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C mutations and metabolic phenotypes in familial partial lipodystrophy
Tuesday June 27, 2000: Workshop Abstracts W:8 Gene Therapy and Other New Treatments
80
Conclusions: Elevated fatty acid levels modulate PG in hepatic cells. If present in vivo, this could affect the clearance rate of remnant particles in insulin resistance and type 2 diabetes and contribute to its dyslipidemia. References [1] Hennig, B. etal. (1995) Prostagl. Leukot. Essent. FattyAcids 53, 315-24 [2] Olsson, U., Bondjers, G., and Camejo, G. (1999) Diabetes 48, 616-22 [3] Mahley, R. W., and Ji, Z. (1999) J. LipidRes. 40, 1-16
•
Adipose tissue insulin resistance in familial combined hyperlipidemia (FCH), but not type 2 diabetes meilitns (DM2)
C.J.H. van der Kallen, EG. Bouwman, R.W.J. van de Hulst, W.D. Boeckx, T.W.A. de Bruin. Lab. Molecular Metabolism and Endocrinology, Maastricht University, Maastricht, The Netherlands Objective: To test in both DM2 and FCH the hypothesis that in both DM2 and FCH insulin induced suppression of hormone sensitive lipase (HSL) activity is reduced as consequence of insulin resistance. Methods: Subcutaneous adipose tissue biopsies were obtained from healthy controls (C, n = 11), DM2 (n = 12) and FCH (n = 10) subjects. Immediately following isolation, mature adipocytes were incubated with isoprenaline or insulin for 2 hours. Both glycerol and free fatty acids (FFA) levels were measured in the incubation media. Results: Isoprenaline stimulated the release of glycerol as well as FFA in all groups, with the highest release in DM2 (p < 0.05 vs C and FCH). Insulin decreased FFA release in C and DM2, but not in FCH, indicating impaired insulin sensitivity towards fractional FFA re-esterification in the adipocytes. Data in table represent nmol/40,000 cells/2 h (mean + sd).
FFA Glycerol
Basal Isoprenaline Insulin Basal l~prenaline Insulin
Controls 26.6 ::E10.8 137.94- 83.6* 18.84, 8.6* 11.1 4- 5.3 61.6 4-45.9* 11.24- 5.6
DM2 56.2 4-48.9 332.7 4- 176.8 32.6 4- 17.8 34.0 4-43.1 136.84- 95.8* 17.1 4- 10.8
FCH 25.8 4- 9.8 94.0 4- 57.4* 27.3 4- 7.3 9.6 4- 3.1 33.7 4- 17.7" 11.04- 3.9
Conclusions: In DM2 maximum lipolysis activity is high, probably due to larger adipocytes (data not shown). In FCH adipocytes, FFA release did not change under the influence of insulin. This suggests that in FCH the FFA metabolism is disturbed. This may be due to impaired acylation, or TG synthesis, or oxidation, eventually resulting to a higher FFA flux to the liver, contributing to the hypedipidemia.
•
Spectrum of nuclear iamin A/C mutations and metabolic phenotypes in familial partial lipodystrophy
Robert A. Hegele, Carol M. Anderson, Jian Wang, Henian Cat. The John P. Robarts Research Institute, London, Canada We were the first to report that Dunnigan-type familial partial lipodystrophy (FPLD) with insulin resistance, diabetes, hyperlipidemia, hypertension and early atherosclerosis results from a mutation, namely R482Q, in LMNA, the gene that encodes nuclear lamins A and C. We have since identified three novel and extremely rare missense mutations in LMNA, namely V440M, R482W and R584H. Examination of the clinical and biochemical phenotypes in carriers of mutant LMNA revealed that hyperinsulinemia and perturbations in plasma lipids preceded the development of plasma glucose abnormalities and hypertension. Our findings indicate that: 1) a spectrum of LMNA mutations underlies FPLD; 2) aberrant lamin A, and not lamin C, underlies FPLD, since R584H occurs within LMNA sequence that is specific for lamin A; 3) compound heterozygosity for mutant LMNA is associated with a relatively more severe FPLD phenotype, but not with complete lipodystrophy; and 4) environmental factors appeared to be partially related to the variation in phenotype severity in LMNA mutation carriers. Thus, rare mutations in a nuclear structural protein are associated with markedly abnormal qualitative and quantitative metabolic phenotypes. The precise mechanism by which mutant LMNA causes fat-wasting in specific anatomical sites is unknown. It is also not clear whether the metabolic disturbances in FPLD are a direct result of deficient or defective intracellular function due to the mutant LMNA or are merely secondary to the abnormal distribution of adipose tissue. In either event, our genetic analysis has implicated an etiologic role for aberrant nuclear lamin A in FPLD. These naturally occurring LMNA mutations can now be evaluated with in vitro molecular and cellular analyses in order to understand why specific cell types and tissues are selectively affected.
W:8
G E N E T H E R A P Y A N D O T H E R N E W TREATMENTS
~ V E G F gene transfer in the treatment of coronary heart disease and peripheral vascular disease S. Yl~i-Herttuala. A.I. Virtanen Institute and Department of Medicine, Universi~ of Kuopio, Kuopio, Finland Vascular gene therapy is a new area where only a few preliminary results from human trials are available (1). Most of the clinical trials are centered around therapeutic angiogenesis, treatment of restenosis, arterial cytoprotection or a combination of these effects. Intravascular adenoviral gene transfer in human peripheral arteries in the leg has been proved feasible with infusion-perfusion catheters (2). Detectable transgene expression was achieved in a maximum of 5% of arterial cells. Beneficial effects have been reported after intramuscular VEGF gene transfer into the muscle or artery of an ischaemic limb or myocardium (3--6). Several gene therapy trials with various types of VEGF are currently ongoing (1). Results are expected within 1-2 years. Based on current information, gene therapy in the cardiovascular system seems to be safe and well tolerated, although edema has been seen in legs treated with intramuscular VEGF gene therapy and hypertension has been reported in some patients. Even though gene therapy has shown promising results in some areas of cardiovascular diseases, further developments in gene transfer vectors, gene delivery techniques and identification of effective treatment genes will be required before the full therapeutic potential of gene therapy can be assessed.
References [1] Yl~i-Herttuala S, Martin JE Cardiovascular gene therapy. Lancet 2000; 355: 213-222. [2] Laitinen M e t al. Hum. Gene Ther. 1998; 9: 1481-1486. [3] Baumgartner Iet al. Circulation 1998; 97:1114-23. [4] Losordo DW et al. Circulation 1998; 98: 2800-804. [5] Rosengart TK et al. Circulation 1999; 100: 468-74. [6] Laitinen M e t al. Hum. Gene Ther. 2000; 11: 263-270.
~ G e n e
therapy for dyslipidemias
Lawrence Chan. Departments of Molecular & Cellular Biology and Medicine Baylor College of Medicine, Houston, Texas 77030, USA Elevation of atherogenic plasma lipoproteins is a major risk factor for atherosclerosis development. Somatic gene therapy is a novel experimental approach for the treatment of hyperlipoproteinemia and dyslipidemia. Successful gene therapy requires the availability of safe and efficient genedelivery vectors. Although first and second-generation adenoviral vectors are highly efficient in delivering transgenes to the liver, they exhibit substantial toxicity and have been associated with significant morbidity and mortality in clinical trials. A helper-dependent adenovrial vector (HD-Ad) deleted of all viral protein genes was developed at Baylor College of Medicine, and an efficient production system for this vector was developed by Dr. Frank Graham at McMaster University. In collaboration with Dr. Arthur Beaudet in the Department of Molecular & Human Genetics at Baylor, we used HD-Ad to deliver lipid-lowering genes to the liver of mouse and nonhuman primate models of hyperlipidemia. A single injection of HD-Ads for LDL receptor, VLDL receptor, apoA-I or apoE produced long-term (6 months to > 1 yr) hepatic transgene expression with negligible toxicity, reversed dyslipidemia and prevented atherosclerosis development. The data support the feasibility and safety of using HD-Ad vectors for the treatment of lipid disorders in clinical trials.
~ G e n e
therapy for proliferative vascular disease
K. Walsh. Division of Cardiovascular Research, St. Elizabeth's Medical Center, and Program in Cell, Molecular, and Developmental Biology, Sackler School of Biomedical Sciences, Tufts University, Boston, MA, USA I will provide an overview of the candidate genes and delivery systems that we are evaluating for the therapy of proliferative vascular disorders. The promise of gene therapy for post-angioplasty restenosis is that one has the opportunity to deliver genetic material to the site of balloon inflation at the time of intervention. The delivery of genetic material to these sites creates a depot for recombinant protein expression, thereby avoiding limitations imposed on other therapies by the short retention periods experienced with small molecules and macromolecules delivered to the vessel wall. The altered expression of genes within the targeted cells will, in theory, alter the course
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
80
Conclusions: Elevated fatty acid levels modulate PG in hepatic cells. If present in vivo, this could affect the clearance rate of remnant particles in insulin resistance and type 2 diabetes and contribute to its dyslipidemia. References [1] Hennig, B. etal. (1995) Prostagl. Leukot. Essent. FattyAcids 53, 315-24 [2] Olsson, U., Bondjers, G., and Camejo, G. (1999) Diabetes 48, 616-22 [3] Mahley, R. W., and Ji, Z. (1999) J. LipidRes. 40, 1-16
•
Adipose tissue insulin resistance in familial combined hyperlipidemia (FCH), but not type 2 diabetes meilitns (DM2)
C.J.H. van der Kallen, EG. Bouwman, R.W.J. van de Hulst, W.D. Boeckx, T.W.A. de Bruin. Lab. Molecular Metabolism and Endocrinology, Maastricht University, Maastricht, The Netherlands Objective: To test in both DM2 and FCH the hypothesis that in both DM2 and FCH insulin induced suppression of hormone sensitive lipase (HSL) activity is reduced as consequence of insulin resistance. Methods: Subcutaneous adipose tissue biopsies were obtained from healthy controls (C, n = 11), DM2 (n = 12) and FCH (n = 10) subjects. Immediately following isolation, mature adipocytes were incubated with isoprenaline or insulin for 2 hours. Both glycerol and free fatty acids (FFA) levels were measured in the incubation media. Results: Isoprenaline stimulated the release of glycerol as well as FFA in all groups, with the highest release in DM2 (p < 0.05 vs C and FCH). Insulin decreased FFA release in C and DM2, but not in FCH, indicating impaired insulin sensitivity towards fractional FFA re-esterification in the adipocytes. Data in table represent nmol/40,000 cells/2 h (mean + sd).
FFA Glycerol
Basal Isoprenaline Insulin Basal l~prenaline Insulin
Controls 26.6 ::E10.8 137.94- 83.6* 18.84, 8.6* 11.1 4- 5.3 61.6 4-45.9* 11.24- 5.6
DM2 56.2 4-48.9 332.7 4- 176.8 32.6 4- 17.8 34.0 4-43.1 136.84- 95.8* 17.1 4- 10.8
FCH 25.8 4- 9.8 94.0 4- 57.4* 27.3 4- 7.3 9.6 4- 3.1 33.7 4- 17.7" 11.04- 3.9
Conclusions: In DM2 maximum lipolysis activity is high, probably due to larger adipocytes (data not shown). In FCH adipocytes, FFA release did not change under the influence of insulin. This suggests that in FCH the FFA metabolism is disturbed. This may be due to impaired acylation, or TG synthesis, or oxidation, eventually resulting to a higher FFA flux to the liver, contributing to the hypedipidemia.
•
Spectrum of nuclear iamin A/C mutations and metabolic phenotypes in familial partial lipodystrophy
Robert A. Hegele, Carol M. Anderson, Jian Wang, Henian Cat. The John P. Robarts Research Institute, London, Canada We were the first to report that Dunnigan-type familial partial lipodystrophy (FPLD) with insulin resistance, diabetes, hyperlipidemia, hypertension and early atherosclerosis results from a mutation, namely R482Q, in LMNA, the gene that encodes nuclear lamins A and C. We have since identified three novel and extremely rare missense mutations in LMNA, namely V440M, R482W and R584H. Examination of the clinical and biochemical phenotypes in carriers of mutant LMNA revealed that hyperinsulinemia and perturbations in plasma lipids preceded the development of plasma glucose abnormalities and hypertension. Our findings indicate that: 1) a spectrum of LMNA mutations underlies FPLD; 2) aberrant lamin A, and not lamin C, underlies FPLD, since R584H occurs within LMNA sequence that is specific for lamin A; 3) compound heterozygosity for mutant LMNA is associated with a relatively more severe FPLD phenotype, but not with complete lipodystrophy; and 4) environmental factors appeared to be partially related to the variation in phenotype severity in LMNA mutation carriers. Thus, rare mutations in a nuclear structural protein are associated with markedly abnormal qualitative and quantitative metabolic phenotypes. The precise mechanism by which mutant LMNA causes fat-wasting in specific anatomical sites is unknown. It is also not clear whether the metabolic disturbances in FPLD are a direct result of deficient or defective intracellular function due to the mutant LMNA or are merely secondary to the abnormal distribution of adipose tissue. In either event, our genetic analysis has implicated an etiologic role for aberrant nuclear lamin A in FPLD. These naturally occurring LMNA mutations can now be evaluated with in vitro molecular and cellular analyses in order to understand why specific cell types and tissues are selectively affected.
W:8
G E N E T H E R A P Y A N D O T H E R N E W TREATMENTS
~ V E G F gene transfer in the treatment of coronary heart disease and peripheral vascular disease S. Yl~i-Herttuala. A.I. Virtanen Institute and Department of Medicine, Universi~ of Kuopio, Kuopio, Finland Vascular gene therapy is a new area where only a few preliminary results from human trials are available (1). Most of the clinical trials are centered around therapeutic angiogenesis, treatment of restenosis, arterial cytoprotection or a combination of these effects. Intravascular adenoviral gene transfer in human peripheral arteries in the leg has been proved feasible with infusion-perfusion catheters (2). Detectable transgene expression was achieved in a maximum of 5% of arterial cells. Beneficial effects have been reported after intramuscular VEGF gene transfer into the muscle or artery of an ischaemic limb or myocardium (3--6). Several gene therapy trials with various types of VEGF are currently ongoing (1). Results are expected within 1-2 years. Based on current information, gene therapy in the cardiovascular system seems to be safe and well tolerated, although edema has been seen in legs treated with intramuscular VEGF gene therapy and hypertension has been reported in some patients. Even though gene therapy has shown promising results in some areas of cardiovascular diseases, further developments in gene transfer vectors, gene delivery techniques and identification of effective treatment genes will be required before the full therapeutic potential of gene therapy can be assessed.
References [1] Yl~i-Herttuala S, Martin JE Cardiovascular gene therapy. Lancet 2000; 355: 213-222. [2] Laitinen M e t al. Hum. Gene Ther. 1998; 9: 1481-1486. [3] Baumgartner Iet al. Circulation 1998; 97:1114-23. [4] Losordo DW et al. Circulation 1998; 98: 2800-804. [5] Rosengart TK et al. Circulation 1999; 100: 468-74. [6] Laitinen M e t al. Hum. Gene Ther. 2000; 11: 263-270.
~ G e n e
therapy for dyslipidemias
Lawrence Chan. Departments of Molecular & Cellular Biology and Medicine Baylor College of Medicine, Houston, Texas 77030, USA Elevation of atherogenic plasma lipoproteins is a major risk factor for atherosclerosis development. Somatic gene therapy is a novel experimental approach for the treatment of hyperlipoproteinemia and dyslipidemia. Successful gene therapy requires the availability of safe and efficient genedelivery vectors. Although first and second-generation adenoviral vectors are highly efficient in delivering transgenes to the liver, they exhibit substantial toxicity and have been associated with significant morbidity and mortality in clinical trials. A helper-dependent adenovrial vector (HD-Ad) deleted of all viral protein genes was developed at Baylor College of Medicine, and an efficient production system for this vector was developed by Dr. Frank Graham at McMaster University. In collaboration with Dr. Arthur Beaudet in the Department of Molecular & Human Genetics at Baylor, we used HD-Ad to deliver lipid-lowering genes to the liver of mouse and nonhuman primate models of hyperlipidemia. A single injection of HD-Ads for LDL receptor, VLDL receptor, apoA-I or apoE produced long-term (6 months to > 1 yr) hepatic transgene expression with negligible toxicity, reversed dyslipidemia and prevented atherosclerosis development. The data support the feasibility and safety of using HD-Ad vectors for the treatment of lipid disorders in clinical trials.
~ G e n e
therapy for proliferative vascular disease
K. Walsh. Division of Cardiovascular Research, St. Elizabeth's Medical Center, and Program in Cell, Molecular, and Developmental Biology, Sackler School of Biomedical Sciences, Tufts University, Boston, MA, USA I will provide an overview of the candidate genes and delivery systems that we are evaluating for the therapy of proliferative vascular disorders. The promise of gene therapy for post-angioplasty restenosis is that one has the opportunity to deliver genetic material to the site of balloon inflation at the time of intervention. The delivery of genetic material to these sites creates a depot for recombinant protein expression, thereby avoiding limitations imposed on other therapies by the short retention periods experienced with small molecules and macromolecules delivered to the vessel wall. The altered expression of genes within the targeted cells will, in theory, alter the course
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000