The present study identifies a deleterious potentiation of impaired glucose homeostasis, β-cell dysfunction and chronic tissue inflammation by the combination of obesity and ageing. Data from our HFD/ageing mouse model support the concept that obesity with ageing leads to further deterioration in blood glucose regulation, which is likely due to a reduced capacity to balance inflammatory genes at an older age.
While our previous studies investigated the effect of chronic HFD feeding on glucose homeostasis and stages of β-cell adaptation, compensation and failure46, in this study we exposed the mice with the HFD for a relatively short term of 8 weeks, which only slightly impaired glucose and insulin tolerance in young mice of around 14 weeks, an age when they are usually investigated, but this was apparently exacerbated in older mice of 14 months of age.
Results from the in vivo GSIS displayed that β-cell function was impaired by the HFD regardless of age, but the definite amount of secreted insulin was only reduced in aged mice, while young mice could compensate for the increased insulin demand at a mildly insulin resistant stage. This suggests that β-cells from young mice keep a sufficient plasticity to maintain its insulin secretion function. This is also reflected by the changes in β-cell mass and in line with previous data showing that the ability of β-cells to proliferate is lost during ageing47,48. Young but not old mice respond to a HFD with β-cell mass expansion to meet the increased insulin demand. Such differences in the β-cell expansion capacity are not only observed with ageing but also with duration of diet. While high fat diet feeding for up to 8 weeks results in β-cell mass expansion, such adaptive increase in β-cell mass is not observed any more after 12 weeks38. This also correlates with β-cell apoptosis, which is only seen after longer periods of HFD feeding45, while after 8 weeks, HFD feeding does not result in changes of β-cell survival in young mice, but induces β-cell apoptosis in old mice. Such effects are certainly also dependent on the diet composition, as an even higher carbohydrate content in the diet (35% of calorie intake) can already severely impair glucose homeostasis in young mice49. While the diabetogenic “Surwit diet” of 58, 16 and 26% calories from fat, protein and carbohydrate, respectively44, disrupts insulin secretion later in life, β-cells can compensate for this in young mice. Their survival is not significantly affected yet, and there is only minor deterioration in glucose and insulin tolerance. This is reminiscent of our earlier study in human islets ex vivo, which shows that β-cell survival per se is not impaired in older individuals, but in response to diabetogenic stimulation, such as HFD or hyperglycemia, apoptosis is accelerated47. In aged HFD mice, there is a significantly elevated basal insulin secretion, and subsequently insulin secretion cannot be further induced in response to glucose during in vivo GSIS, which was just recently confirmed50. Such elevated basal insulin level is mainly due to insulin resistance and elevated FFA levels during an obese insulin resistant stage. The improvement in the β-cell stimulatory index by TLR4 deficiency is mainly attributed to the normalization in basal secretion. Interestingly, despite no obvious impairment in insulin sensitivity by the HFD in TLR4-KO mice, there is a compensatory increase in β-cell mass and insulin content, which suggests that such adaptation may be independent of insulin sensitivity.
In an in vitro GSIS assay, in which the effect of insulin resistance can be ruled out, basal insulin levels were similar and glucose stimulated insulin secretion reduced in young HFD mice, and fully abolished in old HFD mice, suggesting the secretory function is also compromised, while TLR4 deficiency protected the islets from such functional depletion. Such obvious loss in the secretory function was also observed in islets from senescent (21–22-month old) Fisher rats, compared to young rats (4–5-month old)51. Also in 7–8-month old Wistar rats, insulin production as well as secretion is impaired47, which is attributed at least in part to the reduction in PDX147,52, the factor for glucose mediated insulin production in mature β-cells. Other factors, which lead to an almost complete decline in β-cell regeneration in ageing are the increased expression of the cell cycle inhibitor P1648, which is initiated by decreased Bmi-1 binding to the Ink4a/Arf locus53 and by decreased Ezh254; both increase P16, and thus disable β-cell proliferation. As cell cycle and senescence markers have been identified in islets during ageing, we specifically focused here on markers of the inflammatory response; not only in islets but also in insulin responsive tissues. Overall, our study indicates that a mild ageing itself doesn’t induce β-cell functional impairment and survival, whereas it can potentiate the adverse effects of a short-term HFD.
“Sterile” chronic, low-grade inflammation without any obvious infection is a common feature of ageing, and people over the age of 65 have increased serum levels of IL-6, TNF, and IL-1855,56. Similarly, in rodent models of ageing, IL-1β, IL-6, MCP-1 (CCL2), TNF and IL-12b increase in fat and liver31,33,34. With respect to the pancreas, oxidative stress increases in aged mouse pancreases per se at the age of 14–16 months32 and TNF expression is elevated in pancreatic acinar cells in female mice aged 18–19 months35. In the present study, however, a pro-inflammatory cytokine expression by ageing alone was only seen in fat, but not in liver and islets, though differences in age, species and strains exist among this and other studies. We show that ageing alone neither induced metabolic deterioration nor an overall activated inflammatory state in metabolically active tissues.
Along with inflammatory cytokines, lipotoxicity contributes to insulin resistance and β-cell dysfunction through oxidative stress57. Free fatty acids activate TLR4, which further downstream leads to ROS/RNS production58. This is likely another mechanism, besides the inhibition of inflammation, by which TLR4 depletion ameliorated glucose homeostasis, β-cell function and survival in aged HFD fed mice in this study, even though we have not addressed such possible mechanism.
To the question whether the number of macrophages increase in tissues during ageing, several studies reported macrophage accumulation in fat, liver and/or pancreas of aged mice or rats30,31,34, although this was not confirmed by others32,33. In our current study, based on gene expression of accepted markers, neither macrophage accumulation nor their polarization status was changed in older mice in any of the three tissues, which is in line with the unchanged tissue inflammation. Mutually contradictive results were obtained from various studies, where down-regulation in both M1 and M2 polarization markers59, increased M2 macrophages60, or an overall macrophage polarization towards the M1 type during aging were observed33. The problem is that different age groups were used in these studies, with young animals ranging from 1–6 months and old animals ranging from 12–24 months age, thereby no corresponding correlation between an older age and worsening of the inflammatory phenotype could be drawn.
HFD feeding in the young mice for a short period of 8 weeks did not induce a full cytokine response at the mRNA level. The first elevated cytokines in response to the HFD were Il1b in fat and Tnf in islets. Since we didn’t observe compromised glucose tolerance, the very low-grade inflammation in young mice is consistent with the consensus that inflammation precedes hyperglycemia. In contrast, insulin secretion in young mice, tested by GSIS in vivo as well as in vitro, was already affected at this stage, together with the β-cell mass compensation response. Tnf was the only cytokine, which was induced in pancreatic islets by the short HFD feeding in the young mice, and this could act as mediator of β-cell dysfunction. This is in line with a previous study; while TNF does not affect β-cell apoptosis, it blunts GSIS from sorted β-cells61. Such results point to the possibility of early detrimental effects on β-cell function mediated by TNF. Especially, TNF is known to trigger insulin resistance and was also highly elevated at an insulin resistant stage in liver and fat in the old HFD mice. Thus, the results of this study also support the strategy to target TNF for the treatment of insulin resistance and β-cell failure62.
Neither ageing nor short term HFD itself induced severe hyperglycemia or massive changes in the cytokine pattern. But the combination of both synergistically induced inflammation in all insulin responsive and insulin secreting tissues- fat, liver and pancreatic islets. Along with hyperglycemia, insulin resistance, fully abolished insulin secretion and β-cell apoptosis, tissues were more inflamed in old HFD mice than in young, including a more and stronger pro-inflammatory and reduced anti-inflammatory cytokine expression. One limitation of this study is, that we only assessed mRNA levels of inflammatory products, which allowed quantitative analysis of cytokines at a very low expression levels. Moreover, cytokines are unstable and degrade rapidly, and thus often lay under the assay detection range, which makes their assessment on a protein level in tissues difficult.
Consistent with cytokine profiles, we found that only the combination of ageing and HFD feeding could increase the overall macrophage accumulation, again in line with the finding that ageing could potentiate HFD-induced gene expression of inflammatory cytokines, of markers of pro-inflammatory macrophages, along with a reduction in anti-inflammatory macrophage markers in fat and islets, metabolic dysfunction and β-cell failure.
A scenario emerges, how the HFD-ageing duo affects glucose metabolism: young mice are responsive to HFD with mildly increased macrophages in metabolism active tissues. This contributes to a mild inflammatory cytokine production and, in turn, results in an impairment of β-cell function. However, β-cells are still resilient to maintain compensation and glucose homeostasis. When mice get older, the same short-term diet stress not only increases M1-like macrophages but also attenuates M2-like macrophage activation, which further imbalances the macrophage phenotype and brings deterioration in the inflammatory status with more pro- and less anti-inflammatory cytokines. This then may lead to insulin resistance and compromised β-cell function, and in combination with reduced β-cell proliferation and increased β-cell apoptosis during ageing, it will finally result in definite insulin deficiency and concomitant hyperglycemia.
Being a crucial pattern recognition receptor and key player in inflammation, TLR4 is involved in many aspects of the pathogenesis of T2D, at the level of both β-cells and insulin responsive tissues7,8,10,13. As we aimed to identify the contribution of TLR4 on whole body glucose metabolism, we used whole-body TLR4-KO mice. This strategy, however, restrained the identification of the primary tissues affected by TLR4 signals. The generation of mouse models with tissue specific TLR4 re-expression in adipocytes/hepatocytes/β-cells/macrophages, respectively, on a TLR4-KO C57BL/10ScCr background would allow characterization of tissue specific effects, as well as a proof of a TLR4 specific effect upon its re-expression.
Tissue specific TLR4 effects have been studied in the past and confirmed observation from global deletions, e.g. myeloid-specific (as well as global) TLR4-deficiency improves insulin sensitivity and inhibits obesity-induced tissue inflammation in HFD and lipid infusion models7,11,22,63,64. However, in TLR4-deficienct mice, both reduced7,11,65 and unchanged ATM accumulation has been reported9,64. Notably, Orr et al. found that TLR4-depletion promoted M2 polarization in fat9. Similarly, Jia et al. observed that myeloid-specific Tlr4−/− had a trend to promote macrophage alternative activation in fat together with induced IL-10 production12.
Featuring the combinational effect of a mild HFD and ageing, our results in insulin-responsive and insulin-producing tissues from old HFD mice indicate an overall trend that TLR4-deficiency reduces mRNA expression of inflammatory cytokines and M1 macrophage markers, and additionally promotes alternative macrophage activation specifically in the liver. This in vivo study is also in line with previous ex vivo studies13,66; TLR4 activation in isolated islets induces cytokine expression, impairs glucose-stimulated insulin secretion and increases β-cell apoptosis. This is again supportive for the role of TLR4 activation in diabetes progression.
In summary, we found that ageing aggravated diet-induced impairment on glucose homeostasis, pancreatic β-cell function and survival and enhanced gene expression of inflammatory products in fat, liver and pancreatic islets in a HFD-fed mouse model. TLR4-deficiency exhibited protection against such deleterious effects through inhibiting pro-inflammatory cytokine expression and modulating tissue macrophage activation to a more anti-inflammatory phenotype. Ageing and obesity synergistically induce diabetes through TLR4, supporting the therapeutic potential of TLR4 inhibition to treat T2D.