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John Howland Cochrane explains the American Bureaucracy

John Howland Cochrane was born 26 November 1957, an economist specializing in business, economics, and economics; QAR assets administration Distinguished academic of economics at the University of Chicago Both schools of Business, served as vice-president of the American Finance Association in 2008 and was elected the president for the 2010 term.  He has his specialization in financial economics.



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The mammalian circadian clock research

The mammalian circadian clock regulates the timing of numerous physiological functions. Host circadian clock function relies on a variety of factors, including sleep-wake cycles, timing of meals, and dietary composition. Voigt et al. (2014) demonstrates that a high-fat diet disrupts host circadian clock function as well as diurnal oscillations in gut microbial structure and function. Little is known about the causal relationship between a high-fat diet, gut microbial community composition and function, and host circadian clock function. This paper reviews the effect of altered diurnal oscillations in microbial structure and function on circadian clock dysfunction under high-fat diet conditions.     

Circadian rhythms synchronize a number of important bodily functions such as sleep-wake cycles, hormone release, and body temperature. The core circadian clock is located in the mediobasal hypothalamus, a region of the brain responsible for maintaining homeostasis of essential physiological functions such as body temperature, sleep cycles, heart rate, and hormones (Johnson, 2018). The core circadian clock is regulated endogenously by a transcriptional autoregulatory loop made up of master genes, Clock, Bmal1, Cry, and Per (Buhr & Takahashi, 2013). The core circadian clock is also regulated exogenously by light-dark cycles. Together, they maintain a daily oscillation of approximately 24 hours, to drive vital physiological functions.

The core circadian clock also entrains peripheral circadian clocks, which are found in almost every tissue throughout the body, including liver and adipose tissue. Peripheral clocks play an integral role in driving gene expression involved in a variety of bodily functions in each of their respective tissues. Peripheral clocks are regulated by external stimuli such as, light-dark cues sensed by the core circadian clock and internal stimuli such as, feeding cues in the liver, pancreas, kidney, and heart (Richard & Gumz, 2012). Like the core circadian clock, peripheral clocks exhibit rhythmic expression of the CLOCK, BMAL1, CRY, and PER autoregulatory loop.

Disruption of host circadian clock function is associated with cancer, obesity, cardiovascular dysfunction, and immune dysregulation (Evans, 2013). Dysfunction can be caused by a variety of factors such as abnormal sleep-wake cycles, diet, and time of meals (Voigt et al., 2016). Recent research has found that animals fed a high-fat diet exhibit disrupted host circadian clock gene expression and microbial structure and function, resulting in an obese phenotype, however, the mechanism connecting these systems is unknown. (Kohsaka et al., 2007). 

This paper reviews the literature relevant to the effects of high-fat feeding on the gut microbiome and circadian clock function. I will begin by outlining the consequences of a high-fat diet on host circadian clock gene expression. I will then turn to the effect of the presence of the gut microbiome on circadian clock gene expression. This is followed by a detailed discussion of how a high-fat diet affects gut microbiome structure and function, and subsequently host circadian clock function. This review concludes with a discussion of possible interventional strategies for patients with diet-induced obesity.

A high-fat diet disrupts circadian clock gene expression

Previous studies have determined that deletion of Clock and Bmal1 genes results in not only host circadian clock disruptions, but also in metabolic irregularities, such as obesity and decreased insulin secretion  (Staels, 2006). Given these results, Kohsaka et al. (2007), sought to determine the bidirectional relationship between obesity and circadian clock function. To test this, the authors fed mice either a high-fat diet, that was known to induce obesity, or a low-fat diet. Then, the abundance of circadian clock mRNA gene expression in the core circadian clock, within the mediobasal hypothalamus and peripheral clocks within fat and liver tissues was measured using real-time PCR. In adipose and liver tissue, mice fed a high-fat diet exhibit attenuated Clock and Bmal1 expression compared to their low-fat diet counterparts, whereas Per2 expression is attenuated during the dark period. In the mediobasal hypothalamus, a high-fat diet did not appear to affect the expression or rhythmicity of core circadian clock genes (Figure 1). These results suggest that diet plays a vital role in maintaining circadian clock function in peripheral tissues. We know from previous research that diet also plays a vital role in maintaining equilibrium of the gut microbiota (Ley et al., 2005). 

Gut microbes are key mediators in circadian clock function

To test gut microbiome contributions to circadian clock function, Leon et al. (2015) fed germ-free mice and specific-pathogen-free mice either a high-fat diet or a regular chow diet and measured circadian clock gene expression in the mediobasal hypothalamus and liver using real-time PCR. In the mediobasal hypothalamus, specific-pathogen-free mice fed a high-fat diet have induced Bmal1, Clock, and Cry1 gene expression, and the opposite effect is seen in the liver. Instead, a high-fat diet attenuates clock gene expression in specific-pathogen-free mice, particularly in the dark period (Figure 2). This result again suggests that a high-fat diet induces circadian clock dysfunction. In addition, in germ-free mice, there is reduced rhythmicity and abundance of circadian clock mRNA expression regardless of diet. Bmal1 and Clock gene expression is induced in the mediobasal hypothalamus of specific-pathogen-free mice in the dark phase, but not in germ-free mice. Germ-free mice also exhibit strikingly lower Per2  gene expression compared to specific-pathogen free mice, suggesting that gut microbes are key mediators in circadian clock gene expression. 

Furthermore, specific-pathogen-free mice exhibit an obese phenotype when fed a high-fat diet, whereas germ-free mice exhibit a lean phenotype in both high-fat and regular-chow diets, suggesting that the gut microbiome also influences host physiology. In germ-free mice, circadian clock dysfunction should result in an obese phenotype, however, this is not the case. Because these results suggest that gut microbes are key mediators in circadian clock function, this brings into question the mechanism by which a high-fat diet influences the structure and function of the gut microbiome and how it subsequently impacts circadian clock gene expression and host physiology. 

A high-fat diet reduces gut microbiome diversity

Further experiments with mice fed either a high-fat diet or regular-chow diet demonstrate that a high-fat diet significantly alters microbial community composition (Leone et al., 2015). Fecal pellets and cecal contents were collected from specific-pathogen-free mice after being fed a high-fat or regular-chow diet. Then, DNA was isolated from fecal and cecal samples, and 16S rRNA was used to characterize the microbial composition in the gut. In both fecal and cecal samples, mice fed a high-fat diet exhibited reduced microbial diversity (Figure 3). 

To determine the effect of a high-fat diet on diurnal rhythmicity of gut microbiota abundance, microbes identified using 16S rRNA were classified into groups by operational taxonomic units (OTU). Then, oscillations of gut microbe abundance were measured using a computer algorithm used to characterize and identify oscillations in genome data sets. These data suggest that operational taxonomic unit oscillations in the gut microbiome are diet-dependent (Figure 4). In a high-fat diet, diurnal rhythmicity of the relative abundance of specific operational taxonomic units was lost despite a significant increase of relative abundance in cecal samples and reduced relative abundance in fecal samples, with the exception of Lactococcus, OTU #1100972. Under a regular-chow diet, the majority of oscillating operational taxonomic units in both fecal and cecal samples of specific-pathogen-free mice belonged to the family, Lachnospiraceae. However, this family of microbes was mostly absent in mice fed a high-fat diet. Lachnospiraceae has been shown to protect mice against obesity and insulin resistance by producing butyrate and propionate, which in turn promotes a healthy gut microbiome (Sadeghi et al., 2018). Ultimately, this data suggests that gut microbe composition and oscillations are diet-dependent, however, it does not explain how these structural changes can affect the gut microbiome function.

A high-fat diet alters diurnal oscillations of metabolite production in the gut microbiome

Experiments done by Kasubuchi et al. (2015) reveal that the absence of specific microbial metabolites influences host metabolism resulting in obesity. In particular, hydrogen sulfide and butyrate are microbe-derived metabolites that have a known impact on host physiology. Increased hydrogen sulfide and reduced butyrate concentrations in adipose tissue have been linked to weight gain and obesity (Chakraborti, 2015 & Katsouda et al., 2018). 

To determine how structural changes in the gut microbiome effect gut microbiome function in a high-fat diet, Leone et al. (2015), fed specific-pathogen free mice a high-fat diet and measured butyrate and hydrogen sulfide conditions in cecal and fecal contents. Analysis of gut microbe metabolic activity in cecal and fecal contents under high-fat conditions demonstrate altered diurnal oscillations in microbial metabolites. Mice exhibited reduced concentrations and oscillations of fecal and cecal butyrate (Figure 5). 

This result was expected because under regular-chow conditions, many of the oscillatory operational taxonomic units present, belonged to the family Lachnospiraceae, a potent short-chain fatty acid producer. In high-fat diets, Lachnospiraceae microbes are mostly absent, resulting in reduced butyrate concentrations. Conversely, analysis of fecal and cecal  hydrogen sulfide concentrations in mice under regular-chow conditions exhibit higher concentrations and diurnal patterns that are absent in mice fed a high-fat diet (Figure 5) . This result differs from previous research measuring hydrogen sulfide concentrations in mice fed a high-fat diet (Katsouda et al., 2018). Katsouda et al. (2018) have demonstrated that in obese mice, hydrogen sulfide concentrations in adipose tissue are significantly reduced. This discrepancy between studies could be due to high-fat induced alterations in the gut microbiome. In brief, analysis of fecal and cecal butyrate and hydrogen sulfide concentrations under high-fat conditions demonstrate that changes in microbial community composition caused by a high-fat diet subsequently impact microbial metabolite production.

Gut-derived metabolites alter circadian clock gene expression in the liver 

 To examine the impact of butyrate and hydrogen sulfide on microbiome diurnal oscillations and abundance of circadian clock genes in the liver, Leone et al. (2015) exposed liver-like organoids to butyrate and sodium hydrosulfide, an exogenous hydrogen sulfide donor. Using real-time PCR, the investigators established that circadian clock gene expression shifted in the absence of butyrate and hydrogen sulfide. Bmal1 and Per2 expression shifted in both butyrate and sodium hydrosulfide conditions. Under basal conditions, Per2 and Bmal1 exhibit minimal rhythmicity. In contrast, under sodium hydrosulfide conditions, Bmal1 and Per2 expression and rhythmicity is nearly lost, while exposure to butyrate induced Bmal1 expression and shifted Per2 oscillations (Figure 6). 

To determine if butyrate has the same effect in vivo, Leone et al. (2015) injected germ-free mice intraperitoneally with either saline or saline and butyrate twice a day for 5 days followed by the harvesting of the mediobasal hypothalamus and liver at ZT2 (light conditions) or ZT14 (dark conditions). Figure 6 shows that the addition of butyrate induces Per2:Bmal1 expression in the liver, but not in the mediobasal hypothalamus. At ZT14, the Per2:Bmal1 mRNA ratio in the mediobasal hypothalamus begins to approach statistical significance, but butyrate does not appear to truly impact circadian gene expression. This result suggests that peripheral tissues are more sensitive to butyrate and is consistent with the results obtained in vitro. Together, this data suggests that there is a direct link between gut microbe-derived metabolites and circadian clock gene expression, therefore illustrating that shifts in microbial composition due to a high-fat diet has downstream effects on circadian clock gene expression.  

Per1/Per2 mutant mice are prone to obesity

Based on the above findings, disruption of circadian clock gene expression in mice fed a high-fat diet can be linked to changes in microbial structure and function. Circadian clock dysfunction can lead to disruption of a variety of physiological functions, including disruption of key metabolic pathways and hormones. Turnbaugh et al. (2006) suggest that shifts in gut microbiota composition cause diet-induced obesity. While this may be true, Kettner et al. (2015) has demonstrated that circadian clock disruption may also play a role in diet-induced obesity. They bred mice lacking Bmal1, Per1, Per2, Cry1, or Cry2 under standard 24 hour light/dark cycles. When mice were fed a standard chow diet, the Per1 and Per2 mutant mice exhibited a significantly higher body weight than wild type controls. Notably, Bmal1, Cry1, and Cry2 mice had a significantly reduced body weight (Figure 7). Kettner et al. (2015)  attribute this to increased serum levels of leptin in Per1 and Per2 mutants and decreased leptin levels in Cry1 and Cry2 mutant mice. Leptin is regulated by the core circadian clock and serves to inhibit hunger responses when the body does not need energy. Decreased leptin production can trigger an increase in appetite. The fact that Per1 and Per2 mutant mice exhibited increased body weight despite elevated leptin levels indicated that the mice are leptin resistant, meaning that the brain is not receiving the leptin signal and as a result, increases food cravings and reduced energy consumption (Frederich et al., 1995). These results provide evidence that disruptions in circadian clock genes, Per1 and Per2, are linked to metabolic disturbances, including diet-induced obesity.

Time-restricted feeding and increased short-chain fatty acid consumption protect against diet-induced obesity

The findings in this review suggest that there are opportunities to treat and prevent diet-induced obesity, ranging from lifestyle changes to altering diet and feeding patterns. Hatori et al. (2012) illustrates time-restricted feeding patterns as a non-pharmacological approach to prevent diet-induced obesity. To test this approach, Hatori et al. (2012) fed mice a high-fat diet and a normal-chow diet under either an ad libitum regimen or a time-restricted regimen. Circadian clock gene expression was measured using real-time PCR. Mice fed a high-fat diet under ad libitum conditions exhibit reduced rhythmicity and expression of Per2, Bmal1, and Cry1 compared to their time restricted counterparts. The data demonstrate that time restricted feeding can reverse the effects of a high-fat diet on circadian clock gene expression (Figure 8). 

Moreover, mice fed a high-fat diet under ad libitum conditions gained more weight than high-fat diet time-restricted mice in 20 weeks, despite equivalent caloric intake in both groups of mice (Figure 9). High-fat diet time-restricted mice were protected from the excessive weight gain seen in high-fat diet ad libitum mice. This illustrates that a time-restricted diet is capable of protecting mice from diet-induced obesity, thus providing a non-pharmacological means of treatment for diet-induced obesity.  

As a second example of treatment for diet-induced obesity, Lin et al. (2012), determined the effects of short chain fatty acids in mice fed a high-fat diet. Mice were put on a high-fat diet supplemented with butyrate, propionate, or acetate and were weighed every week for four weeks. Mice with diets supplemented with propionate, butyrate, or acetate gained significantly less weight than control mice. Pointedly, dietary supplementation with propionate or butyrate resulted in almost 0% weight gain after four weeks of being fed a high-fat diet (Figure 10). These mice exhibited significantly reduced food intake and improved glucose tolerance. Though the mechanism behind glucose tolerance and obesity is poorly understood, improved glucose tolerance has been associated with suppression of diet-induced obesity (Nolan et al., 1994). A high-fat diet supplemented with short-chain fatty acids results in protection from diet-induced obesity, therefore supporting the notion that short chain fatty acids particularly, propionate and butyrate can be used to treat and prevent diet-induced obesity. 

Conclusion

This review highlights the relationship between gut microbial structure and function, and dietary consumption, as well as the effects of host circadian clock gene expression on host physiology (Figure 11). In the presence of gut microbes, mice fed a high-fat diet exhibit altered host circadian clock rhythm in central and peripheral tissues, resulting in an obese phenotype. Leone et al. (2015) demonstrates that shifts in microbial structure and function generate disruptions in host circadian clock rhythmicity and subsequently diet-induced obesity. In mice fed a high-fat diet, Lachnospiraceae, a potent short-chain fatty acid producer was largely absent. Because of significant shifts in Lachnospiraceae under high fat diet conditions, diurnal oscillations of butyrate were significantly reduced. In vitro and in vivo exposure of butyrate in mice and liver-like organoids, respectively, restored host circadian clock function, therefore demonstrating that butyrate is a key metabolite for proper host circadian clock function and protection from diet-induced obesity.

Under germ-free conditions, cues from the gut microbes are absent, which results in reduced circadian clock gene expression and rhythmicity in both central and peripheral tissues regardless of diet. Despite this, germ-free mice maintain a lean phenotype. Bäckhed (2007) suggests that germ-free mice are protected from diet-induced obesity because they have increased levels of an AMP-activated protein kinase in their skeletal muscle. This AMP-activated protein kinase has direct downstream effects on fatty acid oxidation, resulting in a lean phenotype. In contrast, specific-pathogen-free mice only exhibit disrupted circadian clock function when fed a high-fat diet, indicating that the gut microbiota is a key mediator in circadian clock function. 

Finally, the results outlined in Hatori et al. (2012) and Lin et al. (2012) experiments demonstrate opportunities for treatment and prevention of diet-induced obesity, such as increased short chain fatty acid consumption, manipulation of gut microbial function, and time-restricted feedings. Hatori et al. (2012) demonstrated that mice fed a high-fat diet under time-restricted conditions had reduced weight gain compared to their ad libitum counterparts. In addition, Lin et al. (2012) demonstrated that mice fed a high-fat diet supplemented with short-chain fatty acids gained significantly less weight than mice given a high-fat diet with short-chain fatty acid supplementation. About 40% of U.S. adults are obese, thus the development of  interventional strategies to treat and prevent obesity is critical. Further research on the effectiveness of these strategies would be important in obesity prevention.