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Leptin reverses insulin resistance and hepatic steatosis in patients
with severe lipodystrophy (HIV negative)
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Kitt Falk Petersen1, Elif Arioglu Oral2, Sylvie Dufour3, Douglas Befroy3, Charlotte Ariyan4, Chunli Yu1, Gary W. Cline1, Alex M. DePaoli5, Simeon I. Taylor2, Phillip Gorden2 and Gerald I. Shulman1,3,6
1 Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA 2 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA 3 The Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA 4 Department of Surgery, Yale University School of Medicine, New Haven, Connecticut, USA 5 Amgen Inc., Thousand Oaks, California, USA 6 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA
J Clin Invest, May 2002, Volume 109, Number 10, 1345-1350
Abstract
Lipodystrophy is a rare disorder that is characterized by selective loss of subcutaneous and visceral fat and is associated with hypertriglyceridemia, hepatomegaly, and disordered glucose metabolism. It has recently been shown that chronic leptin treatment ameliorates
these abnormalities. Here we show that chronic leptin treatment improves insulin-stimulated hepatic and peripheral glucose metabolism in severely insulin-resistant lipodystrophic patients. This improvement in insulin action was associated with a marked reduction in hepatic and
muscle triglyceride content. These data suggest that leptin may represent an important new therapy to reverse the severe hepatic and muscle insulin resistance and associated hepatic steatosis in patients with lipodystrophy.
-- There was a marked reduction in the fasting plasma glucose concentration
in patients NIH-3 and NIH-6 after 3 months of leptin treatment and in NIH-1 after 8 months of leptin treatment
--after leptin therapy there was an increase in whole-body insulin sensitivity
--Hepatic insulin responsiveness also improved, as reflected by an increase of insulin suppression of glucose production during the clamp to 82% ± 5% compared with 40% ± 6% prior to leptin treatment
--These changes in hepatic and peripheral insulin sensitivity were associated with an 86% ± 8% reduction in hepatic triglyceride content (P =0.008 compared with before leptin treatment) and a 33% ± 3% decrease in muscle triglyceride content
-- The leptin treatment-induced reduction in muscle triglyceride content was matched by an approximately 30% decrease in muscle total fatty acyl CoA concentrations
-- Energy expenditure was assessed by indirect calorimetry in all patients before and after leptin treatment and was found to be slightly, but not significantly, lower in
all patients after leptin treatment
Introduction
Lipodystrophy is a rare disorder that is characterized by selective loss of subcutaneous and visceral fat. This disease is associated with hypertriglyceridemia, hepatic steatosis, and severe insulin resistance that often results in diabetes (1-3). Shimomura et al. have demonstrated
that leptin treatment reversed insulin resistance in a fat-specific aP2-SREBP-1c knockout mouse model of congenital generalized lipodystrophy (4). More recently, Arioglu Oral et al. found that human recombinant leptin therapy reduced hyperglycemia and hypertriglyceridemia and
increased the rate of glucose disappearance during an intravenous insulin tolerance test in nine lipodystrophic patients (5). In order to definitively examine whether or not leptin treatment might improve insulin sensitivity in these patients, as well as the potential mechanism, we
studied a subset of three of these patients before and after leptin treatment. Insulin sensitivity in liver and muscle was assessed by a hyperinsulinemic-euglycemic clamp study performed in conjunction with [6,6-2H2]glucose turnover measurements, and patient responses were compared with those of a group of age- and weight-matched control subjects. In addition, the effects of leptin treatment on fat me-tabolism were assessed by measuring rates of whole-body lipolysis, as assessed by [2H5]glycerol turnover measurements before and after leptin treatment, along with 1H NMR measurements of liver and muscle triglyceride content.
Methods
Subjects. Three patients with severe, generalized lipodystrophy (fasting leptin concentration less than 4 ng/ml) associated with diabetes were studied. Six healthy nonsmoking women of similar age, weight, and body mass index were studied as normal control subjects.
Lipodystrophic patient NIH-1. The patient designated NIH-1 was a 17-year-old Caucasian female with a severe form of acquired lipodystrophy associated with type 2 diabetes and severe hypertriglyceridemia (plasma triglyceride concentrations greater than 15,000 mg/dl). She exhibited hyperphagia, irregular menses, massive hepatomegaly, multiple xanthomas, and lack of any subcutaneous fat or features of secondary sexual development.
Lipodystrophic patient NIH-3. A 26-year-old Caucasian female with congenital generalized lipodystrophy and moderate hypertriglyceridemia (plasma triglycerides approximately 1,200 mg/dl). Her diabetes was treated with metformin (500 mg twice a day) and insulin (25 units Humulin 70/30 subcutaneously at bed time). She had no history of hyperphagia and reported normal menstrual periods.
Lipodystrophic patient NIH-6. A 35-year-old African-American female with a congenital form of lipodystrophy. The patient was diagnosed with lipodystrophy at age 9 and with type 2 diabetes at age 12. Two siblings were also diagnosed with lipodystrophy. The patient's diabetes was treated with metformin (850 mg three times a day). She had a history of hyperphagia and reported irregular menses as a teenager prior to undergoing partial hysterectomy for uterine
fibroids.
Recombinant methionyl human leptin was given subcutaneously every 12 hours in doses to achieve near-physiological plasma concentrations of leptin (5). The treatment was given to all patients for 3 months and to patient NIH-1 for an additional 5 months.
Body fat composition. Body fat composition was determined using a dual-energy x-ray absorptiometer (model QDR 4500; Hologic Inc., Bedford, Massachusetts, USA).
Muscle biopsy studies. Muscle biopsy samples for fatty acyl CoA were obtained before and after leptin treatment in patient NIH-6. The biopsy was obtained after the subject had been supine and resting quietly for 60 minutes as previously described.
Results
Prior to leptin treatment, all patients had poorly controlled diabetes (as reflected by fasting hyperglycemia and increased glycosylated hemoglobin), and hyperlipidemia. Rates of fasting glucose production were higher in the lipodystrophic subjects than in the control
subjects (lipodystrophic, 3.6 ± 0.8 mg/kg/min versus control, 1.9 ± 0.1 mg/kg/min; P = 0.019). The lipodystrophic patients were severely insulin resistant, as reflected by the very low rates of glucose infusion required to maintain euglycemia during the hyperinsulinemic-euglycemic clamp (lipodystrophic, 1.2 ± 0.2 mg/kg/min versus control, 12.7 ± 0.9 mg/kg/min; P < 0.0001) as well as a marked reduction in insulin-stimulated whole-body glucose metabolism. Furthermore, the lipodystrophic patients had severe hepatic insulin resistance, as reflected by decreased insulin suppression of glucose production during the hyperinsulinemic clamp (40% ± 6%) compared with the control subjects (92% ± 7% suppression, P = 0.0004). These alterations were associated with severe hepatic steatosis in all of the patients: NIH-1, 48% liver triglyceride content, NIH-3, 4.6% liver triglyceride content, and NIH-6, 26% liver triglyceride content. In the control subjects, liver triglyceride content was less
than 1%.
Basal rates of glycerol turnover were markedly increased in the lipodystrophic patients, reflecting very high rates of lipolysis per kg fat tissue, and there was a tendency for energy expenditure to be higher in the lipodystrophic subjects (lipodystrophic, 1,660 ± 108 kcal/day versus control subjects, 1,368 ± 96 kcal/day; P = 0.10), which is consistent with the findings in a previous case report (16).
There was a marked reduction in the fasting plasma glucose concentration in patients NIH-3 and NIH-6 after 3 months of leptin treatment and in NIH-1 after 8 months of leptin treatment.
At the time of the postleptin treatment study, all patients had their antidiabetic medication discontinued (5). This improvement in glycemic control could be attributed to a large increase in whole-body insulin sensitivity, as reflected by an approximately fourfold increase in the rate of glucose infusion required to maintain euglycemia (5.4 ± 0.9 mg/kg/min after treatment versus 1.2 ± 0.2 mg/kg/min before treatment; P = 0.04) and an almost twofold increase in the rate of insulin-stimulated whole-body glucose metabolism (6.1 ± 1.0 mg/kg/min after leptin versus 3.5 ± 0.3 before leptin; P = 0.08). Hepatic insulin responsiveness also improved, as reflected by an increase of insulin suppression of glucose production during the clamp to 82% ± 5% compared with 40% ± 6% prior to leptin treatment (P < 0.05) (Figure 4). These changes in hepatic and peripheral insulin sensitivity were associated with an 86% ± 8% reduction in hepatic triglyceride content (P = 0.008 compared with before leptin treatment) and a 33% ± 3% decrease in muscle triglyceride content (P = 0.006 compared with before leptin treatment).
The leptin treatment-induced reduction in muscle triglyceride content was matched by an approximately 30% decrease in muscle total fatty acyl CoA concentrations. Leptin treatment also had a tendency, but not significant (refer to P value), to decrease glycerol turnover (78 ± 36 µmol/kg fat mass/min before leptin treatment versus 25 ± 7 µmol/kg fat mass/min after leptin treatment; P = 0.23).
Discussion
The lipodystrophic patients manifested severe hepatic and peripheral insulin resistance associated with diabetes, hyperlipidemia, and hepatic steatosis. These patients also had increased rates of glycerol turnover, reflecting increased rates of lipolysis in their residual fat mass. Given their almost complete lack of subcutaneous and visceral fat, these data suggest that there is a significant amount of intrahepatic or intravascular triglyceride lipolysis occurring in these individuals. Chronic leptin treatment caused a marked improvement in glycemic control in all three lipodystrophic patients and reduced their need for both oral hypoglycemic agents and insulin, and is con-sistent with the results of Shimomura et al. in aP2-SREBP-1c knockout lipodystrophic mice (4). The improved glycemic control could be attributed to a marked increase in their insulin responsiveness, as reflected by an almost fourfold increase in the rate of glucose infusion required to maintain euglycemia during the clamp, and these data
are consistent with the improved response to insulin tolerance testing seen in an entire cohort (5). In order to ascertain the mechanism for the improved insulin responsiveness, we also assessed rates of hepatic and peripheral glucose metabolism and found that leptin therapy caused an approximately twofold increase in insulin suppression of hepatic glucose production and an almost twofold increase in insulin-stimulated peripheral glucose disposal. These changes were associated with an approximately 85% reduction of hepatic triglyceride content and an approximately 30% reduction in intramyocellular triglyceride and fatty acyl CoA content.
Previous studies by our group (9, 17-20) and others (7, 21, 22) have demonstrated a strong relationship between triglyceride content in liver and muscle and insulin resistance in these tissues. These data support the hypothesis that a similar mechanism for insulin resistance might occur in patients with severe lipodystrophy and more common forms of insulin resistance associated with obesity and type 2 diabetes.
In summary, this report demonstrates that patients with severe lipodystrophy are severely insulin resistant, which can be attributed to defects in insulin action in both liver and muscle. Chronic leptin treatment caused a marked improvement in whole-body insulin-stimulated glucose metabolism, which could be attributed to both improved insulin sensitivity in the liver (as reflected by improved insulin suppression of glucose production) and in the muscle (as reflected by an increase in insulin-stimulated peripheral glucose uptake). These changes were associated with a marked reduction in hepatic and muscle triglyceride content. These data suggest that leptin therapy may represent an important new therapy to treat insulin resistance, hepatic steatosis, and the associated diabetes in patients with severe lipodystrophy.
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