A Langerhans Islet with insulin-producing cells (in green), and glucagon-producing cells (in red). Cell nuclei in blue. [© UNIGE, Dibner Lab]

Nearly all the cells in our body have molecular clocks that regulate and synchronize metabolic functions to a 24-hour cycle of day-night changes. Researchers at the University of Geneva (UNIGE) and the University Hospitals of Geneva (HUG) have now linked disrupted clock function in human pancreatic islets, with type 2 diabetes (T2D). Their studies showed that pancreatic islet cells derived from T2D human donors had compromised circadian oscillators, and indicated a functional link between pancreatic islet clock disruption and insulin and glucagon secretion. Treating human T2D islet cells with a clock-modulating drug boosted circadian gene expression, which resulted in increased insulin secretion.

“This is the first proof of principle that repairing compromised circadian clocks may help to improve the function of the pancreatic islet hormone secretion,” said Charna Dibner, PhD, principle investigator in the departments of medicine and of cell physiology and metabolism, and Diabetes Centre at UNIGE Faculty of Medicine, and at HUG. Dibner hopes that the findings could translate into new therapeutic approaches to T2D. “By re-synchronizing the perturbed molecular clocks, either by personalized eating and exercise schedules or with the help of clock modulator molecules, we hope to ultimately be able to provide an innovative solution to an epidemical metabolic problem affecting an ever-increasing proportion of the world’s population.”

Dibner and colleagues reported on their studies in the Proceedings of the National Academy of Sciences (PNAS), in a paper titled, “In pancreatic islets from type 2 diabetes patients, the dampened circadian oscillators lead to reduced insulin and glucagon exocytosis.”

Circadian clocks govern the daily cycles that govern the various cellular functions. “In mammals, this time-keeping system governs most aspects of physiology and behavior,” the authors wrote. “It comprises a master pacemaker, located in the paired suprachiasmatic nuclei (SCN) of the hypothalamus, that on a daily basis synchronizes peripheral oscillators situated in the organs.”

There are several interlocking levels of clock synchronization, but the main one is light, which in particular regulates the central clock in the hypothalamus. Hypothalamic control then feeds down to peripheral clocks present in organs and cells. While these peripheral clocks are under the oversight and control of the hypothalamus, they function differently in each organ, and even in each cell type, depending on their role.

Two years ago, Dibner’s team demonstrated in rodents that perturbation of pancreatic cellular clocks led to disrupted insulin and glucagon secretion, thus promoting the onset of diabetes. “Genetic mouse models with pancreas-specific clock perturbation exhibit a phenotype of strongly disrupted insulin secretion, severe glucose intolerance, and all of the features of T2D from an early age, strongly suggesting implication of the islet clocks in T2D development,” the team wrote. Increasing evidence also indicates that disturbances in internal clocks in humans, which may result from frequent time zone changes, irregular working schedules, or aging, may also have a significant impact on the development of metabolic diseases, including T2D. Such disturbances seem to prevent the proper functioning of the cells in the pancreatic islet cells that secrete insulin and glucagon, the hormones that regulate blood sugar levels. “… epidemiological studies in humans strongly suggest that circadian misalignment may lead to the development of metabolic diseases such as obesity and type 2 diabetes,” the authors commented.

“Pancreatic cells are also subject to the rhythm of fasting and food intake, and to tight hormonal regulation,” commented Dibner. “Coordinating all levels of regulation, therefore, allows the optimization of metabolic functions. Clocks deregulation in pancreatic islet leads to a compromised function: they are not anymore anticipating food-derived signals. Indeed, if you eat the same food but at night rather than during the day, you may gain weight much faster, due to a suboptimal response of your metabolism.”

Volodymyr Petrenko, PhD, a researcher in the Dibner lab and the first author of the paper in PNAS, further commented, “We had also previously observed that if the clocks of human pancreatic cells were artificially disrupted in the cell culture in vitro, secretion of the key islet hormones—insulin and glucagon—was compromised. Hence our next step, that we reported here, was to unravel whether the circadian rhythms were perturbed in human pancreatic islets in type 2 diabetes, and, if so, how would this perturbation affect the islet function.”

Using a technique known as combined bioluminescence-fluorescence time-lapse microscopy, which makes it possible to track the molecular clock activity in living cells very precisely over time, the scientists compared the behavior of pancreatic cells derived from donors with T2D, with those from healthy subjects, throughout the day. The results showed that biological rhythms of the islet cells in T2D exhibited both reduced amplitudes of circadian oscillations and poor synchronization capacity.

“Strikingly,” the investigators stated, “our study reveals that the circadian clockwork is compromised in human alpha- and beta-cells in T2D, evidenced by the altered temporal profiles of insulin, proinsulin, and glucagon secreted by T2D human islets … Utilizing time-lapse microscopy, we now report that within intact human islets, alpha- and beta-cells possess oscillators with comparable circadian properties ….” Dibner further stated, “The verdict is indisputable … Moreover, the defects in temporal coordination of insulin and glucagon secretion observed in patients with type 2 diabetes were comparable to those measured in healthy islet cells with the artificially-disrupted circadian clock.” The scientists’ studies indicated that disrupted islet clocks impacted on exocytosis, the process by which insulin and glucagon are released by their islet cell types. “… our experiments suggest that functional islet clocks are indispensable for a proper docking and exocytosis of insulin and glucagon granules in Hunan and in mice,” they wrote.

The Geneva team next investigated the effects of Nobiletin, a small clock modulator molecule that is a natural ingredient of lemon peel, and whose impact on circadian clocks has been recently discovered, on resynchronizing islet cell clocks. The results indicated that treating the cells using Nobiletin increased clock gene oscillations, boosted insulin secretion by non-diabetic (ND) islets, and led to increased glucose-stimulated insulin secretion (GSIS) in T2D islet cells. “Overall, Noobiletin partly restored compromised basal and GSIS human T2D islets,” the team noted. “By acting on one of the core-clock components, it resets efficiently the amplitude of the oscillations in the human islets,” said Petrenko. “And as soon as we got the clocks back in sync, we also observed an improvement in insulin secretion.” Experiments, in addition, showed that Nobiletin induced glucagon secretion in ND and T2D islets during glucagon release test in the presence of glucose.

The authors concluded, “Our study emphasizes a link between the circadian clockwork and T2D and proposes that clock modulators hold promise as putative therapeutic agents for this frequent disorder.” Dibner added, “Our society experiences epidemic growth in metabolic diseases, concomitant with shifted working and eating schedules, and lack of sleep. We will continue by exploring this repair mechanism in vivo, first in animal models.”

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