Agrowing literature suggests that impaired cardiac

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1 ORIGINAL ARTICLE Fatty Acids and Insulin Modulate Myocardial Substrate Metabolism in Humans With Type 1 Diabetes Linda R. Peterson, 1,2,3 Pilar Herrero, 3 Janet McGill, 4 Kenneth B. Schechtman, 5 Zulfia Kisrieva-Ware, 3 Donna Lesniak, 3 and Robert J. Gropler 1,3 OBJECTIVE Normal human myocardium switches substrate metabolism preference, adapting to the prevailing plasma substrate levels and hormonal milieu, but in type 1 diabetes, the myocardium relies heavily on fatty acid metabolism for energy. Whether conditions that affect myocardial glucose use and fatty acid utilization, oxidation, and storage in nondiabetic subjects alter them in type 1 diabetes is not well known. RESEARCH DESIGN AND METHODS To test the hypotheses that in humans with type 1 diabetes, myocardial glucose and fatty acid metabolism can be manipulated by altering plasma free fatty acid (FFA) and insulin levels, we quantified myocardial oxygen consumption (MVO 2 ), glucose, and fatty acid metabolism in nondiabetic subjects and three groups of type 1 diabetic subjects (those studied during euglycemia, hyperlipidemia, and a hyperinsulinemic-euglycemic clamp) using positron emission tomography. RESULTS Type 1 diabetic subjects had higher MVO 2 and lower myocardial glucose utilization rate/insulin than control subjects. In type 1 diabetes, glucose utilization increased with increasing plasma insulin and decreasing FFA levels. Myocardial fatty acid utilization, oxidation, and esterification rates increased with increasing plasma FFA. Increasing plasma insulin levels decreased myocardial fatty acid esterification rates but increased the percentage of fatty acids going into esterification. CONCLUSIONS Type 1 diabetes myocardium has increased MVO 2 and is insulin resistant during euglycemia. However, its myocardial glucose and fatty acid metabolism still responds to changes in plasma insulin and plasma FFA levels. Moreover, insulin and plasma FFA levels can regulate the intramyocardial fate of fatty acids in humans with type 1 diabetes. Diabetes 57: 32 40, 2008 From the 1 Cardiovascular Division, Department of Internal Medicine, Mallinckrodt Institute, Washington University School of Medicine, St. Louis, Missouri; the 2 Division of Geriatrics and Nutritional Sciences, Department of Internal Medicine, Mallinckrodt Institute, Washington University School of Medicine, St. Louis, Missouri; the 3 Mallinckrodt Institute, Department of Radiology, Washington University, St. Louis, Missouri; the 4 Division of Endocrinology, Department of Medicine, Washington University, St. Louis, Missouri; and the 5 Division of Biostatistics, Washington University School of Medicine, St. Louis, Missouri. Address correspondence and reprint requests to Linda R. Peterson, MD, Campus Box 8086, 660 S. Euclid Ave., St. Louis, MO lpeterso@im.wustl.edu. Received for publication 24 August 2007 and accepted 1 October Published ahead of print at on 3 October DOI: /db FFA, free fatty acids; HIEG, hyperinsulinemic euglycemic; MVO 2, myocardial oxygen consumption; PET, positron emission tomography by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Agrowing literature suggests that impaired cardiac metabolism in diabetes contributes to cardiac dysfunction, leading to overt heart failure, a so-called diabetic cardiomyopathy (1 4). Results from studies in animal models of type 1 diabetes show that myocardial substrate metabolic alterations may play a key role in the development of diabetic cardiomyopathy (5). Specifically, in diabetes, there is increased lipid delivery to the myocardium, myocardial fatty acid utilization and oxidation, and decreased myocardial glucose uptake and triglyceride hydrolysis (2,6,7). This excessive myocardial fatty acid utilization may be detrimental to cardiac function via impairing glucose utilization (particularly when glucose is required, e.g., ischemia), decreasing transsarcolemmal calcium flux induced contractility and/or increasing free radical production (8 11). Moreover, decreasing free fatty acid (FFA) delivery to the myocardium in animal models decreases its fat storage and improves function (12). These results suggest that metabolic modulation of myocardial substrate metabolism may be a new paradigm for treatment of diabetic cardiomyopathy (12,13). There is little data on the effects of altering FFA or hormonal delivery to the myocardium on myocardial fatty acid utilization in humans with type 1 diabetes. Furthermore, these alterations effect on the fate of intramyocellular fatty acids is unknown. Thus, we hypothesized that although overdependence on myocardial fatty acid metabolism was present in humans with type 1 diabetes, the myocardium would still be responsive metabolically to the prevailing plasma hormonal and substrate milieu. To prove or disprove this hypothesis, we quantified myocardial fatty acid and glucose metabolism using positron emission tomography (PET) in nondiabetic control subjects and three groups of subjects with diabetes: those studied during euglycemia, hyperlipidemia, and during a hyperinsulinemic euglycemic (HIEG) clamp. RESEARCH DESIGN AND METHODS We studied 34 type 1 diabetic subjects and 12 nondiabetic control subjects. We chose to study patients with type 1 diabetes to avoid the possible confounding effects of obesity and hypertension that often accompany type 2 diabetes (14,15). Type 1 diabetes classification was based on the need for supplemental insulin during the first year, history of ketoacidosis, and plasma C-peptide level 0.50 mol/ml. No subject had active retinopathy, clinically significant autonomic neuropathy, or serum creatinine 1.5 mg/dl. Sedentary subjects were chosen to minimize confounding effects of training-induced adaptations on substrate metabolism (16). All were nonsmokers and normotensive. Cardiac disease was excluded by a normal physical exam and normal rest/exercise echocardiograms. Subjects were not taking any vasoactive medications at the time of the study and did not have other systemic illnesses. Type 1 diabetic subjects were randomly assigned to one of three groups. Twelve were studied under resting euglycemic conditions, 10 during an Intralipid infusion, and 12 during an HIEG clamp. The nondiabetic control 32 DIABETES, VOL. 57, JANUARY 2008

2 L.R. PETERSON AND ASSOCIATES TABLE 1 Demographics, metabolic characteristics, hemodynamics, and cardiac structure and function of nondiabetic versus type 1 diabetic euglycemic subjects Nondiabetic Type 1 diabetic/euglycemic P n Age (years) Sex (men) Race (white) BMI (kg/m 2 ) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) Heart rate (bpm) Systolic blood pressure (mmhg) Diastolic blood pressure (mmhg) Average rate-pressure product during MVO 2 (mmhg bpm) LV mass index (g/m 2 ) Ejection fraction (%) Data are means SD or percentages unless otherwise indicated. LV, left ventricular. subjects were age, sex, and BMI matched with the euglycemic type 1 diabetic subjects. The study was approved by the Human Studies Committee and the Radioactive Drug Research Committee at the Washington University School of Medicine. Written informed consent was obtained from all subjects before enrollment in the study. All studies were performed on a Siemens tomograph (ECAT 962 HR ; Siemens Medical Systems, Iselin, NJ). All subjects were admitted overnight to the General Clinical Research Center and fasted for 12 h before the study. Two intravenous catheters were placed: one for infusion and one for blood sampling. All were studied at 8:00 A.M. to avoid circadian variations in metabolism (17). In the euglycemia group, the substrate environment was standardized using a low-dose insulin infusion with or without D5W infusion to maintain blood glucose level in the 80- to 120-mg/dl range. The Intralipid subjects were given a heparin bolus intravenously (7.0 units/kg heparin) followed by a continuous infusion of heparin/intralipid mixture (5, 000 units heparin in 500 cc Intralipid) at 0.7 ml kg 1 h 1. They also received a regular insulin infusion with or without D5W infusion to maintain blood glucose in the 80- to 120-mg/dl range. Subjects in the HIEG clamp group were started on the clamp 2 h before the PET imaging session (18). Nondiabetic control subjects were studied after the same fasting period as that undergone by the type 1 diabetic subjects. All subjects were on telemetry and had blood pressures obtained throughout the study. Rate-pressure product was calculated as systolic blood pressure heart rate. Myocardial oxygen consumption (MVO 2 ), blood flow, fatty acid, and glucose metabolism were measured after injections of 15 O-water, 11 C-acetate, 1-11 C-palmitate, and 1-11 C-glucose, respectively. During the study, plasma substrates and insulin levels were measured. 11 CO 2 and 11 C-lactate activity were also measured to correct the PET data as required for compartmental modeling of the myocardial kinetics of the metabolic tracers (19,20). PET image analysis. Myocardial 15 O-water, 11 C-acetate, 1-11 C-palmitate, and 1-11 C-glucose images were generated. Blood and myocardial time-activity curves were generated as reported previously (19 22). The curves were used in conjunction with well-established kinetic models to quantify myocardial blood flow, MVO 2, fatty acid, and glucose extraction fractions (20,22). Myocardial fatty acid/glucose utilization was calculated as the product of myocardial fatty acid/glucose extraction fraction myocardial blood flow (plasma FFA/glucose). Myocardial fatty acid extraction fraction was also divided into the portion of extracted fatty acids oxidized and the portion that entered slow turnover pools or esterification. Myocardial fatty acid oxidation was calculated as the fraction of the oxidized myocardial fatty acids myocardial blood flow (plasma FFAs). Myocardial fatty acid esterification was calculated as the fraction of the myocardial fatty acids that entered slow-turnover pools myocardial blood flow (plasma FFAs). Measurement of plasma insulin and substrates. Plasma insulin levels were measured by radioimmunoassay. Plasma glucose and lactate levels were measured using a glucose-lactate analyzer (Yellow Springs Instruments, Yellow Springs, OH). Plasma FFA level was determined by capillary gas chromatography and high-performance liquid chromatography. Echocardiography. During the PET study, subjects had a complete twodimensional and Doppler echocardiographic examination using a Sequoia- C256 (Acuson-Siemens, Mountain View, CA). Left ventricular mass index, ejection fraction, relative wall thickness, and Em were obtained as previously described (14,23). Statistical analysis. SAS software (SAS Institute) was used for statistical analyses. Data are expressed as means SD. Comparisons of continuous and categorical variables between nondiabetic control subjects and the euglycemic type 1 diabetes group were made using unpaired Student s t and 2 tests, respectively. Comparisons among the three type 1 diabetic groups were made using ANOVA for continuous variables and the 2 test for categorical variables. Pearson correlations were performed to determine the univariate relationships between the independent variables (age, BMI, diabetes duration, A1C, rate-pressure product, and plasma insulin level) and the predetermined dependent variables (myocardial glucose extraction fraction and utilization; myocardial fatty acid utilization, oxidation, and esterification; percentage of myocardial fatty acid utilization oxidized; and percentage of myocardial fatty acid utilization esterified). A Pearson correlation was also used to determine the relationship between plasma insulin and plasma FFAs and the relationship between plasma FFA level (during the 1-C 11 -glucose imaging) and myocardial glucose extraction fraction and utilization. (The relationship between plasma FFA and the dependent variables regarding myocardial fatty acid utilization and metabolism was not analyzed because plasma FFA is used in their calculation.) For entry into multivariate analyses, an independent variable was required to have a significant relationship with the dependent variables at the P 0.10 level. Type III sum of squares multivariate analyses were used to determine the independent predictors of dependent variables. RESULTS Patients in the euglycemic type 1 diabetic group and nondiabetic control subjects were not different in terms of age, sex, race, metabolic profiles, left ventricular structure, or function. Only diastolic blood pressure was higher, and rate-pressure product trended toward being higher, in the type 1 diabetic subjects (Table 1 ). As shown in Table 2, the three type 1 diabetic groups were also well matched in demographics, metabolic characteristics, hemodynamics, and left ventricular structure and function. Myocardial blood flow and MVO 2. Myocardial blood flow between the euglycemic type 1 diabetic group and the nondiabetic control subjects did not differ. However, MVO 2 was higher in the type 1 diabetic group, and their MVO 2 per rate-pressure product was trending toward being higher than that in nondiabetic control subjects (Table 3). There were no differences in myocardial blood flow among the euglycemia ( ml g 1 min 1 ), Intralipid ( ml g 1 min 1 ), and HIEG clamp ( ml g 1 min 1 ) groups (P 0.83). There were no differences in DIABETES, VOL. 57, JANUARY

3 MYOCARDIAL METABOLISM MANIPULATION IN DIABETES TABLE 2 Demographics, metabolic characteristics, hemodynamics, and cardiac structure and function in the three groups of type 1 diabetic subjects Euglycemia Intralipid HIEG clamp P n Age (years) Sex (men) Race (white) BMI (kg/m 2 ) Type 1 diabetes duration (years) A1C (%) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) Heart rate (bpm) Systolic blood pressure (mmhg) Diastolic blood pressure (mmhg) Average rate-pressure product (mmhg bpm) LV mass index (g/m 2 ) Ejection fraction (%) Data are means SD or percentages unless otherwise indicated. LV, left ventricular. MVO 2 among the same groups ( [Intralipid] and mol g 1 min 1 [HIEG clamp], P 0.47). Plasma substrates, insulin, and myocardial glucose metabolism. The type 1 diabetic euglycemic group had higher plasma glucose levels and higher insulin levels compared with those of control subjects. However, plasma FFA levels did not differ between the two groups. Myocardial glucose extraction fraction and utilization were not different between the two groups, but myocardial glucose utilization/plasma insulin level was lower in type 1 diabetic compared with control subjects (Table 3). There were no differences in plasma glucose concentrations at the time of the PET 1-11 C-glucose imaging among the three groups ( , , and mmol l 1 l 1 for euglycemia, Intralipid, and HIEG clamp groups, respectively; P 0.67). There were marked differences in plasma insulin levels (Fig. 1A), with the HIEG clamp group having higher levels than the other two groups. Myocardial glucose extraction fraction and utilization paralleled the changes in insulin, with the HIEG clamp group having the highest myocardial glucose extraction fraction and utilization levels (Fig. 1B and C). Furthermore, the higher the plasma insulin level, the higher the myocardial glucose extraction fraction (r 0.53, P 0.005) and glucose utilization (r 0.48, P 0.005). Conversely, the higher the plasma FFA levels, the lower the myocardial glucose extraction fraction and utilization (Fig. 2). Interestingly, in the multivariate analyses only plasma FFA level was an independent predictor of myocardial glucose extraction fraction and utilization (P 0.05 and P 0.01; correlation between plasma FFAs and insulin levels was r 0.65, P ). Plasma substrates and insulin levels and myocardial fatty acid metabolism. Table 3 shows that neither plasma FFA nor any of the measures of myocardial fatty acid uptake or metabolism was different between the euglycemic type 1 diabetic subjects and fasting nondiabetic control subjects. Plasma FFA levels were markedly different among the three type 1 diabetic subject groups, with the HIEG clamp group having the lowest levels (Fig. 3A). Plasma FFA levels were higher in the Intralipid group than in the euglycemia group. Of note, myocardial fatty TABLE 3 Measurements of plasma substrate, insulin levels, and myocardial perfusion and metabolism in nondiabetic control versus type 1 diabetic euglycemic subjects Nondiabetic Type 1 diabetic/euglycemic P n Myocardial blood flow (ml g 1 min 1 ) MVO 2 ( mol g 1 min 1 ) MVO 2 ( 1,000)/rate-pressure product ( mol g 1 min 1 / bpm mmhg ) Plasma glucose ( mol/ml) Plasma insulin ( mol/ml) Myocardial glucose utilization (nmol g 1 min 1 ) Myocardial glucose utilization/plasma insulin ( nmol g 1 min 1 / mol/ml ) Plasma FFA (nmol/ml) Myocardial FA utilization (nmol g 1 min 1 ) Myocardial FA oxidation (nmol g 1 min 1 ) Myocardial FA esterification (nmol g 1 min 1 ) Myocardial FA oxidation (%) Data are means SD unless otherwise indicated. FA, fatty acid. 34 DIABETES, VOL. 57, JANUARY 2008

4 L.R. PETERSON AND ASSOCIATES FIG. 1. The data in the three groups of type 1 diabetic patients are shown as means SD. The P values for the difference among the three groups by ANOVA are in bold. The unadjusted pairwise comparisons are in regular type. f, euglycemia;, Intralipid plus heparin; o, HIEG clamp. acid extraction fraction was fairly constant among the three groups (Fig. 3B). In contrast, myocardial fatty acid utilization was markedly lower in the HIEG clamp group compared with that in either the euglycemia or Intralipid group (Fig. 3C), paralleling the differences in plasma FFA levels. Myocardial fatty acid utilization was strongly and negatively correlated with plasma insulin level (r 0.60, P ), and it was the only independent predictor of myocardial fatty acid utilization (P ). The change in fatty acid oxidation also mirrored the differences in plasma FFA levels and myocardial fatty acid utilization, with a difference among the three groups and significant differences between the HIEG clamp group and the other two (Fig. 4A). Myocardial fatty acid oxidation, FIG. 2. The negative effects of plasma FFA levels on myocardial glucose metabolism in patients with type 1 diabetes, consistent with the Randle cycle. A: The higher the plasma FFA, the lower the myocardial glucose extraction fraction. B: Similarly, the higher the plasma FFA level, the lower the myocardial glucose utilization. DIABETES, VOL. 57, JANUARY

5 MYOCARDIAL METABOLISM MANIPULATION IN DIABETES FIG. 3. The effects of changing plasma FFA and insulin levels on myocardial fatty acid extraction and utilization in patients with type 1 diabetes. The data are shown as means SD. The P values are in bold for the difference among the three groups by ANOVA. Unadjusted pairwise comparisons are in regular type. f, euglycemia;, Intralipid plus heparin; o, HIEG clamp. like fatty acid utilization, inversely correlated with insulin level (r 0.57, P 0.001), and it was the only independent predictor of fatty acid oxidation in a multivariate model (P 0.001). To evaluate the differences in fractional myocardial fatty acid oxidation among the groups apart from the influence of plasma FFA levels, we also evaluated the differences in percentage of oxidation among the three groups. The FIG. 4. Changing plasma FFA and insulin levels modulates the fate of fatty acids after they enter the myocardium in type 1 diabetic patients. The data are shown as means SD. The P values for the difference among the three groups by ANOVA are in bold. Unadjusted pairwise comparisons are in regular type. f, euglycemia;, Intralipid plus heparin; o, HIEG clamp. 36 DIABETES, VOL. 57, JANUARY 2008

6 L.R. PETERSON AND ASSOCIATES FIG. 5. Changing plasma FFA and insulin levels modulates fatty acid esterification within the myocardium. A: Increasing plasma FFA levels increases the percent shunted to nonoxidative, slow turnover pathways, grouped under the term esterification. High plasma insulin decreases the absolute amount of myocardial fatty acid esterification because of the marked decrease in plasma FFA delivered to the myocardium. B: An increase in plasma FFAs (Intralipid and heparin group) is trending toward increasing the percentage of the myocardial fatty acid utilization accounted for by esterification; the highest increases in the anabolic hormone insulin (HIEG clamp group) have the highest percentage of fatty acids that are shunted toward esterification within the myocardium. The data are shown as means SD. The P values for the difference among the three groups by ANOVA are in bold. The significant unadjusted pairwise comparisons are in regular type. f euglycemia;, Intralipid plus heparin; o, HIEG clamp. percentage of the total myocardial fatty acid utilization accounted for by oxidation was highest in the euglycemic group and lowest in the HIEG clamp group (Fig. 4B). There were also significant differences in myocardial fatty acid esterification among the three groups. In this comparison, the Intralipid group had the highest level compared with the other two groups (Fig. 5A). As with myocardial fatty acid utilization, esterification was inversely related to plasma insulin levels (Fig. 6); myocardial fatty acid esterification also correlated significantly but FIG. 6. Contrasting relationships between plasma insulin level and total amount of myocardial fatty acid esterification and between plasma insulin level. The higher the plasma insulin level, the lower the total amount of myocardial fatty acid esterification, paralleling the inverse relationship between plasma insulin and FFA levels (r 0.65, P < ). positively with age (r 0.39, P 0.05) and duration of diabetes (r 0.38, P 0.05). Multivariate analysis demonstrated that both plasma insulin and age were independent predictors of myocardial fatty acid esterification (P 0.05 and P 0.01, respectively). Since the percentage of myocardial fatty acid utilizationaccounted for by myocardial fatty acid esterification is by definition the percentage of fatty acid not oxidized, the HIEG clamp group had the highest percentage of myocardial fatty acid utilization going to esterification and the euglycemic group had the lowest (Fig. 5B). Since in the calculation of the percentage of oxidation or esterification the concentration of plasma FFAs drops out, this suggests that the differences in percentage of myocardial fatty acid oxidation and esterification cannot be explained solely by differences in plasma FFA levels. Thus, although overall myocardial esterification correlated inversely with plasma insulin levels, the percentage of myocardial utilization accounted for by esterification correlated directly with plasma insulin level (r 0.39, P 0.05), and it was the only independent predictor of the percentage of myocardial esterification (P 0.05). DISCUSSION Results of prior studies have shown that the myocardium in humans with type 1 diabetes relies more heavily on myocardial fatty acid (as opposed to glucose) metabolism than that in nondiabetic subjects (7,24,25). The results of our study further extend this concept. First, our results show that when type 1 diabetic subjects are euglycemic and fasting and have similar plasma FFA levels, they have myocardial fatty acid metabolism similar to that in nondiabetic fasting control subjects. This occurs despite type 1 diabetic subjects exhibiting higher plasma insulin levels. In our previous study of type 1 diabetes, we sought to match insulin and glucose (rather than FFA) levels between nondiabetic and type 1 diabetic subjects, so the DIABETES, VOL. 57, JANUARY

7 MYOCARDIAL METABOLISM MANIPULATION IN DIABETES control subjects were fed. In that study, myocardial fatty acid utilization increased in the type 1 diabetic subjects primarily as a result of the increase in plasma FFA levels (7). Taken together, these results further highlight the importance of increased plasma FFA delivery in determining the myocardial metabolic pattern in type 1 diabetes. Furthermore, the presence of similar rates of myocardial glucose use and fatty acid oxidation in type 1 diabetic compared with nondiabetic subjects despite higher plasma insulin levels suggests that myocardial insulin resistance is present in type 1 diabetes under these conditions. Second, our results show that although the myocardium in type 1 diabetes is overly dependent on fatty acid metabolism, both myocardial glucose and fatty acid uptake can be manipulated by altering plasma hormonal and substrate milieu. Last, we have shown that alterations in plasma FFA and insulin levels can change the fate of FFA extracted by the heart in humans with type 1 diabetes. Our findings that myocardial fatty acid utilization may be manipulated by increasing/decreasing FFA delivery extend the findings of R.J. Bing and others, who showed that substrate delivery to the heart is an important determinant of myocardial substrate utilization in humans without type 1 diabetes, to those with type 1 diabetes (26,27). Thus, although the myocardium in type 1 diabetes relies on fatty acid utilization for generation of ATP for contractile function, it can still be manipulated. Our data also show that increasing plasma free FFA levels increase the rates of myocardial fatty acid oxidation above the high baseline levels seen in euglycemic type 1 diabetic subjects. Thus, the euglycemic level of fatty acid oxidation in type 1 diabetes is not at its maximal oxidative capacity and may be further increased. This may have detrimental consequences. Results of studies of animal models show that high fatty acid oxidation occurs early in diabetes before marked changes are seen in cardiac function (28). Increased production of reactive oxygen species with increased oxidation may impair efficient calcium handling (10,29,30). Our finding that increased plasma FFA (and hence increased myocardial fatty acid oxidation) was associated with a decrease in myocardial glucose utilization would also be detrimental during ischemia (when the myocardium prefers glucose use). These observations also extend those of Nuutila et al. (31), who demonstrated Randle cycle operation in nondiabetic human myocardium, to type 1 diabetes myocardium. Despite the increase in myocardial fatty acid oxidation with the increase in plasma FFAs, it appeared that the oxidative capacity of the myocardium can be overwhelmed, as evidenced by a trend toward a decrease in the percentage oxidized and an increase in the percentage esterified in the Intralipid group. Although increase in esterification rate does not necessarily translate to increase in chronic lipid deposition, it agrees with findings of studies in animal models of diabetes and human autopsies, which demonstrated increased cardiac triglyceride content in diabetic compared with control subjects (13,32 34). This increase in esterification may be due to fatty acid uptake in excess of oxidation and/or an increase in the amount or activity of triglyceride synthesis enzymes in response to increased fatty acid availability (35). This fatty acid esterification increase in humans with type 1 diabetes may be detrimental, based on the results of studies in animals and humans suggesting that excessive myocardial fatty acid storage may result in apoptosis, oxidative stress, abnormal energy metabolism, fibrosis, and contractile dysfunction (12,36 40). Future imaging and/or pathological sample studies of lipid accumulation and function are necessary to confirm this hypothesis in humans. Effects of plasma insulin levels. Increasing plasma insulin in type 1 diabetes has a more complicated effect on myocardial glucose and fatty acid utilization and on the metabolic fate of fatty acids within the myocardium. Our results are consistent with those of Monti et al. (41), who found that increased plasma insulin resulted in increased myocardial glucose extraction fraction and utilization in patients with type 1 diabetes. We also demonstrated for the first time in humans with type 1 diabetes that increasing plasma insulin levels decreases rates of myocardial fatty acid utilization. This decrease is likely due to insulin s inhibition of peripheral lipolysis and the resultant decrease in plasma FFA, since myocardial fatty acid extraction fraction was not different in the HIEG clamp group compared with the euglycemic group. In addition, we showed in humans with type 1 diabetes that increase in plasma insulin and myocardial glucose utilization impaired myocardial fatty acid oxidation proportionately more than other fatty acid processes, such as myocardial fatty acid extraction and esterification (Figs. 3B, 4B, and 5B). This decrease in myocardial fatty acid oxidation with an increase in glucose metabolism, previously demonstrated in animal models, has not heretofore been demonstrated in humans with type 1 diabetes (42,43). The net result of the HIEG clamp is that plasma FFA decreases to such a degree that myocardial fatty acid esterification rates decrease, but insulin s anabolic effect on FFAs that do enter the myocardium encourages a high proportion to enter esterification processes in lieu of oxidation. Clinical implications. Because our data demonstrate that it is possible to manipulate myocardial glucose and fatty acid metabolism by manipulating plasma substrate and hormone levels in humans with type 1 diabetes and because animal data demonstrate deleterious effects of excessive fatty acid oxidation and/or storage on the myocardium, it may be desirable to decrease excessive delivery of FFAs to the myocardium in humans with type 1 diabetes, particularly during ischemia, when the myocardium needs to switch fuel sources and utilize predominantly glucose. Thus, our data, although not obtained in patients with ischemia, indirectly support the utility of glucose-insulin-potassium therapy or other treatments for decreasing fatty acid and increasing glucose utilization in patients with type 1 diabetes and ischemia. Exogenous insulin therapy, clearly necessary in subjects with type 1 diabetes for myocardial glucose metabolism, may also have a beneficial effect on decreasing plasma FFA levels, thereby ameliorating the tendency for excessive fatty acid utilization. The myocardium in type 1 diabetes may be especially prone to excessive dependence on fatty acid metabolism and its potential deleterious effects (particularly during ischemia) because it appears to be somewhat insulin resistant (although it still responds to high doses of insulin during the HIEG clamp) (12,44). It appears that insulin resistance based on our data showing that the myocardial glucose utilization/plasma insulin was lower in subjects with euglycemic type 1 diabetes than in normal control subjects. Although requiring further study, these findings may be very applicable to patients with type 2 diabetes, in whom insulin resistance predominates. Limitations. Our results may not be extrapolated to subjects who do not fit our inclusion/exclusion criteria. We did not study nondiabetic control subjects under all of 38 DIABETES, VOL. 57, JANUARY 2008

8 L.R. PETERSON AND ASSOCIATES the same conditions as those for the type 1 diabetic subjects, although how the nondiabetic myocardium s substrate choice is modified by substrates, insulin, and condition is known (45,46). Myocardial metabolism of other substrates (e.g., lactate) were not measured; however, since subjects were not ketotic, ischemic, or exercising at the time of the study, these should be minor contributors to metabolism. Myocardial glucose oxidation and myocardial metabolism or deposition of endogenous substrates were not quantified. Based on previous studies, endogenous triglycerides would be expected to contribute less to fatty acid oxidation when plasma FFA levels are high, and glycogen should contribute more during adrenergic stimulation (not an intervention in our study) (47,48). Also, Intralipid infusion and an HEIG clamp are not physiological conditions; however, high levels of FFAs (as evoked by Intralipid in our study) may be seen in obese subjects or those with poorly controlled type 1 diabetes, and very low levels (e.g., 100 nmol/ml) may be seen after a high-carbohydrate meal (plus insulin) in type 1 diabetic subjects. Thus, our interventions mimicked these physiologic conditions without altering the fasting status or glucose and ketone levels of our subjects. Moreover, although a HIEG clamp is not a physiologic state, it is being clinically tested as a therapy for patients with ischemia, including those with type 1 diabetes, and therefore is a relevant intervention. Humans with type 1 diabetes have higher MVO 2 than nondiabetic subjects, which is mostly but perhaps not completely accounted for by increased cardiac work. In humans with type 1 diabetes, myocardial utilization and the metabolic fate of substrates can be manipulated by altering plasma FFA and insulin levels, although some degree of myocardial insulin resistance is present. Alterations in myocardial glucose and fatty acid metabolism affected by increasing plasma FFA levels conform to Randle s hypothesis, with increasing fatty acid utilization and oxidation decreasing myocardial glucose extraction fraction and utilization. Furthermore, short-term increases in plasma FFA can increase myocardial fatty acid oxidation rates but also overwhelm the already overtaxed myocardial oxidative capacity and lead to increased myocardial fatty acid esterification rates. Insulin therapy increases myocardial glucose extraction and utilization; decreases myocardial fatty acid utilization, oxidation, and esterification; and yet increases the percentage of the myocardial fatty acid extraction fraction directed to esterification. These findings fine-tune our notions of myocardial metabolism in humans with type 1 diabetes and support the theory that metabolic manipulation of the myocardium is feasible and may have benefit in humans with type 1 diabetes. ACKNOWLEDGMENTS This work was supported by National Institutes of Health grants (PO1-HL13581, MO1-RR00036, RO1-HL073120, and P60-DK020579) and a grant from the Barnes-Jewish Hospital Foundation. Part of this work was presented in abstract form in J Nucl Cardiol 10:S3 S14, We extend special thanks to the participants in this study and to the staff of the General Clinical Research Center and our laboratory for their help with data collection and technical assistance. We thank Jean E. Schaffer, MD, for critical reading of the manuscript and Ava Ysaguirre for secretarial assistance. REFERENCES 1. Vadlamudi RV, Rodgers RL, McNeill JH: The effect of chronic alloxan- and streptozotocin-induced diabetes on isolated rat heart performance. 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