Effects of Insulin Treatment in Type 2 Diabetic Patients on Intracellular Lipid Content in Liver and Skeletal Muscle

Christian Anderwald 0 Elisabeth Bernroider 0 Martin Krssa k 0 Harald Stingl 0 Attila Brehm 0 Martin G. Bischof 0 Peter Nowotny 0 Michael Roden 0 Werner Waldh ausl 0 0 From the Division of Endocrinology and Metabolism, Department of Internal Medicine III, University of Vienna , Vienna , Austria . Endocrinology and Metabolism , Department of Internal Medicine III , Univer- sity of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria Insulin resistance is frequently associated with increased lipid content in muscle and liver. Insulin excess stimulates tissue lipid accumulation. To examine the effects of insulin and improved glycemia on insulin sensitivity and intracellular lipids, we performed stepped (1, 2, and 4 mU min1 kg1 ) hyperinsulinemiceuglycemic clamps in eight type 2 diabetic and six nondiabetic control subjects at baseline and after 12 and 67 h of insulin-mediated near-normoglycemia (118 7 mg/dl). Intrahepatocellular lipids (IHCLs) and intramyocellular lipids (IMCLs) of soleus (IMCL-S) and tibialis anterior muscle (IMCL-TA) were measured with 1H nuclear magnetic resonance spectroscopy. At baseline, nondiabetic subjects had an approximate twofold higher insulin sensitivity (P < 0.02) and lower IHCLs than diabetic patients (5.8 1.2 vs. 18.3 4.2%, P < 0.03), in whom IMCL-TA negatively correlated with insulin sensitivity (r 0.969, P < 0.001). After a 67-h insulin infusion in diabetic patients, IMCL-S and IHCLs were increased (P < 0.05) by 36 and 18%, respectively, and correlated positively with insulin sensitivity (IMCL-S: r 0.982, P < 0.0005; IHCL: r 0.865, P < 0.03), whereas fasting glucose production, measured with D-[6,6-2H2]glucose, decreased by 10% (P < 0.04). In conclusion, these results indicate that IMCLs relate to insulin resistance in type 2 diabetic patients at baseline and that insulin-mediated near-normoglycemia for 3 days reduces fasting glucose production but stimulates lipid accumulation in liver and muscle without affecting insulin sensitivity. Diabetes 51:3025-3032, 2002 - Iskeletal muscle and fat, and represents the major nsulin resistance is commonly defined by reduced insulin sensitivity of peripheral tissues, such as abnormality of the metabolic syndrome and type 2 diabetes (1). Likewise, impaired suppression by insulin of endogenous glucose production (EGP) in these disorders is considered to indicate hepatic insulin resistance (2), which correlates with plasma free fatty acids (FFAs) (3) and intrahepatocellular lipids (IHCLs) (4 6). Increased plasma FFAs also reflect skeletal muscle insulin resistance (7,8) and induce a rise in intramyocellular lipid (IMCL) content at high but not low plasma insulin concentrations (9 11). In some muscles, IMCLs correlate negatively with whole-body glucose disposal in both insulin-sensitive and -resistant nondiabetic subjects (1214). Recently, evidence was provided that subjects with increased IMCLs (15) exhibit impaired insulin signal transduction in skeletal muscle (7,8,11,14,16). However, it is not clear whether muscle lipid accumulation primarily induces insulin resistance by defects in insulin signaling or is simply a secondary phenomenon of impaired insulin sensitivity due to reduced lipid oxidation (17). In insulin-resistant states, including type 2 diabetes, abnormalities in insulin signaling (18) coexist with increased plasma insulin concentrations (19), but with progression of the disease, impaired insulin secretion fails to compensate for the decrease in insulin sensitivity (20). The resulting rise in plasma glucose can be normalized by insulin treatment at the expense of peripheral hyperinsulinemia (21). Thereby, insulin not only stimulates lipogenesis, but could also increase IMCLs and IHCLs and alter whole-body insulin sensitivity (2124). This study was therefore designed to examine the relationship between whole-body insulin sensitivity and EGP with IMCLs and IHCLs before as well as after short-term (12-h) and prolonged (67-h) insulin-mediated near-normoglycemia in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS Study participants. Eight type 2 diabetic patients with known duration of disease 10 years were recruited. They were hyperglycemic, and most of them were overweight and dyslipidemic (Table 1). None had been treated with insulin before or presented with type 1 diabetesrelated antibodies (25) or any liver or kidney diseases. Type 2 diabetic patients with hypertension, cardiovascular diseases, or diabetic late complications were excluded from the study. Female patients were postmenopausal. Treatment with diuretics was TABLE 1 Baseline clinical characteristics as well as fasting plasma/serum laboratory parameters of eight type 2 diabetic patients ALT, alanine transaminase; AST, aspartate transaminase. discontinued for at least 1 week and any other medication, including oral hypoglycemic agents, for at least 3 days before the study. The control group consisted of healthy nondiabetic subjects (four men and two women, aged 57 2 years, body weight 78 6 kg, BMI 25.7 1.1 kg/m2; NS vs. type 2 diabetic patients) exhibiting normal fasting and 2-h plasma glucose during a standard 75-g oral glucose challenge. Fasting plasma glucose (99 1 mg/dl), HbA1c (5.5 0.1%), FFAs (556 46 mol/l), and triglycerides (122 27 mg/dl) were lower (each P 0.02), whereas serum cholesterol (total 202 14 mg/dl, HDL 55 9, LDL 123 12) was similar to type 2 diabetic patients (Table 1). Control subjects also had normal liver and kidney function (serum creatinine 1.1 0.0 mg/dl, aspartate transaminase 10 0 units/l, alanine transaminase 10 1 units/l). All participants gave informed consent to the protocol, which was approved by the institutional ethics board. Study design. Type 2 diabetic and control subjects were instructed to ingest an isocaloric carbohydrate-rich diet from day 7 onwards. Type 2 diabetic patients were admitted to the hospital at least 1 day before the studies and given an isocaloric diet (25 kcal day1 kg1 body wt; 50% carbohydrate, 15% protein, and 35% fat) divided into five meals (7:30 A.M., 11:00 A.M., 12:30 P.M., 4:30 P.M., and 7:00 P.M.) until day 6. Intracellular lipids, EGP, and whole-body insulin sensitivity were measured after a fast for at least 10 h on study days 1 (P-D1), 3 (P-D3), and 6 (P-D6) for type 2 diabetic patients and on study day 1 for control subjects (C-D1). At 5:50 A.M., two catheters (Vasofix; Braun, Melsungen, Germany) were inserted into one antecubital vein of the left and right arm for blood sampling and infusions, respectively. Starting at 6:00 A.M., a primed-continuous infusion (5 min: 3.6 mg kg body wt fasting glucose [mg/dl]/90 [mg/dl]; 475 min: 0.036 mg/min kg body wt) of D-[6,6-2H2]glucose (98% enriched; Cambridge Isotope Laboratories, Andover, MA) was performed until the end of the clamp to determine EGP (26). At 7:00 A.M., all participants were transported by wheelchair to the MR Unit to measure IHCLs and IMCLs. From 9:00 A.M. to 2:00 P.M., three s (...truncated)