842 ± 376
to 558 ± 187 pmol/L (P < 0.02) after placebo and
from 758 ± 276 to 559 ± 134 pmol/L (P <
0.1) after Ro 45-2081. Plasma FFA concentrations decreased similarly in
both conditions (Fig. 1). Therefore, in the two experimental
conditions, insulin was equally effective in suppressing pancreatic
B-cell function and controlling lipolysis.
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), confirming that all patients selected for the study were clearly
insulin resistant. No significant change occurred in GIR and
MCRG after injection of Ro 45-2081 (Fig. 1 and
Table 1). The reproducibility of the GIR value between the two clamps
was excellent, with a coefficient of variation of 5.0 ± 5.7%. No
significant changes in glucose, lipid and protein oxidation, as well as
in glucose storage were observed after administration of Ro 45-2081
when compared with the results measured after placebo. An OGTT was performed at the inclusion and 3 days after administration of Ro 45-2081. No significant difference in plasma glucose (0 min, 5.9 ± 1.2 vs. 6.2 ± 1.4 mmol/L; 120 min, 8.7 ± 3.6 vs. 10.1 ± 3.2 mmol/L) and insulin (0 min, 122 ± 72 vs. 86 ± 52 pmo/L; 120 min, 1845 ± 1493 vs. 1608 ± 1422 pmol/L) levels were observed between the two conditions. Therefore, glucose tolerance was not improved and reactive hyperinsulinemia was not reduced after administration of the drug.
Discussion
TNF- is expressed by human adipocytes, and TNF-
messenger RNA expression and TNF- protein production occur at a
higher rate in obese than in lean human subjects, resulting in a
greater (2- to 3-fold) serum concentration of TNF- in obese subjects
(5, 7, 8). The increase in TNF- messenger RNA levels is positively
correlated to the degree of hyperinsulinemia (5), and weight loss is
accompanied by a decrease in serum TNF- concentrations, as well as
an increase in insulin sensitivity (7, 8). In addition, a much higher
expression of TNF- in the muscle tissue and in the cultured muscle
cells from insulin-resistant and diabetic subjects and an inverse
relationship between muscle TNF- expression and insulin-mediated
glucose disposal rate assessed during a euglycemic hyperinsulinemic
clamp were observed (6). All these data suggest that TNF- may play a
role in human insulin resistance, as demonstrated in various animal
models (3, 4). Indeed, in genetically obese rats, injection of a
soluble TNF-receptor-IgG fusion protein has been shown to result in a
2- to 3-fold increase in insulin-stimulated glucose utilization during
a euglycemic hyperinsulinemic clamp, without significant effect on
hepatic glucose production (3).
The potential to interfere with TNF- overexpression and/or action
may, thus, open new perspectives for the treatment of insulin-resistant
states. However, the single human study investigating the effects of
neutralizing TNF- failed to demonstrate any significant effect on
insulin sensitivity and glycemic control of obese patients with type 2
diabetes (14). In that study, insulin sensitivity was assessed during
an iv insulin tolerance test, using the percentage rate of glucose
clearance per minute (KITT), rather than during a
euglycemic hyperinsulinemic clamp, which is now considered as the gold
standard method (15). In addition, subjects had long-lasting diabetes
(known duration of the disease averaging 9 yr) and rather severe
hyperglycemia (mean fasting blood glucose levels above 10 mmol/L) (14).
In such patients, various other mechanisms could contribute to the
reduced insulin action, including hyperglycemia, which has been shown
to affect insulin signaling in a different way than that of TNF-
(21). Finally, the TNF- neutralization was obtained by a
recombinant-engineered human antibody, whereas a soluble
TNF-receptor-IgG fusion protein was used for TNF neutralization in
animal models (3).
Our study is the first one in which a TNF- neutralizing agent (Ro
45-2081, a soluble TNF-receptor-IgG fusion protein) has been given to
patients with insulin resistance, and in whom insulin sensitivity has
been assessed with the euglycemic hyperinsulinemic glucose clamp. In
contrast to the previous study performed in man (14), we tested android
obese patients with no or only very mild hyperglycemia to avoid the
potential interference of glucose toxicity (21). By comparison with
placebo, we did not observe any significant influence of the drug on
the various indices of insulin sensitivity, substrate oxidation and
glucose storage measured during the clamp. Thus, the negative results
of the present study are in agreement with those reported in patients
with overt type 2 diabetes (14). They did not confirm the significant
2- to 3-fold increase in insulin-stimulated peripheral glucose uptake
observed in genetically obese fa/fa rats treated with a similar TNF-
neutralizing agent (3).
Pharmacological and clinical data obtained in healthy volunteers and
rheumatoid arthritis patients treated with a recombinant soluble TNF
receptor (p80) fusion protein have demonstrated that a single dose of
50 mg completely binds and neutralizes TNF- for approximately 4
weeks (22). In our study, at the time when the second glucose clamp was
performed, the circulating plasma levels of the drug (around 3 µg/mL)
were well above the reported effective concentration attained in
studies on experimental animals (around 0.5 µg/mL) (3). However,
because it is likely that TNF- can work in an autocrine or paracrine
fashion, neutralization of circulating TNF- may not be sufficient
for observing improvements in insulin action.
The reported differences between animal and human studies may result from fundamental differences in the etiopathogenesis of insulin resistance. In contrast to the monogenic form of rodent obesity, insulin resistance associated to obesity in man is a multifactorial phenomenon (2). Although a few individuals with diabetes may have similar defects as in animal models, this does not include the majority of the affected patients.
In conclusion, under the conditions used in this study, no improvement
in insulin sensitivity and glucose tolerance has been observed in obese
insulin-resistant patients following a single iv injection of 50 mg Ro
45-2081, a soluble TNF-receptor-IgG fusion protein neutralizing
endogenous TNF- for several weeks. These negative results contrast
with those of the only study in animals demonstrating a marked
increased in insulin sensitivity of fa/fa rats after a similar drug
treatment and confirm those of the single study in humans showing the
absence of effects of an anti-TNF- antibody in patients with type 2
diabetes. Clearly, additional studies are needed to determine the role
of TNF- in human insulin resistance associated with obesity, and the
potential interest of anti-TNF- therapeutic strategies remains to be
demonstrated in humans.
Acknowledgments
We gratefully acknowledge Dr. N. Wood and Ms. S. Maledrie for helpful discussion and Dr. H. Birnböck for the assay of Ro 45-2081.
Footnotes
1 This study was initiated and financially supported by the
manufacturers of the Ro 45-2081 product, Hoffman-La Roche Ltd. 
Received May 6, 1999.
Revised November 2, 1999.
Accepted November 8, 1999.
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) and after administration of Ro
45-2081 (•). Results are expressed as mean ± SD,
n = 7.