A network of cellular clocks adapts physiology and behavior to a 24-h rhythmic environment. Epidemiological evidence suggests a strong association between circadian rhythm disruption—e.g., in shift workers—and metabolic disorders (1). The development of genetic rodent models has been essential in deciphering the mechanisms linking clock dysfunction and the pathogenesis of metabolic diseases, highlighting the therapeutic potential of circadian rhythm manipulation in this context. Here, we provide a brief overview of the metabolic consequences of circadian disruption derived from rodent model studies and discuss potential and pitfalls of emerging genetic techniques in studying clock-metabolism crosstalk.

Early approaches describing the interplay between circadian clock function and metabolism were based on models of non-genetic circadian disruption (Table 1). Lesion experiments identified the suprachiasmatic nucleus (SCN) as the central circadian pacemaker (2, 3). In rats, SCN lesions abolish plasma rhythms of glucose, insulin (4), and leptin (5). In terms of general energy homeostasis, there are differences between studies that range from slight weight reductions (6) to no effect (5) and marked obesity (7). In the latter study on mice, increased weight is accompanied by hepatic insulin resistance. In sum, these studies illustrate that the SCN regulates metabolic hormones and tissue physiology either directly or via its control on food intake rhythms.

In an alternative paradigm, exposure to altered light regimens leads to disruption of behavioral circadian rhythms and energy metabolism. Mice exposed to bright day/dim night illumination lose their feeding rhythm along with reduced glucose tolerance and increased body weight (8). Constant light (LL)-exposed mice develop a higher body weight together with a profound loss in insulin sensitivity (7). In rats, LL exposure disrupts pancreatic beta cell clocks, correlating with reduced glucose-stimulated insulin secretion (9). In type-2 diabetes-prone rats, LL accelerates disease development due to a 50% reduction in beta cell mass (10). Most of these studies interpret their findings as a consequence of the disruptive effects of abnormal light exposure on SCN function. However, light can alter various centrally controlled functions such as mood and appetite independent of a clock effect (11, 12). Moreover, even though irregular light regimens are common for many animals and humans, there are only very few occasions where continuous light exposure above a certain threshold level may occur, making LL a highly artificial paradigm.

Several studies have outlined shift-working as a risk factor for the development of metabolic diseases (13), and various animal models have been developed to mimic this condition. Mice exposed to repeated 6-h advances of the light/dark (LD) cycle gain significantly more body weight than stable-LD controls (14). Interestingly, when mice are exposed to paradigms mimicking even more rapidly rotating weekly shift-work patterns, there is no or only a moderate effect on body weight (15). Sleep-restricting mice during their normal rest phase for 2 weeks leads to significant alterations in liver clock gene expression rhythms associated impaired pyruvate-stimulated gluconeogenesis, but no significant alterations in body weight (16). A similar approach in rats further yielded body weight increases and impaired glucose tolerance (17).

The mixed metabolic outcomes in the mentioned shift-work studies are likely directly related to the ability of the chosen paradigms to provoke food intake during the normal rest phase. Rest phase-restricted food access in mice promotes weight gain without increased energy intake (18). In line with that, while both the rapid-shift (15) and our sleep-restriction study in mice (16) showed no change in food intake rhythms, the 6-h shift model (14) and the rat sleep-restriction study (17) reported misaligned food intake. Considering the reciprocal interaction between energy intake and clock function, it has been speculated that epigenetic programming may be an important mechanism in the circadian regulation of energy metabolism—even across generations [reviewed in Ref. (19, 20)].

The development of clock gene mutant mouse models has provided a substantial tool to understand clock-metabolism interaction at the molecular level. Following the identification of circadian locomotor output cycles kaput (Clock) (21), further mammalian clock genes have been cloned and functionally characterized in corresponding mouse mutants (22–25). So far, brain and muscle ARNT-like 1 (BMAL1) has been identified as the only essential component of the molecular clockwork in mammals. Deletion of Bmal1 in mice abolishes behavioral circadian rhythms in constant environmental conditions (25). Gene expression profiling experiments show that the rate-limiting steps of various metabolic pathways are subject to circadian regulation (26). Bmal1 knockout (KO) mice show impaired glucose metabolism and insulin hypersensitivity (27, 28). At young age, they also gain weight more rapidly than wild-type littermates (28). Recently, an inducible global Bmal1 KO mouse model has been developed. Unexpectedly, adult-onset Bmal1 KOs do not suffer from many metabolic abnormalities described in the standard Bmal1 KOs (29).

Mice expressing a dominant-negative CLOCK variant (CLOCK-Δ19) on a C57BL/6J genetic background display altered 24-h feeding patterns (30). Clock-Δ19 mice are hyperphagic and show reduced energy expenditure. They also develop hyperglycemia, hyperlipidemia, hyperleptinemia, hypoinsulinemia, and increase in body weight and visceral adiposity under different diet conditions. These phenotypes may be partly explained by a reduced lipolytic capacity of white adipose tissue (31). By contrast, on an ICR genetic background, the same Clock-Δ19 mutation leads to a reduction in body weight and impaired dietary lipid absorption, suggesting that the genetic background influences clock-metabolism interaction (32).

Inconsistent findings were also reported for the metabolic consequences of mutations in another clock gene, Period (Per) 2. Per2^Brdm1 mutant mice (24) show hyperphagy, diet-induced obesity (33), hyperinsulinemia with altered insulin sensitivity, hypoglycemia, and low fasting hepatic glycogen content (34). By contrast, Per2^ldc mutants (35) show reduced adiposity, increased fatty acid oxidation, and hypotriglyceridemia (36), but are normoglycemic with improved clearance after glucose challenge (37). Of note, while genetic background may also play a role here, residual protein-coding transcripts have been detected in both mutants that may yield biologically active peptides (35, 38). Male, but not female, Per1/2/3 triple mutants become obese under an HFD (39). A Per3 KO alone has even stronger effects on male diet-induced weight gain, suggesting a genetic interaction of different Per genes in metabolic regulation (39).

Liver transcriptomic analyses from mice carrying Rev-Erbα loss- or gain-of-function alleles have identified it as a circadian regulator of cholesterol/lipid and bile acid homeostasis (40). In line with this, mice with adult-onset global loss of Rev-Erbα/β show deregulated glucose and lipid metabolism (41). Moreover, administration of REV-ERB agonists in mice induces body weight loss and decreased lipogenesis in liver and white adipose tissue, increased lipid and glucose oxidation in skeletal muscle, and elevated energy expenditure (42).

Together, these studies provide evidence that the circadian clock plays a fundamental role in energy homeostasis and that different clock components have specific functions in this context. However, because the mice used in these studies carry clock gene mutations in all tissues including the SCN, much like the non-genetic models discussed above, metabolic phenotypes may be confounded by systemic abnormalities such as altered sleep patterns, activity and feeding behaviors, or counteractive consequences of clock disruption in different tissues.

Several techniques have been developed to study the physiology of tissue circadian clocks (Table 1). In vitro experiments allow the study of tissue rhythms in the absence of external influences (43). However, such approaches have only limited potential for predicting the impact of tissue clock disruption on complex physiological processes such as energy metabolism. Transplantations have been used to study tissue clock function (44, 45). However, such techniques are highly invasive and only applicable for few tissues. Instead, conditional CRE-loxP-based gene targeting has been widely used for tissue-specific deletion of clock function in vivo (in most cases by targeting Bmal1) (46, 47). Here, we highlight some studies which have provided important insights into the contribution of different tissue clocks to metabolism.

Hepatocyte-specific deletion of Bmal1 (L-Bmal1 KO) improves glucose tolerance—the opposite effect of a global Bmal1 KO—which was attributed to reduced hepatic glucose export during the fasting phase via glucose transporter 2 (28). By contrast, mice with pancreatic beta cell-specific Bmal1 deletion (P-Bmal1 KOs) show hyperglycemia and impaired glucose tolerance (48) due to decreased insulin exocytosis (49). Further, while mice with muscle-specific deletion of Bmal1 (M-Bmal1 KO) show no significant change in systemic glucose regulation, impaired myocyte glucose uptake, and metabolism have been reported (50). Finally—and similar to global Bmal1 KOs and Clock-Δ19 mutants—mice with adipocyte-specific Bmal1 deletion (A-Bmal1 KOs) become obese (ca. 20% body weight) under HFD but not normal chow conditions. This phenotype correlates with misaligned food intake and blunted secretion rhythms of appetite-regulating polyunsaturated fatty acids from adipocytes (51).

Clock gene rhythms have been reported in central energy regulatory circuits (52). So far, few studies have addressed the biological function of specific brain clocks. With regard to metabolism, deletion of Bmal1 in steroidogenic neurons of the ventromedial hypothalamus reduces sympathetic activation of thermogenesis in brown adipose tissue (53).

These few examples make clear that tissue-specific dissection of clock gene function has provided a much more detailed picture on how circadian clocks affect energy metabolism in vivo. Nevertheless, conclusions drawn from these experiments are often confounded by intrinsic drawbacks of the classical CRE-loxP system which novel genetic tools may help to overcome.

Some of the emerging novel genetic techniques may help to overcome the issues discussed above. Below we will highlight some methodologies that may help improving our understanding of the role of tissue clocks in complex physiological contexts.

In the last decades, the tools for studying the mechanisms underlying organismal circadian timekeeping and its role in metabolic regulation have been constantly refined (Figure 1). The network structure of the circadian system represents an important challenge in this context, posing high demands on both tempo-spatial control and recombination efficiency in genetic experiments. Novel genetic approaches may help to overcome these issues and provide a clearer picture of the complex interaction of different tissue clocks in the regulation of energy metabolism.

AT, MA, BL, and HO discussed the concept, complied the literature, and wrote the paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.