OXIDATION OF FFA is a fundamental biochemical

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1 /02/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 87(5): Printed in U.S.A. Copyright 2002 by The Endocrine Society Metabolic Characterization of a Woman Homozygous for the Ser113Leu Missense Mutation in Carnitine Palmitoyl Transferase II MICHAEL HAAP, CLAUS THAMER, JÜRGEN MACHANN, OTTO TSCHRITTER, KLAUS LÖBLEIN, MONIKA KELLERER, FRITZ SCHICK, STEPHAN JACOB, HANS-ULRICH HÄRING, AND MICHAEL STUMVOLL Department of Endocrinology, Metabolism and Pathobiochemistry Eberhard-Karls-Universität Tübingen, Tübingen D-72076, Germany Carnitine palmitoyl transferase (CPT) II is a key enzyme in transporting FFA into the mitochondrial matrix for oxidation. The clinical manifestation of CPT II deficiency is characterized mainly by myopathic symptoms. Conceivably, the inability of skeletal muscle to oxidize (long-chain) FFAs could also have far-reaching metabolic consequences, such as insulin resistance secondary to increased muscle lipids, about which relatively little is known. We therefore performed a series of metabolic studies in a 43-yr-old woman homozygous for the Ser113Leu mutation in the CPT II gene, the single most common genetic cause of CPT II deficiency, and compared the results with data from a male and female control group taken from the Tübingen family study database. The metabolic studies included oral glucose tolerance test (OGTT), euglycemic hyperinsulinemic clamp to measure insulin sensitivity, indirect calorimetry to measure substrate oxidation, stable isotopes for determination of glycerol turnover, and magnetic Abbreviations: BMI, Body mass index; CPT, carnitine palmitoyl transferase; EC 50, effective concentration for half-maximal suppression; GIR, glucose infusion rate; IMCL, increased intramyocellular lipids; MRS, magnetic resonance spectroscopy; OGTT, oral glucose tolerance test; Ra, rate of appearance; SOL, soleus; SS, steady-state; TA, tibialis anterior. resonance spectroscopy for measurement of intramyocellular lipids. Compared with the female control group, the patient was normal glucose tolerant but severely insulin resistant, basal lipolysis was markedly reduced, and carbohydrate oxidation was maximally increased in the basal state and did not increase further during insulin stimulation. Conversely, lipid oxidation was virtually absent and did not decrease during insulin stimulation. Surprisingly, intramyocellular lipids were well within the range of the control group. In conclusion, genetic CPT II deficiency is characterized by insulin resistance, which is not explained by increased intramyomellular lipids. However, it may be partially explained by glucose oxidation already maximally increased in the basal state, which cannot be increased any further by insulin. Reduced basal lipolysis may represent a compensatory mechanism for the reduced oxidative FFA disposal characteristic for this disease. (J Clin Endocrinol Metab 87: , 2002) OXIDATION OF FFA is a fundamental biochemical pathway generating large amounts of energy in tissues such as cardiac and striated muscle. The enzymes carnitine palmitoyl transferase (CPT) I and II play a rate-limiting role in transporting long-chain fatty acids into the mitochondrium where the enzymes of -oxidation are located. Medium- and short-chain fatty acids, in contrast, can freely cross the mitochondrial membrane. Whereas CPT I is located on the outer mitochondrial membrane, CPT II transports acylcarnitine across the inner mitochondrial membrane and initiates activation to acyl-coa (1, 2). Genetic defects impairing activity of the CPT primarily affect liver (mainly CPT I deficiency) and muscle (mainly CPT II deficiency). Genetic defects in CPT II (3) are clinically characterized by myalgia, muscle weakness, and rhabdomyolysis, and therefore have been the focus primarily of myopathy research (4, 5). However, muscle tissue is of pivotal importance also for glucose homeostasis (6) and imbalance of muscle substrate utilization (carbohydrates vs. lipids) particularly in the insulin-stimulated state leads to insulin resistance and ultimately type 2 diabetes (7). Moreover, increased intramyocellular lipids (IMCL), which one would expect in CPT deficiency (8), have been associated (though not necessarily causally) with insulin resistance (9 14). This should make CPT II deficiency a scientific target also for the metabolism and diabetes field. To the authors knowledge, nothing is known about insulin sensitivity, substrate oxidation in the insulin-stimulated state, or lipolysis in patients with CPT II deficiency. It is also unknown whether the prevalence of type 2 diabetes is increased in patients with CPT II deficiency. In our department, we collect family members of patients with type 2 diabetes (and control subjects without family history) in an ongoing study with the primary purpose to relate genetic polymorphisms to pathomechanisms (for example, insulin resistance of lipolysis) relevant for type 2 diabetes. For this purpose, we established a series of clinical experimental procedures in our laboratory for use on a nearly routine basis. These techniques include OGTT, euglycemic hyperinsulinemic clamp, indirect calorimetry, stable isotopes for determination of glycerol turnover, magnetic resonance spectroscopy (MRS) for measurement of intramyocellular lipids. For the present studies, we used this panoply of investigative tools to characterize metabolic pathways in a 43-yr-old woman homozygous for the Ser113Leu missense mutation in the CPT II gene presenting the full-blown clinical picture of CPT II deficiency. This specific mutation has been 2139

2 2140 J Clin Endocrinol Metab, May 2002, 87(5): Haap et al. Metabolic Characterization of CPT II Deficiency estimated to account for 50% of all mutations involved in CPT II deficiency (15). Subjects Materials and Methods Woman with CPT II deficiency. A 43-yr-old Caucasian patient was admitted to our department for metabolic assessment. The clinical and molecular diagnosis of CPT II deficiency had been previously established in the department of neurology. It was based on the clinical presentation of recurrent episodes of myalgia, rhabdomyolysis, and myoglobinuria with and without renal failure beginning in early adolescence. The diagnosis was confirmed by demonstrating homozygosity of the Ser113Leu mutation in the CPT II gene in DNA obtained from a muscle biopsy. For several years, she has been on a fat-restricted diet (10% of total calories from fat) rich in medium- and short-chain fatty acids. Control group. A healthy control group was created from a preexisting database to establish a normal range for each parameter. For this purpose, male and female subjects were separately recruited to primarily match age and body mass index (BMI) of the patient. Eight subjects in each gender group were eligible (the subjects undergoing the lipolysis studies were different ones, and their characteristics are given separately). The clinical characteristics are shown in Table 1. All protocols were approved by the local ethics committee. After the nature of the study was explained, all subjects gave informed written consent. The primary recruitment purpose for the control group was presence (or absence) of a first-degree relative with type 2 diabetes within the ongoing Tübingen family study. The control group was asked to maintain their regular diet (containing approximately 50% of calories from carbohydrates, 20% from protein and 30% from fat) 3 d before the study days. OGTT A standard 75 g WHO OGTT was performed with determination of glucose and insulin at 0, 30, 60, 90, and 120 min. Glucose tolerance was classified as normal with a 120-min plasma glucose less than 140 mg/dl (7.8 mm). Hyperinsulinemic-euglycemic clamp TABLE 1. Baseline characteristics of the CPT II-deficient patient and the female and male control groups CPT IIdeficient patient Female controls Male controls Age (yr) a 39 3 a BMI (kg/m 2 ) a a WHR a a % Body fat a 18 1 a Fasting glucose (mmol/liter) a a Fasting insulin (pmol/liter) a 37 6 a Fasting FFA ( mol/liter) a a Data of the control groups are means SEM. a Indicates values for control group undergoing antilipolysis studies that contained different subjects. The hyperinsulinemic-euglycemic clamp was performed as previously described using an insulin infusion rate of 1.0 mu kg 1 min 1 for 2 h (16). The insulin sensitivity index (in mol kg 1 min 1 pm ) for systemic glucose uptake was calculated as mean infusion rate of exogenous glucose [glucose infusion rate (GIR) in mol kg 1 min 1 ] necessary to maintain euglycemia during the last 60 min of the clamp divided by the SS insulin concentration. The metabolic clearance rate of glucose was calculated as GIR divided by the SS glucose concentration. Indirect calorimetry Oxygen consumption and carbon dioxide production was measured using a Deltatrac II Metabolic Monitor (Deltatrac, Helsinki, Finland). Basal measurements were performed during the 20 min preceding the clamp, SS measurement in the insulin-stimulated state during the last 30 min of the clamp. Substrate oxidation rates were calculated as described previously (17). For urinary nitrogen excretion, a value of 11.5 g/24 h was assumed. Lipolysis studies The hyperinsulinemic-euglycemic clamp procedure was combined with the infusion of [ 2 H 5 ]glycerol (Cambridge Isotope Laboratories, Andover, MA; 1 mol/kg, 0.4 mol/min, started 2 h before the clamp) to determine glycerol rate of appearance. For these purpose arterialized blood samples were obtained at 20, 10, and 0 min before the start of the insulin infusion and at 30, 60, 90, 100, 110, and 120 min for determination of plasma [ 2 H 5 ]glycerol enrichments, plasma glycerol concentrations, serum insulin, and FFA concentrations. The plasma rate of appearance of glycerol (Ra) calculated by standard equations as previously described was used as index for systemic lipolysis (16). The effective insulin concentration for half-maximal suppression of lipolysis (EC 50 ) was estimated using the following equation: EC minus glycerol Ra 60 min (% decrease from baseline) times 0.79, which had been previously validated against the three-step hyperinsulinemic clamp (18). MRS Proton MRS enables differentiation between neutral lipids located inside muscle cells (intramyocellular lipids, IMCL) and those interlaced between the muscle fibers (extramyocellular lipids) due to their different geometrical arrangement. Localized image guided proton spectra with a voxel size of 2.4 cm 3 of the soleus muscle (SOL) representing a muscle of type I fibers with high oxidative capacity and M. tibialis anterior (TA) representing a muscle of mixed type I and II fibers were acquired on a 1.5 T whole body imager (Magnetom Vision, Siemens, Erlangen, Germany). IMCL and EMCL were quantified as previously described (19, 20). Analytical procedures Serum insulin was measured with a microparticle enzyme immunoassay (Abbott, Wiesbaden, Germany), serum FFA with an enzymatic method (WAKO chemicals, Neuss, Germany), plasma glycerol with an enzymatic method (Sigma Diagnostics, Deisenhofen, Germany). Plasma [ 2 H 5 ]glycerol enrichment was determined by gas chromatography massspectrometry using the trimethylsilyl derivative of glycerol. Electron impact ionization was applied and the mass-to-charge ratios 205 and 208 were monitored (21). Results The results are summarized in Table 2. The patient was normal glucose tolerant. The 4- to 5-fold increase in insulin by the end of the OGTT compared with the control group indicates that insulin resistance was present and endogenous hyperinsulinemia was necessary to maintain normal glucose tolerance. Consistent with this interpretation, during the hyperinsulinemic euglycemic clamp the GIR was only 50% of the female control group resulting in a very low insulin sensitivity index of about 25% of the control group. This reflects a high degree of insulin resistance. Basal glycerol Ra, the index of whole body lipolysis, was 35% lower than in the control group, probably as a consequence of the hyperinsulinemia. The steady-state (SS) glyc-

3 Haap et al. Metabolic Characterization of CPT II Deficiency J Clin Endocrinol Metab, May 2002, 87(5): TABLE 2. Results of metabolic studies in the CPT II-deficient patient and the female and male control groups CPT II Deficient patient Female controls Male controls Glucose tolerance Glucose 120 min OGTT (mmol/liter) Insulin 120 min OGTT (pmol/liter) Insulin sensitivity ISI ( mol kg 1 min 1 pm 1 ) GIR ( mol kg 1 min 1 ) MCR (ml kg 1 min 1 ) Antilipolysis Basal glycerol Ra ( mol kg 1 min 1 ) SS glycerol Ra ( mol kg 1 min 1 ) Suppression lipolysis, insulin EC 50 (pmol/liter) Substrate oxidation Basal carbohydrate oxidation (mg kg 1 min 1 ) SS carbohydrate oxidation (mg kg 1 min 1 ) Basal lipid oxidation (mg kg 1 min 1 ) SS lipid oxidation (mg kg 1 min 1 ) Basal respiratory quotient SS respiratory quotient Muscle MR spectroscopy IMCL SOL (a.u.) IMCL TA (a.u.) Data of the control groups are means SEM. SS, Steady-state during the hyperinsulinemic euglycemic clamp. erol Ra during hyperinsulinemia and the insulin concentration resulting in half-maximal suppression of lipolysis (insulin EC 50 ) was comparable to the mean of the control groups. Interestingly, in the basal state carbohydrate oxidation was increased 2-fold, whereas lipid oxidation was virtually absent. During insulin stimulation, carbohydrate oxidation in the patient was not increased any further, whereas it nearly doubled in the controls. Analogously, basal lipid oxidation was only one fifth of the mean control value and did not decrease any further during insulin stimulation. Also, the respiratory quotient which is independent of any estimates for protein oxidation (i.e. urinary nitrogen excretion) confirmed the predominant oxidation of carbohydrates that could not be increased further by insulin. Intramyocellular lipid contents in SOL and TA muscle determined by MRS were well within the range of the control group and not substantially different from their mean values. Discussion The purpose of the present studies was to identify metabolic abnormalities in a patient with the single most common genetic form of CPT II deficiency. Because formal statistical comparisons were not possible for the interpretation of the patient s data we related the results to means sem of a control group (women and men matched for BMI and age) and assumed those to be a representative of the normal range. The normal weight patient was characterized by normal glucose tolerance despite severe insulin resistance reflected by the low glucose infusion rate during the euglycemic clamp. At the present stage, the cell function could obviously compensate for the insulin resistance. Pronounced hyperinsulinemia was present in the basal state and at 120 min during the OGTT. Interestingly, in the basal fasting state carbohydrate oxidation was substantially increased, whereas lipid oxidation was nearly absent (also reflected by the RQ close to 1). Most notably, in the insulin-stimulated state glucose oxidation did not increase any further and the RQ remained unchanged. This strongly indicates that carbohydrate oxidation was maximally turned on and could not be stimulated any more by insulin. Even with glucose oxidation maximally turned on in the basal state, insulin should still stimulate glycogen synthesis during the euglycemic clamp resulting in an increase in glucose infusion rate. The fact that this was not the case we believe has two explanations: for some unknown reason (possible related to the primary defect) glycogen-synthesis in muscle is indeed impaired. Alternatively, the explanation could reside extramuscularly. The glucose infusion rate during the euglycemic clamp not only reflects stimulation of peripheral (mainly muscle) glucose disposal but also suppression of endogenous (mainly hepatic) glucose production. It is of note in this context that rats treated with the CPT I inhibitor etomoxir had a paradoxic increase in endogenous glucose production in the insulin-stimulated state (8). This observation could not be satisfactorily explained by the authors. An analogous mechanism might have been operative in our patient and the reduced or absent suppression of endogenous glucose production during the clamp could explain the reduced requirements for exogenous glucose. Basal glycerol rate of appearance was only 65% of the mean control value indicating a substantial reduction in fasting lipolysis. In the insulin-stimulated state glycerol Ra was within the normal range. The insulin EC 50 for suppression of lipolysis was only marginally greater than the mean of the control group indicating only slightly reduced insulin sensitivity of lipolysis. The reduced basal lipolysis rate may reflect a compensatory mechanism operative in a patient with CPT II deficiency to reduce availability of FFAs. Con-

4 2142 J Clin Endocrinol Metab, May 2002, 87(5): Haap et al. Metabolic Characterization of CPT II Deficiency sistent with this explanation, circulating FFAs were not elevated although disposal of FFA (at least of long-chain FFA) by definition was. It is unclear by which signal reduced FFA oxidation mediates reduced FFA generation via lipolysis. Conceivably, the absolute concentration of long-chain FFAs or the proportion relative to medium- and short-chain FFAs could be involved. In addition to serving as metabolic substrate FFAs have been shown to interfere with insulin signaling (11) and to act as ligand for transcription factors such as the PPAR family (22). A correlation between increased muscle lipids and insulin resistance has been known for many years (7, 9 14, 23 27). More, recently MRS has facilitated the noninvasive study of intramyocellular lipids in humans in vivo. This method in fact allows distinction between triglycerides located within the muscle cell, i.e. intramyocellularly vs. triglycerides surrounding muscle cells i.e. extramyocellularly (19). Data obtained by this elegant method indicate that a correlation exists also between increased intramyocellular lipids and insulin resistance in otherwise healthy subjects (13, 14). It is unclear, however, whether this relationship is causal in nature or whether it merely reflects a common primary defect resulting in both increased IMCL and reduced insulin-stimulated glucose disposal. Because FFA oxidation is one important pathway of intracellular lipid disposal in muscle its inhibition could well result in excessive lipid accumulation. Moreover, inhibition of CPT I by etomoxir in rats resulted in increased intramyocellular lipids accompanied by insulin resistance (8). In our patient with CPT deficiency, however, IMCL in both TA and SOL muscle was well within the range of the control groups. The very low lipids diet of the patient may provide a partial explanation for this observation. It is also possible that despite the normal IMCL content the proportion of longchain acyl CoAs is increased. This cannot be differentiated by our method. In addition, negative feedback of intracellular FFAs on transport of FFAs into the cell may exist protecting muscle from excess lipid accumulation. Our data also indicate that the amount of IMCL per se does not necessarily predict insulin resistance. This suggests that the link between increased IMCL and insulin resistance is rather complex and its understanding requires a much better insight into the regulation of IMCL turnover. It is necessary to point out that our control group was not on the same diet as the patient. The control group maintained a diet containing about 20% of fat, whereas the patient s diet was essentially fat free. The normal IMCL values may well reflect the fact that the patient did indeed strictly adhere to this diet. And the control group might have substantially lower IMCL contents when subjected to a very low fat diet. Nevertheless, it is still interesting to note that despite this severe defect the high carbohydrate diet prevented muscle from being overloaded with intracellular triglycerides. In conclusion, genetic CPT II deficiency is characterized by insulin resistance, which is not explained by increased intramyocellular lipids. However, it may be partially explained by glucose oxidation already maximally increased in the basal state and unable to increase any further upon insulin stimulation. Reduced basal lipolysis may represent a compensatory mechanism for the reduced oxidative FFA disposal characteristic for this disease. Acknowledgments We want to thank our laboratory staff for the excellent technical support. 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