Conceived and designed the experiments: HH NB AF OT HP KP. Performed the experiments: OT KP SS. Analyzed the data: AF OT HP. Contributed reagents/materials/analysis tools: NB FM AH WL. Wrote the paper: HH AF AH OT. Other: Study supervision: HH NB.
A.F. and H.-U.H. obtained the grant of the DFG and H.-U.H. obtained the grant of NovoNordisk. A.F. and O.T. obtained travel cost reimbursement by NovoNordisk for attending scientific meetings.
Insulin stimulates cerebrocortical beta and theta activity in lean humans. This effect is reduced in obese individuals indicating cerebrocortical insulin resistance. In the present study we tested whether insulin detemir is a suitable tool to restore the cerebral insulin response in overweight humans. This approach is based on studies in mice where we could recently demonstrate increased brain tissue concentrations of insulin and increased insulin signaling in the hypothalamus and cerebral cortex following peripheral injection of insulin detemir.
We studied activity of the cerebral cortex using magnetoencephalography in 12 lean and 34 overweight non-diabetic humans during a 2-step hyperinsulinemic euglycemic clamp (each step 90 min) with human insulin (HI) and saline infusion (S). In 10 overweight subjects we additionally performed the euglycemic clamp with insulin detemir (D). While human insulin administration did not change cerebrocortical activity relative to saline (p = 0.90) in overweight subjects, beta activity increased during D administration (basal 59±3 fT, 1st step 62±3 fT, 2nd step 66±5, p = 0.001, D vs. HI). As under this condition glucose infusion rates were lower with D than with HI (p = 0.003), it can be excluded that the cerebral effect is the consequence of a systemic effect. The total effect of insulin detemir on beta activity was not different from the human insulin effect in lean subjects (p = 0.78).
Despite cerebrocortical resistance to human insulin, insulin detemir increased beta activity in overweight human subjects similarly as human insulin in lean subjects. These data suggest that the decreased cerebral beta activity response in overweight subjects can be restored by insulin detemir.
The role of insulin signaling to the brain in normal physiology and pathophysiology is so far only incompletely understood. The majority of data on insulin action in the brain was obtained in animal models, only few studies characterize insulin action in human brain.
Peripherally injected insulin crosses the blood-brain barrier
In a previous study we measured insulin effects on neuronal activity of the human cerebral cortex using magnetoencephalography (MEG) during a 2-step hyperinsulinemic euglycemic clamp
Modern insulin therapy regimens in type 1 and type 2 diabetes aim to mimick the patterns of physiologic insulin secretion. For this purpose, long- and short-acting insulin analogues have been designed in recent years. Insulin detemir is a long-acting insulin analogue and its delay of action is achieved by acylation of the B-chain with myristic acid and reversible albumin binding
It has been shown that the transport of insulin across the blood-brain barrier is reduced in dogs with obesity induced by a high-fat diet
Based on these findings in mice, we hypothesized that insulin detemir might restore the decreased cerebral insulin response in overweight human subjects. Therefore, we designed a 2-step hyperinsulinemic euglycemic clamp with i.v. infusion of insulin detemir or human insulin and simultaneous MEG recording. To ensure that potential cerebral effects are not a spill over from peripheral insulin effects, we applied clamp conditions which avoided overstimulation of peripheral tissues with insulin detemir.
Here we selected 10 overweight or slightly obese subjects who were otherwise healthy and normal glucose tolerant according to WHO criteria. As indicator of overweight, a percentage body fat above the sex-specific normal range (male >19%, female >28%) was taken. Body fat was measured by bioelectrical impedance analysis using a BIA101A analyzer (RJL systems, Clinton Twp., MI 48035 USA). Differentiation of overweight from normal body weight by percent body fat content resulted in inclusion of two female subjects with a BMI slightly below 25 kg/m2 but increased body fat. Severe obesity (BMI >40 kg/m2) and/or psychiatric disorders represented exclusion criteria. No subject took any medication except for hormone substitution (like thyroid hormones).
In these 10 subjects we studied the effect of insulin detemir and human insulin on cerebrocortical activity. The effect of insulin detemir on cerebrocortical function was then compared to the human insulin effect measured in 12 lean and 34 obese non-diabetic subjects. The subject characteristics of all three groups are shown in
Lean | Overweight | Overweight, insulin detemir | ||
Mean±SEM | Mean±SEM | Mean±SEM | Range | |
N | 12 | 34 | 10 | - |
Sex (M/F) | 4/8 | 19/15 | 5/5 | - |
Age (years) | 29±2 | 36±2 | 30±2 | [23 … 42] |
Weight (kg) | 62±3 | 88±2 | 88±6 | [67 … 119] |
BMI (kg/m2) | 22±1 | 29±3 | 29±1 | [23 … 36] |
Percent body fat (%) | 19±2 | 31±1 | 30±2 | [21 … 42] |
Percent body fat, females (%) | 24±1 | 37±1 | 35±4 | [30 … 42] |
Percent body fat, males (%) | 10±1 | 26±5 | 25±4 | [21 … 29] |
Waist-hip-ratio | 0.80±0.02 | 0.91±0.01 | 0.88±0.04 | [0.66 … 0.98] |
Fasting plasma glucose (mmol/L) | 4.7±0.1 | 5.0±0.1 | 5.2±0.1 | [4.6 … 5.7] |
2 Hr plasma glucose (mmol/L) | 5.6±0.4 | 6.4±0.2 | 5.8±0.4 | [4.1 … 7.7] |
Fasting plasma insulin (pmol/L) | 37±3 | 61±6 | 52±10 | [24 … 108] |
2 Hr plasma insulin (pmol/L) | 195±56 | 468±65 | 360±99 | [69 … 989] |
Oral glucose tolerance test;
subset of the overweight group
After a 10-hour overnight fast the subjects ingested a solution containing 75 g of dextrose and venous blood samples were obtained at 0, 30, 60, 90 and 120 minutes for determination of plasma glucose and plasma insulin.
The human insulin experiment and the saline experiment with simultaneous MEG recordings have already been established and used in a previous study
Human insulin (HI) and insulin detemir (D) were applied as a 2-step infusion. Each infusion step was primed with a bolus. Blood glucose was monitored every 5–10 min between minute 0 and minute 180, and a variable glucose infusion was titrated to maintain euglycemia (blood glucose 5 mmol/l). Cerebrocortical activity was measured with magnetoencephalography (MEG) during the baseline period and during the last 30 minutes of each insulin infusion step. In the control experiment, a comparable volume of saline solution (S) was infused instead of HI, D and glucose. The MEG measurement, glucose monitoring and blood sampling were performed analogously to the clamp experiments.
We chose MEG parameters that permitted sensitive assessment of both spontaneous cortical activity and stimulated cortical activity (discrimination between two sound qualities, auditory mismatch negativity). Data were recorded in a continuous mode (sampling rate 312.5 Hertz [Hz]) starting with eyes open and closed (counterbalanced over sessions and subjects) for 1.5 minutes each for analysis of spontaneous cortical activity, followed by the auditory mismatch experiment. Auditory mismatch negativity is independent of alertness or attention and is considered to be a robust parameter of preconscious cortical information processing
Plasma glucose was determined using the glucose-oxidase method (YSI, Yellow Springs Instruments, Yellow Springs, CO, USA). Blood glucose was determined by a HemoCue blood glucose photometer using the glucose dehyrogenase method (HemoCue AB, Aengelholm, Sweden). Plasma insulin levels were determined by microparticle enzyme immunoassay (Abbott Laboratories, Tokyo, Japan).
MEG data was analyzed by a repeated measure ANOVA containing the condition factor (SUBSTANCE, e.g. human insulin and insulin detemir) and the repeated measure factor level (baseline, 1st step and 2nd step of insulin infusion). To visualize the relative changes under human insulin and insulin detemir MEG data of the insulin/detemir experiment were divided by data of the saline experiment. MEG parameters were calculated using SPSS 12.0 (SPSS, Chicago, IL) incorporating Greenhouse-Geisser correction. For analysis of metabolic data the software package JMP 4.0 (SAS Institute, Cary, NC) was used. Non-normally distributed variables were logarithmically transformed, when necessary. Correlations were calculated using least square regression analysis. A p-value of <0.05 was considered to indicate statistical significance.
A: Plasma human insulin and total serum insulin detemir concentrations. Similar profiles of plasma/serum levels (Mean±SE) of human insulin and insulin detemir were achieved with both insulins. Approximately 98% of serum insulin detemir is bound to albumin. Therefore, total insulin detemir concentrations are substantially higher than human insulin concentrations at corresponding time points. Human insulin was determined by microparticle enzyme immunoassay (Abbott Laboratories, Tokyo, Japan) and insulin detemir by a modified radioimmunoassay (Capio Diagnostik, Copenhagen, Denmark). B: Blood glucose concentrations. Blood glucose was measured twice at baseline and every 5–10 minutes during the infusion of human insulin, insulin detemir or saline. Blood glucose concentrations were not different between the experiments at baseline (all p>0.1) and did not change significantly during the experiments (all p>0.2). C: Glucose infusion rates. In the saline experiment no glucose was infused. Despite much higher insulin detemir concentrations, the glucose infusion rate was lower in the detemir experiment, indicating a lower peripheral effect (1st step: insulin 11±1 µmol kg−1 min−1, insulin detemir 9±1 µmol kg−1 min−1, p = 0.01; 2nd step: insulin 36±3 µmol kg−1 min−1, insulin detemir 26±3 µmol kg−1 min−1, p = 0.003), as intended by the experimental protocol. D: Changes in beta activity during the experiments. During human insulin (HI) and saline (S) infusion, beta activity decreased slightly (p = 0.70, HI vs. S). During insulin detemir (D) infusion beta activity increased compared to HI (p = 0.001, repeated measures ANOVA, adjusted for multiple testing in all frequency bands).
In the human insulin experiment (HI) and the insulin detemir experiment (D) basal plasma insulin concentration were not different from S (HI 48±6 pM, p = 0.91; D 57±10 pM, p = 0.16). During both clamps, plasma insulin and total serum insulin detemir concentrations displayed similar time profiles (
Blood glucose concentrations were not different at baseline (S 4.8±0.4 mM, HI 5.0±0.3 mM, D 5.0±0.5 mM, all p>0.1) and did not change significantly during the experiments (all p>0.2) (
Though the total insulin detemir concentrations were higher than the human insulin concentrations, the glucose infusion rate required to maintain euglycemia was lower in the insulin detemir experiment (
As we screened all frequency bands of spontaneous cerebrocortical activity for differences between HI, S and D, we used a randomization approach to adjust for multiple testing as previously described
To compare the effect of insulin detemir on beta activity with the effect of human insulin, we subtracted the beta activity measured during the saline experiment from beta activity measured during the human insulin experiment or the insulin detemir experiment in the same subject. At baseline, there was no difference between lean and overweight subjects studied with human insulin or overweight subjects studied with insulin detemir (all p>0.6). During the second step of infusion beta activity was increased in the lean group with human insulin compared to overweight individuals (p = 0.031) and with insulin detemir compared to human insulin in the overweight group (p = 0.040), while there was no difference between the human insulin effect in lean and the insulin detemir effect in obese subjects (p = 0.78) (
A: Comparison of the insulin detemir effect on beta activity with the effects human insulin in lean and overweight subjects. The figure shows beta activity from human insulin experiments in 12 lean and 34 overweight subjects and from insulin detemir experiments in 10 overweight subjects. Data from corresponding saline experiments have been subtracted. In the second step of the clamps beta activity was significantly higher in lean than in overweight subjects (p = 0.031) and higher with insulin detemir than with human insulin in overweight subjects (p = 0.040). B: Relationship between body-mass-index (BMI) and cerebral effects of insulin. The change in beta activity induced by human insulin (filled circles and open rhombs) was negatively correlated with BMI (r = −0.38, p = 0.0086) under hyperinsulinemic euglycemic clamped conditions as previously published
In order to review the effect of insulin detemir in the context of the biological variation of cerebrocortical insulin effects, we show all data together in
Insulin receptors in the brain play an important role in a variety of physiologic functions (memory, cognitive function, control of appetite, energy homeostasis, and endogenous glucose production). Cerebral insulin receptors seem to be involved in neurodegenerative diseases such as Alzheimer's disease and metabolic diseases such as obesity and type 2 diabetes. The prevalence of these diseases increases
In a previous study, in which we established the detection of insulin effects on cerebrocortical activity with MEG, we have shown that human obesity is characterized by a reduced cerebral insulin response
As proposed in the mouse study
Beta activity and other frequency bands are very unspecific measures of cerebrocortical activity. Changes in this parameter may reflect multiple functions and at the current stage no specific function can be assigned to the insulin effect. Therefore, it is unclear whether the increase of beta activity by insulin in lean subjects or by insulin detemir in overweight subjects is directly involved in body weight regulation, glucose tolerance or neuroprotection and whether it reflects a beneficial effect on brain function. However, we have some circumstantial evidence of a functional link as we recently found that a polymorphism in the
In conclusion, we demonstrate that insulin detemir acts in the human brain more efficiently than human insulin at comparable or even lower peripheral metabolic effects. This tissue selectivity has already been demonstrated in mice and might be explained by the pharmacokinetic properties of insulin detemir. In the present study we could stimulate cerebrocortical beta activity in subjects who displayed no effect of human insulin in the brain. Therefore, insulin detemir seems to be a tool to restore at least in part the cerebrocortical insulin response in overweight humans. This principle may become a new therapeutic paradigm in obesity, type 2 diabetes and neurodegenerative diseases and might be applicable to other peptides which act in peripheral tissues and the brain.
We gratefully acknowledge the superb technical assistance of Anna Bury, Heike Luz and Gabi Walker.