Objective

Dual amylin and calcitonin receptor agonists (DACRAs) are novel therapeutic agents that not only improve insulin sensitivity, but also work as an adjunct to established T2DM therapies. DACRAs are currently administered once daily, and the question remains whether DACRAs with increased plasma half-life can be developed as once weekly therapy.

Methods

in vitro potencies of the KBP-066A and KBP-066 (non-acylated) were assessed using reporter assays. Acylation functionality was investigated by a combination of pharmacokinetics and acute food intake in rats. in vivo efficacies were investigated head-to-head in obese (HFD) and T2D (ZDF) models.

Results

In vitro, KBP-066A activated the CTR and AMY-R potently, with no off target activity. Acylation functionality was confirmed by the acute tests as KBP-066A demonstrated a prolonged PK and PD response compared to KBP-066. Both compounds induced a potent and dose-dependent weight loss in the HFD rat model. In ZDF rats, fasting blood glucose/fasting insulin levels (tAUC) were reduced by 39%/50% and 36%/47% for KBP-066 and KBP-066A, respectively. This resulted in 31% and 46% vehicle-corrected reduction in HbA1c at study end for KBP-066 and KBP-066A, respectively.

Conclusions

We here present preclinical data on an acylated DACRA, KBP-066A. The in vivo efficacy of KBP-066A is significantly improved compared to its non-acylated variant in terms of weight loss and glycemic control in obese (HFD), and in obese, diabetic rats (ZDF). This compendium of pre-clinical studies highlights KBP-066A as a promising, once-weekly, therapeutic agent for treating T2DM and obesity.

Objective

Brown adipose tissue (BAT) is critical for thermogenesis and glucose/lipid homeostasis. Exploiting the energy uncoupling capacity of BAT may reveal targets for obesity therapies. This requires a greater understanding of transcriptional mechanisms underlying BAT function. One potential regulator of BAT is the transcriptional co-regulator LIM domain-binding protein 1 (LDB1), which acts as a dimerized scaffold allowing for the assembly of transcriptional complexes. Utilizing a global LDB1 heterozygous mouse model, we recently reported that LDB1 may have novel roles in regulating BAT function. However, direct evidence for LDB1 regulation of BAT thermogenesis and substrate utilization has not been elucidated. We hypothesize that brown adipocyte-expressed LDB1 is required for BAT function.

Methods

To define the brown adipose-specific roles, LDB1 deficient primary cells and brown adipocyte cell lines were assessed via qRT-PCR and western blotting for altered mRNA and protein levels. We conducted chromatin immunoprecipitation with primary BAT tissue and immortalized cell lines. Potential transcriptional partners of LDB1 were revealed by conducting LIM factor surveys via qRT-PCR in mouse and human brown adipocytes. To test LDB1 function in vivo, we developed a Ucp1-Cre driven LDB1-deficiency mouse model, termed Ldb1^ΔBAT. Glucose tolerance and uptake were assessed at thermoneutrality via intraperitoneal glucose challenge and glucose tracer studies. Insulin tolerance was measured at thermoneutrality and after stimulation with cold or administration of the β3-adrenergic receptor (β3-AR) agonist CL316,243. Additionally, we analyzed plasma insulin via ELISA and insulin signaling via western blotting. Lipid metabolism was evaluated via BAT weight, histology, lipid droplet morphometry, and examination of lipid associated mRNA. Finally, energy expenditure and cold tolerance were evaluated via indirect calorimetry and cold challenges.

Results

Reducing Ldb1 both in vitro and in vivo resulted in altered BAT-selective mRNA, including Ucp1, Elovl3, and Dio2, plus a reduced Ucp1 induction in vitro. Impacts on gene expression may be due in part to LDB1 occupying Ucp1 upstream regulatory domains. We also identified BAT-expressed LIM-domain factors Lmo2, Lmo4, and Lhx8, which may partner with LDB1 to mediate activity in brown adipocytes. Additionally, we observed LDB1 enrichment in human brown adipose. In vivo analysis revealed LDB1 is required for whole body glucose and insulin tolerance, in part through reduced glucose uptake into BAT. In Ldb1^ΔBAT tissue, we found significant alterations in insulin signaling effectors. Assessment of brown adipocyte morphology and lipid droplet size revealed larger and more unilocular brown adipocytes in Ldb1^ΔBATmice, particularly after a cold challenge. Alterations in lipid handling was further supported by reductions in mRNA associated with fatty acid oxidation and mitochondrial respiration. Finally, LDB1 is required for energy expenditure and cold tolerance in both male and female mice.

Conclusions

Our findings support LDB1 as a regulator of BAT function. Furthermore, given LDB1 enrichment in human brown adipose, this co-regulator may have conserved roles in human BAT.

Background

While insulin has been central to the pathophysiology and treatment of patients with diabetes for the last 100 years it has only been since 2007 that genetic variation in the INS gene has been recognised as a major cause of monogenic diabetes. Both dominant and recessive mutations in the INS gene are now recognised as important causes of neonatal diabetes and offer important insights both into the structure and function of insulin. It is also recognised that in rare cases, mutations in the INS gene can present in patients with diabetes diagnosed outside the first year of life.

Scope of review

This review examines the genetics and clinical features of monogenic diabetes resulting from INS gene mutations from the first description in 2007 and includes information from 389 patients from 292 families diagnosed in Exeter with INSgene mutations. We discuss the implications for diagnosing and treating this subtype of monogenic diabetes.

Major conclusions

The dominant mutations in the INS gene typically affect the secondary structure of the insulin protein usually by disrupting the 3 disulfide bonds in mature insulin. The resulting misfolded protein results in ER stress and beta-cell destruction. In contrast recessive INS gene mutations typically result in no functional protein being produced as a result of reduced insulin biosynthesis or loss-of-function mutations of the insulin protein. There are clinical differences between the two genetic aetiologies, between the specific mutations, and finally variation within patients with identical mutations.

Objective

The glucose tolerance test (GTT) is widely used in human and animal biomedical and pharmaceutical research. Despite its prevalent use, particularly in mouse metabolic phenotyping, to the best of our knowledge we are not aware of any studies that have attempted to qualitatively compare the metabolic events during a GTT in mice with those performed in humans.

Methods

To provide greater resolution into postprandial glucose kinetics, stable isotope labelled oral glucose tolerance tests (siOGTTs; [6,6-²H₂]glucose) were performed in both human and mouse cohorts. The siOGTT allows the partitioning of circulating glucose into that derived from exogenous and endogenous sources. Young adults spanning the spectrum of normal glucose tolerance (n=221), impaired fasting (n=14), and impaired glucose tolerance (n=19) underwent a 75g siOGTT, while a 50 mg siOGTT was performed on chow (n=43) and high-fat high-sucrose fed C57Bl6 male mice (n=46).

Results

In humans during the siOGTT, there is a long period (>3hr) of glucose absorption and accordingly a large and sustained insulin response as well as robust suppression of lipolysis and endogenous glucose production (EGP), even in the presence of glucose intolerance. In contrast, mice appear to be highly reliant on glucose effectiveness to clear exogenous glucose and experience only modest and transient insulin responses with little, if any, suppression of EGP. In addition to impaired stimulation of glucose uptake, mice with the worst glucose tolerance appear to have a paradoxical and persistent rise in EGP during the OGTT which is likely related to handling stress.

Conclusions

The metabolic response to the OGTT in mice and humans is highly divergent. The potential reasons for these differences and the impact this could have on the interpretation of mouse glucose tolerance data and their translation to humans is discussed.

Objective

Insulin regulates mitochondrial function, thereby propagating efficient metabolism. Conversely, diabetes and insulin resistance are linked to mitochondrial dysfunction with decreased expression of the mitochondrial chaperone HSP60. The aim of this investigation was to determine the effect of a reduced HSP60 expression on the development of obesity and insulin resistance.

Methods

Control and heterozygous whole-body HSP60 knock-out (Hsp60^+/-) mice were fed a high-fat diet (HFD, 60% of calories from fat) for 16 weeks and subjected to extensive metabolic phenotyping. To understand the effect of HSP60 on white adipose tissue, microarray analysis of gonadal WAT was executed, ex vivo experiments performed and a lentiviral knockdown of Hsp60 in 3T3-L1 cells conducted to gain detailed insights into the effect of reduced Hsp60 levels on adipocyte homeostasis.

Results

Male Hsp60^+/- mice exhibited lower body weight with lower fat mass. These mice exhibited improved insulin sensitivity compared to control as assessed by Matsuda Index and HOMA-IR. Accordingly, insulin levels were significantly reduced in Hsp60^+/- mice in a glucose tolerance test. Yet, Hsp60^+/- mice exhibited an altered adipose tissue metabolism with an elevated insulin-independent elevated glucose uptake, adipocyte hyperplasia in the presence of mitochondrial dysfunction, altered autophagy and local insulin resistance.

Conclusion

We discovered that the reduction of HSP60 in mice predominantly affects adipose tissue homeostasis, leading to beneficial alterations in body weight, body composition, and adipocyte morphology albeit exhibiting local insulin resistance.

Objective

Immature CD11b ⁺ Gr1⁺ myeloid cells that acquire immunosuppressive capability, also known as myeloid-derived suppressor cells (MDSCs), are a heterogeneous population of cells that regulate immune responses. Our study's objective was to elucidate the role of ovarian cancer microenvironment in regulating the immunosuppressive function of CD11b⁺Gr1⁺ myeloid cells.

Methods

All studies were performed using the intraperitoneal ID8 syngeneic epithelial ovarian cancer mouse model. Myeloid cell depletion and immunotherapy were carried out using anti-Gr1 mAb, gemcitabine treatments, and/or anti PD1 mAb. The treatment effect was assessed by a survival curve, in situ luciferase-guided imaging, and histopathologic evaluation. Adoptive transfer assays were carried out between congenic CD45.2 and CD45.1 mice. Immune surface and intracellular markers were assessed by flow cytometry. ELISA, western blot, and RT-PCR techniques were employed to assess the protein and RNA expression of various markers. Bone marrow-derived myeloid cells were used for ex vivo studies.

Results

The depletion of Gr1⁺ immunosuppressive myeloid cells alone and in combination with anti-PD1 immunotherapy inhibited ovarian cancer growth. In addition to the adoptive transfer studies, these findings validate the role of immunosuppressive CD11b⁺Gr1⁺ myeloid cells in promoting ovarian cancer. Mechanistic investigations showed that ID8 tumor cells and their microenvironments produced recruitment and regulatory factors for immunosuppressive CD11b⁺Gr1⁺ myeloid cells. CD11b⁺Gr1⁺ myeloid cells primed by ID8 tumors showed increased immunosuppressive marker expression and acquired an energetic metabolic phenotype promoted primarily by increased oxidative phosphorylation fueled by glutamine. Inhibiting the glutamine metabolic pathway reduced the increased oxidative phosphorylation and decreased immunosuppressive markers’ expression and function. Dihydrolipoamide succinyl transferase (DLST), a subunit of α-KGDC in the TCA cycle, was found to be the most significantly elevated gene in tumor-primed myeloid cells. The inhibition of DLST reduced oxidative phosphorylation, immunosuppressive marker expression, and function in myeloid cells.

Conclusion

Our study shows that the ovarian cancer microenvironment can regulate the metabolism and function of immunosuppressive CD11b ⁺ Gr1⁺ myeloid cells and modulate its immune microenvironment. Targeting glutamine metabolism via DLST in immunosuppressive myeloid cells decreased their activity, leading to a reduction in the immunosuppressive tumor microenvironment. Thus, targeting glutamine metabolism has the potential to enhance the success of immunotherapy in ovarian cancer.

Objective

Retinal ischemic disease is a major cause of vision loss. Current treatment options are limited to late-stage diseases, and the molecular mechanisms of the initial insult are not fully understood. We have previously shown that the deletion of the mitochondrial arginase isoform, arginase 2 (A2), limits neurovascular injury in models of ischemic retinopathy. Here, we investigated the involvement of A2-mediated alterations in mitochondrial dynamics and function in the pathology.

Methods

We used wild-type (WT), global A2 knockout (A2KO-) mice, cell-specific A2 knockout mice subjected to retinal ischemia/reperfusion (I/R), and bovine retinal endothelial cells (BRECs) subjected to an oxygen-glucose deprivation/reperfusion (OGD/R) insult. We used western blotting to measure levels of cell stress and death markers and the mitochondrial fragmentation protein, dynamin related protein 1 (Drp1). We also used live cell mitochondrial labeling and Seahorse XF analysis to evaluate mitochondrial fragmentation and function, respectively.

Results

We found that the global deletion of A2 limited the I/R-induced disruption of retinal layers, fundus abnormalities, and albumin extravasation. The specific deletion of A2 in endothelial cells was protective against I/R-induced neurodegeneration. The OGD/R insult in BRECs increased A2 expression and induced cell stress and cell death, along with decreased mitochondrial respiration, increased Drp1 expression, and mitochondrial fragmentation. The overexpression of A2 in BREC also decreased mitochondrial respiration, promoted increases in the expression of Drp1, mitochondrial fragmentation, and cell stress and resulted in decreased cell survival. In contrast, the overexpression of the cytosolic isoform, arginase 1 (A1), did not affect these parameters.

Conclusions

This study is the first to show that A2 in endothelial cells mediates retinal ischemic injury through a mechanism involving alterations in mitochondrial dynamics and function.

Objective

NAD⁺ is a co-factor and substrate for enzymes maintaining energy homeostasis. Nicotinamide phosphoribosyltransferase (NAMPT) controls NAD⁺ synthesis, and in skeletal muscle, NAD⁺ is essential for muscle integrity. However, the underlying molecular mechanisms by which NAD⁺ synthesis affects muscle health remain poorly understood. Thus, the objective of the current study was to delineate the role of NAMPT-mediated NAD⁺ biosynthesis in skeletal muscle development and function.

Methods

To determine the role of Nampt in muscle development and function, we generated skeletal muscle-specific Nampt KO (SMNKO) mice. We performed a comprehensive phenotypic characterization of the SMNKO mice, including metabolic measurements, histological examinations, and RNA sequencing analyses of skeletal muscle from SMNKO mice and WT littermates.

Results

SMNKO mice were smaller, with phenotypic changes in skeletal muscle, including reduced fiber area and increased number of centralized nuclei. The majority of SMNKO mice died prematurely. Transcriptomic analysis identified that the gene encoding the mitochondrial permeability transition pore (mPTP) regulator Cyclophilin D (Ppif) was upregulated in skeletal muscle of SMNKO mice from 2 weeks of age, with associated increased sensitivity of mitochondria to the Ca²⁺-stimulated mPTP opening. Treatment of SMNKO mice with the Cyclophilin D inhibitor, Cyclosporine A, increased membrane integrity, decreased the number of centralized nuclei, and increased survival.

Conclusions

Our study demonstrates that NAMPT is crucial for maintaining cellular Ca²⁺homeostasis and skeletal muscle development, which is vital for juvenile survival.

Objective

Glucagon is secreted by pancreatic α-cells in response to hypoglycemia and its hyperglycemic effect helps to restore normal blood glucose. Insulin and somatostatin (SST) secretions from β- and δ-cells, respectively, are stimulated by glucose by mechanisms involving an inhibition of their ATP-sensitive K⁺ (K_ATP) channels, leading to an increase in [Ca²⁺]_c that triggers exocytosis. Drugs that close K_ATP channels, such as sulfonylureas, are used to stimulate insulin release in type 2 diabetic patients. α-cells also express K_ATP channels. However, the mechanisms by which sulfonylureas control glucagon secretion are still largely debated and were addressed in the present study. In particular, we studied the effects of K_ATP channel blockers on α-cell [Ca²⁺]_c and glucagon secretion in the presence of a low (1 mM) or a high (15 mM) glucose concentration and evaluated the role of SST in these effects.

Methods

Using a transgenic mouse model expressing the Ca²⁺-sensitive fluorescent protein, GCaMP6f, specifically in α-cells, we measured [Ca²⁺]_c in α-cells either dispersed or within whole islets (by confocal microscopy). By measuring [Ca²⁺]_c in α-cells within islets and glucagon secretion using the same perifusion protocols, we tested whether glucagon secretion correlated with changes in [Ca²⁺]_c in response to sulfonylureas. We studied the role of SST in the effects of sulfonylureas using multiple approaches including genetic ablation of SST, or application of SST-14 and SST receptor antagonists.

Results

Application of the sulfonylureas, tolbutamide, or gliclazide, to a medium containing 1 mM or 15 mM glucose increased [Ca²⁺]_c in α-cells by a direct effect as in β-cells. At low glucose, sulfonylureas inhibited glucagon secretion of islets despite the rise in α-cell [Ca²⁺]_c that they triggered. This glucagonostatic effect was indirect and attributed to SST because, in the islets of SST-knockout mice, sulfonylureas induced stimulation of glucagon secretion which correlated with an increase in α-cell [Ca²⁺]_c. Experiments with exogenous SST-14 and SST receptor antagonists indicated that the glucagonostatic effect of sulfonylureas mainly resulted from the inhibition of the efficacy of cytosolic Ca²⁺ on exocytosis. Although SST-14 was also able to inhibit glucagon secretion by decreasing α-cell [Ca²⁺]_c, no decrease in [Ca²⁺]_coccurred during sulfonylurea application because it was largely counterbalanced by the direct stimulatory effect of these drugs on α-cell [Ca²⁺]_c. At high glucose, i.e., in conditions where glucagon release was already low, sulfonylureas stimulated glucagon secretion because their direct stimulatory effect on α-cells exceeded the indirect effect by SST. Our results also indicated that, unexpectedly, SST-14 poorly decreased the efficacy of Ca²⁺ on exocytosis in β-cells.

Conclusions

Sulfonylureas exert two opposite actions on α-cells: a direct stimulation as in β-cells and an indirect inhibition by SST. This suggests that any alteration of SST paracrine influence, as described in diabetes, will modify the effect of sulfonylureas on glucagon release. In addition, we suggest that δ-cells inhibit α-cells more efficiently than β-cells.

Objective

Throughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. Therefore, we sought to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB).

Methods

Using targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, and complementary physiological approaches in novel cell type-specific knockout mice, we quantified the roles of hepatocyte D-β-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD⁺/NADH-dependent oxidation/reduction of ketone bodies.

Results

Exogenously administered AcAc is reduced to D-βOHB, which increases hepatic NAD⁺/NADH ratio and reflects hepatic BDH1 activity. Livers of hepatocyte-specific BDH1-deficient mice did not produced D-βOHB, but owing to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-βOHB interconversion. Compared to littermate controls, hepatocyte-specific BDH1 deficient mice exhibited diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis, but normal hepatic energy charge overall. Glycemic recovery after acute insulin challenge was impaired in knockout mice, but they were not more susceptible to starvation-induced hypoglycemia.

Conclusions

Ketone bodies influence liver homeostasis. While liver BDH1 is not required for whole body equilibration of AcAc and D-βOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production. Therefore, through oxidation/reduction of ketone bodies, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis.

Objective

Obesity, in particular visceral obesity, and insulin resistance emerged as major risk factors for severe Coronavirus Disease 2019 (COVID-19), which is strongly associated with hemostatic alterations. Since obesity and insulin resistance predispose to thrombotic diseases, we investigated the relationship between hemostatic alterations and body fat distribution in subjects at risk for type 2 diabetes.

Subjects and methods

Body fat distribution (visceral and subcutaneous abdominal adipose tissue) and liver fat content of 150 subjects with impaired glucose tolerance and/or impaired fasting glucose was determined using magnetic resonance imaging and spectroscopy. Participants underwent precise metabolic characterization and major hemostasis parameters were analyzed.

Results

Procoagulant factors (FII, FVII, FVIII and FIX) and anticoagulant proteins (antithrombin, protein C and protein S) were significantly associated with body fat distribution. In subjects with fatty liver, fibrinogen (298 mg/dl vs. 264 mg/dl, p=0.0182), FVII (99% vs. 90%, p=0.0049), FVIII (114% vs. 90 %, p=0.0098), protein C (124% vs. 111%, p=0.0006) and protein S (109% vs. 89%, p<0.0001) were higher than in controls. In contrast, antithrombin (97% vs. 102%, p=0.0025) was higher in control subjects. In multivariate analyses controlling for insulin sensitivity, body fat compartments and genotype variants (PNPLA3^I148MM/MI/TM6SF2^E167KK/KE), only protein C and protein S remained significantly increased in fatty liver.

Conclusions

Body fat distribution is significantly associated with alterations of procoagulant as well as anticoagulant parameters. Liver fat plays a key role for the regulation of protein C and protein S suggesting a potential counteracting mechanism to the prothrombotic state in subjects with prediabetes and fatty liver.

Background

A strong association of obesity and insulin resistance with increased circulating levels of branched-chain and aromatic amino acids and decreased glycine levels has been recognized in human subjects for decades.

Scope of Review

More recently, human metabolomic and genetic studies have confirmed and expanded upon these observations, accompanied by a surge in preclinical studies that have identified mechanisms involved in perturbation of amino acid homeostasis, how these events are connected to dysregulated glucose and lipid metabolism, and how elevations in branched-chain amino acids (BCAA) may participate in development of insulin resistance, type 2 diabetes (T2D) and other cardiometabolic diseases and conditions.

Major Conclusions

In human cohorts, BCAA and related metabolites are now well established as among the strongest biomarkers of obesity, insulin resistance, T2D, and cardiovascular diseases. Lowering of BCAA and branched-chain ketoacid (BCKA) levels by feeding of a BCAA-restricted diet or by activation of the rate limiting enzyme in BCAA catabolism, branched-chain ketoacid dehydrogenase (BCKDH) in rodent models of obesity has clear salutary effects on glucose and lipid homeostasis, but BCAA restriction has more modest effects in shorter-term studies in human T2D subjects. Feeding of rats with diets enriched in sucrose or fructose results in induction of the ChREBP transcription factor in liver to increase expression of the BCKDH kinase (BDK) and suppress expression of its phosphatase (PPM1K) resulting in inactivation of BCKDH and activation of the key lipogenic enzyme ATP-citrate lyase (ACLY). These and other emergent links between BCAA, glucose and lipid metabolism motivate ongoing studies of possible causal actions of BCAA and related metabolites in development of cardiometabolic diseases.

Objective

Skeletal muscle is an attractive target for blood glucose-lowering pharmacological interventions. Oral dosing of small molecule direct pan-activators of AMPK, that bind to the Allosteric Drug and Metabolite (ADaM) site, lowers blood glucose through effects in skeletal muscle. The molecular mechanisms responsible for this effect are not described in detail. The aim of this study was to illuminate the mechanism by which the ADaM-site activators of AMPK increase glucose uptake in skeletal muscle. Further, we investigated the consequence of co-stimulating muscles with two types of AMPK activators i.e. ADaM-site binding small molecules and the prodrug AICAR.

Methods

The effect of the ADaM-site binding small molecules (PF739 and 991), AICAR or co-stimulation with PF739 or 991 and AICAR on muscle glucose uptake was investigated ex vivo in m. extensor digitorum longus (EDL) excised from muscle-specific AMPKα1α2-as well as whole-body AMPKγ3-deficient mouse models. In vitrocomplex-specific AMPK activity was measured by immunoprecipitation and molecular signaling was assessed by Western Blotting in muscle lysate. To investigate the translatability of our studies, we treated mice in vivo with PF739 and measured complex-specific AMPK activation in skeletal muscle.

Results

Incubation of skeletal muscle with PF739 or 991 increased skeletal muscle glucose uptake in a dose-dependent manner. Co-incubating PF739 or 991 with a maximal dose of AICAR, increased glucose uptake to a greater extent than any of the treatments alone. Neither PF739 nor 991 increased AMPKα2β2γ3 activity to the same extent as AICAR, while co-incubation led to potentiated effects on AMPKα2β2γ3 activation. In muscle from AMPKγ3 KO mice, AICAR-stimulated glucose uptake was ablated. In contrast, the effect of PF739 or 991 on glucose uptake was not different between WT and AMPKγ3 KO muscle. In vivo PF739 treatment lowered blood glucose levels and increased muscle AMPKγ1-complex activity 2-fold, while AMPKα2β2γ3 activity was not affected.

Conclusions

ADaM-site binding AMPK activators increase glucose uptake independently of AMPKγ3. Co-incubation with PF739 or 991 and AICAR potentiates the effects on muscle glucose uptake and AMPK activation. In vivo, PF739 lowers blood glucose and selectively activates muscle AMPKγ1-complexes. Collectively, this suggests that pharmacological activation of AMPKγ1-containing complexes in skeletal muscle can increase glucose uptake and can lead to blood glucose lowering.

Background

Insulin's discovery 100 years ago and its ongoing use since that time to treat diabetes belies the molecular complexity of its structure and that of its receptor. Advances in single-particle cryo-electron microscopy have over the past three years revolutionized our understanding of the atomic detail of insulin-receptor interactions.

Scope of review

This review describes the three-dimensional structure of insulin and its receptor and details on how they interact. This review also highlights the current gaps in our structural understanding of the system.

Major conclusions

A near-complete picture has been obtained of the hormone receptor interactions, providing new insights into the kinetics of the interactions and necessitating a revision of the extant two-site cross-linking model of hormone receptor engagement. How insulin initially engages the receptor and the receptor's traversed trajectory as it undergoes conformational changes associated with activation remain areas for future investigation.

Background

The insulin-like growth factor family of ligands (IGF-I, IGF-II, and insulin), receptors (IGF-IR, M6P/IGF-IIR, and insulin receptor [IR]), and IGF-binding proteins (IGFBP-1-6) play critical roles in normal human physiology and disease states.

Scope of review

Insulin and insulin receptors are the focus of other chapters in this series and will therefore not be discussed further. Here we review the basic components of the IGF system, their role in normal physiology and in critical pathology's. While this review concentrates on the role of IGFs in human physiology, animal models have been essential in providing understanding of the IGF system, and its regulation, and are briefly described.

Major conclusions

IGF-I has effects via the circulation and locally within tissues to regulate cellular growth, differentiation, and survival, thereby controlling overall body growth. IGF-II levels are highest prenatally when it has important effects on growth. In adults, IGF-II plays important tissue-specific roles, including the maintenance of stem cell populations. Although the IGF-IR is closely related to the IR it has distinct physiological roles both on the cell surface and in the nucleus. The M6P/IGF-IIR, in contrast, is distinct and acts as a scavenger by mediating internalization and degradation of IGF-II. The IGFBPs bind IGF-I and IGF-II in the circulation to prolong their half-lives and modulate tissue access, thereby controlling IGF function. IGFBPs also have IGF ligand-independent cell effects.

Background

Non-alcoholic fatty liver disease, or as recently proposed ‘metabolic-associated fatty liver disease’ (MAFLD), is characterized by pathological accumulation of triglycerides and other lipids in hepatocytes. This common disease can progress from simple steatosis to steatohepatitis, and eventually end-stage liver diseases. MAFLD is closely related to disturbances in systemic energy metabolism, including insulin resistance and atherogenic dyslipidemia.

Scope of review

The liver is the central organ in lipid metabolism by secreting very low density lipoproteins (VLDL) and, on the other hand, by internalizing fatty acids and lipoproteins. This review article discusses recent research addressing hepatic lipid synthesis, VLDL production, and lipoprotein internalization as well as the lipid exchange between adipose tissue and the liver in the context of MAFLD.

Major conclusions

Liver steatosis in MAFLD is triggered by excessive hepatic triglyceride synthesis utilizing fatty acids derived from white adipose tissue (WAT), de novo lipogenesis (DNL) and endocytosed remnants of triglyceride-rich lipoproteins. In consequence of high hepatic lipid content, VLDL secretion is enhanced, which is the primary cause of complex dyslipidemia typical for subjects with MAFLD. Interventions reducing VLDL secretory capacity attenuate dyslipidemia while they exacerbate MAFLD, indicating that the balance of lipid storage versus secretion in hepatocytes is a critical parameter determining disease outcome. Proof of concept studies have shown that promoting lipid storage and energy combustion in adipose tissues reduces hepatic lipid load and thus ameliorates MAFLD. Moreover, hepatocellular triglyceride synthesis from DNL and WAT-derived fatty acids can be targeted to treat MAFLD. However, more research is needed to understand how individual transporters, enzymes, and their isoforms affect steatosis and dyslipidemia in vivo, and whether these two aspects of MAFLD can be selectively treated. Processing of cholesterol-enriched lipoproteins appears less important for steatosis. It may, however, modulate inflammation and consequently MAFLD progression.

Background

Insulin has been demonstrated to exert direct and indirect effects on vascular tissues. Its actions in vascular cells are mediated by two major pathways: the insulin receptor substrate 1/2-phosphoinositide-3 kinase/Akt (IRS1/2/PI3K/Akt) pathway and the Src/mitogen-activated protein kinase (MAPK) pathway, both of which contribute to the expression and distribution of metabolites, hormones, and cytokines.

Scope of review

In this review, we summarize the current understanding of insulin's physiological and pathophysiological actions and associated signaling pathways in vascular cells, mainly in endothelial cells (EC) and vascular smooth muscle cells (VSMC), and how these processes lead to selective insulin resistance. We also describe insulin's potential new signaling and biological effects derived from animal studies and cultured capillary and arterial EC, VSMC, and pericytes. We will not provide a detailed discussion of insulin's effects on the myocardium, insulin's structure, or its signaling pathways' various steps, since other articles in this issue discuss these areas in depth.

Major conclusions

Insulin mediates many important functions on vascular cells via its receptors and signaling cascades. Its direct actions on EC and VSMC are important for transporting and communicating nutrients, cytokines, hormones, and other signaling molecules. These vascular actions are also important for regulating systemic fuel metabolism and energetics. Inhibiting or enhancing these pathways leads to selective insulin resistance, exacerbating the development of endothelial dysfunction, atherosclerosis, restenosis, poor wound healing, and even myocardial dysfunction. Targeted therapies to improve selective insulin resistance in EC and VSMC are thus needed to specifically mitigate these pathological processes.

Background

The brain was once thought of as an insulin-insensitive organ. We now know that the insulin receptor is present throughout the brain and serves important functions in whole body metabolism and brain function. Brain insulin signaling is involved in not only brain homeostatic processes, but also neuropathological processes such as cognitive decline and Alzheimer’s disease.

Scope of review

In this review, we provide an overview of insulin signaling within the brain, the metabolic impact of brain insulin resistance, and discuss Alzheimer’s disease, one of the neurologic diseases most closely associated with brain insulin resistance.

Major conclusions

While brain insulin signaling plays only a small role in central nervous system glucose regulation, it has a significant impact on the metabolic health of the brain. Normal insulin signaling is important for mitochondrial functioning and normal food intake. Brain insulin resistance contributes to obesity and may also play an important role in neurodegeneration.

Dominant mutations in the human insulin gene (INS) lead to pancreatic β-cell dysfunction and diabetes mellitus (DM) due to toxic misfolding of a mutant proinsulin. Analogous to a classical mouse model of monogenic DM (“Akita”), this syndrome highlights the susceptibility of β-cells to endoreticulum (ER) stress due to protein misfolding and aberrant aggregation. Diverse clinical mutations directly or indirectly perturb native disulfide pairing. Whereas most introduce or remove a cysteine (Cys; leading in either case to an unpaired thiol group), non-Cys-related mutations identify key determinants of folding efficiency. Studies of such mutations suggest that the hormone’s evolution has been constrained not only by structure-function relationships, but also by the susceptibility of its single-chain precursor to impaired foldability. An intriguing hypothesis posits that INS overexpression in response to peripheral insulin resistance likewise leads to chronic ER stress and β-cell dysfunction in the natural history of non-syndromic Type 2 DM. Cryptic contributions of conserved residues to folding efficiency, as uncovered by rare genetic variants, define molecular links between biophysical principles and the emerging paradigm of Darwinian medicine: Biosynthesis of proinsulin at the edge of non-foldability provides a key determinant of “diabesity” as a pandemic disease of civilization.

Objective

The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and also potentially in BAT. Here, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed if γ3 plays a role in adipose thermogenesis and browning.

Methods

Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo, as well as blood glucose clearance in response to small molecule AMPK activators that target nucleotide-binding domain of γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and β subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a β3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics and function.

Results

Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and β2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and β2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice.

Conclusions

These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for β3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT.

The 100th anniversary of the discovery of insulin in Toronto in 1921 is an important moment in medical and scientific history. The demonstration that an extract of dog pancreas reproducibly lowered blood glucose, initially in diabetic dogs and then in humans with type 1 diabetes, was a medical breakthrough that changed the course of what was until then a largely fatal disease. The discovery of the “activity”, soon named “insulin”, was widely celebrated, garnering a Nobel Prize for Banting and McLeod in 1923. Over the ensuing 100 years, research on insulin has advanced on many fronts, producing insights that have transformed our understanding of diabetes and our approach to its treatment. However, research on insulin had another consequence of far broader scientific significance, serving as a pacesetter and catalyst to bioscience research across many fields. Some of this was directly insulin-related and was also recognized by the Nobel Prize. Equally important, however, was research stimulated by the discovery of insulin that has profoundly influenced biomedical research, sometimes also recognized by the Nobel Prize and sometimes without this recognition. In this paper, we review some of the most notable examples of both insulin-related and insulin-stimulated research to illustrate the impact of this discovery on the course of modern bioscience.

Non-alcoholic fatty liver disease (NAFLD) is an important component of metabolic syndrome and one of the most prevalent liver diseases worldwide. This disorder is closely linked to hepatic insulin resistance, lipotoxicity, and inflammation. Although the mechanisms that cause steatosis and chronic liver injury in NAFLD remain unclear, a key component of this process is the activation of stress-activated kinases (SAPKs), including p38 and JNK in the liver and immune system. This review summarizes findings which indicate that the dysregulation of stress kinases plays a fundamental role in the development of steatosis and are important players in inducing liver fibrosis. To avoid the development of steatohepatitis and liver cancer, SAPK activity must be tightly regulated not only in the hepatocytes but also in other tissues, including cells of the immune system. Possible cellular mechanisms of SAPK actions are discussed.

Background

Non-alcoholic fatty liver disease (NAFLD) is a clinicopathologic entity that requires a liver biopsy assessment to diagnose the progressive form of NAFLD called non-alcoholic steatohepatitis (NASH). Liver biopsy is invasive, subject to sampling and interobserver variability, and impractical to scale to the affected population of up to 1 billion affected individuals worldwide. Non-invasive imaging biomarkers have emerged as a key modality to address the major unmet need to diagnose, stage, and longitudinally monitor NAFLD.

Scope of review

In this review, we critically examine the use of non-invasive imaging biomarkers to diagnose NAFLD, NASH, and fibrosis stage.

Major Conclusions

Ultrasound and magnetic resonance imaging (MRI) biomarkers of liver fat can diagnose NAFLD. MRI proton density fat fraction (MRI-PDFF) is better than liver biopsy, particularly for following longitudinal changes in liver fat in clinical trials. Imaging biomarkers to reliably diagnose NASH are under investigation, but when used alone, continue to have only modest diagnostic accuracy. However, the fibrosis stage has the strongest association with liver decompensation and mortality, and elastography has emerged as a reliable biomarker for liver fibrosis. We review the combination of biomarkers to risk stratify patients and identify individuals needing treatment and the implications of longitudinal changes in liver stiffness measurement.

Background

Metabolic-associated fatty liver disease (MAFLD), also known as non-alcoholic fatty liver disease, has become the leading cause of chronic liver disease worldwide. In addition to hepatic accumulation of triglycerides, dysregulated cholesterol metabolism is an important contributor to the pathogenesis of MAFLD. Maintenance of cholesterol homeostasis is highly dependent on cellular cholesterol uptake and, subsequently, cholesterol transport to other membrane compartments, such as the endoplasmic reticulum (ER).

Scope of review

The endolysosomal network is key for regulating cellular homeostasis and adaptation, and emerging evidence has shown that the endolysosomal network is crucial to maintain metabolic homeostasis. In this review, we will summarize our current understanding of the role of the endolysosomal network in cholesterol homeostasis and its implications in MAFLD pathogenesis.

Major conclusions

Although multiple endolysosomal proteins have been identified in the regulation of cholesterol uptake, intracellular transport, and degradation, their physiological role is incompletely understood. Further research should elucidate their role in controlling metabolic homeostasis and development of fatty liver disease.

Background

The incidence of non-alcoholic fatty liver disease (NAFLD) is rapidly increasing worldwide parallel to the global obesity epidemic. NAFLD encompasses a range of liver pathologies and most often originates from metabolically driven accumulation of fat in the liver, or non-alcoholic fatty liver (NAFL). In a subset of NAFL patients, the disease can progress to non-alcoholic steatohepatitis (NASH), which is a more severe form of liver disease characterized by hepatocyte injury, inflammation, and fibrosis. Significant progress has been made over the past decade in our understanding of NASH pathogenesis, but gaps remain in our mechanistic knowledge of the precise metabolic triggers for disease worsening.

Scope of review

The transition from NAFL to NASH likely involves a complex constellation of multiple factors intrinsic and extrinsic to the liver. This review focuses on early metabolic events in the establishment of NAFL and initial stages of NASH. We discuss the association of NAFL with obesity as well as the role of adipose tissue in disease progression and highlight early metabolic drivers implicated in the pathological transition from hepatic fat accumulation to steatohepatitis.

Major conclusions

The close association of NAFL with features of metabolic syndrome highlight plausible mechanistic roles for adipose tissue health and the release of lipotoxic lipids, hepatic de novo lipogenesis (DNL), and disruption of the intestinal barrier in not only the initial establishment of hepatic steatosis, but also in mediating disease progression. Human genetic variants linked to NASH risk to date are heavily biased toward genes involved in the regulation of lipid metabolism, providing compelling support for the hypothesis that NASH is fundamentally a metabolic disease.

Background

Mitochondrial oxidative function plays a key role in the development of non-alcoholic fatty liver disease (NAFLD) and insulin resistance (IR). Recent studies reported that fatty liver might not be a result of decreased mitochondrial fat oxidation caused by mitochondrial damage. Rather, NAFLD and IR induce an elevation in mitochondrial function that covers the increased demand for carbon intermediates and ATP caused by elevated lipogenesis and gluconeogenesis. Furthermore, mitochondria play a role in regulating hepatic insulin sensitivity and lipogenesis by modulating redox-sensitive signaling pathways.

Scope of review

We review the contradictory studies indicating that NAFLD and hyperglycemia can either increase or decrease mitochondrial oxidative capacity in the liver. We summarize mechanisms regulating mitochondrial heterogeneity inside the same cell and discuss how these mechanisms may determine the role of mitochondria in NAFLD. We further discuss the role of endogenous antioxidants in controlling mitochondrial H₂O₂ release and redox-mediated signaling. We describe the emerging concept that the subcellular location of cellular antioxidants is a key determinant of their effects on NAFLD.

Major conclusions

The balance of fat oxidation versus accumulation depends on mitochondrial fuel preference rather than ATP-synthesizing respiration. As such, therapies targeting fuel preference might be more suitable for treating NAFLD. Similarly, suppressing maladaptive antioxidants, rather than interfering with physiological mitochondrial H₂O₂-mediated signaling, may allow the maintenance of intact hepatic insulin signaling in NAFLD. Exploration of the subcellular compartmentalization of different antioxidant systems and the unique functions of specific mitochondrial subpopulations may offer new intervention points to treat NAFLD.

Background

Nonalcoholic fatty liver disease (NAFLD) comprises hepatic alterations with increased lipid accumulation (steatosis) without or with inflammation (nonalcoholic steatohepatitis, NASH) and/or fibrosis in the absence of other causes of liver disease. NAFLD is developing as a burgeoning health challenge, mainly due to the worldwide obesity and diabetes epidemics.

Scope of review

This review summarizes the knowledge on the pathogenesis underlying NAFLD by focusing on studies in humans and on hypercaloric nutrition, including effects of saturated fat and fructose, as well as adipose tissue dysfunction, leading to hepatic lipotoxicity, abnormal mitochondrial function, and oxidative stress, and highlights intestinal dysbiosis. These mechanisms are discussed in the context of current treatments targeting metabolic pathways and the results of related clinical trials.

Major conclusions

Recent studies have provided evidence that certain conditions, for example, the severe insulin-resistant diabetes (SIRD) subgroup (cluster) and the presence of an increasing number of gene variants, seem to predispose for excessive risk of NAFLD and its accelerated progression. Recent clinical trials have been frequently unsuccessful in halting or preventing NAFLD progression, perhaps partly due to including unselected cohorts in later stages of NAFLD. On the basis of this literature review, this study proposed screening in individuals with the highest genetic or acquired risk of disease progression, for example, the SIRD subgroup, and developing treatment concepts targeting the earliest pathophysiolgical alterations, namely, adipocyte dysfunction and insulin resistance.

Background

As a result of a sedentary lifestyle and excess food consumption in modern society, non-alcoholic fatty liver disease (NAFLD) characterized by fat accumulation in the liver is becoming a major disease burden. Non-alcoholic steatohepatitis (NASH) is an advanced form of NAFLD characterized by inflammation and fibrosis that can lead to hepatocellular carcinoma and liver failure. Nuclear receptors (NRs) are a family of ligand-regulated transcription factors that closely control multiple aspects of metabolism. Their transcriptional activity is modulated by various ligands, including hormones and lipids. NRs serve as potential pharmacological targets for NAFLD/NASH and other metabolic diseases.

Scope of review

In this review, we provide a comprehensive overview of NRs that have been studied in the context of NAFLD/NASH with a focus on their transcriptional regulation, function in preclinical models, and studies of their clinical utility.

Major conclusions

The transcriptional regulation of NRs is context-dependent. During the dynamic progression of NAFLD/NASH, NRs play diverse roles in multiple organs and different cell types in the liver, which highlights the necessity of targeting NRs in a stage-specific and cell-type-specific manner to enhance the efficacy and safety of treatment methods.

Background

Non-alcoholic fatty liver disease (NAFLD) is defined by the abundance of lipid droplets (LDs) in hepatocytes. While historically considered simply depots for energy storage, LDs are increasingly recognized to impact a wide range of biological processes that influence cellular metabolism, signaling, and function. While progress has been made toward understanding the factors leading to LD accumulation (i.e. steatosis) and its progression to advanced stages of NAFLD and/or systemic metabolic dysfunction, much remains to be resolved.

Scope of review

This review covers many facets of LD biology. We provide a brief overview of the major pathways of lipid accretion and degradation that contribute to steatosis and how they are altered in NAFLD. The major focus is on the relationship between LDs and cell function and the detailed mechanisms that couple or uncouple steatosis from the severity and progression of NAFLD and systemic comorbidities. The importance of specific lipids and proteins within or on LDs as key components that determine whether LD accumulation is linked to cellular and metabolic dysfunction is presented. We discuss emerging areas of LD biology and future research directions that are needed to advance our understanding of the role of LDs in NAFLD etiology.

Major conclusions

Impairments in LD breakdown appear to contribute to disease progression, but inefficient incorporation of fatty acids (FAs) into LD-containing triacylglycerol (TAG) and the consequential changes in FA partitioning also affect NAFLD etiology. Increased LD abundance in hepatocytes does not necessarily equate to cellular dysfunction. While LD accumulation is the prerequisite step for most NAFLD cases, the protein and lipid composition of LDs are critical factors in determining the progression from simple steatosis. Further defining the detailed molecular mechanisms linking LDs to metabolic dysfunction is important for designing effective therapeutic approaches targeting NAFLD and its comorbidities.

Background

Hepatic steatosis is a common chronic liver disease that can progress into more severe stages of NAFLD or promote the development of life-threatening secondary diseases for some of those affected. These include the liver itself (nonalcoholic steatohepatitis or NASH; fibrosis and cirrhosis, and hepatocellular carcinoma) or other organs such as the vessels and the heart (cardiovascular disease) or the islets of Langerhans (type 2 diabetes). In addition to elevated caloric intake and a sedentary lifestyle, genetic and epigenetic predisposition contribute to the development of NAFLD and the secondary diseases.

Scope of review

We present data from genome-wide association studies (GWAS) and functional studies in rodents which describe polymorphisms identified in genes relevant for the disease as well as changes caused by altered DNA methylation and gene regulation via specific miRNAs. The review also provides information on the current status of the use of genetic and epigenetic factors as risk markers.

Major conclusion

With our overview we provide an insight into the genetic and epigenetic landscape of NAFLD and argue about the applicability of currently defined risk scores for risk stratification and conclude that further efforts are needed to make the scores more usable and meaningful.

Objective

The prevalence of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NAFLD/NASH) is increasing. NAFLD/NASH may progress to cirrhosis and hepatocellular carcinoma. However, most patients with NAFLD/NASH will die from a vascular cause. There are no approved pharmacological treatments for NASH/NAFLD. Many clinical trials have been, or are being, undertaken; however, the challenge is the assessment of the clinical endpoint. The main objective of this narrative review was to evaluate the efficacy of drugs used in clinical trials for the treatment of NAFLD/NASH that included a liver biopsy as the gold standard.

Methods

A literature search was conducted using 3 databases (PubMed, Scopus, and Google Scholar) to identify the clinical trials that included liver biopsy assessment before and after treatment.

Results

Interventional clinical trials (n = 33) involving 18 different agents, alone and in combination, were identified. Pioglitazone is the only agent that has shown consistent benefit and efficacy in clinical trials. Pentoxifylline, rosiglitazone, and ursodeoxycholic acid had both positive and negative results from clinical trials. There is also evidence for vitamin E and metformin. Other drugs, including bicyclol, cysteamine bitartrate, l-carnitine, liraglutide, obeticholic acid, oligofructose, selonsertib, silymarin, and statins, each had a single clinical study.

Conclusions

In summary, the available molecules demonstrated a significant improvement in NASH and/or liver fibrosis in a minority of patients; thus, other drugs should be identified, possibly those acting on alternative pathophysiological pathways, and tested for their safety and efficacy.