New Insights into the Regulation of Leptin Gene Expression

Abstract

Leptin is a key hormone in the homeostatic regulation of body weight. While past research focused mainly on overall leptin actions, a recent study by Dallner et al. (2019) takes a fresh look at the regulatory elements of the leptin gene locus, providing new insights into processes that modulate leptin levels.

The discovery of adipocyte-derived leptin in 1994 quickly led to the recognition of this hormone as a key to understanding the homeostatic regulation of body weight (Zhang et al., 1994). Leptin is expressed in proportion to body fat, suppresses food intake, and prevents low metabolism, thereby promoting weight loss overall. In turn, negative energy balance suppresses leptin levels enabling weight gain (Schwartz et al., 2000). Yet, the initial high hopes that leptin treatment could cure obesity were not fulfilled due to the paradoxical observation that obesity is already associated with high leptin levels (Considine et al., 1996), which can cause leptin resistance (El-Haschimi et al., 2000), while lack of leptin also results in severe obesity (Pelleymounter et al., 1995).

The dynamic range of circulating leptin levels is well known in response to energy states, wherein insufficient energy intake during fasting causes a drop in leptin levels (Trayhurn et al., 1995), while overfeeding and obesity result in increased leptin levels (Kolaczynski et al., 1996). Despite this knowledge, most experiments have aimed to understand overall leptin action rather than the processes that are involved in modulating the dynamic changes in leptin levels.

However, in a recent issue of Nature Medicine, Dallner et al. took a fresh look at the leptin gene locus and its regulatory promotor elements (Dallner et al., 2019). Indeed, the authors found novel enhancer sites and a non-coding RNA sequence that are relevant to fat-specific leptin expression and possibly to the dynamic changes in leptin expression during fasting and high-fat-diet-induced obesity. The authors made use of an in vivo luciferase expression assay, in which the leptin gene locus with extended DNA segments, beyond the known proximal promoter and exons (de la Brousse et al., 1996; Mason et al., 1998), was coupled to luciferase expression, and full-body images were analyzed in anesthetized mice. This in vivo live-imaging approach not only allowed for comparison of expression levels across different tissues (e.g., skeletal muscle, brain, kidney, liver, and different adipose tissue depots) but also facilitated monitoring of expression during physiological challenges, such as fasting or high-fat-diet feeding.

Systematic deletions of the leptin locus up- and downstream of the transcription start site resulted in the discovery of two novel enhancer sites for leptin gene expression, referred to as LE1 (located upstream of the transcription start site) and LE2 (located downstream of the transcription start site). Both enhancers interact with the proximal leptin promoter, which was found by others to be required for proper leptin expression (de la Brousse et al., 1996; Mason et al., 1998). However, the authors clarify that the proximal promoter is not sufficient for fat-specific leptin expression, and at least one enhancer (LE1 or LE2) is necessary to maintain adipose tissue leptin expression.

The authors further identified diverse binding factors for LE1 and LE2 that led them to suspect that interactions with non-coding RNA segments might be involved in the regulation of leptin gene expression. Indeed, they identified a long non-coding RNA that is transcribed even further upstream of the LE1 enhancer that they refer to as lncOb (Figure 1). However, IncOb exclusively binds to the proximal promoter and not to the LE1 or LE2 enhancers.

Figure 1. Hypothetical Schematic Organization of the Regulatory Units within the Leptin Gene Locus.

A high-fat diet (HFD) and fasting lead to positive and negative energy balance, respectively, with corresponding moderation of leptin expression. LE1 and LE2 are two novel enhancer sites for leptin gene expression that are located upstream and downstream of the transcription start site, respectively. Two proteins, EBF1 (early B cell factor 1) and NF1 (nuclear factor 1), bind to the leptin enhancers and may play a role regulating leptin transcription. lncOb is long non-coding RNA in the leptin locus.

In subsequent experiments, the authors focus on the function of IncOb due to its relevance to humans where a corresponding RNA was identified, with fatspecific expression that correlated with leptin gene expression, even though the evolutionary conservation of IncOb was weaker than the proximal promoter and the two enhancers, LE1 and LE2.

The authors provided further confirmation of the physiological importance of IncOb by generating KO mice with CRISPR technology. Lack of IncOb significantly decreased leptin gene expression, supporting a role for IncOb in ensuring full capacity of leptin expression. Interestingly, in chow-fed or high-fat-diet obese animals, suppressed leptin levels caused an increase in body weight despite an increase in leptin sensitivity.

This is an important finding, as hyperleptinemia and associated leptin resistance in people who are obese are thought to enable, or at least promote, obesity. Hyperleptinemia is therefore generally considered a pathological state. The finding that reduced hyperleptinemia and enhanced leptin sensitivity might limit weight gain during high-fat-diet feeding further indicates that hyperleptinemia is, in fact, a physiological response to overfeeding and suppresses weight gain. Indeed, the authors provide additional evidence that mutations in a region overlapping with the IncOb locus in humans are associated with decreased leptin levels and increased body weight in children and adults, highlighting the relevance of these findings for obesity in humans

Recent studies of leptin function have focused on the hormone’s action in the brain via its receptors (Münzberg and Morrison, 2015), while the regulatory elements that enable dynamic changes in leptin gene expression have received less attention. The findings from Dallner et al. (2019) refocus our attention on the regulatory elements that modulate leptin gene expression in the adipocyte.

Several questions that have plagued the leptin research field remain unanswered by this research, although the observations of Dallner et al. (2019) may provide new avenues to explore. Despite their capacity for examining dynamic changes in leptin expression (i.e., fasting, high-fat diet), Dallner et al. (2019) did not investigate if the enhancer and/or IncOb mediate these dynamics of leptin expression; their role may be limited to overall leptin expression levels, while dynamic changes (fasting-induced hypoleptinemia, high-fat-diet-induced hyperleptinemia) may be maintained. Similarly, it remains unclear how IncOb blunts leptin sensitivity. This may largely be explained by the well-known effect of leptin signaling to induce negative feedback molecules in leptin-receptor-expressing target cells in the brain and suppress leptin sensitivity (Myers et al., 2008). Where are dynamic changes in leptin levels induced? Earlier research showed that sympathetic activation via beta-3 adrenergic receptors in adipose tissue is critical for facilitating the fasting-induced drop in leptin levels (Commins et al., 1999; Swoap et al., 2006) and is induced by central leptin target sites in the arcuate nucleus (Caron et al., 2018). Is IncOb expression-dependent on the same inputs? It will be critical to explore if hyperleptinemia in individuals who are obese indeed prevents additional weight gain. The research from Dallner et al. (2019) reminds us that we currently do not have a consensus whether obesity-associated hyperleptinemia should be viewed as a pathological condition causing obesity or if we should approach hyperleptinemia as a homeostatic beneficial adaptation to overfeeding.

The findings by Dallner et al. (2019) provide us with new tools for exploring these questions that once answered will improve our understanding of the dynamic changes in leptin expression for body weight and overall homeostatic regulatory processes.