These studies describe a new form of in vivo lipotoxicity: inhibition of glucose-stimulated β-cell proliferation. If FFAs restrict β-cell proliferation in response to other stimuli, such as obesity and insulin resistance, this process could influence β-cell mass accrual and type 2 diabetes risk. This finding could also be relevant to type 1 diabetes; when immune-protective tools become sufficiently developed to pursue β-cell regenerative treatments, the elevated FFAs that occur with acute insulin insufficiency could impair the β-cell proliferative response to treatment.
Strengths of this study include the use of a carefully controlled intravenous infusion system to directly assess the impact of elevating FFAs under basal and proliferation-stimulatory conditions in vivo, in vitro studies to verify that FFAs act directly on the β-cell, and the novel identification of two cell cycle inhibitors required for the antiproliferative effect. Our infusion system allows manipulation of a single variable in an in vivo setting; interventions such as genetic alteration or diet-induced obesity introduce multiple variables. Although it is not yet known whether the mechanism of adaptive β-cell proliferation in response to hyperglycemia is similar to obesity or insulin resistance, various parallels, such as increased insulin secretory load on the β-cell, downstream signals such as insulin receptor substrate 2 and cyclin D2, and a role for intracellular glucose metabolism in obesity-related proliferation, suggest areas of potential overlap (4
Although an antiproliferative effect of FFAs on β-cells has been hypothesized for a decade (17
) and was observed in vitro (18
), in vivo evidence to date has not supported this hypothesis (22
). An interesting distinction raised by our data set is the difference between basal- and proliferation-stimulated settings. Our findings suggest that FFAs do not alter basal proliferation, but prevent glucose-stimulated proliferation. Several experiments have observed a proproliferative effect of FFAs on β-cells under basal nutrient conditions, both in vitro (19
) and in vivo (24
). In our experiments, glucose and insulin levels were lower in LIP mice than SAL mice, possibly due to reduced chow intake and lower overall caloric and carbohydrate load in this group, raising the possibility that a proproliferative effect of lipids in the basal state was counteracted by an antiproliferative effect of hypoglycemia, hypoinsulinemia, or undernutrition.
Several studies have examined the effect of elevated lipids on β-cell proliferation in rats; however, none allows direct comparison of stimulated proliferation without and with elevated FFAs. One important study found robust β-cell mass recovery after pancreatectomy in Zucker fatty rats, which have elevated plasma lipids (22
). However, β-cell proliferation was not increased by pancreatectomy, and islet neogenesis was thought to be responsible for the β-cell mass expansion, limiting conclusions regarding the effect of FFAs on β-cell proliferation. Three studies have measured β-cell proliferation in rats after intravenous infusion of lipids. In one, lipid effects on proliferation were examined only in the unstimulated state (24
). In the second, cyclical glucose exposure did not induce proliferation, preventing conclusions regarding the effect of cyclical FFA exposure on stimulated proliferation (25
). In the third study, lipids were continuously coinfused with glucose into 2- or 6-month-old rats for 72 h (23
). Intriguingly, β-cell proliferation in L+G rats was equivalent to saline controls at 2 months of age, similar to our present mouse data, but was increased relative to saline controls at 6 months of age. Since a glucose-alone control was not presented, effects of coinfusion of lipid on glucose-stimulated proliferation cannot be inferred. Therefore, our study is the first to isolate lipids as a variable and compare in vivo stimulated β-cell proliferation in low and high FFA states.
Acute elevation of FFAs causes insulin resistance (rev. in 15
); insulin resistance is a potent stimulus for β-cell proliferation (35
). Although we have not measured insulin sensitivity in this study, the antiproliferative effect of FFAs cannot be related to changes in peripheral insulin resistance, because the effect occurs when β-cells are exposed to FFAs in culture and because insulin resistance would be expected to increase rather than decrease β-cell proliferation. The intriguing hypothesis that FFAs might induce insulin resistance at the level of the β-cell, and thereby block a proliferative effect of insulin, remains to be tested. Since the proliferative effect of glucose requires glucose metabolism (8
), the observed negative impact of lipids on glucose-induced proliferation may be related to the metabolic impact of FFAs, which includes inhibition of glucose oxidation (16
At first glance, an antiproliferative effect of FFAs seems inconsistent with the marked proproliferative effect of high-fat diet exposure. However, overnutrition is associated with other potent proliferation drivers (4
). Elevated circulating FFA levels are not a consistent feature of early, compensated high-fat diet exposure, since insulin potently suppresses lipolysis (45
). Teleogically, one might predict no evolutionary pressure to expand β-cell mass in the setting of high FFAs, which would occur during nutrient deprivation on a hunter-gatherer diet.
β-Cell death was not increased by combined hyperglycemia and FFAs in vivo in our model. This may be due to the degree of hyperglycemia, which is lower than glucose concentrations used to elicit glucolipotoxicity in vitro (11
), or to the timing. The deleterious effects of FFAs on β-cells vary with exposure duration (11
); 4 days represents an intermediate duration which may produce different effects than either acute or chronic exposure.
How FFAs increase expression of INK4 family cell cycle inhibitors remains unknown; however, the observation that genomic polymorphisms near the p16 locus predict risk of type 2 diabetes adds importance to our finding (46
). Peroxisome proliferator–activated receptors, nuclear receptors activated by lipids, increase p16 expression in mesenchymal cells (47
). Intriguingly, p16 is a primary mediator of senescence, and senescence was observed in β-cells after prolonged high-fat diet exposure (48
). On the other hand, after 8 weeks of high-fat diet, at a time when obesity- or insulin resistance–mediated β-cell proliferation was markedly increased, islet p16 levels were reduced (49
). Future studies are needed to dissect the mechanisms of the antiproliferative effect of FFAs.
Surprisingly, knockdown of either p16 or p18 was sufficient to relieve proliferation repression by FFAs, suggesting that both are required for the antiproliferative effect. We speculate that signaling downstream of p16 and p18 interacts in such a way that reduction of one disallows action of the other, resulting in the apparent requirement for both. Intriguingly, glucose-stimulated proliferation lost statistical significance in INS-1 cells after p16 knockdown, suggesting a possible basal suppression of proliferation in low glucose by p16. Based on the modest increase in INK4 expression by palmitate and the modest reduction by siRNA, it seems that relatively small changes in cellular INK4 content are able to significantly influence the rate of proliferation. In mice, loss of either p16 or p18 is compensated by the presence of the other, as measured by islet cell proliferation under basal conditions (50
). Future experiments will determine whether both p16 and p18 are required for the antiproliferative effect of FFAs in β-cells in vivo.
In conclusion, modest elevations of FFAs and glucose cause in vivo glucolipotoxicity with respect to adaptive β-cell proliferation. If FFAs also restrict proliferation in response to obesity and insulin resistance, this may be an important mechanism driving failure of β-cell mass expansion in prediabetes. A logical extension of our findings is to speculate that the antiproliferative effect of FFAs might represent the elusive connection between p16 and genetic risk of type 2 diabetes. It is our hope that this work leads to therapeutic interventions that expand β-cell mass to prevent diabetes.