Intestinal heat shock proteins in metabolic syndrome: Novel mediators of obesity and its comorbidities resolution after metabolic surgery

Abstract

Over the past 40 years, the prevalence of obesity has risen dramatically, reaching epidemic proportions. Metabolic surgery has proven to be highly effective in treating obesity, leading to significant improvements or complete resolution of obesity-related comorbidities.

Research conducted in both animals and humans suggests that the metabolic benefits achieved through metabolic surgery cannot be solely attributed to weight loss. Indeed, there has been an increasing recognition of intestinal inflammation as a novel factor influencing obesity. The gastrointestinal tract is continuously exposed to dietary components, particularly diets rich in saturated fats, which are known to contribute to obesity. It is now widely accepted that heat shock proteins can be released from various cells including intestinal epithelial cells and act as proinflammatory signals. Several studies have shown that circulating levels of glucose-regulated protein 78 (GRP78) are increased in subjects with obesity and correlate with the severity of the disease. Moreover, mice with a partial knockout of GRP78 are protected from diet-induced obesity.

In this review, we discuss the role of GRP78 in the development of obesity. Several evidence suggests that GRP78 can influence adipogenesis, lipid droplets stabilization, insulin resistance, and liver steatosis. We also provide an update on GRP78 regulation following metabolic surgery, focusing on the bypass of the small intestine as a key factor for GRP78 secretion. Finally, we discuss the potential role of monoclonal antibodies against GRP78 as a treatment for obesity.

Introduction

Obesity has reached epidemic proportions, with the World Health Organization estimating that nearly 39% of adults are overweight or living with obesity.¹

For decades, obesity has been viewed as the result of a long-term energy imbalance, but emerging evidence has revealed that its pathogenesis is much more complex. Indeed, obesity is the result of a complex interaction between genetic, epigenetic, and environmental factors like diet and lifestyle.²

Conventional treatments for obesity and its comorbidities include lifestyle interventions (i.e., physical activity and caloric restriction), medical therapy, and metabolic surgery. However, lifestyle interventions and medical therapy have been shown to be partially effective in inducing weight loss in the long term.³

Metabolic surgery leads to the improvement and remission of many obesity-related comorbidities, resulting in sustained weight loss, improved quality of life, and prolonged survival.

Indeed, metabolic surgery can induce durable (10-year and beyond) remission of type 2 diabetes in addition to the reversal of chronic inflammation, hypertension, and metabolic dysfunction-associated steatotic liver disease.⁴

Several studies in animals and humans suggest that the metabolic improvement achieved with metabolic surgery cannot be exclusively associated with weight loss.7, 8, 5, 6 While metabolic surgery often results in the remission of obesity-related comorbidities patient’s body mass index (BMI) rarely returns to normal. Interestingly, a greater improvement of insulin resistance has been reported for those metabolic procedures that bypass large portions of the jejunum, despite inducing similar weight loss.9, 8

Recently, intestinal inflammation has emerged as a novel mediator of obesity. The gastrointestinal tract is continuously exposed to dietary components, such as diets rich in saturated fats known to promote obesity. Furthermore, it has been reported that gut inflammation precedes adipose tissue inflammation in animal model of diet-induced obesity.¹⁰

The 70-kDa heat shock protein (HSP70) is a family of ubiquitous and highly conserved molecular chaperones known for their cytoprotective activities in response to various cellular insults, including oxidative stress.¹¹ Since their discovery, HSPs have been regarded as intracellular molecules controlling the folding of nascent proteins, protein transport to organelles, and protein degradation.13, 12

It is now widely accepted that, following cellular stress, HSPs can be released into the extracellular compartment from a variety of cells, including intestinal epithelial cells,¹⁴ and act as a proinflammatory signal.15, 11

GRP78, a member of the HSP70 family, serves as a major endoplasmic reticulum (ER) chaperone, maintained within the ER lumen by the KDEL motif. Recent evidence shows that GRP78 can also exist outside the ER, and cellular stress promotes GRP78 secretion. Moreover, GRP78 has been recently implicated in the pathogenesis of obesity and type 2 diabetes.¹⁶

In this review, we summarize the main evidence, from human and animal studies, linking GRP78 to obesity and its comorbidities. We then discuss how metabolic surgery can modulate GRP78 release and provide novel insights into a promising therapeutic approach that could mimic the effect of metabolic surgery.

GRP78 roles beyond the ER

HSP70 is a highly conserved and ubiquitous protein family consisting of 13 gene products that vary in expression level, subcellular location, and amino acid sequence.¹¹ One of the key members of the HSP70 family is GRP78, also known as immunoglobulin heavy chain-binding protein. Unlike other members of the HSP70 family, GRP78 is encoded by a single copy gene in the eukaryotic genome, and its induction is primarily regulated at the transcriptional level.¹⁷

In humans, the gene responsible for GRP78 encoding is located on chromosome number 9 and spans 4532 nucleotides.¹⁸

GRP78 structure

GRP78 consists of three main domains: a N-terminal Adenosine triphosphate (ATP)ase domain, a substrate-binding domain, and a C-terminal lid domain¹⁹ (Figure 1).

Fig. 1.

Functional domain structure of GRP78. GRP78 consists of three main domains: a N-terminal ATPase domain, a substrate-binding domain, and a C-terminal lid domain. Abbreviation used: ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78; (ATP) Adenosine triphosphate.

The N-terminal ATPase domain binds and hydrolyzes ATP, providing the energy needed for GRP78 chaperone function. The ATPase domain also interacts with co-chaperones and client proteins, facilitating their folding and assembly.¹⁹

The substrate-binding domain binds to unfolded or misfolded proteins through a hydrophobic pocket, preventing the aggregation and misfolding of client proteins.¹⁹

Finally, the C-terminal lid domain regulates the interaction between GRP78 and client proteins. The lid domain can adopt different conformations that allow GRP78 to bind and release client proteins in a controlled manner.¹⁹

GRP78 cellular location

GRP78 is primarily located in the ER, where it plays a crucial role in facilitating the transport of newly synthesized proteins into the ER lumen and their subsequent trafficking or secretion (Figure 2). The retention of GRP78 in the ER is mediated by a signal sequence known as the KDEL motif. The KDEL motif is a tetrapeptide sequence (Lys-Asp-Glu-Leu) located at the C-terminus of GRP78.

Fig. 2.

Functions of GRP78 within different subcellular compartments. GRP78, traditionally considered a component of the endoplasmic reticulum, has also been identified in other cellular locations, including the cytosol and extracellular space, where it displays novel functions that affect the pathophysiology of several diseases. Abbreviation used: ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78.

The KDEL retrieval system relies on specific receptor in the Golgi apparatus, known as KDEL receptors. These receptors recognize the KDEL motif and facilitate the retrograde transport of GRP78 from the Golgi apparatus to the ER²⁰ maintaining an adequate level of the chaperone in the ER lumen.²¹ This process helps maintain ER protein folding capacity, mitigate ER stress, and prevent the accumulation of misfolded proteins in the ER, thereby preventing the activation of the unfolded protein response (UPR).

In addition to its involvement in protein folding, GRP78 plays a crucial role in regulating UPR. Under normal ER conditions, GRP78 keeps the UPR transmembrane sensor inositol requiring enzyme-1 inactive and activates transcription factor 6 and protein kinase RNA-like endoplasmic reticulum kinase.²² However, during ER stress, when unfolded proteins accumulate within the ER lumen, GRP78 dissociates from inositol requiring enzyme-1 and activates the UPR. The activated UPR mitigates ER stress by reducing protein synthesis and enhancing ER folding capacity, including an increased expression of GRP78.²³

GRP78 outside the ER

Emerging research suggests that GRP78 can also be found outside the ER (Figure 2). Notably, GRP78 has been observed in the cytoplasm, where it plays several roles, including the regulation of UPR signaling. A recent study²⁴ has identified an isoform of GRP78 called GRP78va, which is expressed specifically in the cytosol and is produced by alternative splicing. The messenger RNA of GRP78va retains intron 1 of the Grp78 gene and encodes a truncated form of GRP78. Despite lacking a signal peptide, GRP78va retains the essential functional domains of GRP78, allowing it to reside in the cytosol and potentially interact with numerous client proteins. The expression of GRP78va is upregulated during ER stress, promoting protein kinase RNA-like endoplasmic reticulum kinase signaling, and enhancing cell survival under ER stress.²⁴

In the past, HSPs were considered to be intracellular proteins with functions restricted to the intracellular environment. An increasing number of observations indicate that several HSPs, including GRP78, can be released into the extracellular space, where they may exert influence on various tissues and cells.

Immunogold electron microscopy studies conducted in the early 1990s provided evidence that several ER proteins, including GRP78, can be exported from the ER to other organelles within exocrine pancreatic cells.²⁵ Following these observations, secretion of GRP78 was also detected in the extracellular milieu.26, 27

The presence of the KDEL retention signal suggests that GRP78 should be tethered to the ER lumen. However, it has been suggested that in cells with high demands for protein synthesis and maturation, the saturation of KDEL receptors or deficiencies in the protein sorting system could hinder the retrieval of KDEL-bearing proteins to the ER.

Indeed, ER stress has been shown to induce the secretion of a group of ER proteins, including GRP78, when induced by ER Ca²⁺ depletion or by the inhibition of glycosylation.²⁸

Furthermore, rodent models of obesity display increased levels of GRP78 in the jejunal secretome, indicating that intestinal epithelial cells may be the primary source of GRP78 secretion in response to lipid overload.¹⁴

The understanding of the precise mechanisms and functions of extracellular GRP78 is still an active area of research. However, it is believed to play a role in intercellular communication, modulation of the immune system, and the pathophysiology of various diseases, including cancer, metabolic disorders, and cardiovascular diseases. Further investigations are needed to elucidate its precise role in different biological processes.

GRP78 secretion

Several potential pathways for GRP78 secretion have been proposed, such as exosome-mediated secretion and shedding from the cell surface. Indeed, it has been suggested that cells can actively secrete GRP78 through exosome-mediated secretion. This process involves the association of GRP78 with secretory vesicles, which are then transported to the cell membrane and released into the extracellular space.²⁹

Exosomes are small membrane-bound vesicles released by cells that can contain a variety of cellular components, including proteins and nucleic acids. Some studies have shown that GRP78 can be loaded into exosomes and released into the extracellular environment, where the exosomes act as vehicles delivering GRP78 to cells or tissues.³⁰

Another proposed mechanism is the shedding of GRP78 from the cell surface. It has been suggested that GRP78 can be cleaved or released from the plasma membrane through proteolytic processes, generating soluble fragments that are then released into the extracellular space.³¹ Despite several potential pathways for GRP78 secretion have been proposed, further research is needed to gain a comprehensive understanding of its functional implications in physiological and pathological contexts.

GRP78 in the pathogenesis of obesity

The link between obesity and GRP78

The rates of obesity and type 2 diabetes have dramatically escalated over the past four decades. Projections indicate that by 2030, approximately 50% of the US population will be affected by obesity, and the prevalence of diabetes is expected to increase by 54%, with over 54.9 million Americans projected to have diabetes.32, 33 Obesity and type 2 diabetes share numerous pathological characteristics, such as a chronic state of low-grade inflammation, hepatic insulin resistance, ectopic fat accumulation, and an elevated risk of developing metabolic dysfunction-associated steatotic liver disease, including the more severe form known as metabolic dysfunction-associated steatohepatitis (MASH).35, 36, 34, 37

The role of GRP78 in the context of obesity is an area of ongoing research, and our understanding of the mechanisms involved is still evolving. Nevertheless, several studies (Table 1) suggest that GRP78 can influence various pathways associated with the development of obesity.

Table 1.

An overview of the role of GRP78 in obesity pathogenesis.

Pathway/disease	Experimental model	Mechanism of action	Reference
Adipogenesis	GRP78 conditional KO (mouse model)	Increased expression of PPARγ Stabilization of lipid droplets	Zhu et al.38
Insulin resistance	In vitro (hepatocytes and myocytes) and in vivo (rat model)	Reduced phosphorylation of Akt, FoxO1, and GSK3αβ	Angelini et al.14, 39
Hyperglycemia	GRP78 KO (mouse model)	Reduced phosphorylation of Akt	Ye et al.40
MASH	Continuous infusion of GRP78 (rat model)	Increased de novo lipogenesis, inflammation, fibrosis	Angelini et al.14

Abbreviations used: Akt, protein kinase B; FoxO1, Forkhead Box O1; GRP78, glucose-regulated protein 78; GSK3αβ, glycogen synthase kinase-3αβ KO, knock-out; MASH, metabolic dysfunction-associated steatohepatitis; PPARγ, peroxisome proliferator-activated receptor γ.

Adipogenesis

Adipogenesis, induced by a high-energy diet, is a key contributor of adipose tissue expansion and its inhibition has been shown to be effective in preventing obesity. Peroxisome proliferator-activated receptor γ plays a crucial role in adipogenesis and regulates important molecular targets such as perilipin, hormone-sensitive lipase, and glucose transporter type 4.

These targets are involved in adipogenesis, lipid metabolism, and glucose metabolism.41, 42 Notably, GRP78 overexpression has been shown to enhance the expression of peroxisome proliferator-activated receptor γ, indicating its potential role in adipogenesis. Additionally, GRP78 plays a vital role in several processes, including lipogenesis, metabolic homeostasis, and mouse growth during both fetal and postnatal stages.³⁸ Indeed, GRP78 knockout in mouse adipose tissue results in a significant reduction of plasma triglyceride levels by approximately 40–60% compared to wild-type mice.³⁸ Similarly, in individuals with obesity, serum level of GRP78 was significantly higher and correlated with low-density lipoprotein and triglycerides.²⁶ Moreover, GRP78 levels are increased in the adipose tissue of individuals with obesity.⁴³

Lipid droplets

Triglycerides and other lipids are primarily stored in cellular compartments called lipid droplets. The fusion and growth of lipid droplets are tightly regulated by proteins that coat the droplets, adapting to cellular energy requirements.⁴⁴ Proteomic studies have identified several HSPs, including GRP78, as major structural proteins coating lipid droplets.⁴⁵ Under stress conditions, GRP78 accumulates on the surface of lipid droplets, potentially playing a role in stabilizing the phospholipid monolayer of the droplets.

Insulin resistance and liver steatosis

Insulin resistance, which is considered a key factor in the development of metabolic syndrome and obesity, has been found to be positively correlated with GRP78 levels in both human and animal studies.26, 46 A significant improvement in insulin sensitivity and glycemic control has been demonstrated in murine models by the downregulation of GRP78.²⁷ Notably, when subjected to a high-fat diet, mice with partial knockout of GRP78 (Grp78+/−) were protected from the development of hyperglycemia, insulin resistance, liver steatosis, and adipose tissue inflammation.⁴⁰

Furthermore, GRP78 can modulate the insulin signaling pathway by reducing the phosphorylation of protein kinase B in primary hepatocytes³⁹ and long-term continuous infusion of GRP78 in rats on a standard diet promotes insulin resistance, hyperglycemia, and MASH, mimicking the metabolic effects of a high-fat diet.¹⁴

While these findings suggest a potential involvement of GRP78 in obesity, further research is needed to fully elucidate the mechanisms and functional implications of GRP78 in this complex disease. Understanding the specific role of GRP78 in obesity-related metabolic dysfunction could provide insights into novel therapeutic strategies for managing obesity and its associated comorbidities.

GRP78 modulation after metabolic surgery

Metabolic surgery: An overview

One of the conventional treatments for obesity is metabolic surgery, which leads to sustained weight loss and the improvement and remission of many obesity-related comorbidities. The most common types of metabolic surgical techniques include vertical sleeve gastrectomy (VSG), laparoscopic Roux-en-Y gastric bypass (RYGB), and biliopancreatic diversion (BPD) (Figure 3).

Fig. 3.

Graphical representation of metabolic surgeries. Vertical sleeve gastrectomy (VSG) is a restrictive procedure that reduces the gastric volumes to 100–150 mL. Roux-en-Y gastric bypass (RYGB) involves both gastric reduction (15–30 mL volume) and intestinal rearrangement. This procedure creates a nutrient limb of 100 cm and biliopancreatic limb of 100 cm, while biliopancreatic diversion (BPD) creates a nutrient limb of 200 cm and biliopancreatic limb of 300–500 cm. The major difference between RYGB and BPD derives from the bypass of both duodenum and jejunum in BPD.

In VSG, a large portion of the stomach is removed along the greater curvature, reducing gastric volume by approximately 70–80%.⁴⁷

RYGB involves both gastric reduction and intestinal rearrangement, rapidly transporting nutrients from the stomach to the proximal jejunum while bypassing the duodenum.⁴⁸

BPD creates a gastric pouch similar to RYGB but bypasses both the duodenum and proximal jejunum, delivering nutrients directly to the distal jejunum.⁸

Metabolic surgery: Mechanisms of action

Metabolic surgery is known to have profound effects on various metabolic parameters, including insulin sensitivity and glucose homeostasis.⁴

Preliminary studies suggested that the main mechanism of action of VSG was the reduction of gastric volume; nevertheless, recent studies have shown that VSG can also affect hormone secretion.

Indeed, it has been shown that gastric fundus removal following VSG reduces the production of ghrelin, a hormone associated with weight loss.⁴⁹ VSG could also increase the intestinal expression of glucagon-like peptide-1, an anorexigenic hormone that promotes gastric emptying and decreases hepatic glucose production.⁵⁰ Additionally, VSG increases the expression of peptide YY and pancreatic polypeptide, leading to reduced hunger and food intake.⁵¹

It has been suggested that positive changes in gut hormones,⁵² bile acids,⁵³ and intestinal microbiota⁵⁴ contribute to the improved metabolic regulation following RYGB and BPD.

The “anti-incretin theory” suggests that gastrointestinal signals induced by nutrients transiting through the upper small bowel can lead to insulin resistance.⁵⁵ Therefore, the surgical exclusion of the duodenum and jejunum from nutrient transit could reduce such signals, explaining the reversal of diabetes and insulin resistance.

Insulin resistance, which is the primary contributor to type 2 diabetes can be improved within days through glycemic control, even without significant weight loss.56, 57 This suggests that glycemic control may result directly from surgical procedures rather than being a secondary outcome of weight loss procedures. Interestingly, the extent of jejunum bypassed during metabolic surgery correlates with the degree of improvement in insulin sensitivity.⁸ Both RYGB and BPD, which exclude a portion of the upper gastrointestinal tract, lead to the rapid improvement of insulin sensitivity shortly after the operation and are associated with higher rates of type 2 diabetes remission compared to restrictive procedures such as VSG.⁵⁸ Notably, BPD has shown greater rates of type 2 diabetes remission than RYGB.4, 3 Similarly, severe caloric restriction in individuals with obesity also results in a rapid improvement in glucose control.⁵⁹ Considering that the primary distinction between RYGB and BPD lies in the exclusion of the jejunum in the latter, it is reasonable to speculate that the jejunum may secrete one or more factors leading to insulin resistance. In support of this notion, the direct infusion of nutrients into the distal jejunum has been shown to substantially enhance insulin sensitivity in individuals with normal glucose tolerance or those with type 2 diabetes.⁶⁰

GRP78 expression after metabolic surgery in subject with obesity

Recent studies have shown that metabolic surgery can affect GRP78 expression and activity.14, 39, 9 In a recent study, circulating levels of GRP78 were found to be higher in people affected by obesity and insulin resistance compared to lean subjects and decreased following BPD.³⁹

In another study, GRP78 levels were compared in patients with class III obesity who were scheduled to undergo RYGB, BPD, or VSG.¹⁴ Circulating GRP78 decreased after all surgical procedures, but the effect of VSG in reducing the circulating levels of GRP78 was significantly lower compared to RYGB and BPD. Moreover, the reduction of circulating GRP78 tended to be larger after bypassing both the duodenum and jejunum in BPD, than after bypassing the duodenum alone with RYGB. These results point toward a key role of the upper gut in GRP78 secretion under fat meal stimulation and its therapeutic potential to reverse obesity comorbidities.

An interesting finding was that recombinant GRP78 reversed the metabolic benefits of metabolic surgery, indicating that GRP78 may play a key role in the development of obesity.¹⁴ While the precise mechanisms underlying the changes in GRP78 expression and its impact on metabolic improvements following metabolic surgery are still under investigation, recent findings in human and animal models suggest that GRP78 may be involved in the metabolic changes associated with these surgical interventions. Further research is needed to elucidate the therapeutic implications of GRP78 in the context of metabolic surgery and metabolic disorders. In fact, blocking circulating or intestinal GRP78 might be a valuable treatment option for obesity and its comorbidities.

Targeting GRP78 in obesity

As a result of the promising data linking metabolic surgery and GRP78, the modulation of GRP78 in obesity has emerged as a potential therapeutic approach. Ongoing research is focused on the discovery and development of novel therapeutic agents that specifically target GRP78. These may include small molecule compounds, peptides, or antibodies designed to modulate GRP78 activity or expression in a more selective manner.

Monoclonal antibody against GRP78

The development of monoclonal antibodies targeting GRP78 for the treatment of obesity is an area of active research. Monoclonal antibodies are designed to bind target proteins with high affinity.⁶¹

To date, GRP78 antibodies have been developed and patented for cancer therapy⁶² but PAT-SM6 is the only one that has been evaluated in clinical trials.⁶³ These antibodies can be also used to target GRP78 in the context of obesity.

Indeed, preclinical studies using monoclonal antibodies targeting GRP78 have shown promising results.64, 65, 14 These studies have demonstrated that monoclonal antibodies against GRP78 can reduce ER stress, improve insulin sensitivity, reverse MASH, and ameliorate metabolic dysregulation in animal models of obesity (Figure 4). Indeed, the use of monoclonal antibodies to block circulating GRP78 in rats reversed the metabolic derangement consequent to a high-fat diet mimicking the effects of metabolic surgery.¹⁴

Fig. 4.

Monoclonal antibodies targeting GRP78 for the treatment of obesity. An illustration of the potential mechanism of action of monoclonal antibodies targeting GRP78. Continuous infusion of GRP78 monoclonal antibody reverses the metabolic derangement consequent to high-fat diet affecting several pathways linked to obesity and MASH. Abbreviation used: GRP78, glucose-regulated protein 78; MASH, metabolic dysfunction-associated steatohepatitis.

Monoclonal antibodies that mimic the effects of metabolic surgery could be a valuable nonsurgical alternative for individuals affected by obesity or for patients who are ineligible for metabolic surgery. Indeed, despite its effectiveness, metabolic surgery is chosen by only 1% of eligible patients due to risks, limited access, costs, and patient preferences.

Despite preclinical studies have shown promising results further investigations between rodents and humans are highly warranted. Further studies are needed to fully understand their benefits, safety, and long-term effects in the human population.

Conclusion

A growing body of evidence indicates that extracellular GRP78 can disrupt metabolic processes, contributing to obesity, insulin resistance, and MASH. So far it has been demonstrated that GRP78 role in obesity extends beyond its chaperone function, influencing adipogenesis, stabilizing lipid droplets, and contributing to insulin resistance and liver steatosis.

Metabolic surgery, known to impact various metabolic pathways, has recently been associated with decreased GRP78 expression. Interestingly, the extent of jejunum bypassed during the surgery is correlated with a more significant decrease in GRP78 expression, suggesting that the upper gut plays a pivotal role in GRP78 secretion.

Understanding the intricate relationship between GRP78 and obesity may pave the way for novel therapeutic strategies and interventions targeting this chaperone to treat obesity and its related comorbidities. One promising strategy is the use of monoclonal antibodies against GRP78. Indeed, monoclonal antibodies targeting GRP78 can reverse the metabolic derangements caused by a high-fat diet, mimicking the effects of metabolic surgery. These results show the potential role of monoclonal antibodies as a nonsurgical alternative for individuals affected by obesity.

Future research should address various research gaps, including understanding the precise mechanism of GRP78 secretion from the jejunum, evaluating the long-term effects of monoclonal antibodies against GRP78 in humans, and assessing their safety and potential benefits.

Author contribution

GA and SR contributed to article collection and data analysis. GA and SR contributed to figure preparation and table organization. GA and GM wrote the draft.

All the authors approved this submission.

Declarations of interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Giulia Angelini reports a relationship with Metadeq Inc and GHP Scientific Ltd that includes: consulting or advisory. Geltrude Mingrone reports consulting fees from Novo Nordisk, Fractyl Inc, Recor Inc. She is also Scientific Advisor of Keyron Ltd, Metadeq Inc, GHP Scientific Ltd, and Jemyll Ltd. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.