Type 2 diabetes (T2D) and the clock

concentration (hyperglycemia), that induces insulin secretion by the pancreatic β-cells, further stimulated by the insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), released from the intestine in a nutrient-dependent manner, whereas glucagon is used to promote releasing of glucose into the blood stream under hypoglycemia (Figure 6). Glucose is absorbed mostly by the liver, the adipose tissue and skeletal muscle, with simultaneous inhibition of hepatic glucose production (210). The type 2 diabetes (T2D) is characterized by an incapacity to regulate blood glucose concentration, especially after food intake,

due to insulin resistance in target organs, and impaired insulin secretion caused by pancreatic β-cell dysfunction (211). Insulin normally induces increase in glucose transporters recruited to the membrane in peripheral metabolic organs, mostly skeletal muscle and adipose tissue, via the AKT-phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling pathway, allowing to absorb blood glucose to the organs, and thus to decrease systemic glucose levels (212). Despite enormous long-standing effort undertaken in the field, etiology of T2D stays not entirely understood now days. It is believed to comprise genetic predisposition along with environmental factors, such as alimentation, stress, or physical inactivity. T2D is the most spread form of diabetes worldwide, with 422 million of people affected in 2014 (213), and up to 642 million predicted for 2040 (211). Moreover, the age of the affected population is constantly decreasing, as reported in the United Kingdom (UK), where 5% of the T2D population were <30 years in 2003, increasing to 12% by 2006 (214), and reaching 24% of the T2D subjects younger than 40 years old in 2008 (215).

Figure 6. (Adopted from (210)). Peripheral regulation of glucose homeostasis. See text for detailed explanations.

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We, and others, have demonstrated that peripheral clocks operative in metabolic organs are regulating glucose homeostasis (Figure 7) (131). Of note, cell-autonomous clocks are ticking in the endocrine pancreas of mammals (8, 9, 138), and our group has published several papers describing the role of the circadian clock in human islet cells (8, 16, 158). Moreover, we have recently found that mouse and human islet cells secrete basal insulin in a circadian manner in vitro (16, 158). Strikingly, this secretion profile was disrupted, and absolute levels of both acute and chronic glucose-induced insulin secretion were decreased upon siCLOCK-mediated clock disruption in adult human islet cells. Using high throughput RNA sequencing (RNA-seq) analysis, alteration of

gene expression involved in vesicle trafficking and secretion mechanisms have been identified, providing a plausible hypothesis for a mechanism of the observed phenomenon (158). Furthermore, upon T2D in humans, mRNA levels of PER2, PER3, and CRY2 were significantly lower, and correlated with insulin content and HbA1c levels in the same human donors (216). In line with these findings, mRNA levels of BMAL1, PER1 and PER3 appeared to be reduced also in leucocytes from T2D human subjects (217).

Several studies reported that disruption of the circadian rhythm leads to T2D and insulin resistance development in both rodents (9, 139, 157) and humans (158). First demonstration of a such an important direct link came out with the finding that Clock^∆19 mutant and Bmal1 KO mice exhibited smaller islet size, together with an altered function, which resulted in hyperglycemia and hypoinsulinemia. Reduced levels of insulin secretion might be linked to defect of insulin exocytosis, as several genes coding for SNAREs and transporters have a modified expression (139, 157). Even stronger phenotype was reported in the

Figure 7. (Adopted from (131)). Circadian system regulates glucose homeostasis. Local clocks are required for proper organ function and inter-organ crosstalk.

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islets-specific Bmal1 KO mice (157), resulting in the development of overt T2D already at the age of two months (9). Moreover, the Rev-Erbα agonists were shown to increase energy expenditure, improve glucose concentration in blood, decrease body weight and fat mass in WT mice fed with both regular chow or HFD (218) and regulate adipogenesis (219). Collectively, these data suggest that the intact molecular clock is required for proper endocrine pancreas functions.

Liver is an important regulator of the glucose homeostasis, as it produces glucose during the starvation and stores its excess as glycogen and lipids in the fed state (212). Reports arguing that functional liver clock is necessary for regulation of glucose homeostasis have been recently published.

Hepatic glucose export follows daily oscillations, with liver-specific Bmal1 KO mice displaying hypoglycemia during the fasting phase, a tendency for a hypoinsulinemia with no difference in body weight and fat percentage, which was similar to the Per1^-/- Per2^-/- mice phenotype (220). The same study has shown that total Bmal1 KO leads to weight gain with more fat accumulation, a clear reduction of the insulin plasma level, and hyperglycemia (220). In line with these results, another study has demonstrated that Bmal1 KO mice do not exhibit rhythmic insulin action profile as compared to WT mice. Moreover, rescue experiment with overexpressing Bmal2 successfully restored insulin action and activity patterns in these mice (221). HFD causes hyperinsulinemia, and alters the expression of core clock genes and lipogenic genes in mouse liver (222). In addition, CRY1 and CRY2 proteins inhibit cAMP accumulation and CREB activity, thus resulting in the arrest of the glucagon-induced gluconeogenesis and regulation of the fasting glucose concentration by the liver. Overexpression of Cry1 in liver leads to lower blood glucose levels and insulin resistance in the db/db mouse model (223).

In humans, the link between disturbed circadian rhythm in peripheral organs and T2D development has emerged but it is far to be totally understood. Diurnal variations in glucose tolerance have been described (224), however the underlying molecular mechanism remains unknown. Moreover, genetic linkage analyses in the human population have shown that BMAL1 (225), CRY2 (226), NPAS2 and PER2 (227) genetic variants might be related to high fasting plasma glucose levels.

Metformin is a common anti-diabetic drug used for T2D treatment, mediating its effect via activation of AMPK, leading to the reduction of hepatic glucose production (228, 229). Since AMPK plays a role in the regulation of the circadian clock, it is most likely that metformin might have an impact on peripheral oscillators. Indeed, metformin blocks the mitochondrial Complex I which triggers elevation

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of NADH and AMP levels. The decrease of the NAD⁺/NADH ratio increases CLOCK:BMAL1-mediated transcription resulting in a high amplitude rhythms. The increased AMP/ATP ratio promotes AMPK phosphorylation, which activates SIRT1 and NAMPT in order to restore NAD+ levels. This reduces CLOCK:BMAL1 DNA binding activity and promotes a phase delay (230, 231). Interestingly, in white adipose tissue (WAT) of db/db mice, and in WAT from WT mice fed with HFD, mRNA expression and/or protein levels of Nampt, Sirt1, Clock and Bmal1 are decreased. Treatment with metformin restored mRNA/protein levels of these four genes (232). In addition, AMPK phosphorylates CKIα (liver), and CKIε on Ser-389 (muscle), causing the degradation of PERs protein and release of CLOCK:BMAL1 inhibition, leading to a phase advance in expression of clock and metabolic genes or protein, such as Pgc1α and Pparα (230, 233). Taken together, these results indicate that balance between NAMPT and AMPK activities determines the clock phase shift in a tissue-specific manner (230), and that the phase shifting effects of metformin on peripheral clocks observed in rodent models should be studied in humans, in view of its utmost clinical importance for T2D treatment.