AJP - Endo Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 292: E549-E560, 2007. First published September 26, 2006; doi:10.1152/ajpendo.00255.2006
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/2/E549    most recent
00255.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Goh, T. T.
Right arrow Articles by Giacca, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Goh, T. T.
Right arrow Articles by Giacca, A.

Lipid-induced beta-cell dysfunction in vivo in models of progressive beta-cell failure

Tracy T. Goh,1 Timothy M. Mason,1 Neehar Gupta,1 Abby So,1 Tony K. T. Lam,1 Loretta Lam,1 Gary F. Lewis,2,1 Andrea Mari,3 and Adria Giacca1,2

Departments of 1Physiology and 2Medicine, University of Toronto, Toronto, Ontario, Canada; and 3Institute of Biomedical Engineering, National Research Council, Padua, Italy

Submitted 26 May 2006 ; accepted in final form 19 September 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
 GRANTS
 REFERENCES
 
We determined the effect of 48-h elevation of plasma free fatty acids (FFA) on insulin secretion during hyperglycemic clamps in control female Wistar rats (group a) and in the following female rat models of progressive beta-cell dysfunction: lean Zucker diabetic fatty (ZDF) rats, both wild-type (group b) and heterozygous for the fa mutation in the leptin receptor gene (group c); obese (fa/fa) Zucker rats (nonprediabetic; group d); obese prediabetic (fa/fa) ZDF rats (group e); and obese (fa/fa) diabetic ZDF rats (group f). FFA induced insulin resistance in all groups but increased C-peptide levels (index of absolute insulin secretion) only in obese prediabetic ZDF rats. Insulin secretion corrected for insulin sensitivity using a hyperbolic or power relationship (disposition index or compensation index, respectively, both indexes of beta-cell function) was decreased by FFA. The decrease was greater in normoglycemic heterozygous lean ZDF rats than in Wistar controls. In obese "prediabetic" ZDF rats with mild hyperglycemia, the FFA-induced decrease in beta-cell function was no greater than that in obese Zucker rats. However, in overtly diabetic obese ZDF rats, FFA further impaired beta-cell function. In conclusion, 1) the FFA-induced impairment in beta-cell function is accentuated in the presence of a single copy of a mutated leptin receptor gene, independent of hyperglycemia. 2) In prediabetic ZDF rats with mild hyperglycemia, lipotoxicity is not accentuated, as the beta-cell mounts a partial compensatory response for FFA-induced insulin resistance. 3) This compensation is lost in diabetic rats with more marked hyperglycemia and loss of glucose sensing.

free fatty acid; obesity; insulin secretion; hyperglycemic clamp; Zucker diabetic fatty rat

TYPE 2 DIABETES IS CHARACTERIZED by impaired insulin secretion and action (19, 24). An early characteristic of the defect in insulin secretion is selective impairment of glucose-stimulated insulin secretion (GSIS). More than 80% of type 2 diabetic individuals are obese, and obese individuals often have elevated plasma levels of free fatty acids (FFA) (4) because of their expanded (3, 4) and more lipolytically active (41) adipose tissue stores. It is well known that FFA can impair insulin action (4); however, it is less clear whether FFA can also impair beta-cell function, thus playing a role in the defect in GSIS that characterizes type 2 diabetes. Acutely, FFA increase insulin secretion both in vitro (13, 46) and in vivo (15, 21, 45). However, in vitro and ex vivo studies (8, 28, 43, 47, 48) have shown that, chronically, FFA impair GSIS. The effect of prolonged FFA elevation on GSIS in vivo is more controversial. We have found that prolonged elevation of plasma FFA results in decreased GSIS in vivo in normal female Wistar rats (34). The decrease was marked with oleate infusion but was also seen at high glucose levels with infusion of Intralipid + heparin (IH). In that study, a mild elevation of FFA was obtained that did not significantly decrease insulin sensitivity during a hyperglycemic clamp. However, a comparable elevation of FFA induced insulin resistance in male humans. After 1 h of IH infusion, insulin secretion during a hyperglycemic clamp increased, so that the disposition index (DI, which is insulin secretion corrected for insulin resistance) was unaffected. However, after 48 h of IH, insulin secretion failed to increase so that DI decreased. We concluded that prolonged elevation of FFA has an impairing (lipotoxic) effect on beta-cell function (11). Other studies in rats (30) and humans (5, 26, 31, 35, 36, 44) reported increased (5, 30, 31), decreased (35, 36), and unchanged (44) absolute insulin secretion after prolonged IH infusion; however, insulin secretion was generally found to be inadequate to compensate for FFA-induced insulin resistance (35, 44), at least in predisposed individuals (26).

Because the majority of obese individuals with elevated FFA do not become diabetic, it is important to investigate the conditions that accentuate the impairing effect of FFA on GSIS. Prolonged in vitro exposure to FFA decreased GSIS in islets of obese prediabetic male Zucker diabetic fatty (ZDF) rats and heterozygous lean ZDF rats (22, 27) to a greater extent than in islets of control Wistar rats. This is consistent with a genetic susceptibility to FFA-induced beta-cell impairment in the ZDF strain, which may or may not be dependent on the leptin receptor mutation. However, dietary models of insulin resistance such as the fructose-fed rat also showed accentuation of FFA-induced impairment of GSIS in in vitro studies (12). Although in cultured human islets lipotoxicity also occurs at normal basal glucose concentrations (16), high-fat feeding decreased GSIS in islets of hyperglycemic Goto-Kakizaki (GK) rats but not in control Wistar rats (7), suggesting that hyperglycemia may be a prerequisite for lipotoxicity in this model (38). The term "glucolipoxicity" has been coined to describe synergistic or permissive interactions between glucose and FFA to decrease beta-cell function (38, 39) and is supported by some (6, 7, 9, 23) but not all in vitro and ex vivo studies (43, 47). In vivo studies in obese, insulin-resistant, and diabetic models are few. We have found that, in contrast to the in vitro findings mentioned above, IH decreased absolute GSIS in obese subjects with glucose intolerance but not in type 2 diabetic subjects. However, DI could not be calculated in that study (10).

The objective of the present study is to determine whether models of progressive beta-cell dysfunction with increasing degrees of hyperglycemia are more sensitive to the anticipated inhibitory effect of FFA on beta-cell function in vivo than control Wistar rats.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
 GRANTS
 REFERENCES
 
Animal Models

Six groups of rats were studied. Female rats (Charles River, Wilmington, MA) were chosen to allow for comparison of the effect of FFA on beta-cell function with that in the ZDF female rat (Charles River), a prediabetic rat model that develops diabetes when fed a high-fat diet (14). Thus both prediabetic and diabetic models can be studied differentially.

Group a. Female Wistar rats at 11 wk of age served as lean controls.

Groups b and c. Age-matched female lean ZDF littermates of obese ZDF rats, either wild-type (+/+; model b) or heterozygous (+/fa; model c) for the normal allele of the leptin receptor gene, served as lean controls for the ZDF strain of rats. The rats were genotyped by the supplier. The lean ZDF rats have the same genetics as the obese prediabetic ZDF strain, except for the double copy of the mutated fa gene. A reduction in insulin gene expression has been found in the ZDF strain that segregates independently from the fa locus (20). This mutation, perhaps together with other genetic defects, may explain why the ZDF strain is susceptible to diabetes in the presence of a mutated leptin receptor.

Group d. Age-matched female Zucker obese rats (fa/fa) were used as obese nonprediabetic controls. The Zucker obese rats are outbred rats derived from the Wistar rat that have a mutated leptin receptor. These rats are obese and insulin resistant but do not develop overt diabetes (42).

Groups e and f. Age-matched female obese ZDF (fa/fa) rats were used as both a prediabetic (group e) and diabetic obese model (group f) (14). The ZDF rat is a model obtained by inbreeding occasional descendents of Zucker rats with diabetic characteristics. Male ZDF rats fed a standard rodent diet (Purina 5008) spontaneously become diabetic. Female ZDF rats only become overtly diabetic (fed glucose levels >15 mM) when fed the Gmi diabetogenic diet (14). Obese ZDF rats have a mutated leptin receptor similar to obese Zucker rats. However, as explained above, other genetic defects account for the propensity of ZDF rats to develop diabetes when obese, since overt diabetes does not develop in obese Zucker rats.

Surgical Procedures

On arrival, all rats were housed in the Department of Comparative Medicine of the University of Toronto and exposed to a 12:12-h light-dark cycle. They were kept on the same diet as previously fed by the supplier (diabetogenic Gmi/RD 13004 diet from Charles River, 47.9% fat, for diabetic ZDF rats; and standard Purina 5008 from Ralston Purina, St. Louis, MO, 16.7% fat, for all other groups) with water ad libitum. All procedures were approved by the Animal Care Committee of the University of Toronto. Procedures for surgery and preparation for infusion have been described previously (34). In brief, the left carotid artery and right jugular vein were cannulated under anesthesia with ketamine-xylazine-acepromazine (87:1.7:0.4 mg/ml, 1 µl/g body wt), and indwelling catheters were inserted to reach the aortic arch and right atrium, respectively. The catheters were fed through a subcutaneous intrascapular implant and exteriorized. To maintain patency, the catheters were filled with a mixture of 60% polyvinylpyrrolidone and heparin and flushed every 2–3 days.

Preclamp (48-h Infusion) Period

At least 3 days after vessel cannulation surgery, the rats were randomized to either intralipid plus heparin (IH) or saline (SAL). Intralipid (20%) plus heparin (20 U/ml infusate) was infused (iv) at 7.5 µl/min. SAL was infused at a rate that matched the volume infused in IH. The IH/SAL infusions were given through lines that ran inside a tether that was fitted to the subcutaneous implant. Each rat was placed in a circular cage, and the infusion lines were run through a swivel that was suspended on top of the cage. This procedure protected the infusion tubing and allowed the rat complete freedom of movement. During the infusion, the rat had access to water and food (its usual Purina 5008 diet if nondiabetic or diabetogenic Gmi/RD 13004 diet if diabetic).

Samples for FFA, glucose, and insulin were taken before infusion (fed sample) and at 18, 24, and 46 h after the onset of the IH/SAL infusion, i.e., at –30, –24, and –2 h before the onset of the hyperglycemic clamp (time = 0). Food was removed at 7 PM the day before the two-step hyperglycemic clamp.

Two-Step Hyperglycemic Clamp

GSIS was determined by measuring the insulin and C-peptide response to a two-step (~13 and ~22 mM) hyperglycemic clamp. The two-step hyperglycemic clamp was performed in overnight-fasted conscious rats. Two basal samples were taken at –20 and 0 min, after which an infusion of 37.5% glucose was started (time = 0 min) to approximately double the plasma glucose levels (first step of the hyperglycemic clamp). The target plasma glucose level of 13 mM (upper physiological for rats) was achieved and maintained by adjusting the rate of the glucose infusion according to frequent (every 5–10 min) plasma glucose determinations. At 120 min, the glucose infusion was again raised to achieve and maintain plasma glucose levels of ~22 mM (second step of the hyperglycemic clamp), which are maximum stimulatory for insulin release, until the end of the experiment (time = 240 min). The SAL/IH infusion was continued throughout the clamp. Samples for insulin, C-peptide, and FFA were taken at regular intervals.

Laboratory Methods

Plasma glucose concentrations were measured by the glucose oxidase method using a Beckman Glucose Analyzer II (Beckman Instruments, Fullerton, CA). Plasma FFA levels were analyzed enzymatically using a colorimetric kit (Wako Chemicals, Neuss, Germany) with a coefficient of variation of <14.5%. Plasma insulin and C-peptide levels were determined by radioimmunoassay using kits specific for rat insulin and C-peptide from Linco Research (St. Charles, MO), with coefficients of variation of less than 9 and 10.5%, respectively.

Calculations

C-peptide levels during the last 40 min of each step of the clamp were taken as indexes of absolute secretion at the corresponding glucose levels, as the insulin secretion rate cannot be calculated in rats. This is because the parameters of C-peptide kinetics cannot be determined, as rat C-peptide is unavailable for injection. Notably, IH has not been found to affect C-peptide kinetics in humans (1). The slope of the C-peptide vs. glucose curve, which is an index of the beta-cell sensitivity to glucose, was calculated as in APPENDIX A. Insulin secretion in vivo has to be evaluated in the context of insulin sensitivity, since the normal beta-cell compensates for insulin resistance by increasing secretion, independent of plasma glucose. Bergman et al. (2) and Kahn et al. (25) introduced the concept that the quantitative nature of the relationship between insulin sensitivity and insulin secretion in normal subjects is hyperbolic, i.e., the product of insulin sensitivity and insulin secretion is a constant that they defined as DI (disposition index) and considered a measure of beta-cell function (including beta-cell ability to compensate for insulin resistance). The hyperbolic relationship yields a slope of –1 on linear regression after logarithmic transformation of the variables [i.e., DI = secretion index x sensitivity index yields ln(DI) = ln(secretion index) + ln(sensitivity index), where ln is the natural logarithm, and, after rearrangement, ln(secretion index) = ln(DI) (i.e., a constant) – 1x ln (sensitivity index)]. In the present study, insulin sensitivity was first calculated using the simple and established M/I index, i.e., the glucose infusion rate (Ginf) necessary to maintain the hyperglycemic clamp in the last 40 min of the clamp divided by the insulin level. Since, in our control group (SAL-treated Wistar rats), we found an inverse relationship between the ln-transformed M/I and C-peptide levels (see RESULTS) with a slope not significantly different from –1 (confidence limits encompassing –1, see RESULTS), we calculated a DI.

To evaluate insulin sensitivity, we also used an alternative index based on the glucose infusion and insulin concentration data during the whole hyperglycemic clamp. This insulin sensitivity index (ISI; APPENDIX B) quantifies the increment in glucose flux relative to the increment in insulin concentration. Because ISI uses all the hyperglycemic clamp data, it is less prone than M/I to intrinsically correlate with insulin secretion assessed during the same experiment (32). In fact, ISI was inversely related to C-peptide levels only when all data of the SAL-treated groups were considered, whereas in the SAL-treated Wistar control group, the inverse relationship did not reach significance, presumably because of the low n. Because the slope of the inverse relationship in all SAL-treated groups was different from –1 (i.e., –0.743, see RESULTS), when using ISI, we calculated a compensation index (CI = C-peptide x ISI0.743) as in Refs. 32 and 33. [Note that this index is based on the inverse relationship ln(C-peptide) = ln(CI) (i.e., a constant) – 0.743 x ln(ISI) that, after rearrangement to lnCI = ln(C-peptide) + 0.743 x ln(ISI) and exponential transformation, yields CI = C-peptide x ISI0.743.]

Statistical Analysis

All data are presented as means ± SE. In case of unequal variance, the data were logarithmically transformed. The effect of treatment (IH vs. SAL) was compared within each group using one-way ANOVA for repeated measures. Two-way ANOVA with interaction followed by a linear contrast statement was used to determine whether the effect of the treatment was different between groups. The statistical calculations were performed using Statistical Analysis System software (SAS, Cary, NC). Significance was assumed when the two-tailed P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 APPENDIX A
 APPENDIX B
 GRANTS
 REFERENCES
 
Body Weight

Body weight was lower (P < 0.001) than in control Wistar rats (SAL = 262 ± 6 g, n = 9; IH = 264 ± 8, n = 8) in both groups of lean ZDF rats (wild-type, SAL = 202 ± 4 g, n = 6; IH = 192 ± 4, n = 6; and heterozygous, SAL = 200 ± 4 g, n = 7; IH = 210 ± 5, n = 9), whereas it was higher (P < 0.001), as expected, in all obese groups (obese Zucker rats, SAL = 412 ± 16 g, n = 7; IH = 381 ± 8, n = 9; obese prediabetic ZDF rats, SAL = 340 ± 10 g, n = 6; IH = 330 ± 14, n = 6; and obese diabetic ZDF rats, SAL = 345 ± 11 g, n = 8; IH = 348 ± 15, n = 6). Body weight was greater in obese Zucker rats (P < 0.001) than in obese prediabetic and diabetic ZDF rats. As expected, there was no significant difference in body weight in the SAL- vs. IH-treated groups.

Preclamp (48-h Infusion) Period

Food intake on the first day of infusion (when rats were not fasted) was less (P < 0.05) in control Wistar rats (SAL = 13.3 ± 0.6 g, IH = 6.0 ± 3.2) and lean ZDF rats (wild-type, SAL = 12.2 ± 1.0 g, IH = 7.4 ± 2.2; and heterozygous, SAL = 13.3 ± 1.6 g, IH = 5.7 ± 2.0) than in obese diabetic ZDF rats (SAL = 22.4 ± 1.4 g, IH = 12.7 ± 4.2). Food intake was similar in all obese groups (obese Zucker rats, SAL = 16.7 ± 2.3 g, IH = 12.8 ± 3.4; and obese prediabetic ZDF rats, SAL = 21.0 ± 2.9 g, IH = 7.7 ± 2.8). IH treatment tended to decrease food intake in all groups and did so significantly in heterozygous lean ZDF rats and in obese prediabetic and diabetic ZDF rats (all P < 0.05). The IH-induced trend toward a decrease in food intake was different from our previous observations in Wistar rats, where a lower rate of IH infusion did not affect food intake (34).

The values in Fig. 1 refer to the plasma FFA concentrations at time = –48 h (baseline fed preinfusion levels), –30 h, –24 h (fed levels during active feeding in the dark cycle), and –2 h (fasting sample) before the onset of the clamp (time = 0). All obese groups had elevated baseline FFA compared with the nonobese groups (P < 0.01). There were no significant differences in the baseline plasma FFA between IH- and SAL-treated rats in any groups. The effect of IH to increase FFA (P < 0.01) appeared to be less in obese prediabetic ZDF rats and greater in obese diabetic ZDF rats than in control Wistar rats; however, the differences among groups were not significant.


Figure 1
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 1. Plasma free fatty acid (FFA) levels (means ± SE) during the 48-h infusion period before the 2-step hyperglycemic clamp (time = 0) in rats infused with Intralipid + heparin (IH; 20% Intralipid + 20 U/ml heparin, 7.5 µl/min) or equivolume saline (SAL). The –48-h (baseline preinfusion), –30-h, and –24-h samples were fed, and the –2-h sample was a fasting sample. ZDF rat, Zucker diabetic fatty rat. Significances between groups are reported in text for all figures.

 
Lean ZDF and obese Zucker rats had baseline (–48 h) fed plasma glucose levels similar to those of control Wistar rats (Fig. 2). Obese prediabetic ZDF rats exhibited mild hyperglycemia (P < 0.001 vs. controls), whereas obese diabetic ZDF rats were markedly hyperglycemic (P < 0.001 vs. obese prediabetic ZDF rats). After overnight fasting, plasma glucose declined to almost normal levels in SAL-treated obese prediabetic rats and, surprisingly, also in SAL-treated obese diabetic ZDF rats. There was no significant difference in baseline plasma glucose between the IH and the SAL groups, except for the control Wistar rats, where glucose was slightly higher in the IH group (P < 0.01). IH infusion did not affect plasma glucose in the nonobese groups. IH raised fed plasma glucose levels in Zucker obese rats and obese prediabetic ZDF rats (P < 0.05). In obese diabetic ZDF rats, IH elevated (P < 0.05) fasting plasma glucose (–2 h).


Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 2. Plasma glucose levels (means ± SE) during the 48-h infusion period before the 2-step hyperglycemic clamp (time = 0) in rats infused with IH (20% Intralipid + 20 U/ml heparin, 7.5 µl/min) or equivolume SAL. The –48-h (baseline preinfusion), –30-h, and –24-h samples were fed, and the –2-h sample was a fasting sample. No. of rats in each group is shown in Fig. 1. Insets: the same data on an expanded scale. Significances between groups are reported in text for all figures.

 
In control Wistar and lean ZDF rats, plasma insulin levels during the preclamp period were similar and low (Fig. 3). All obese groups were markedly hyperinsulinemic. In the nondiabetic rats but not in the diabetic rats, insulin levels increased during the dark cycle (feeding period). Baseline insulin levels were not significantly different in the IH- vs. SAL-treated rats in all groups. IH did not significantly affect the preclamp fed insulin levels. However, fasting plasma insulin levels (–2 h) were higher in IH than in SAL groups in heterozygous lean ZDF rats (P < 0.05) and in obese prediabetic (P < 0.001) and diabetic ZDF rats (P < 0.05).


Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 3. Plasma insulin levels (means ± SE) during the 48-h infusion period before the 2-step hyperglycemic clamp (time = 0) in rats infused with IH (20% Intralipid + 20 U/ml heparin, 7.5 µl/min) or equivolume SAL. The –48-h (baseline preinfusion), –30-h, and –24-h samples were fed, and the –2-h sample was a fasting sample. No. of rats in each group is shown in Fig. 1. Insets: the same data on an expanded scale. Significances between groups are reported in text for all figures.

 
Two-Step Hyperglycemic Clamp

As reported above (–2 h data are shown in Fig. 2), fasting plasma glucose levels (–20 to 0 min data are averaged as "basal" in Table 1) were mildly elevated in obese prediabetic ZDF rats and in SAL-treated diabetic ZDF rats (both P < 0.001 vs. controls), whereas IH-treated diabetic ZDF rats had marked hyperglycemia (P < 0.05 vs. SAL). Basal glucose levels tended to be higher in IH- than in SAL-treated rats in most groups (Table 1). Glucose levels were elevated to 13 mM during the first step and to 22 mM during the second step of the hyperglycemic clamp, with no difference between IH and SAL in the nonobese groups. Glucose levels tended to be higher during the clamp in IH-treated than in SAL-treated obese groups (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Plasma glucose and FFA levels during the 2-step (13 and 22 mM) hyperglycemic clamp

 
Fasting plasma FFA were elevated in all obese groups (Table 1). The effect of IH to increase fasting plasma FFA was less (P < 0.05) in obese prediabetic ZDF rats than in obese diabetic ZDF rats. In obese Zucker rats and obese prediabetic ZDF rats, the IH-induced elevation of FFA was actually not significant in the basal state, which may be attributed to the preexisting FFA elevation and/or to the elevation in plasma insulin in obese prediabetic ZDF rats (see below). Consistent with the insulin secretory response during the hyperglycemic clamp, FFA levels decreased in all groups except in IH-treated obese prediabetic ZDF rats and SAL-treated obese diabetic ZDF rats, which may be attributed to insulin resistance/insulin lack in these groups (Table 1).

Also as reported above, fasting plasma insulin was markedly elevated in all obese groups (Fig. 4). During the hyperglycemic clamp, insulin levels rose in all groups except for the diabetic rats; however, the rise was blunted in wild-type lean ZDF rats. IH did not affect the insulin levels in any group, except in mildly hyperglycemic obese prediabetic ZDF rats, where IH induced an elevation of basal (P < 0.05) and glucose-stimulated insulin levels (P < 0.01 at 13 mM glucose).


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 4. Plasma insulin levels (means ± SE) during the 2-step hyperglycemic clamp in rats infused with IH (20% Intralipid + 20 U/ml heparin, 7.5 µl/min) or equivolume SAL. No. of rats in each group is shown in Fig. 1. From –20 min to 0 min is basal. At time = 0 (i.e., 48 h after the start of the SAL/IH infusion), the plasma glucose levels were raised to 13 mM; at time = 120 min, the plasma glucose levels were further raised to 22 mM. The SAL/IH infusion was continued throughout the clamp. Significances between groups are reported in text for all figures.

 
Fasting C-peptide levels were similar in control Wistar and lean ZDF rats, whereas they were elevated in all obese groups (Fig. 5). Interestingly, both groups of lean ZDF rats exhibited minimal stimulation of C-peptide levels by hyperglycemia, i.e., the slope of the C-peptide vs. glucose curve calculated as in APPENDIX A was decreased (P < 0.01) in these rats. This slope tended to be decreased (P = 0.06) in obese Zucker rats vs. Wistar controls and was decreased in obese prediabetic ZDF rats (P < 0.05) vs. Wistar controls but not wild-type lean ZDF rats. C-peptide response to glucose was absent in obese diabetic ZDF rats. IH tended to increase C-peptide levels at 22 mM glucose in Wistar controls, but the effect was not significant (P = 0.19) and there was no difference between SAL- and IH-treated rats in any other group except the mildly hyperglycemic obese prediabetic ZDF rats. In these rats, IH induced a striking elevation of basal (P < 0.01) and glucose-stimulated C-peptide levels (P < 0.001 at 13 mM glucose and P < 0.05 at 22 mM glucose) that paralleled the IH-induced elevation in insulin levels. Notably, IH did not increase C-peptide levels in obese diabetic rats who had marked hyperglycemia and loss of the C-peptide response to glucose. The slope of the C-peptide vs. glucose curve was increased significantly by IH (P < 0.05) only in control Wistar rats (SAL = 16.6 ± 1.1 nM/mM x 103 vs. IH = 21.2 ± 1.9) but was unaffected by IH in all other groups.


Figure 5
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 5. Plasma C-peptide levels (means ± SE) during the 2-step hyperglycemic clamp in rats infused with IH (20% Intralipid + 20 U/ml heparin, 7.5 µl/min) or equivolume SAL. No. of rats in each group is shown in Fig. 1. From –20 min to 0 min is basal. At time = 0 (i.e., 48 h after the start of the SAL/IH infusion), the plasma glucose levels were raised to 13 mM; at time = 120 min, the plasma glucose levels were further raised to 22 mM. The SAL/IH infusion was continued throughout the clamp. Significances between groups are reported in text for all figures.

 
The C-peptide-to-insulin ratio, which is an approximate index of insulin clearance, was lower (P < 0.05) in heterozygous SAL-treated lean ZDF rats (1.4 ± 0.3 pmol C-peptide/pmol insulin in the basal state) and obese prediabetic (1.2 ± 0.2) and diabetic ZDF rats (1.6 ± 0.2) than in control Wistar rats (2.9 ± 0.5). These ratios did not change during the clamp. IH tended to decrease these ratios in the basal state or during the clamp, but its effect was not significant.

Figure 6 shows the glucose infusion rate (Ginf). During the hyperglycemic clamp, Ginf is a measure of glucose tolerance, which includes both the capacity to dispose of the exogenously infused glucose at a predetermined insulin level (i.e., insulin sensitivity) and the capacity to increase the insulin level (by increased secretion and/or decreased insulin clearance) to accelerate this disposal. In the absence of significant changes in insulin clearance, Ginf mainly reflects the ability of the beta-cell to compensate for insulin resistance. Thus, because IH did not appear to significantly affect insulin clearance in our study (see above), the IH-induced differences in Ginf were expected to be consistent with those obtained using calculated indexes of beta-cell function. Ginf was similar in SAL-treated control Wistar and lean ZDF rats but was markedly reduced in all obese groups (P < 0.001), especially in obese diabetic rats at 22 mM glucose. IH decreased Ginf in most groups at 13 mM glucose and in all groups at 22 mM glucose (Fig. 6). In heterozygous lean ZDF rats, the IH-induced decrease in Ginf was markedly greater than in control Wistar rats or wild-type lean ZDF rats (P < 0.001). In obese diabetic ZDF rats, the IH-induced decrease in Ginf was greater than in any other obese group (P < 0.05).


Figure 6
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 6. Glucose infusion rate (Ginf; mean ± SE) necessary to achieve and maintain the 2-step hyperglycemic clamp in rats infused with IH (20% Intralipid + 20 U/ml heparin, 7.5 µl/min) or equivolume SAL. No. of rats in each group is shown in Fig. 1. From –20 min to 0 min is basal. At time = 0 (i.e., 48 h after the start of the SAL/IH infusion), the plasma glucose levels were raised to 13 mM; at time = 120 min, the plasma glucose levels were further raised to 22 mM. The SAL/IH infusion was continued throughout the clamp. Significances between groups are reported in text for all figures.

 
Indexes of Insulin Sensitivity and beta-Cell Function

As described in METHODS, the M/I index of insulin sensitivity was calculated first (Table 2). Lean ZDF rats were the most insulin sensitive (P < 0.05) followed by control Wistar rats. All obese groups were markedly (P < 0.001) insulin resistant. IH decreased M/I in all groups, although the decrease failed to reach significance in wild-type lean ZDF rats (P = 0.06 at 13 mM glucose, and P = 0.07 at 22 mM glucose). The effect of IH was greater in heterozygous lean ZDF rats (single copy of the fa gene) and obese Zucker rats than in control Wistar rats at 13 mM glucose (both P < 0.05). The IH-induced decrease in M/I was greater than in obese Zucker rats (P < 0.05), wild-type lean ZDF rats (P < 0.05), and control Wistar rats (P < 0.01) in both obese hyperglycemic groups (prediabetic and diabetic ZDF rats).


View this table:
[in this window]
[in a new window]

  		Table 2. Insulin sensitivity expressed as M/I index during the 2-step (13 and 22 mM) hyperglycemic clamp

 
Table 3 shows the results of the linear regression between M/I and C-peptide after ln transformation of both variables. It can be seen that both r2 and the slope of the regression were lower with progressive deterioration of glucose tolerance and that, in general, IH decreased them further. This is consistent with impaired beta-cell compensation for insulin resistance. Because the slope of the regression was not significantly different from –1 (confidence interval from –0.584 to –1.130) in SAL-treated control Wistar rats, indicating a hyperbolic relationship, we calculated a DI.


View this table:
[in this window]
[in a new window]

  		Table 3. Results of linear regression analysis between M/I index and C-peptide levels after logarithmic transformation

 
Figure 7 shows the DI (=C-peptide x M/I), which is an index of insulin secretion corrected for insulin sensitivity. DI was lower in lean ZDF rats than in control Wistar rats (P < 0.05) and was much lower (P < 0.001) in all obese groups. In obese prediabetic and diabetic ZDF rats, DI was lower than in obese Zucker rats (P < 0.05). IH decreased DI significantly at 13 mM glucose in heterozygous lean ZDF rats (P < 0.001) and in obese Zucker rats (P < 0.05). At 22 mM glucose, IH decreased DI significantly also in control Wistar rats (P < 0.05) and obese diabetic ZDF rats (P < 0.001). IH failed to decrease DI significantly in both wild-type lean ZDF rats (P = 0.08 at 22 mM glucose) and obese prediabetic ZDF rats (P = 0.11 at 22 mM glucose). Analysis of the interaction between treatment and group further revealed that 1) the IH-induced decrease in DI was greater in heterozygous lean ZDF rats (single copy of the fa gene) (P < 0.01), obese Zucker rats (double copy of the fa gene) (P < 0.05), and obese diabetic ZDF rats (P < 0.05) than in control Wistar rats; and 2) in accordance with the results of the group-by-group analysis reported above, in mildly hyperglycemic obese prediabetic ZDF rats, the IH-induced decrease in DI was not greater than in obese Zucker rats, wild-type lean ZDF rats, or control Wistar rats but was less (P < 0.05) than that observed in markedly hyperglycemic obese diabetic ZDF rats.


Figure 7
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 7. Disposition index (DI, mean ± SE; calculated as plasma C-peptide level multiplied by the sensitivity index M/I reported in Table 2) during the last 40 min of each step of the 2-step hyperglycemic clamp (13 and 22 mM) in rats infused with IH (20% Intralipid + 20 U/ml heparin, 7.5 µl/min) or equivolume SAL. Unit for DI: C-peptide (µmol/l x unit of M/I) No. of rats in each group is shown in Fig. 1. At time = 0 (i.e., 48 h after the start of the SAL/IH infusion), the plasma glucose levels were raised to 13 mM; at time = 120 min, the plasma glucose levels were further raised to 22 mM. The SAL/IH infusion was continued throughout the clamp. Insets: the same data on an expanded scale. Significances between groups are reported in text for all figures.

 
Table 4 shows the alternative index of insulin sensitivity (ISI), which, being based on the whole clamp, is less prone than M/I to intrinsically correlate with the C-peptide level obtained at the same time. As described in METHODS, this index was inversely related to C-peptide only when all SAL-treated groups were combined (r2 = 0.316, P < 0.001) and the slope (–0.743) was different from –1 (confidence interval from –0.597 to –0.889), indicating that the relationship was not hyperbolic. Therefore, the alternative index of beta-cell compensation (CI) was calculated (CI = C-peptide x ISI0.743) (32, 33) using the steady-state C-peptide values of both the first (13 mM) and second (22 mM) steps of the clamp. It can been seen from Table 4 that, when using this method of adjusting insulin secretion for insulin sensitivity, the results were similar to those described in Fig. 7, although the IH-induced decrease in CI in control Wistar rats failed to reach significance. Specifically, a significant IH-induced decrease in CI was seen only in heterozygous lean ZDF rats at 22 mM glucose and in obese diabetic ZDF rats at 13 mM glucose, whereas the decrease approached significance in obese Zucker rats at 13 mM glucose (P = 0.07) and in obese diabetic ZDF rats at 22 mM glucose (P = 0.06). Correspondingly, the IH effect to decrease CI was greater in heterozygous lean ZDF rats (P < 0.05), obese Zucker rats (P < 0.05), and obese diabetic ZDF rats (P < 0.01) than in controls (P < 0.05). Obese prediabetic ZDF rats did not show a greater IH-induced decrease in CI than obese Zucker rats, but they did show less effect of IH on CI than obese diabetic ZDF rats (P < 0.01) during the first step of the clamp.


View this table:
[in this window]
[in a new window]

  		Table 4. ISI and CI

 
Adjustment of insulin secretion for insulin sensitivity could only be performed with an index of absolute insulin secretion (C-peptide level), not with an index of beta-cell step response to glucose, as there was no significant inverse relationship between either index of insulin sensitivity (M/I or ISI) and the slope of the C-peptide vs. glucose curve. This presumably indicates that the step insulin secretory response to glucose is less related to insulin sensitivity than the absolute secretion, as previously reported (18).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
APPENDIX A
 APPENDIX B
 GRANTS
 REFERENCES
 
In the present study, a greater infusion rate of IH than used in our previous rat study (34) did not decrease absolute insulin secretion in control Wistar rats but decreased beta-cell function (i.e., secretion considered in relation to insulin resistance) (analysis based on DI). In other words, in the present rat model, we could reproduce the results we previously found in humans where, similar to the present rat study, IH induced significant insulin resistance (11). The effect of IH to decrease beta-cell function was greater in heterozygous lean ZDF rats that carry a copy of the mutated leptin receptor gene than in control Wistar rats (analysis based on both DI and CI). Moreover, the effect of IH to decrease beta-cell function was greater in obese Zucker rats and in obese diabetic ZDF rats than in control Wistar rats (analysis based on both DI and CI). However, in obese prediabetic ZDF rats with only mild hyperglycemia, the IH-induced decrease in beta-cell function was not greater than that in obese Zucker rats but was less than that in markedly hyperglycemic obese ZDF rats (analysis based on both DI and CI).

Nonobese Rats

Our results in vivo in heterozygous lean ZDF rats (carriers of the fa gene) are in keeping with previous results in isolated islets (22, 27) that have shown an increased susceptibility to the chronic desensitizing effect of FFA on GSIS vs. control Wistar rats. Wildtype lean ZDF rats have not been studied previously.

Interestingly, our lean ZDF rats (both heterozygous and wild-type) exhibited minimal stimulation of insulin secretion throughout the hyperglycemic clamp. However, in heterozygous lean ZDF rats, the rise in insulin was much higher than that in C-peptide, indicating a reduction in insulin clearance. In addition, both groups of lean ZDF rats were likely more insulin sensitive than control Wistar rats (analysis based on M/I index). The reduction in clearance and the increase in insulin sensitivity may be compensatory for the low insulin secretion. The lower body weight of lean ZDF rats than that of control Wistar rats could also contribute to greater insulin sensitivity.

In heterozygous lean ZDF rats, IH induced insulin resistance and decreased beta-cell function to a greater extent than in wild-type lean ZDF rats or control Wistar rats. Lean ZDF rats were normoglycemic; therefore, their hypersensitivity to IH-induced beta-cell dysfunction cannot be attributed to hyperglycemia. These rats may be hypersensitive to lipotoxicity on both insulin sensitivity and secretion because of the presence of one copy of the mutated leptin receptor (49). This hypersensitivity may be explained by an enhanced fatty acid esterification pathway, which would account for the modest elevation in triglyceride content found in insulin target tissues and islets of these rats (27). The latter mechanism would be similar to the postulated mechanism of enhancement of beta-cell lipotoxicity by hyperglycemia (6). In the wild-type lean ZDF rats (without the fa mutation), the IH-induced decrease in beta-cell function measured by DI or CI was not greater than that in control Wistar rats. However, IH increased the slope of the C-peptide vs. glucose curve in control Wistar rats but failed to do so in wild-type lean ZDF rats. This may indicate that low glucose-sensing ability in ZDF rats may somewhat compromise beta-cell adaptation to FFA-induced insulin resistance.

Obese Rats

All groups of obese rats had high FFA levels, were insulin resistant, and had basal hyperinsulinemia that was mainly due to hypersecretion of insulin, as indicated by their elevated basal C-peptide levels. Obese prediabetic and diabetic rats also showed a reduction in insulin clearance, as indicated by their lower C-peptide-to-insulin ratio. Obese Zucker rats were normoglycemic; however, obese prediabetic ZDF rats had mild hyperglycemia. Obese diabetic rats had marked hyperglycemia in the fed state, consistent with their absent glucose-stimulated insulin-secretory response and with the dependence of their diabetic state on ingestion of the diabetogenic diet, but only modest hyperglycemia in the fasting state. All obese groups had a lower Ginf and DI than nonobese groups, indicating that their hypersecretion of insulin was not totally adequate to compensate for their insulin resistance.

It is difficult to compare the response to IH in the obese vs. the nonobese groups because of the greater preexisting FFA levels and the lower preexisting insulin sensitivity. However, the IH-induced decrease in DI (or CI) was greater in obese Zucker rats than in control Wistar rats, consistent with the greater decrease in insulin sensitivity and the double copy of the leptin receptor mutation. In both obese prediabetic and diabetic ZDF rats, IH decreased insulin sensitivity (either M/I or ISI) more than in obese Zucker rats or wild-type lean ZDF rats. This suggests a role for hyperglycemia to enhance the effect of lipotoxicity in insulin-sensitive tissues. As to beta-cell lipotoxicity, however, in obese prediabetic ZDF rats, the IH-induced decrease in DI or CI was not greater than that in obese Zucker rats, and the IH-induced decrease in DI was not greater than that in wild-type lean ZDF rats despite the greater decrease in insulin sensitivity, because absolute basal and glucose-stimulated insulin secretion increased with IH. We cannot exclude that this paradoxical response to IH in obese prediabetic ZDF rats may be somewhat linked to their beta-cell dysfunction, as wild-type lean ZDF rats also showed a nonsignificant decrease in DI. However, wild-type lean ZDF rats did not actually show any IH-induced enhancement of absolute insulin secretion and did not significantly differ from control Wistar rats in their DI (or CI) response to IH. Thus we favor the possibility that the paradoxical insulin-secretory response to IH in obese prediabetic ZDF rats, which was not seen in normoglycemic rats, is due to their preexisting mild hyperglycemia (preexisting because, during the clamp, glycemia was similar in all groups). Our results suggest that mild hyperglycemia, despite interacting with fatty acids to cause increased demands on the beta-cell, did not impart a more negative effect on insulin secretion than that observed in animals with normoglycemia. Prolonged hyperglycemia may initially sensitize the beta-cell compensation for insulin resistance [perhaps by increasing beta-cell glucose influx, for example via glucokinase induction (29)] in the in vivo clamp condition. Interestingly, increased absolute insulin secretion was also observed in nondiabetic humans when IH infusion was combined with a prolonged (48 h) hyperglycemic clamp (5).

Since high-fat feeding precipitates diabetes in obese prediabetic ZDF rats, it is attractive to speculate that, ultimately, however, beta-cell overwork or glucotoxicity related to hyperglycemia-induced increased beta-cell glucose influx may result in beta-cell glucose blindness as seen in obese diabetic ZDF rats. In these rats, further FFA elevation by IH infusion did not stimulate insulin secretion despite aggravating hyperglycemia. The lack of further increase in insulin secretion in the diabetic rats may be a consequence of their more marked hyperglycemia and/or their beta-cell glucose blindness, as glucose sensing may be involved in the poorly understood mechanisms of beta-cell adaptation to insulin resistance.

Comparison with Previous Studies

Our results show that hypersensitivity to beta-cell lipotoxicity characterizes the very early stages of beta-cell dysfunction in vivo. Our studies in humans are also consistent with this notion, as moderately obese individuals with glucose intolerance were hypersensitive to the impairing effect of FFA on GSIS (10), similar to obese Zucker rats. Obese type 2 diabetic subjects with mild hyperglycemia showed a slight enhancement of absolute GSIS after 48-h IH (10), in accord with our results in obese prediabetic ZDF rats. Our results are consistent with those found by other authors, who described an improvement in the insulin-secretory response to glucose after acipimox, a FFA-lowering agent, in well-controlled type 2 diabetes (similar to our findings in obese glucose-intolerant subjects) but not in less well-controlled diabetes (similar to our hyperglycemic models) (40). There are no studies on the beta-cell effect of FFA in type 2 diabetic patients with marked hyperglycemia and absent glucose-stimulated insulin response (similar to our diabetic ZDF rats). In vitro (38, 39, 47) and ex vivo (7, 43) studies have suggested that prolonged exposure of beta-cell lines or pancreatic islets to glucose can have additive (43, 47) or synergistic effects (7, 38, 39) on impairing beta-cell function to those of fatty acids; however, these models eliminate the beta-cell compensatory response to insulin resistance that is observed in vivo and would therefore be expected to yield different results from the in vivo model, if hyperglycemia specifically affects this response. In vitro studies have also suggested synergistic effects of glucose and fatty acids on beta-cell apoptosis (17, 37). Models of more prolonged FFA exposure than 48 h should be used to address these apoptotic effects in vivo.

In conclusion, the present study shows that the lipotoxic effect of fatty acids on beta-cell function is accentuated in the presence of the fa mutation (leptin resistance favoring fat accumulation) not only in vitro, as previously reported, but also in vivo. However, our studies in obese prediabetic ZDF rats suggest that lipid-induced beta-cell dysfunction is not enhanced by a mild chronic elevation of glucose levels in vivo, contrary to previous studies in vitro. In vivo, preexisting mild hyperglycemia may instead sensitize the beta-cell to mount a partial compensatory response to FFA-induced insulin resistance. However, this compensatory response is abolished in overt diabetes, presumably because of a more marked hyperglycemia and/or loss of glucose sensing.


   APPENDIX A
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
APPENDIX B
 GRANTS
 REFERENCES
 
Slope of C-Peptide vs. Glucose Curve

The slope of the C-peptide vs. glucose curve (C-peptide vs. glucose slope, nM/mM) is an index of the sensitivity of the beta-cell to glucose. It was calculated as the ratio of the integral of the C-peptide concentration increment over basal (CP – CPb, nM) to the integral of the glucose concentration increment over basal (G – Gb, mM) during the whole experimental period [from 0 to time (T) = 240 min]

Formula 1(1)


   APPENDIX B
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
GRANTS
 REFERENCES
 
Calculation of ISI

ISI is an insulin sensitivity index that quantifies the increment in glucose flux relative to the increment in insulin concentration. ISI is based on a simplified model of the glucose-insulin system during the hyperglycemic clamp. The mass-balance equation for glucose is

Formula 2(2)
or rearranging

Formula 3(3)
where Q (µmol/kg) is the glucose mass, Ginf (µmol·min–1·kg–1) is the glucose infusion rate during the clamp, EGP (µmol·min–1·kg–1) is endogenous glucose production, and Rd (µmol·min–1·kg–1) is the rate of disappearance of glucose. Integration from 0 (onset of the clamp) to T = 240 min (end of the clamp) of both members of Eq. 3 yields

Formula 4(4)
where V is the glucose volume (200 ml/kg) and G (mM) is glucose concentration. Note that the right side of Eq. 4 can be calculated from the clamp data. In particular, the glucose concentration increment [G(T) – G(0)] is calculated as the difference between mean glucose from time t = 180 min to the end of the clamp and mean glucose in the basal period.

The term "Rd – EGP" is expected to be positive and increase during the clamp as glucose utilization increases and glucose production is suppressed. The integral of Rd – EGP is proportional to insulin sensitivity if glucose and insulin concentrations are the same.

ISI does not take into account the effect of glucose on the integral of Rd – EGP, because the glucose levels were similar in all groups by experimental design (standardized hyperglycemic clamp). It does, however, take into account the effect of insulin on this integral, as the insulin levels vary among groups during the hyperglycemic clamp. ISI (µmol·min–1·kg–1·pM–1) is defined as the ratio of the integral of Rd – EGP to the integral of the insulin concentration increment over basal (I – Ib)

Formula 5(5)
The parameter 240,000 pM in the denominator was empirically chosen to avoid negative values of insulin sensitivity in obese diabetic rats. Note that ISI calculated from Eq. 5 by definition includes both peripheral and hepatic insulin sensitivity.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
 GRANTS
REFERENCES
 
This study was funded by a grant to A. Giacca from the Canadian Institutes of Health Research. Partial support for personnel was also provided by the Heart and Stroke Foundation of Canada and the Canadian Diabetes Association. T. T. Goh was funded by a graduate scholarship from the Banting and Best Diabetes Centre of the University of Toronto.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Giacca, Dept. of Physiology, Univ. of Toronto, Medical Sciences Bldg., Rm. 3336, Toronto, ON, M5S 1A8 Canada (e-mail: adria.giacca{at}utoronto.ca)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 APPENDIX A
 APPENDIX B
 GRANTS
 REFERENCES
 

  1. Balent B, Goswami G, Goodloe G, Rogatsky E, Rauta O, Nezami R, Mints L, Angeletti RH, Stein DT. Acute elevation of NEFA causes hyperinsulinemia without effect on insulin secretion rate in healthy human subjects. Ann NY Acad Sci 967: 535–543, 2002.[Abstract/Free Full Text]
  2. Bergman RN, Phillips LS, Cobelli C. Physiologic evaluation of factors controlling glucose tolerance in man: measurement of insulin sensitivity and beta-cell glucose sensitivity from the response to intravenous glucose. J Clin Invest 68: 1456–1467, 1981.[ISI][Medline]
  3. Bjorntorp P, Bergman H, Varnauskas E, Lindholm B. Lipid mobilization in relation to body composition in man. Metabolism 18: 840–851, 1969.[CrossRef][ISI][Medline]
  4. Boden G. Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes 46: 3–10, 1997.[Abstract]
  5. Boden G, Chen X, Rosner J, Barton M. Effects of a 48-h fat infusion on insulin secretion and glucose utilization. Diabetes 44: 1239–1242, 1995.[Abstract]
  6. Briaud I, Harmon JS, Kelpe CL, Segu VB, Poitout V. Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids. Diabetes 50: 315–321, 2001.[Abstract/Free Full Text]
  7. Briaud I, Kelpe CL, Johnson LM, Tran PO, Poitout V. Differential effects of hyperlipidemia on insulin secretion in islets of langerhans from hyperglycemic versus normoglycemic rats. Diabetes 51: 662–668, 2002.[Abstract/Free Full Text]
  8. Brun T, Assimacopoulos-Jeannet F, Corkey BE, Prentki M. Long-chain fatty acids inhibit acetyl-CoA carboxylase gene expression in the pancreatic beta-cell line INS-1. Diabetes 46: 393–400, 1997.[Abstract]
  9. Carlsson C, Borg LA, Welsh N. Sodium palmitate induces partial mitochondrial uncoupling and reactive oxygen species in rat pancreatic islets in vitro. Endocrinology 140: 3422–3428, 1999.[Abstract/Free Full Text]
  10. Carpentier A, Mittelman SD, Bergman RN, Giacca A, Lewis GF. Prolonged elevation of plasma free fatty acids impairs pancreatic beta-cell function in obese nondiabetic humans but not in individuals with type 2 diabetes. Diabetes 49: 399–408, 2000.[Abstract]
  11. Carpentier A, Mittelman SD, Lamarche B, Bergman RN, Giacca A, Lewis GF. Acute enhancement of insulin secretion by FFA in humans is lost with prolonged FFA elevation. Am J Physiol Endocrinol Metab 276: E1055–E1066, 1999.[Abstract/Free Full Text]
  12. Chen NG, Reaven GM. Fatty acid inhibition of glucose-stimulated insulin secretion is enhanced in pancreatic islets from insulin-resistant rats. Metabolism 48: 1314–1317, 1999.[CrossRef][ISI][Medline]
  13. Chen S, Ogawa A, Ohneda M, Unger RH, Foster DW, McGarry JD. More direct evidence for a malonyl-CoA-carnitine palmitoyltransferase I interaction as a key event in pancreatic beta-cell signaling. Diabetes 43: 878–883, 1994.[Abstract]
  14. Corsetti JP, Sparks JD, Peterson RG, Smith RL, Sparks CE. Effect of dietary fat on the development of non-insulin dependent diabetes mellitus in obese Zucker diabetic fatty male and female rats. Atherosclerosis 148: 231–241, 2000.[CrossRef][ISI][Medline]
  15. Crespin SR, Greenough WB III, Steinberg D. Stimulation of insulin secretion by long-chain free fatty acids. A direct pancreatic effect. J Clin Invest 52: 1979–1984, 1973.[ISI][Medline]
  16. Dubois M, Kerr-Conte J, Gmyr V, Bouckenooghe T, Muharram G, D’Herbomez M, Martin-Ponthieu A, Vantyghem MC, Vandewalle B, Pattou F. Non-esterified fatty acids are deleterious for human pancreatic islet function at physiological glucose concentration. Diabetologia 47: 463–469, 2004.[CrossRef][ISI][Medline]
  17. El Assaad W, Buteau J, Peyot ML, Nolan C, Roduit R, Hardy S, Joly E, Dbaibo G, Rosenberg L, Prentki M. Saturated fatty acids synergize with elevated glucose to cause pancreatic beta-cell death. Endocrinology 144: 4154–4163, 2003.[Abstract/Free Full Text]
  18. Ferrannini E, Gastaldelli A, Miyazaki Y, Matsuda M, Mari A, DeFronzo RA. Beta-cell function in subjects spanning the range from normal glucose tolerance to overt diabetes: a new analysis. J Clin Endocrinol Metab 90: 493–500, 2005.[Abstract/Free Full Text]
  19. Gerich JE. The genetic basis of type 2 diabetes mellitus: impaired insulin secretion versus impaired insulin sensitivity. Endocr Rev 19: 491–503, 1998.[Abstract/Free Full Text]
  20. Griffen SC, Wang J, German MS. A genetic defect in beta-cell gene expression segregates independently from the fa locus in the ZDF rat. Diabetes 50: 63–68, 2001.[Abstract/Free Full Text]
  21. Hennes MM, Dua A, Kissebah AH. Effects of free fatty acids and glucose on splanchnic insulin dynamics. Diabetes 46: 57–62, 1997.[Abstract]
  22. Hirose H, Lee YH, Inman LR, Nagasawa Y, Johnson JH, Unger RH. Defective fatty acid-mediated beta-cell compensation in Zucker diabetic fatty rats. Pathogenic implications for obesity-dependent diabetes. J Biol Chem 271: 5633–5637, 1996.[Abstract/Free Full Text]
  23. Jacqueminet S, Briaud I, Rouault C, Reach G, Poitout V. Inhibition of insulin gene expression by long-term exposure of pancreatic beta cells to palmitate is dependent on the presence of a stimulatory glucose concentration. Metabolism 49: 532–536, 2000.[CrossRef][ISI][Medline]
  24. Kahn SE. The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of Type 2 diabetes. Diabetologia 46: 3–19, 2003.[CrossRef][ISI][Medline]
  25. Kahn SE, Prigeon RL, McCulloch DK, Boyko EJ, Bergman RN, Schwartz MW, Neifing JL, Ward WK, Beard JC, Palmer JP, Porte D Jr. Quantification of the relationship between insulin sensitivity and beta-cell function in human subjects. Evidence for a hyperbolic function. Diabetes 42: 1663–1672, 1993.[Abstract]
  26. Kashyap S, Belfort R, Gastaldelli A, Pratipanawatr T, Berria R, Pratipanawatr W, Bajaj M, Mandarino L, DeFronzo R, Cusi K. A sustained increase in plasma free fatty acids impairs insulin secretion in nondiabetic subjects genetically predisposed to develop type 2 diabetes. Diabetes 52: 2461–2474, 2003.[Abstract/Free Full Text]
  27. Lee Y, Hirose H, Ohneda M, Johnson JH, McGarry JD, Unger RH. Beta-cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: impairment in adipocyte-beta-cell relationships. Proc Natl Acad Sci USA 91: 10878–10882, 1994.[Abstract/Free Full Text]
  28. Liang Y, Buettger C, Berner DK, Matschinsky FM. Chronic effect of fatty acids on insulin release is not through the alteration of glucose metabolism in a pancreatic beta-cell line (beta HC9). Diabetologia 40: 1018–1027, 1997.[CrossRef][ISI][Medline]
  29. Liang Y, Najafi H, Smith RM, Zimmerman EC, Magnuson MA, Tal M, Matschinsky FM. Concordant glucose induction of glucokinase, glucose usage, and glucose-stimulated insulin release in pancreatic islets maintained in organ culture. Diabetes 41: 792–806, 1992.[Abstract]
  30. Magnan C, Collins S, Berthault MF, Kassis N, Vincent M, Gilbert M, Penicaud L, Ktorza A, Assimacopoulos-Jeannet F. Lipid infusion lowers sympathetic nervous activity and leads to increased beta-cell responsiveness to glucose. J Clin Invest 103: 413–419, 1999.[ISI][Medline]
  31. Magnan C, Cruciani C, Clement L, Adnot P, Vincent M, Kergoat M, Girard A, Elghozi JL, Velho G, Beressi N, Bresson JL, Ktorza A. Glucose-induced insulin hypersecretion in lipid-infused healthy subjects is associated with a decrease in plasma norepinephrine concentration and urinary excretion. J Clin Endocrinol Metab 86: 4901–4907, 2001.[Abstract/Free Full Text]
  32. Mari A, Ahren B, Pacini G. Assessment of insulin secretion in relation to insulin resistance. Curr Opin Clin Nutr Metab Care 8: 529–533, 2005.[ISI][Medline]
  33. Mari A, Gastaldelli A, Natali A, Ostergard T, Schmitz O, Ferrannini E. Characterization of beta-cell function impairment in first-degree relatives of type 2 diabetic subjects: modeling analysis of 24-h triple-meal tests. Am J Physiol Endocrinol Metab 288: E541–E546, 2005.[Abstract/Free Full Text]
  34. Mason TM, Goh T, Tchipashvili V, Sandhu H, Gupta N, Lewis GF, Giacca A. Prolonged elevation of plasma free fatty acids desensitizes the insulin secretory response to glucose in vivo in rats. Diabetes 48: 524–530, 1999.[Abstract]
  35. Paolisso G, Gambardella A, Amato L, Tortoriello R, D’Amore A, Varricchio M, D’Onofrio F. Opposite effects of short- and long-term fatty acid infusion on insulin secretion in healthy subjects. Diabetologia 38: 1295–1299, 1995.[ISI][Medline]
  36. Paolisso G, Tagliamonte MR, Rizzo MR, Gualdiero P, Saccomanno F, Gambardella A, Giugliano D, D’Onofrio F, Howard BV. Lowering fatty acids potentiates acute insulin response in first degree relatives of people with type II diabetes. Diabetologia 41: 1127–1132, 1998.[CrossRef][ISI][Medline]
  37. Piro S, Anello M, Di Pietro C, Lizzio MN, Patane G, Rabuazzo AM, Vigneri R, Purrello M, Purrello F. Chronic exposure to free fatty acids or high glucose induces apoptosis in rat pancreatic islets: possible role of oxidative stress. Metabolism 51: 1340–1347, 2002.[CrossRef][ISI][Medline]
  38. Poitout V, Robertson RP. Minireview: Secondary beta-cell failure in type 2 diabetes–a convergence of glucotoxicity and lipotoxicity. Endocrinology 143: 339–342, 2002.[Abstract/Free Full Text]
  39. Prentki M, Joly E, El Assaad W, Roduit R. Malonyl-CoA signaling, lipid partitioning, and glucolipotoxicity: role in beta-cell adaptation and failure in the etiology of diabetes. Diabetes 51, Suppl 3: S405–S413, 2002.[Abstract/Free Full Text]
  40. Qvigstad E, Mostad IL, Bjerve KS, Grill VE. Acute lowering of circulating fatty acids improves insulin secretion in a subset of type 2 diabetes subjects. Am J Physiol Endocrinol Metab 284: E129–E137, 2003.[Abstract/Free Full Text]
  41. Rebuffe-Scrive M, Anderson B, Olbe L, Bjorntorp P. Metabolism of adipose tissue in intraabdominal depots in severely obese men and women. Metabolism 39: 1021–1025, 1990.[CrossRef][ISI][Medline]
  42. Rohner-Jeanrenaud F, Proietto J, Ionescu E, Jeanrenaud B. Mechanism of abnormal oral glucose tolerance of genetically obese fa/fa rats. Diabetes 35: 1350–1355, 1986.[Abstract]
  43. Sako Y, Grill VE. A 48-hour lipid infusion in the rat time-dependently inhibits glucose-induced insulin secretion and B cell oxidation through a process likely coupled to fatty acid oxidation. Endocrinology 127: 1580–1589, 1990.[Abstract]