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Glucose and lipid metabolism in insulin resistance: an experimental study in fat cells
Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type 2 diabetes is usually caused by a combination of pancreatic β-cell failure and insulin resistance in target tissues like liver, muscle and fat. Insulin resistance is characterised by an impaired effect of insulin to reduce hepatic glucose production and to promote glucose uptake in peripheral tissues. The focus of this study was to further elucidate cellular mechanisms for insulin resistance that may be of relevance for type 2 diabetes in humans. We used rat and human adipocytes as an established model of insulin’s target cells.

Glucocorticoids, e.g. cortisol, can induce insulin resistance in vivo. In the present study, pretreatment of rat adipocytes in vitro for 24 h with the cortisol analogue dexamethasone produced a downregulation of glucose uptake capacity as well as a marked depletion of cellular insulin receptor substrate 1 (IRS-1) and protein kinase B (PKB), two proteins suggested to play a critical role in the intracellular signal transduction pathway of insulin. The amount of phosphorylated PKB in response to acute insulin treatment was decreased in parallel to total PKB content. The basal rate of lipolysis was enhanced, but insulin’s antilipolytic effect was not consistently altered following dexamethasone pretreatment.

Alterations in blood glucose as well as insulin levels may be of great importance for cellular as well as whole-body insulin resistance. High glucose (≥15 mM) for 24 h induced a decrease in glucose uptake capacity in rat adipocytes and IRS-1 content was reduced whereas IRS-2 was increased. Long-term pretreatment with a high insulin concentration downregulated insulin binding capacity and when combined with high glucose, it produced a pronounced

reduction of cellular IRS-1 and 2 content together with insensitivity to insulin’s effect to activate PKB and a decrease in glucose uptake capacity. A common denominator for a decrease in glucose uptake capacity in our rat adipocyte studies seems to be a decrease in IRS-1 content.

Adipocytes from type 2 diabetes patients are insulin-resistant, but in our work the insulin resistance could be reversed by incubation of the cells at a physiological glucose level for 24 h. Insulin resistance in fresh adipocytes from type 2 diabetes patients was associated with in vivo insulin resistance and glycemic level and with adipocyte cell size and waist-hip ratio

(WHR).

As a potential mechanism for postprandial dyslipidemia in type 2 diabetes, we examined the nutritional regulation of subcutaneous adipose tissue lipoprotein lipase (LPL) activity. It was upregulated by ~40-50 % after a standardised lipid-enriched meal and this was very similar in type 2 diabetes patients and control subjects, suggesting that the postprandial

hypertriglyceridemia found in type 2 diabetes is not explained by an altered nutritional regulation of LPL in subcutaneous fat.

In conclusion, the present work provides evidence for novel interactions between glucocorticoids and insulin in the regulation of glucose metabolism that may potentially contribute to the development of insulin resistance. High levels of glucose and insulin produce perturbations in the insulin signalling pathway that may be of relevance for human type 2 diabetes. Cellular insulin resistance may be secondary to the diabetic state in vivo, e.g. via glucotoxicity. This is supported by our finding that insulin resistance in adipocytes from type 2 diabetes patients can be reversed after incubation at a physiological glucose level.

Key words: adipocyte, insulin resistance, type 2 diabetes, insulin signalling, glucose uptake,

insulin, glucose, dexamethasone, insulin receptor substrate, protein kinase B, GLUT4,

lipoprotein lipase.

Place, publisher, year, edition, pages
Umeå: Folkhälsa och klinisk medicin , 2003. , 63 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 817
Keyword [en]
Public health, adipocyte, insulin resistance, type 2 diabetes, insulin signalling, glucose uptake, insulin, glucose, dexamethasone, insulin receptor substrate, protein kinase B, GLUT4, lipoprotein lipase
Keyword [sv]
Folkhälsomedicin
National Category
Public Health, Global Health, Social Medicine and Epidemiology
Research subject
Medicine
Identifiers
URN: urn:nbn:se:umu:diva-26ISBN: 91-7305-359-7 (print)OAI: oai:DiVA.org:umu-26DiVA: diva2:142848
Public defence
2002-12-06, Umeå, 13:00
Available from: 2002-12-06 Created: 2002-12-06 Last updated: 2010-06-17Bibliographically approved
List of papers
1. Dexamethasone impairs insulin signalling and glucose transport by depletion of insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase B in primary cultured rat adipocytes.
Open this publication in new window or tab >>Dexamethasone impairs insulin signalling and glucose transport by depletion of insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase B in primary cultured rat adipocytes.
2002 (English)In: European Journal of Endocrinology, ISSN 0804-4643, E-ISSN 1479-683X, Vol. 146, no 3, 419-429 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: Glucocorticoid excess leads to insulin resistance. This study explores the effects of glucocorticoids on the glucose transport system and insulin signalling in rat adipocytes. The interaction between glucocorticoids and high levels of insulin and glucose is also addressed. DESIGN AND METHODS: Isolated rat adipocytes were cultured for 24 h at different glucose concentrations (5 and 15 mmol/l) with or without the glucocorticoid analogue dexamethasone (0.3 micromol/l) and insulin (10(4) microU/ml). After the culture period, the cells were washed and then basal and insulin-stimulated glucose uptake, insulin binding and lipolysis as well as cellular content of insulin signalling proteins (insulin receptor substrate-1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (PKB)) and glucose transporter isoform GLUT4 were measured. RESULTS: Dexamethasone in the medium markedly decreased both basal and insulin-stimulated glucose uptake at both 5 and 15 mmol/l glucose (by approximately 40-50%, P<0.001 and P<0.05 respectively). Combined long-term treatment with insulin and dexamethasone exerted additive effects in decreasing basal, and to a lesser extent insulin-stimulated, glucose uptake capacity (P<0.05) compared with dexamethasone alone, but this was seen only at high glucose (15 mmol/l). Insulin binding was decreased (by approximately 40%, P<0.05) in dexamethasone-treated cells independently of surrounding glucose concentration. Following dexamethasone treatment a approximately 75% decrease (P<0.001) in IRS-1 expression and an increase in IRS-2 (by approximately 150%, P<0.001) was shown. Dexamethasone also induced a subtle decrease in PI3-K (by approximately 20%, P<0.01) and a substantial decrease in PKB content (by approximately 45%, P<0.001). Insulin-stimulated PKB phosphorylation was decreased (by approximately 40%, P<0.01) in dexamethasone-treated cells. Dexamethasone did not alter the amount of total cellular membrane-associated GLUT4 protein. The effects of dexamethasone per se on glucose transport and insulin signalling proteins were mainly unaffected by the surrounding glucose and insulin levels. Dexamethasone increased the basal lipolytic rate (approximately 4-fold, P<0.05), but did not alter the antilipolytic effect of insulin. CONCLUSIONS: These results suggest that glucocorticoids, independently of the surrounding glucose and insulin concentration, impair glucose transport capacity in fat cells. This is not due to alterations in GLUT4 abundance. Instead dexamethasone-induced insulin resistance may be mediated via reduced cellular content of IRS-1 and PKB accompanied by a parallel reduction in insulin-stimulated activation of PKB.

Place, publisher, year, edition, pages
European Society of Endocrinology, 2002
Identifiers
urn:nbn:se:umu:diva-3931 (URN)10.1530/eje.0.1460419 (DOI)
Available from: 2002-12-06 Created: 2002-12-06 Last updated: 2010-06-17Bibliographically approved
2. High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes: possible implications for insulin resistance in type 2 diabetes
Open this publication in new window or tab >>High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes: possible implications for insulin resistance in type 2 diabetes
2003 (English)In: European Journal of Endocrinology, ISSN 0804-4643, E-ISSN 1479-683X, Vol. 148, 157-167 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: The purpose of this study was to investigate the cellular effects of long-term exposure to high insulin and glucose levels on glucose transport and insulin signalling proteins. DESIGN AND METHODS: Rat adipocytes were cultured for 24 h in different glucose concentrations with 10(4) microU/ml of insulin or without insulin. After washing, (125)I-insulin binding, basal and acutely insulin-stimulated d-[(14)C]glucose uptake, and insulin signalling proteins and glucose transporter 4 (GLUT4) were assessed. RESULTS: High glucose (15 and 25 mmol/l) for 24 h induced a decrease in basal and insulin-stimulated glucose uptake compared with control cells incubated in low glucose (5 or 10 mmol/l). Twenty-four hours of insulin treatment decreased insulin binding capacity by approximately 40%, and shifted the dose-response curve for insulin's acute effect on glucose uptake 2- to 3-fold to the right. Twenty-four hours of insulin treatment reduced basal and insulin-stimulated glucose uptake only in the presence of high glucose (by approximately 30-50%). At high glucose, insulin receptor substrate-1 (IRS-1) expression was downregulated by approximately 20-50%, whereas IRS-2 was strongly upregulated by glucose levels of 10 mmol/l or more (by 100-400%). Insulin treatment amplified the suppression of IRS-1 when combined with high glucose and also IRS-2 expression was almost abolished. Twenty-four hours of treatment with high glucose or insulin, alone or in combination, shifted the dose-response curve for insulin's effect to acutely phosphorylate protein kinase B (PKB) to the right. Fifteen mmol/l glucose increased GLUT4 in cellular membranes (by approximately 140%) compared with 5 mmol/l but this was prevented by a high insulin concentration. CONCLUSIONS: Long-term exposure to high glucose per se decreases IRS-1 but increases IRS-2 content in rat adipocytes and it impairs glucose transport capacity. Treatment with high insulin downregulates insulin binding capacity and, when combined with high glucose, it produces a marked depletion of IRS-1 and -2 content together with an impaired sensitivity to insulin stimulation of PKB activity. These mechanisms may potentially contribute to insulin resistance in type 2 diabetes.

Place, publisher, year, edition, pages
European Society of Endocrinology, 2003
Identifiers
urn:nbn:se:umu:diva-3932 (URN)10.1530/eje.0.1480157 (DOI)
Available from: 2002-12-06 Created: 2002-12-06 Last updated: 2010-06-17Bibliographically approved
3. In vitro reversal of hyperglycemia normalizes insulin action in fat cells from type 2 diabetes patients: is cellular insulin resistance caused by glucotoxicity in vivo?
Open this publication in new window or tab >>In vitro reversal of hyperglycemia normalizes insulin action in fat cells from type 2 diabetes patients: is cellular insulin resistance caused by glucotoxicity in vivo?
2002 (English)In: Metabolism: Clinical and Experimental, ISSN 0026-0495, E-ISSN 1532-8600, Vol. 52, no 2, 239-245 p.Article in journal (Refereed) Published
Abstract [en]

Chronic hyperglycemia promotes the development of insulin resistance. The aim of this study was to investigate whether cellular insulin resistance is secondary to the diabetic state in human type 2 diabetes. Subcutaneous fat biopsies were taken from 3 age-, sex-, and body mass index (BMI)-matched groups with 10 subjects in each group: type 2 diabetes patients with either good (hemoglobin A1c [HbA1c] [lt ] 7%, G) or poor (HbA1c [gt ] 7.5%, P) metabolic control and healthy control subjects (C). Insulin action in vitro was studied by measurements of glucose uptake both directly after cell isolation and following a 24-hour incubation at a physiological glucose level (6 mmol/L). The relationship with insulin action in vivo was addressed by employing the euglycemic clamp technique. Freshly isolated fat cells from type 2 diabetes patients with poor metabolic control had [sim ]55% lower maximal insulin response (1,000 [mu ]U/mL) on glucose uptake (P [lt ] .05) compared to C. Cells from P were more insulin-resistant (P [lt ] .05) than cells from G at a low (5 [mu ]U/mL) but not at a high (1,000 [mu ]U/mL) insulin concentration, suggesting insulin insensitivity. However, following 24 hours of incubation at physiological glucose levels, insulin resistance was completely reversed in the diabetes cells and no differences in insulin-stimulated glucose uptake were found among the 3 groups. Insulin sensitivity in vivo assessed with hyperinsulinemic, euglycemic clamp (M-value) was significantly associated with insulin action on glucose uptake in fresh adipocytes in vitro (r = 0.50, P [lt ] .01). Fasting blood glucose at the time of biopsy and HbA1c, but not serum insulin, were negatively correlated to insulin's effect to stimulate glucose uptake in vitro (r = [minus ]0.36, P = .064 and r = [minus ] 0.41, P [lt ].05, respectively) in all groups taken together. In the in vivo situation, fasting blood glucose, HbA1c, and serum insulin were all negatively correlated to insulin sensitivity (M-value; r = [minus ]0.62, P[lt ] .001, r= [minus ]0.61, P[lt ] .001, and r = [minus ]0.56, p [lt ] .01, respectively). Cell size, waist-to-hip ration (WHR), and BMI correlated negatively with insulin's effect to stimulate glucose uptake both in vitro (r = [minus ]0.55, P [lt ] .01, r = [minus ]0.54, P [lt ] .01, and r = [minus ]0.43, P [lt ] .05, respectively) and in vivo (r = [minus ]0.43, P [lt ] .05, r = [minus ]0.50, P [lt ] .01, and r = [minus ]0.36, P [lt ] .05, respectively). Multiple regression analyses revealed that adipocyte cell size and WHR independently predicted insulin resistance in vitro. Furthermore, insulin sensitivity in vivo could be predicted by fasting blood glucose and serum insulin levels. We conclude that insulin resistance in fat cells from type 2 diabetes patients is fully reversible following incubation at physiological glucose concentrations. Thus, cellular insulin resistance may be mainly secondary to the hyperglycemic state in vivo.

Place, publisher, year, edition, pages
Elsevier Science, 2002
Identifiers
urn:nbn:se:umu:diva-3933 (URN)10.1053/meta.2003.50041 (DOI)
Available from: 2002-12-06 Created: 2002-12-06 Last updated: 2010-06-17Bibliographically approved
4. Postprandial regulation of blood lipids and adipose tissue lipoprotein lipase in type 2 diabetes patients and healthy control subjects
Open this publication in new window or tab >>Postprandial regulation of blood lipids and adipose tissue lipoprotein lipase in type 2 diabetes patients and healthy control subjects
Show others...
2003 (English)In: Atherosclerosis, ISSN 0021-9150, E-ISSN 1879-1484, Vol. 166, no 2, 359-367 p.Article in journal (Refereed) Published
Abstract [en]

Background/aim: In type 2 diabetes and other insulin-resistant conditions, postprandial hypertriglyceridaemia is an important metabolic perturbation. To further elucidate alterations in the clearance of triglyceride-rich lipoproteins in type 2 diabetes we focused on the nutritional regulation of adipose tissue lipoprotein lipase (LPL).

Subjects and methods: Eight subjects with type 2 diabetes and eight age-, sex- and body mass index (BMI)-matched control subjects underwent subcutaneous abdominal adipose tissue biopsies in the fasting state and 3.5 h following a standardized lipid-enriched meal. LPL activity and mass were measured in adipose tissue and also in plasma after an intravenous injection of heparin.

Results: Postprandial, but not fasting, triglycerides were significantly higher in the diabetic subjects than in the control subjects (3.0±0.4 vs 2.0±0.2 mmol/l, P=0.028). Adipose tissue LPL activity was increased following the meal test by ∼35–55% (P=0.021 and 0.004, respectively). There was no significant difference between the groups in this respect. The specific enzyme activity of LPL was not altered in the postprandial state. Fasting and postprandial adipose tissue LPL activity as well as post-heparin plasma LPL activity tended to be lower among the diabetes patients (NS). There was a significant and independent inverse association between insulin resistance (homeostasis model assessment insulin resistance (HOMA-IR) index) vs post-heparin plasma LPL activity and postprandial triglyceride levels, respectively. Adipose tissue LPL activity was related to insulin action in vitro on adipocyte glucose transport, but not to HOMA-IR.

Conclusion: Following food intake adipose tissue LPL activity is enhanced to a similar degree in patients with type 2 diabetes and in healthy control subjects matched for BMI, age and gender. If LPL dysregulation is involved in the postprandial hypertriglyceridaemia found in type 2 diabetes, it should occur in tissues other than subcutaneous fat.

Place, publisher, year, edition, pages
Elsevier, 2003
Keyword
Type 2 diabetes, Adipose tissue, Lipoprotein lipase, Insulin resistance, Glucose uptake, Triglycerides, Free fatty acids, Postprandial
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:umu:diva-3934 (URN)10.1016/S0021-9150(02)00366-0 (DOI)
Available from: 2002-12-06 Created: 2002-12-06 Last updated: 2012-05-16Bibliographically approved

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