Metabolic Signalling in Muscle- and Adipose-tissue Following Insulin Withdrawal and Growth Hormone Injection.

Study Overview
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Brief Summary
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Diabetes mellitus type I (DM I) is characterized by lack of endogenous insulin and these patients are 100% dependent on insulin substitution to survive. Insulin is a potent anabolic hormone with its primary targets in the liver, the skeletal muscle-tissue and - adipose-tissue. Severe lack of insulin leads to elevated blood glucose levels, dehydration, electrolyte derangement, ketosis and thus eventually ketoacidosis. Insulin signalling pathways are well-known. Growth hormone (GH) is also a potent anabolic hormone, responsible for human growth and preservation of protein during fasting. GH (in concert with lack of insulin) induces lipolysis during fasting. It is not known how GH exerts its lipolytic actions. The aim is to define insulin and growth hormone (GH) signalling pathways in 3 different states in patients with DM I. And to test whether ATGL-related lipolysis in adipose tissue contributes to the development of ketosis. Good glycemic control Lack of insulin (ketosis/ketoacidosis) Good glycemic control and GH injection

Detailed Description
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Diabetes mellitus type I (DMI ) is characterized by lack of endogenous insulin and these patients are 100% dependent on insulin substitution to survive. Insulin is a potent anabolic hormone with its primary targets in- the liver, -the skeletal muscle-tissue and - fat-tissue. In the liver it enhances glycogenesis and inhibits glycogenolysis and gluconeogenesis. In skeletal muscle-tissue, it enhances glucose transport into the cell, glycogenesis, glycolysis, glucose oxidation and protein synthesis. In fat-tissue, it inhibits lipolysis and enhances lipogenesis. This indicates that a fall in serum insulin levels lead to increased blood glucose and increased levels of FFA's (free fatty acids) in the blood - eventually leading to ketone production. If this condition is not corrected, it will lead to ketoacidosis, which is a potentially life-threatening condition, that is to be corrected under hospital admission with fluid-therapy, electrolyte- and insulin-substitution. Insulin has been studied thoroughly and signalling pathways are well known. An interesting pathway is suppression of lipolysis. The most important and rate-limiting lipase in triglyceride hydrolysis is adipose triglyceride lipase (ATGL)(1-5). A connection between ATGL and G0/G1 switch gene (G0S2) has been shown (6,7). During lipolysis ATGL is up-regulated and G0S2 is down-regulated and the promoter region for G0S2 has binding-sites for glucose, insulin dependent transcription factors and peroxisome proliferator-activated receptors y (PPAR-y)(8). One former study has shown that fasting reduces G0S2 and increases ATGL in humane adipose-tissue(7). The anti-lipolytic effects of insulin, could be thought, to be mediated through increased transcription of G0S2 which then in turn inhibits ATGL. Conversely, increased lipolysis during lack of insulin. Growth hormone and growth hormone dependent synthesis og IGF-1 (Insulin-like growth factor - 1) is crucial for human growth before and during adolescence. As an adult GH and IGF-1 are still potent growth factors and also they exert essential regulatory properties on human metabolism(9,10) GH- signalling pathways go through the GH-receptor, which phosphorylates and thus activates the receptor associated Janus Kinase 2 (JAK2). The signals from this point have been examined in numerous studies. In rodents, the signal has been shown to run three ways (9,10) Studies on human fibroblast cells have been able to support two of these pathways (MAPK - mitogen-activated protein kinase and STAT - signal transducer and activator of transcription), but not through the insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase (PI3-K) pathway. In human (in vivo) studies, GH stimulation and phosphorylation of STAT5 has been evident, however an association between GH stimulation and activation of MAPK and PI3-K has not been shown (11). The latter is interesting and remarkable, considering the insulin-agonistic and antagonistic effects of GH. GH stimulates lipolysis, but exactly how the lipolytic properties of GH are mediated is not fully understood. However, it is shown that GH has an effect on hormone-sensitive lipase (12) (HSL). Other options could be, as found in rodents, interaction via PI3-K signaling pathway or via G0S2/ATGL interaction, either directly or perhaps mediated through IGF-1. Humane intracellular signaling-pathways during development of ketosis/ketoacidosis are not well-known. The investigators believe that understanding these pathways and the exact mechanisms behind the development of ketoacidosis, is of great importance.

Official Title
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Metabolic Signalling in Muscle- and Adipose Tissue Following Insulin Withdrawal and Growth Hormone Injection in Type I Diabetes Mellitus, a Clinical Experimental Study.

Conditions
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Diabetes Mellitus Type I, Ketoacidosis

Intervention / Treatment
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* Drug: Insulin withdrawal
* Drug: Norditropin


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Diagnosis of Diabetes Mellitus Type I, C-peptide negative, 19 < BMI < 26, Written consent - Exclusion Criteria: Ischemic heart disease, Cardiac arrythmia, Epilepsy, Other medical illness -

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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Male

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Factorial Assignment
Masking: Single


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| No Intervention: Insulin<br>good glycemic control: 50 % of the subject's basal insulin dosage will be given as a continuous IV administration of insuman rapid overnight (hospitalized and fasting from 10 p.m.) and on the study-day. Basal period from 7.00 am to 12.00pm. The subject will undergo a hyperinsulinemic euglycemic clamp from 12.00 pm to 2.30 pm. Three muscle- and three fat-biopsies will be obtained. A palmitic-acid tracer, a glucose tracer, urea tracer, tyrosine- and phenylalanine- tracers will be given. | |
| Experimental: Insulin withdrawal<br>10 % of the individual subject's regular insulin dosage will be given as a continuous IV administration of insuman rapid overnight (hospitalized and fasting from 10 p.m.) Basal period from 7.00 am to 12.00 pm (without insulin). The subject will undergo a hyperinsulinemic euglycemic clamp from 12.00 pm to 2.30 pm. Three muscle- and three fat-biopsies will be obtained. A palmitic-acid tracer, a glucose tracer, urea tracer, tyrosine- and phenylalanine- tracers will be given. | Drug: Insulin withdrawal<br>* Withdrawal of usual (evening) insulin, replaced by Insuman Rapid (10% of the amount of usual evening insulin) as a continuous IV- administration overnight until 8 o'clock on the study day.<br>* Other names: Insuman Rapid;|
| Experimental: Norditropin (Growth Hormone)<br>Same amount of insulin administered on the control day (good glycemic control) overnight and on the study day (hospitalized and fasting from 10 p.m.). On the study day, a bolus injection of 0,4 mg of growth hormone (Norditropin) will be administered at 7.05 am. Basal period from 7.00 am to 12.00 pm (good glycemic control).The subject will undergo a hyperinsulinemic euglycemic clamp from 12.00 pm to 2.30 pm. Three muscle- and three fat-biopsies will be obtained. A palmitic-acid tracer, a glucose tracer, urea tracer, tyrosine- and phenylalanine- tracers will be given. | Drug: Norditropin<br>* 0,4 mg of GH administered at 7.05 A.M. on the study day.<br>* Other names: Growth Hormone;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Insulin and growth hormone signalling, expressed as CHANGE in phosphorylation of intracellular target proteins and CHANGE in mRNA expression of target genes in muscle- and fat-tissue. | Change in phosphorylation of target proteins and mRNA (messenger RNA) expression of target genes assessed with western blotting technique. | Muscle and fat biopsies obtained on each study day (arm): t1= 7.00 (0 min) am t2=11.30 (270min) am t3= 13.00 pm (360min) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in Intracellular markers of lipid metabolism in muscle- and fat tissue biopsies. | Assessed by Western blotting. | Muscle and fat biopsies obtained on each study day (arm): t1= 7.00 (0 min) am t2=11.30 (270min) am t3= 13.00 pm (360min) | 
| Metabolism | Change in glucose, fat and protein metabolism assessed by tracer kinetics on every study day (specific times below) and by indirect calorimetry. [3H 3]Glucose tracer from t=80min - 260min. [9,10-3H]Palmitic acid tracer from t=200min - 260min. [13C] Urea tracer from 20min - 260min. 15N-phenylalanine tracer and 2H4-tyrosine tracer from 80 min - 260 min. | Change in glucose, fat and protein metabolism between study days. | 
| Ghrelin | Change in circulating plasma acyl- and desacyl ghrelin levels between study days. | Plasma samples obtained at t=0, t=15, t=30, t=45, t=60, t=75, t=90, t=105, t=120, t=150, t=180, t=210, t=240, t=270, t=300 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Diabetes Mellitus type I, Ketoacidosis, Insulin withdrawal, Growth hormone injection, Lipolysis and ATGL