Insulin secretion in lipodystrophic HIV-infected patients is associated with high levels of nonglucose secretagogues and insulin resistance of
-cells
Steen B. Haugaard,1,2,3
Ove Andersen,1,3
Heidi Storgaard,4
Flemming Dela,5,6
Jens Juul Holst,6
Johan Iversen,1
Jens Ole Nielsen,1 and
Sten Madsbad2
1Department of Infectious Diseases, 2Department of Endocrinology and Internal Medicine, and 3Clinical Trial Unit, Hvidovre University Hospital, DK 2650 Copenhagen; 4Steno Diabetes Center, Gentofte, DK-2820 Copenhagen; and 5Copenhagen Muscle Research Centre and 6Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK 2200 Copenhagen, Denmark
Submitted 7 January 2004
; accepted in final form 6 May 2004
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ABSTRACT
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We examined whether plasma concentrations of nonglucose insulin secretagogues are associated with prehepatic insulin secretion rates (ISR) in nondiabetic, insulin-resistant, human immunodeficiency virus (HIV)-infected, lipodystrophic patients (LIPO). Additionally, the negative feedback of insulin on ISR was evaluated. ISR were estimated by deconvolution of plasma C-peptide concentrations during fasting (basal) and during the last 30 min of a 120-min euglycemic insulin clamp (40 mU·m2·min1). Eighteen normoglycemic LIPO were compared with 25 normoglycemic HIV-infected patients without lipodystrophy (controls). Thirty minutes before start of the clamp, a bolus of glucose was injected intravenously to stimulate endogenous insulin secretion. Insulin sensitivity index (SiRd) was estimated from glucose tracer analysis. LIPO displayed increased basal ISR (69%), clamp ISR (114%), basal insulin (130%), and clamp insulin (32%), all P
0.001, whereas SiRd was decreased (57%, P < 0.001). In LIPO, ISRbasal correlated significantly with basal insulin, alanine, and glucagon (all r > 0.65, P < 0.01), but not with glucose. In control subjects, ISRbasal correlated significantly with insulin, glucagon, and glucose (all r > 0.41, P < 0.05), but not with alanine. In LIPO, ISRclamp correlated significantly with clamp free fatty acids (FFA), alanine, triglyceride, and glucagon (all r > 0.51, P < 0.05). In control subjects, ISRclamp correlated with clamp triglyceride (r = 0.45, P < 0.05). Paradoxically, in LIPO, ISRclamp correlated positively with clamp insulin (r = 0.68, P < 0.01), which suggests an absent negative feedback of insulin on ISR. Our data support evidence that lipodystrophic, nondiabetic, HIV-infected patients exhibit increased ISR, which can be partially explained by an impaired negative feedback of insulin on
-cells and an increased stimulation of ISR by FFA, alanine, triglyceride, and glucagon.
human immunodeficiency virus; nonglucose insulin secretagogues
IN RECENT YEARS, A SYNDROME of fat redistribution in human immunodeficiency virus (HIV)-infected patients on highly active antiretroviral therapy (HAART) has been acknowledged (5, 8, 12, 37). Epidemiological data demonstrate associations between the use of protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors (NRTI) and the prevalence of HIV-associated lipodystrophy syndrome (HALS) (5, 16, 30, 37, 38). Besides the typical lipodystrophic traits in these patients, data have accumulated demonstrating associated insulin resistance (2, 11, 28, 37). Data from major cohorts of HALS patients have revealed increased fasting insulin and C-peptide concentrations, although HALS patients most frequently are normoglycemic (11, 37, 38). After 12 wk of treatment with PI in former PI-naïve HIV-infected subjects, an impairment in
-cell function was shown (44). Interestingly, the PI-induced insulin resistance appears to be reversible within a few hours after discontinuation of the drug (21).
The pancreatic
-cells adapt to peripheral insulin sensitivity to facilitate appropriate insulin secretion rates (ISR) (25). However, in HIV-negative subjects, an early sign of impaired glucose metabolism is impaired insulin secretion relative to insulin sensitivity (35).
-Cell resistance to insulin itself may be a mechanism behind dysregulation of insulin secretion, i.e., an impaired feedback mechanism resulting in inappropriately high ISR (29). Insulin secretagogues other than glucose, e.g., free fatty acids, alanine, triglyceride, and glucagon, have been shown to stimulate insulin secretion (6, 10, 22, 42). Accordingly, an excess of these metabolites may mediate increased ISR.
Upon this background, we first investigated whether plasma levels of the nonglucose insulin secretagogues free fatty acids, alanine, triglyceride, and glucagon are associated with altered basal insulin secretion in normoglycemic lipodystrophic HIV-infected patients. Second, we addressed whether infusion of exogenous insulin may downregulate glucose-stimulated endogenous ISR. Third, we evaluated whether persistently high ISR during infusion of exogenous insulin are associated with elevated plasma concentrations of free fatty acids, alanine, triglyceride and glucagon. To obtain an accurate estimation of insulin secretion, we calculated ISR by deconvolution of plasma C-peptide concentrations. We present evidence suggesting that lipodystrophic, HIV-infected patients display increased prehepatic ISR, which may be due to a defective feedback mechanism of insulin itself on pancreatic
-cells and persistently increased concentrations of insulin secretagogues stimulating the
-cells.
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METHODS
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Subjects.
Eighteen nondiabetic, HIV-1-infected, male patients with fat redistribution and on HAART (LIPO) were recruited consecutively from the outpatient clinic of infectious diseases, Hvidovre Hospital, University of Copenhagen. Likewise, 25 nondiabetic HIV-1-infected male patients without signs of lipodystrophy were recruited (i.e., control subjects). Seven of these 25 control subjects were naïve to antiretroviral therapy (i.e., NAÏVE), whereas 18 were on HAART (i.e., NONLIPO). A detailed description of selection, anthropometry, immunology, and components of HAART of all 18 case subjects and of the 18 control subjects who were on HAART has been provided previously (2). In short, for case subjects, a questionnaire and a physical examination had to be positive for signs of lipodystrophy, whereas for control subjects, both the questionnaire and physical examination had to be negative. All cases, except one who had multiple lipomatosis, displayed lipodystrophy consistent with peripheral fat atrophy and central fat accumulation [i.e., mixed lipodystrophy (30)]. Except for HAART, none of the subjects received medication known to affect glucose metabolism. All participants, except for two control subjects, had a negative family history of diabetes mellitus, and all participants except for two control subjects were of Caucasian ethnicity.
Fifteen HIV-negative individuals, who had participated in an identical study setup/protocol at the same time period as the present study, were included for comparison (40) to evaluate whether basal ISR and suppression of ISR during clamp in LIPO and control subjects would be comparable with HIV-negative individuals.
Subjects gave their written informed consent, and the protocol was approved by the Ethics Committee in Copenhagen, Denmark, and performed in accordance with the Helsinki Declaration II.
Study protocol.
Instructions were given to abstain from strenuous exercise for
3 days before the metabolic assessments. The HIV-infected patients reported to our laboratory at 0800 after a 12-h overnight fast, including a 16-h withdrawal of HAART. At 0830, two intravenous cannulas were inserted, one in a dorsal hand vein and the other in an antecubital vein. The hand vein was used for blood sampling, whereas the antecubital vein was used for infusion. A primed continuous infusion of tritiated glucose {[3-3H]glucose; New England Nuclear, Boston, MA; bolus: 2.05 µCi/mM fasting plasma glucose (p-glucose); continuous: 0.11 µCi/min} was initiated at 0900 at the start of the 150-min basal period (0150 min) and continued throughout the basal period, the 30-min intravenous glucose tolerance test (IVGTT, 150180 min), and the 120-min hyperinsulinemic euglucemic clamp (180300 min). Two steady-state periods were predefined, i.e., at 90120 min as basal and at 270300 min as clamp. The IVGTT was initiated with a 1-min bolus glucose infusion (0.3 g/kg body wt). Blood samples for glucose and insulin were drawn relative to start of the glucose bolus at 4, 2, 0, 2, 4, 6, 8, 10, 15, 20, and 30 min. Thereafter, an insulin (Actrapid; Novo Nordisk, Denmark) infusion was started, with a stepwise reduction in the infusion rate every 3rd min from 100 to 80 to 60 to 40 mU·m2·min1. Thereafter (+9120 min), the insulin infusion rate was fixed at 40 mU·m2·min1. The plasma glucose concentration was kept at
5 mmol/l by adjusting the infusion rate of glucose (180 g/l), which was enriched with tritiated glucose (110 µCi/l). During the clamp, glucose levels were determined every 5 min, and blood samples for assessing plasma insulin and C-peptide levels were taken every 10 min.
Body composition was evaluated by dual-energy X-ray absorptiometry (DEXA; Norland, WI). To estimate the amount of fat in the trunk (chest, abdomen, and pelvis) and in the extremities, a whole body scan was performed. The proximal limitation of the leg region was placed through the hip joints, at an angle of
45°. The DEXA scans were done in random order, and the operator was unaware of the assignment of the patients to study groups. The ratio of limb fat (RLF) was calculated as the sum of the fat mass of the arms and legs divided by the trunk fat mass.
Assays.
Whole blood glucose levels were determined pair-wise on two calibrated HemoCue B-Glucose Analyzers (HemaCue, Angelholm, Sweden) with an intra-analyzer coefficient of variation (CV) of 3.5% and an interanalyzer CV of 3.3%. Plasma glucose was calculated using the equations of Fogh-Andersen and D'Orazio (14) by filling in data on whole blood glucose (the mean of the pair of measurements) and blood hematocrit. Blood samples were centrifuged immediately at 4°C, and plasma was stored at 80°C for later analysis. Plasma insulin and C-peptide concentrations were determined by the 1235 AutoDELPHIA automatic immunoassay system (Wallac Oy, Turku, Finland). The insulin assay had a detection limit of
3 pM. Cross-reactivity with intact proinsulin was 0.1 and 0.4%, with 3233 split proinsulin and 66% with 6465 split proinsulin. The intra-assay CV was 4.5%, and the interassay CV was 7%. The detection limit of the C-peptide assay was 5 pM. The cross-reactivity with intact proinsulin was 51%, 35% with 3233 split proinsulin and 92% with 6465 split proinsulin. There was no detectable cross-reactivity with insulin, and the intra- and interassay variations were 5 and 8%, respectively. Plasma free fatty acids (FFA) were determined using an enzymatic colorimetric method (Wako C test kit; Wako Chemicals, Neuss, Germany) with an interassay CV of 5%. CD4 count determination (flow cytometry, Becton-Dickinson FACscan; Frankling Lakes, NJ, with an interassay CV of 7%) and viral load determination (Roche Amplicor and Roche amplicer ultrasensitive assay with a detection limit of 20 copies/ml of plasma; Roche, Basel, Switzerland) met the requirements of interlaboratory quality control. The glucagon assay (17) is directed against the COOH terminus of the glucagon molecule and therefore measures glucagon of mainly pancreatic origin. The detection limit and intra-assay CV were 1 pmol/l and <6%, respectively. In cases where the measured glucagon concentration was lower than the detection limit, a value of 0.5 pmol/l was noted. Concentrations of alanine were measured by the fluorometric method. Basal plasma concentrations of triglyceride, FFA, glucagon, and alanine were measured at 90 and 120 min, whereas clamp concentrations were measured at 270 and 300 min; means were noted.
Calculations.
Rates of glucose disposal (Rd) and endogenous glucose production (EGP) were estimated during predefined steady-state periods (i.e., basal and clamp periods) at intervals of 10 min by use of Steele's non-steady-state equations, as previously described (18). In the calculations of glucose turnover rates, the distribution volume of glucose was taken as 200 ml/kg body wt, the pool fraction was taken as 0.65, plasma water was assumed to represent 93% of total plasma volume, and total body water mass was set at 65% of body wt (24). As previously suggested, the insulin sensitivity index (SiRd) was calculated as [(Rd clamp Rd basal)/(p-insulin clamp p-insulin basal) x p-glucose clamp] (24). The unit of SiRd is expressed as a microgram of glucose per kilogram FFM per minute per picomole of insulin per millimole of glucose.
Prehepatic ISR were calculated from plasma C-peptide measurements using the ISEC (Insulin SECretion) computer program (20). The model is based on the assumptions that insulin and C-peptide are cosecreted in an equimolar fashion by the
-cells and that C-peptide is not cleared by the liver. ISEC has been validated to calculate ISR during IVGTT (19, 27) and has been applied to calculate ISR during a meal tolerance test, a hyperinsulinemic euglycemic clamp, and during basal conditions (20).
Statistical analysis.
Data are presented as means ± SE, and as medians and ranges when distributions were skewed. Analysis of variance (ANOVA) was performed to compare distribution of data between LIPO and control subjects. Because it appeared that age was increased in LIPO compared with control subjects, we adjusted for age by univariate ANOVA, with patient group (qualitative factor) and age (quantitative factor) as covariates. The Pearson correlation coefficient (r) was applied for measurements of associations between variables for each of the study groups. Partial correlation coefficients were calculated between variables for each of the study groups, controlling for age and body mass index (BMI; see Table 2). Multiple linear regression analyses were performed step-wise to identify significant predictors of ISR. If data distribution was skewed, data were log transformed before applying ANOVA and t-tests and before calculation of a correlation coefficient. Calculations were performed by SPSS (SPSS version 12.0; SPSS, Chicago, IL). Two-sided P values <0.05 were defined as statistically significant. A trend was noted for P values, 0.05
P < 0.2.
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Table 2. Correlations between basal and clamp prehepatic ISR vs. plasma concentrations of insulin, insulin secretagogues, age, BMI, duration of HIV infection, insulin sensitivity, and measures of fat distribution
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RESULTS
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Insulin action, prehepatic insulin secretion, anthropometry, immunology, and insulin secretagogues.
LIPO were older than control subjects; accordingly, an adjustment for age was performed in the comparison of distribution of variables between LIPO and control subjects (Table 1). Peripheral insulin sensitivity was highly significantly decreased in LIPO compared with control subjects, whereas basal and clamp prehepatic ISR were increased twofold in LIPO (Table 1). Within the group of LIPO, the clamp ISR of LIPO tended to be higher than the basal ISR (P = 0.11), whereas the basal ISR and the clamp ISR of the control subjects were similar. BMI and total fat mass were not statistically significantly different between groups, although BMI, after adjustment for age, appeared to be increased in LIPO. The RLF was highly significantly reduced in LIPO, confirming that cases were lipodystrophic. RLF was not significantly different between the subgroups of the control subjects, i.e., NONLIPO, 89 ± 7% and NAÏVE, 92 ± 5% (data not shown).
All LIPO and all NONLIPO were treated with NRTI as part of HAART, with a mean duration of NRTI treatment of 47 ± 7 and 42 ± 6 mo [P = nonsignificant (NS)]. Frequently used NRTI in LIPO and NONLIPO were Lamivudine (83%), Zidovudine (50%), and Stavudine (47%). As part of HAART, PI were used by 90% of LIPO and NONLIPO, and duration of PI treatment was comparable in these groups (32 ± 4 and 25 ± 4 mo, P = NS). Frequently used PI were Indinavir (33%), Ritonavir (31%), and Nelfinavir (22%). Components of HAART did not differ between LIPO and NONLIPO. CD4 cell counts were similar in LIPO (427 ± 45 cells/µl), NONLIPO (352 ± 46 cells/µl), and NAÏVE (521 ± 90 cells/µl, P = NS), whereas LIPO and NONLIPO displayed a lower number of HIV particles than NAÏVE (median <20 copies per ml vs. 14,900, P < 0.05). Duration of HIV infection was comparable in LIPO (99 ± 14 mo), NONLIPO (72 ± 11 mo), and NAÏVE (61 ± 25 mo, P = NS).
All participants were normoglycemic (p-glucose
6.1 mmol/l), with a similar basal p-glucose in LIPO and control subjects. Basal and clamp p-insulin and p-C-peptide were significantly increased in LIPO. Basal p-alanine and p-triglyceride, clamp p-alanine, p-FFA, and p-triglycerides were significantly increased in LIPO. Clamp p-glucagon was increased in LIPO compared with control subjects, although the differences did not reach statistical significance. Basal p-FFA and p-glucagon were not different between groups.
Plasma glucose, insulin, C-peptide, and prehepatic ISR throughout the study.
No significant differences in p-glucose concentrations throughout the study period were found between LIPO and control subjects or within the group of control subjects [i.e., n = 18 control subjects on HAART (NONLIPO) vs. n = 7 control subjects naïve to HAART (NAÏVE); Fig. 1]. p-Insulin, p-C-peptide, and ISR in LIPO were increased in the basal state and during clamp compared with control subjects, whereas these variables did not differ between NONLIPO and NAÏVE groups of control subjects (Fig. 1). Although endogenous insulin secretion was stimulated by an intravenous glucose bolus 30 min before the start of the 120-min clamp, steady-state levels of ISR and p-glucose were reached within 30 min after the start of exogenous insulin infusion in LIPO and control subjects (LIPO: at 210225 min, 4.0 ± 0.6 pmol·min1·kg1 and 5.7 ± 0.3 mmol/l compared with clamp steady-state period, 3.4 ± 0.5 pmol·min1·kg1 and 5.1 ± 0.1 mmol/l, P = NS; control subjects: at 210225 min, 1.6 ± 0.2 pmol·min1·kg1 and 5.2 ± 0.2 mmol/l compared with clamp steady-state period, 1.6 ± 0.2 pmol·min1·kg1 and 5.2 ± 0.2 mmol/l, P = NS).

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Fig. 1. Plasma concentrations of glucose, insulin, C-peptide, and prehepatic insulin secretion rates of the 3 study groups throughout the study. The mark " " indicates a statistically significant difference (i.e., P < 0.05) between lipodystrophic patients (LIPO; , n = 18) control subjects on highly active antiretroviral therapy (NONLIPO; , n = 18); *P < 0.05, LIPO vs. control subjects naïve to antiretroviral therapy (NAÏVE; , n = 7). Note that NONLIPO and NAÏVE establish the combined group of control subjects (n = 25). Significance tests were performed at the start of the study (0 min), during basal steady-state period (90120 min), and at the clamp steady-state period (270300 min). Please note the log scale of the y-axis for prehepatic insulin secretion rates.
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Correlations between basal and clamp prehepatic ISR, insulin secretagogues, glucagon, age, BMI, duration of HIV infection, and measures of fat distribution.
Basal ISR correlated significantly with basal p-insulin in both groups (Table 2) and Fig. 2). We found strong positive correlations between basal ISR and clamp ISR in both study groups (Table 2) and Fig. 3). In LIPO, but not in control subjects, clamp ISR and clamp insulin correlated strongly and positively. In other words, an increased plasma insulin concentration did not downregulate ISR in LIPO. In each study group, basal ISR and clamp ISR correlated positively. In LIPO, but not in control subjects, significant positive correlations were found between basal ISR and basal p-alanine, and during the clamp, between ISR and p-alanine and p-FFA. During the clamp, p-triglyceride concentrations were positively correlated with ISR in both groups, albeit more strongly in LIPO. In control subjects, but not in LIPO, a positive correlation was found between p-glucose and ISR. During the basal period, the positive correlation between ISR and p-glucagon in LIPO was stronger than that in control subjects. During the clamp, a significant correlation between ISR and p-glucagon was found in LIPO only. The aforementioned correlations between ISR and the nonglucose insulin secretagogues were robust after adjustment for age and BMI (Table 2).
In control subjects, BMI and percent total fat mass correlated positively with basal and clamp ISR, whereas RLF correlated with basal ISR (Table 2). Significant correlation between these parameters of fat content and distribution vs. ISR was not found in LIPO. Age and duration of HIV infection did not correlate significantly with ISR in LIPO and control subjects (Table 2). Duration of PI and NRTI treatment, CD4 count, and number of HIV-RNA copies did not correlate significantly with ISR in LIPO and control subjects (data not shown).
Multiple regression analysis and colinearity of nonglucose secretagogues.
To examine the relative importance of each of the nonglucose secretagogues alanine, glucagon, FFA, and triglyceride on ISR in LIPO, a multiple linear regression analysis was performed, with the secretagogues as predictors. During the basal period, a model including p-glucagon explained 56% (R2= 0.56, P < 0.001) of the variability of basal ISR in LIPO. During the clamp, a model including log10 p-triglyceride and p-glucagon explained 63% (R2 = 0.63, P < 0.001) of the variability of clamp ISR in LIPO. We performed similar calculations in control subjects. During the basal period, a model including p-glucose and the nonglucose secretagogues as putative predictors in the calculation, including p-glucose and p-glucagon, explained 48% (R2 = 0.48, P < 0.001) of variability of basal ISR in control subjects. Because the significant correlation during clamp between p-glucose and ISR in control subjects was based upon a single outlier, we included only the nonglucose secretagogues in the multiple regression analysis. A model including log10 p-triglyceride explained 19% (R2 = 0.19, P < 0.05) of variability of clamp ISR in control subjects. During the basal period, in LIPO, p-alanine correlated positively with log10 p-triglyceride and p-glucagon (both r = 0.61, P < 0.01), whereas no significant colinearity was found between other nonglucose secretagogues in LIPO and control subjects. During the clamp steady-state period, however, multiple significant correlations were found between the nonglucose secretagogues alanine, glucagon, FFA, and triglyceride in LIPO and control subjects (Table 3).
High and low ISR in the LIPO group during clamp.
The LIPO group was stratified arbitrarily into the nine patients with the lowest ISR during the clamp and the nine patients exhibiting the highest ISR during the clamp (Table 4). Within the subgroup with the highest ISR, the ISR during the clamp was significantly increased compared with basal ISR, whereas within the subgroup with the lowest ISR, basal and clamp ISR did not differ. Plasma concentrations of triglyceride, FFA, alanine, and glucagon were increased in the subgroup with high clamp ISR compared with the subgroup with the relatively low clamp ISR. In contrast, no significant differences in peripheral insulin sensitivity, p-glucose, age, duration of HIV, PI treatment, NRTI treatment, CD4 count, BMI, fat mass, or RLF were observed.
Clamp ISR of LIPO and control subjects compared with clamp ISR of HIV-negative individuals.
We calculated the clamp ISR of HIV-negative individuals who had participated in an identical study setup/protocol as the present study (40) and who appeared to display similar basal ISR to LIPO and to the control subjects (Fig. 4).Seven HIV-negative, glucose intolerant, obese men (BMI 32.7 ± 1.4 kg/m2) of mean age 57 ± 2 yr, basal p-glucose 6.6 ± 0.2 mmol/l, and basal p-insulin 98 ± 32 pmol/l, displayed a basal ISR of 3.3 ± 0.6 pmol·min1·kg1, which matched the basal ISR of LIPO (2.7 ± 0.3 pmol·min1·kg1, P = NS). During the clamp steady-state period, the glucose-intolerant, obese, HIV-negative men matched LIPO in plasma insulin (503 ± 60 vs. 535 ± 35 pmol/l, P = NS) and SiRd (1.2 ± 0.3 vs. 1.5 ± 0.3 µg glucose·kg FFM1·min1·pM insulin1·mM glucose1, P = NS), whereas their ISR was significantly lower (1.9 ± 0.4 vs. 3.4 ± 0.5 pmol·min1·kg1, P < 0.05). Furthermore, within the group of HIV-negative obese men with impaired glucose tolerance (IGT), clamp ISR was decreased compared with the basal ISR (P < 0.01). Eight HIV-negative obese men (BMI 32.7 ± 1.4 kg/m2) with normal glucose tolerance, mean age 53 ± 2 yr, basal p-glucose 5.9 ± 0.1 mmol/l, and basal p-insulin 38 ± 5 pmol/l displayed a basal ISR of 1.5 ± 0.2 pmol·min1·kg1 and a peripheral insulin sensitivity (SiRd) of 3.5 ± 0.6 µg glucose·kg FFM1·min1·pM insulin1·mM glucose1, matching the basal ISR (1.6 ± 0.1 pmol·min1·kg1, P = NS) and SiRd (3.5 ± 0.5 µg glucose·kg FFM1·min1·pM insulin1·mM glucose1, P = NS) of the HIV-infected control subjects. During the clamp steady-state period, the glucose-tolerant obese HIV-negative men displayed similar ISR and p-insulin compared with the HIV-infected control subjects (1.3 ± 0.2 and 431 ± 25 pmol/l vs. 1.6 ± 0.1 and 407 ± 14 pmol/l, respectively, all P = NS). Importantly, a steady state of ISR was achieved within 30 min after start of the 120-min clamp period in both groups of HIV-negative obese men.

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Fig. 4. ISR during basal steady-state period (basal) and during clamp steady-state period (clamp) in LIPO (solid bars, n = 18), control subjects (i.e., combined groups of NONLIPO and NAÏVE, open bars, n = 25), obese human immunodeficiency virus (HIV)-negative subjects with impaired glucose tolerance (IGT non-HIV, gray bars, n = 7), and obese HIV-negative subjects with normal glucose tolerance (NGT non-HIV, stippled bars, n = 8), respectively. Values are means + SE.
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DISCUSSION
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A major finding of the present study was the strong positive associations between plasma concentrations of the nonglucose secretagogues (alanine, FFA, triglyceride, and glucagon) and ISR in insulin-resistant, normoglycemic, lipodystrophic, HIV-infected patients (LIPO) on combined antiretroviral therapy. ISR were increased twofold in LIPO compared with HIV-infected patients without lipodystrophy (control subjects), in whom only weak or no significant correlations between ISR and plasma alanine, plasma FFA, plasma triglyceride, and plasma glucagon were observed. Our data are consistent with a persistent stimulatory effect of alanine, FFA, triglyceride, and glucagon on ISR in a subgroup of LIPO. Two hours after an intravenous glucose bolus and 90 min of continuous exogenous insulin infusion (40 mU·m2·min1), including 60 min of euglycemia, increased endogenous ISR were correlated positively with increased plasma insulin concentrations in LIPO, which suggests an absent feedback of insulin on
-cells in those LIPO with high ISR. Accordingly, multiple factors other than glucose concentrations seem to play a role in the regulation of ISR in HIV-lipodystrophy.
In physiological settings, alanine, glucagon, FFA, and triglyceride have been shown to stimulate ISR. Thus, in healthy individuals with a family history of diabetes mellitus, an increased rate of prehepatic insulin secretion was associated with increased levels of plasma alanine (34). Alanine has been shown to increase ISR at physiological glucose concentrations (1, 10). Glucagon is known to stimulate insulin secretion, likely through a combined paracrine and direct effect on the
-cell (22, 32). FFA have long been known to acutely stimulate insulin secretion in humans (4, 15, 31), and very low levels of plasma FFA have been shown to attenuate glucose-stimulated insulin secretion (39). In contrast, in healthy individuals, a prolonged (96-h) lipid infusion in physiological concentrations has been shown to impair insulin secretion, demonstrating a toxic effect of prolonged high plasma FFA concentrations on the
-cells (26). However, a sustained stimulatory effect of lipid infusion for 48 h on ISR has also been demonstrated (7).
High fasting plasma triglyceride is associated with increased ISR in prediabetic individuals (42), whereas in the Zucker fatty rat, increased concentrations of islet triglyceride stimulate
-cell growth and ISR. Ultimately, however, lipoapoptosis of
-cells ensues, producing glucose intolerance and diabetes (43). In lipodystrophic HIV-infected patients, Sutinen et al. (41) demonstrated increased plasma triglycerides and increased liver triglyceride, the latter being shown to be associated with increased plasma levels of insulin and C-peptide. Speculatively, lipodystrophic HIV-infected patients, in addition to increased plasma and liver triglyceride concentrations, display increased islet triglyceride concentrations, which interfere with insulin secretion.
In the present study, the clamp ISR was not suppressed below the basal ISR in LIPO or control subjects. It has been shown that euglycemic clamps, with identical insulin infusion rates as used in the present study, suppress ISR by 2050% (3, 9, 13). Such study protocols, however, did not include prestimulation of insulin secretion by intravenous glucose, which induces a first- and second-phase insulin response (33). Most likely, the absent partial suppression of ISR during exogenous insulin infusion is explained by the preceding glucose infusion inducing a second-phase insulin release.
We present data from obese, glucose-intolerant, HIV-negative men, who displayed similar basal ISR as LIPO. During the clamp, these obese men suppressed ISR more than LIPO, and below their own basal ISR. This was achieved at similar clamp p-insulin concentrations as in LIPO. These data lend support to the notion that LIPO display defects in downregulation of endogenous ISR after intravenous infusion of insulin. The strong positive correlation between plasma insulin and ISR during the clamp in LIPO suggests an absent negative feedback of insulin on the
-cells in those LIPO patients with high endogenous ISR. Absent negative feedback might be caused by early lipotoxicity of the
-cells, inducing overproduction of intracellular nitric oxide (31) and/or influence directly on the insulin receptor of
-cells (29). Supporting the hypothesis of a state of "early" lipotoxicity of the
-cells in our lipodystrophic HIV-infected patients, the subgroup of LIPO with the highest ISR exhibited 100% higher p-FFA during the clamp and 100% higher p-triglyceride during basal and clamp periods compared with the subgroup of LIPO with the lowest ISR.
Multiple regression analyses suggested that clamp plasma glucagon and triglyceride concentrations were strong independent predictors of ISR during the clamp, explaining nearly two-thirds of the variability of clamp ISR. However, correlation analyses revealed strong colinearity of the nonglucose secretagogues during clamp. Accordingly, it would be an overinterpretation to suggest that p-alanine and p-FFA do not play roles in the stimulation of ISR during the clamp in LIPO. Most likely, the nonglucose secretagogues exhibit additive stimulatory effects on ISR in LIPO.
Interestingly, the subgroups of HIV-positive control subjects, i.e., NONLIPO and NAÏVE, displayed similar basal and clamp ISR, which was unexpected, inasmuch as PI treatment itself has been shown to increase fasting plasma insulin concentrations and decrease insulin sensitivity (44). An explanation could be that antiretroviral therapy was discontinued for more than 16 h before metabolic examinations in all subjects that had been on antiretroviral therapy in the present study, suggesting that the acute effect of PI on insulin secretion might have vanished (21). Accordingly, we hypothesize that, after abstinence from antiretroviral treatment for more than 16 h, the differences in ISR between NONLIPO and LIPO are explained by different fat distribution rather than by markers of immunologic status, e.g., CD4 and HIV-RNA, which did not correlate with ISR, and duration and modality of the antiretroviral treatment. Our observations do not contradict earlier findings linking PI treatment, lipodystrophy, and high plasma insulin (11, 37, 38), but they suggest that the aberrations in insulin secretion in HIV-lipodystrophy prevail after attenuation of the acute effect of PI on ISR.
The present data emphasize that more attention should be paid to high plasma triglyceride concentrations in HIV-lipodystrophy, because this metabolite might be related to impaired regulation of insulin secretion, implying stress on the
-cell. Furthermore, quantification of the impact of glucagon and other nonglucose secretagogues on insulin secretion in HIV-infected subjects compared with the impact of antiretroviral therapy, especially PI, merits further research.
We sought to take account of the differences in age between LIPO and control subjects in the present study in our statistical analysis. A multi-center study, including
1,000 HIV-negative nondiabetic subjects, demonstrated that such age differences do not significantly influence insulin sensitivity and have a very limited impact on posthepatic insulin delivery rate (23). A control group of healthy, weight-matched subjects was not included in the present study, but when identical techniques were used to calculate prehepatic insulin secretion rates in healthy humans, a basal ISR of
1.6 pmol·kg1·min1 was determined (36). This nicely matches the basal ISR of our HIV-infected control subjects and emphasizes that the basal ISR of 2.7 pmol·kg1·min1 in LIPO was increased.
In conclusion, our data suggest that normoglycemic, insulin-resistant, HIV-infected male patients with fat redistribution display increased baseline prehepatic ISR compared with HIV patients without lipodystrophy who were matched for total body fat and level of fasting plasma glucose. The increased ISR seemed to be explained by a combination of increased plasma levels of nonglucose secretagogues and absent negative feedback of insulin on
-cells.
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GRANTS
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This study was supported financially by The Danish AIDS Foundation, The Novo Nordisk Foundation, The Copenhagen Hospital Cooperation Foundation, and the Danish Medical Research Council Foundation.
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ACKNOWLEDGMENTS
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We thank Susanne Reimer, Lena Hansen, and Anne Louise Sørensen for excellent technical assistance.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. B. Haugaard, Clinical Trial Unit 136, Hvidovre Univ. Hospital, DK 2650 Hvidovre, Copenhagen, Denmark (E-mail: sbhau{at}dadlnet.dk)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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