Pancreatic steatosis: harbinger of type 2 diabetes in obese rodents

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1 (2) 3, 39 & 2 Macmillan Publishers Limited All rights reserved 37-55/ $32. ORIGINAL ARTICLE Pancreatic steatosis: harbinger of type 2 diabetes in obese rodents Y Lee 1,2, I Lingvay 3, LS Szczepaniak,5, M Ravazzola, L Orci and RH Unger 1,2 1 Touchstone Center for Diabetes Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA; 2 Division of Research, VA North Texas Health Care System, Dallas, TX, USA; 3 Division of Endocrinology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA; Division of Hypertension, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA; 5 Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX, USA and Department of Cell Physiology and Metabolism, University Medical Center, Geneva, Switzerland Objective: The aim of this study was to determine if the fat accumulation in the exocrine pancreas fat of obese Zucker diabetic fatty (ZDF) rodents, like that in their endocrine pancreas, precedes the onset of type 2 diabetes mellitus (T2DM). As the fat content of whole pancreas, but not islets, can now be measured in humans by magnetic resonance spectroscopy (MRS), such measurements could be used as a predictor of impending T2DM and an indication for preventive intervention. Animals: Obese ZDF (fa/fa) rats and lean ( þ / þ ) controls on a % fat diet were killed at time points from to 1 weeks and total pancreatic fat was measured biochemically and electronmicroscopic examination of tissue for fat droplets was carried out. Results: Compared to lean ZDF controls, pancreatic fat was elevated above lean controls from to 1 weeks of age, peaking at weeks of age when hyperglycemia first appeared. The pancreatic profile of fat content in whole pancreas paralleled that of islets. Electronmicroscopic examination identified the acinar location of the fat droplets and ruled out a major contribution of intrapancreatic adipocytes. Conclusion: The almost identical pattern of triglyceride overaccumulation in the exocrine and endocrine pancreas of obese rodents before the onset of T2DM suggests that MRS of the human pancreas might predict T2DM in obese subjects and permit timely interventions to prevent the disease. (2) 3, 39 ; doi:.3/ijo.29.25; published online 15 December 29 Keywords: fatty pancreas; triglycerides; lipotoxicity; metabolic syndrome; type 2 diabetes prevention Introduction Obesity and its life-threatening comorbidities are rising at alarming rates throughout the world. Type 2 diabetes mellitus (T2DM) has increased almost threefold since 195, and is now estimated at 7.% of the American population. 1 Its pathogenesis appears closely related to chronic caloric surplus, which initially leads to obesity with compensated resistance to insulin. Both the insulin resistance 2 and the accompanying compensatory hyperinsulinemia 3 have been attributed to ectopic lipid overload. 3, Fatty acids interfere with the action of insulin on peripheral target cells, such as myocytes and hepatocytes, 5 while they promote b-cell hyperplasia and Correspondence: Dr RH Unger, Touchstone Center for Diabetes Research, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX , USA. roger.unger@utsouthwestern.edu Received 5 August 29; revised October 29; accepted 1 October 29; published online 15 December 29 hypersecretionfat least during the initial period of caloric surplus. 3 Ultimately, however, increased lipid accumulation in islets causes dysfunction and death of nonadipocytes through lipoapoptosis. The loss of b-cells ultimately leads to overt T2DM. 7 In Zucker diabetic fatty (ZDF) rats, a model of genetic obesity with T2DM, b-cell loss and hyperglycemia appear within weeks of the accumulation of excess islet fat. 9, Any intervention that prevents the increase in lipid content of islets, whether by reducing caloric intake or by increasing fatty acid oxidation, will prevent T2DM. 11 Although the T2DM of obese humans resembles that of obese rodents in many respects, a lipotoxic etiology cannot be directly established in humans because noninvasive quantification of triglycerides (TG) content of human islets is not possible. However, it is now possible to measure the TG content of the whole pancreas noninvasively using proton-localized magnetic resonance spectroscopy (MRS).,13 If the TG content of the endocrine and exocrine pancreas rise in parallel, MRS assay of fat in the exocrine pancreas might serve as a surrogate noninvasive index of

2 lipid overaccumulation in the endocrine pancreas and a harbinger of impending lipid-mediated b-cell destruction. If so, it would enable timely lipid-lowering interventions designed to prevent this increasingly prevalent disorder and its comorbidities. Unfortunately, in all of the studies of islet fat, the exocrine tissue was discarded in the process of islet isolation and its fat content was never measured. This study was intended to compare the whole pancreas fat content with islet fat content before and during the development of T2DM in ZDF rats. Materials and methods Animal studies ZDF rats (fa/fa), a model of obesity and T2DM, were housed in individual cages at 22 1C on a -h light dark cycle with free access to % fat chow (Harlan Teklad, Madison, WI, USA) and water. Obese ZDF male rats (fa/fa) develop T2DM after weeks of age, whereas lean ZDF ( þ / þ ) rats remain normoglycemic at all ages and were used as controls. Pancreases were resected under pentobarbital anesthesia and all visible adipose tissue was removed. In brief, 2 lean ( þ / þ ) and 2 obese ZDF (fa/fa) rats were killed in groups of at,,,,, 1 and 1 weeks of age. Tail vein blood was collected weekly at 9 hours for measurement of insulin and glucose levels. Animal experiments were approved by the animal studies committee at the University of Texas Southwestern Medical Center, following Animal Welfare Act Guidelines. with the Linco sensitive rat insulin kit (Linco Research, St Charles, MO, USA). Statistical analysis Pancreatic TG content was compared using analysis of variance. Data were reported as mean and standard error unless otherwise indicated. A P-value of o.5 was considered significant. Results and discussion Chronology of pancreatic steatosis relative to islet steatosis To determine if the fat content of the entire pancreas parallels that of isolated islets, which increases before the onset of T2DM, we quantified fat in pancreata of obese ZDF rats. Earlier studies showed a progressive increase in TG content of pancreatic islets before the onset of hyperglycemia. We observed a parallel progressive, age-related increase in biochemically assayed TG content of whole pancreas, which was remarkably similar to the rise in TG noted previously in the islets of ZDF rats. 9 At weeks of age and all subsequent time points, it far exceeded the TG content of age-matched lean littermates (Po.1; Figure 1a). Pancreas tg (mg/g) 2 Prediabetic Diabetic 1..5 µg/islet 397 Biochemical determination of TG content of pancreas Pancreatic tissue was excised and immediately frozen and stored in liquid nitrogen after careful removal of all identifiable peripancreatic adipose tissue. Neutral lipids were extracted from each aliquot using the Folch procedure 1 with slight modifications. Samples (2 ml) were dried and TG content was measured in triplicate using Sigma TG kit (Sigma-Aldrich, St Louis, MO, USA). Plasma insulin (ng/ml) Electron microscopy of pancreas Pancreatic fragments were fixed in %, cacodylate-buffered (.2 M) glutaraldehyde (ph 7.), for 2 h at 1C, rinsed in Millonig s phosphate buffer and postfixed in buffered osmic acid (1%, 2 h at 2 1C). The specimens then were processed for epoxy embedding (Polybed R, Polysciences, Warrington, PA, USA; Fluka). Thin sections were stained with uranyl acetate and lead citrate and were photographed by Philips LS2 electron microscope (Philips, Eindhoven, the Netherlands). Analytical assays Blood glucose was measured with a glucose meter (Bayer Health Care, Mishawka, IN, USA). Plasma insulin was assayed Plasma glucose (mg/ml) Age (weeks) Figure 1 Temporal relationships between (a) pancreatic fat content; (b) plasma insulin; (c) plasma glucose levels in obese ZDF (fa/fa) rats (&) before and after the development of hyperglycemia and in nonobese ZDF ( þ / þ ) controls (K). The shaded area in the upper panel shows the time course of islet fat content in a previously published study. 1 (Pp.1 vs level at weeks of age; Pp.5 vs level at weeks of age; w Pp.1 vs peak insulin level at weeks of age.) 1 1

3 39 Chronology of pancreatic steatosis relative to plasma insulin levels and T2DM The previously reported increase in islet TG began at weeks of age and reached a peak at weeks, the age at which nonfasting hyperglycemia first appeared. This pattern of TG accumulation and the age of onset of T2DM were virtually identical to the earlier study of islet TG content. 9 In our earlier study of islet fat in obese rodents before and after the onset of T2DM, a negative relationship between lipid accumulation and the number of b-cells and the glucosestimulated insulin response was reported. 9,15 The T2DM has been ascribed to lipid-induced loss of b-cells, with the surviving cells incapable of meeting the high demand for insulin required to compensate for the obesity-related insulin resistance. 1 To determine if there is a similar temporal relationship between fat accumulation in the exocrine pancreas, the function of the endocrine pancreas and T2D, we measured 3 h postprandial plasma insulin levels in the obese ZDF rats (fa/fa) and lean ZDF controls ( þ / þ ) from to 1 weeks of age (Figure 1b). Insulin levels were at least 1 times higher than in normal lean rats throughout the 1-week period of observation. Whereas in lean rats insulin levels remained unchanged throughout this period, in the obese rats they rose to a peak at weeks that was three times their level at weeks of age (Po.1). Thereafter, they began a gradual decline that was statistically significant at, 1 and 1 weeks of age (Po.2.5), at which point they were 3% below the peak insulin level. As the first statistically significant decline in plasma insulin followed the increase in pancreatic fat content by weeks (Figure 1b) and coincided with hyperglycemia averaging over 2 mg per ml (Figure 1c), the results were consistent with the lipotoxic concept of rodent T2DM. Electron microscopy of steatotic pancreata Electronmicroscopic examination revealed fat droplets in acinar cells. They were very scarce in the pancreas of a 1-week-old nondiabetic lean ZDF rat (Figure 2a), but were more abundant in the pancreas of a -week-old obese, new-onset diabetic rat (Figure 2b). They were much more abundant in the portion of the pancreas of a 1-week-old diabetic rat shown in Figure 2c, even though no increase in mean triglyceride content of the entire group of 1-weekold rats was noted. As no attempt was made to correlate fat droplets with chemically quantified triglyceride levels of the pancreata of the entire group, it must be assumed that the fat-laden cells chosen for display in Figure 2c are not representative of the lipid content of the entire pancreas of the group as a whole. Intrapancreatic adipocytes, which are present in older obese rodents fed a high-fat diet, 17 were uncommon in the age range of this study (Table 1). These studies in obese rodents raised the possibility of a similar lipotoxic etiology in obese humans. If so, the T2DM might also be prevented by timely intervention in the Figure 2 Electron micrographs of the exocrine pancreas showing the presence of lipid droplets in the acinar cells of (a) 1-week-old normal lean ZDF ( þ / þ ) rat (a few small lipid droplets are indicated by arrows); (b) -week-old newly obese diabetic ZDF (fa/fa) rat; (c) 1-week-old obese diabetic ZDF rat (large accumulations of lipid droplets are seen in the basal cytoplasm). Table 1 Volume density (%) of adipose tissue per section of pancreas of ZDF and ZL rats Intralobular adipose tissue Interlobular adipose tissue Male ZL weeks old Male ZDF weeks old Male ZDF 35 weeks old Abbreviations: ZDF, Zucker diabetic fatty; ZL, Zucker lean ( þ / þ ) rats.

4 prediabetic phase of the disease. In 199, our group reported that T2DM in ZDF rats is preceded by fat accumulation in their pancreatic islets 9 and is prevented by measures that reduce the lipid overload. Because noninvasive measurement of islet fat content is not now feasible in humans, this study was undertaken to determine if accumulation of fat in the whole pancreas, which can now be quantified by MRS,,13 also predicts T2DM. If fat content of the exocrine pancreas parallels that in the endocrine pancreas, it could provide a noninvasive predictor of impending T2DM in humans and an indication for prophylactic intervention. 1,19 This study was conducted to determine if MRS measurement of the TG content of the whole pancreas might provide a useful surrogate marker for islet fat content. This premise is based on the demonstration in two rodent models of obesity associated with T2DM, the ZDF rat, and db/db mouse, 2 that TG accumulate in the pancreatic islets at least 2 weeks before b-cell apoptosis becomes apparent and hyperglycemia appears. Prevention of this islet steatosis, whether by caloric restriction, 21 AICAR, 22 metformin 23 or thiazolidinedione administration, 23,2 will prevent the apoptosis, the loss of b-cells, the decline in insulin and the hyperglycemia. We show here that in ZDF rats TG in the exocrine pancreas rises in parallel with the previously reported TG accumulation in the endocrine pancreas; TG accumulation in the exocrine pancreas also precedes the onset of overt diabetes. Consequently, the evaluation of MRS measurements of pancreatic fat in humans as a possible predictor of T2DM seems justified. Published results in humans reveal that pancreatic fat measured by MRS is increased in normoglycemic subjects with an elevated body mass index and is further elevated in subjects with impaired glucoregulation and T2DM. These results, which resemble those in rodents, are consistent with a lipotoxic etiology for human T2DM, as has been suggested in rodents. 23 These observations constitute presumptive evidence that increased pancreatic fat is a predictor of impending T2DM in rats. The facts that lifestyle modification, thiazolidinediones and metformin, measures that reduce pancreatic fat content in rodents, 25 are known to reduce the incidence of T2DM in humans, 2,27 suggest a lipotoxic etiology for T2DM in both species. This sets the stage for prospective clinical studies to determine if interventions that reduce pancreatic TG levels will preserve b-cell function and reduce the incidence of T2DM in humans as effectively as they do in rodents. However, it remains to be determined if pancreatic steatosis in humans occurs independently of fat accumulation in other organs, in which case it would become a valuable clinical procedure. Conflict of interest The authors declare no conflict of interest. Acknowledgements We thank the Pole Facultaire de Microscopie Ultrastructurale and the Service Facultaire d Histologie of Geneva Medical School who kindly provided access to microscopy equipment. Part of this work was financially supported by NIH CTSA Grant UL1 RR292. ILL was supported by a Doris Duke Charitable Foundation clinical research fellowship. LSS was supported by NIH K25 HL-73. YL and RHU were supported by NIH Grant R1 DK27 and the Department of Veterans Affairs Merit Review. LO was supported by the Swiss National Science Foundation. S Kay McCorkle and Peggy McCravy prepared the paper. References 1 Ong KL, Cheung BM, Wong LY, Wat NM, Tan KC, Lam KS. 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