Objective

A vast majority of studies are routinely using overnight or 6h of fasting before metabolic testing of glucose homeostasis in mice. Other studies have empirically been using shorter fasting time (<6h). We aimed here to determine the shortest fasting time required for optimal insulin responsiveness while minimizing metabolic stress.

Methods

A time-course of fasting up to 24h (0, 2, 4, 6, 12 and 24h) was performed in C57Bl/6J male mice. Body weight, metabolic parameters and insulin tolerance were measured in each experimental group. Organs were collected at the same time point on a separate occasion and glycogen and metabolic gene expression measured in liver and skeletal muscle.

Results

Our data show that blood glucose levels do not significantly change during a 6h fast, while plasma insulin levels decrease to similar levels between 2h and 6h fast. During overnight (12h) and 24h fast, a robust decrease of blood glucose and plasma insulin is observed along with a profound depletion in liver glycogen content. Insulin tolerance was comparable between baseline and 6h fast while 4h and 6h fast was associated with a greater depletion of liver glycogen than 2h fast impacting the glucose counter-regulatory response. Fasting induced progressive weight loss which was attenuated at thermoneutrality. Above 4h, fasting induced major body weight loss (>5%) and significant changes in catabolic gene expression in liver and skeletal muscle.

Conclusion

Collectively, these data suggest that 2h fasting appears optimal for the assessment of insulin tolerance in mice as this duration minimizes major metabolic stress and weight loss.

Objective

Cholesterol plays a pivotal role in mitochondrial steroidogenesis, membrane structure, and respiration. Mitochondrial membranes are intrinsically low in cholesterol content, and therefore must be replenished with cholesterol from other subcellular membranes. However, the molecular mechanisms underlying mitochondrial cholesterol transport remains poorly understood. The Aster-B gene encodes a cholesterol binding protein recently implicated in cholesterol trafficking from the plasma membrane to the ER. In this study, we investigated the function and underlying mechanism of Aster-B in mediating mitochondrial cholesterol transport.

Methods

CRISPR/Cas9 gene editing was carried out to generate cell lines deficient in Aster-B expression. The effect of Aster-B deficiency on mitochondrial cholesterol transport was examined by both confocal imaging analysis and biochemical assays. Deletion mutational analysis was also carried out to identify the function of a putative mitochondrial targeting sequence (MTS) at the N-terminus of Aster-B for its role in targeting Aster-B to mitochondria and in mediating mitochondrial cholesterol trafficking.

Results

Ablation of Aster-B impaired cholesterol transport from the ER to mitochondria, leading to a significant decrease in mitochondrial cholesterol content. Aster-B is also required for mitochondrial transport of fatty acids derived from hydrolysis of cholesterol esters. A putative MTS at the N-terminus of Aster-B mediates the mitochondrial cholesterol uptake. Deletion of the MTS or ablation of Arf1 GTPase which is required for mitochondrial translocation of ER proteins prevented mitochondrial cholesterol transport, leading to mitochondrial dysfunction.

Conclusions

We identified Aster-B as a key regulator of cholesterol transport from the ER to mitochondria. Aster-B also coordinates mitochondrial cholesterol trafficking with uptake of fatty acids derived from cholesterol ester, implicating the Aster-B protein as a novel regulator of steroidogenesis.

Objective

Elevations in pancreatic α-cell intracellular Ca²⁺ ([Ca²⁺]_i) lead to glucagon (GCG) secretion. Although glucose inhibits GCG secretion, how lactate and pyruvate control α-cell Ca²⁺ handling is unknown. Lactate enters cells through monocarboxylate transporters (MCTs) and is also produced during glycolysis by lactate dehydrogenase A (LDHA), an enzyme expressed in α-cells. As lactate activates ATP-sensitive K⁺ (K_ATP) channels in cardiomyocytes, lactate may also modulate α-cell K_ATP. Therefore, this study investigated how lactate signalling controls α-cell Ca²⁺ handling and GCG secretion.

Methods

Mouse and human islets were used in combination with confocal microscopy, electrophysiology, GCG immunoassays, and fluorescent thallium flux assays to assess α-cell Ca²⁺ handling, V_m, K_ATP currents, and GCG secretion.

Results

Lactate inhibited mouse (75±25%) and human (47±9%) α-cell [Ca²⁺]_i fluctuations only under low glucose (1mM) conditions but had no effect on β- or δ-cell [Ca²⁺]_i. Glyburide inhibition of K_ATP channels restored α-cell [Ca²⁺]_i fluctuations in the presence of lactate. Lactate transport into α-cells, via MCTs, hyperpolarized mouse (14±1mV) and human (12±1mV) α-cell V_m and activated K_ATP channels. Interestingly, pyruvate showed a similar K_ATP activation profile and α-cell [Ca²⁺]_iinhibition as lactate. Lactate-induced inhibition of α-cell [Ca²⁺]_i influx resulted in reduced GCG secretion in mouse (62±6%) and human (43±13%) islets.

Conclusions

These data demonstrate for the first time that lactate entry into α-cells through MCTs results in K_ATP activation, V_m hyperpolarization, reduced [Ca²⁺]_i and inhibition of GCG secretion. Thus, taken together these data illuminate that lactate either within α-cells and/or elevated in serum could serve as important modulators of α-cell function.

Objective

Estrogen protects animals against obesity through estrogen receptor α (ERα), partially by inhibiting overeating in animals fed ad libitum. However, effects of estrogen on feeding behavior in hungry animals remain unclear. Here we examined the roles of 17β-estradiol (E2) and ERα in the regulation of feeding in hungry female animals and explored the underlying mechanisms.

Methods

Wild type female mice with surgical depletion of endogenous estrogens were used to examine effects of E2 supplementation on acute refeeding behavior after starvation. ERα-C451A mutant mice deficient in membrane-bound ERα activity and ERα-AF2⁰ mutant mice lacking ERα transcriptional activity were used to further examine mechanisms underlying the acute feeding triggered by either fasting or central glucopenia (induced by intracerebroventricular injections of 2-Deoxy-D-glucose). We also used electrophysiology to explore the impact of these ERα mutations on the neural activities of ERα neurons in the hypothalamus.

Results

In wild type female mice, ovariectomy reduced fasting-induced refeeding, which was restored by E2 supplementation. The ERα-C451A mutation, but not the ERα-AF2⁰ mutation, attenuated acute feeding induced by either fasting or by central glucopenia. Consistently, the ERα-C451A mutation impaired the neural responses of hypoglycemic ERα neurons to hypoglycemia.

Conclusion

In addition to previous evidence that estrogen reduces the deviation of the energy balance by inhibiting eating at satiated state, our findings demonstrate the unexpected role of E2 that promotes eating in hungry mice, also contributing to the stability of energy homeostasis. This latter effect specifically requires the membrane-bound ERα activity.

Objectives

Diet-driven obesity is increasingly widespread. Its consequences pose major challenges both to human health and health-care systems. There are two MAP kinase-interacting kinases (MNKs) in mice, MNK1 and MNK2. Previous studies showed that mice lacking either MNK1 or MNK2 were partially protected against high-fat diet (HFD)-induced weight gain and insulin resistance. The aims of this study were to evaluate the phenotype of mice lacking both MNKs when given a HFD, to assess whether pharmacological inhibition of MNK function also protects against diet-induced obesity (DIO) and its consequences and to probe the mechanisms underlying such protection.

Methods

Male wild-type (WT) C57Bl6 mice or mice lacking both MNK1 and MNK2 (double knockout, DKO) were fed HFD or control diet (CD) for up to 16 weeks.

In a separate study, WT mice were also given HFD for 6 weeks, after which half were treated with the recently-developed MNK inhibitor ETC-206 daily for 10 more weeks, whilst maintained on a HFD. Metabolites and other parameters were measured, and the expression of selected mRNAs and proteins was assessed.

Results

MNK-DKO mice were almost completely protected from HFD-induced obesity. Higher energy expenditure (EE) in MNK-DKO mice was observed, which likely reflects the changes in a number of genes or proteins linked to lipolysis, mitochondrial function/biogenesis, oxidative metabolism and/or ATP consumption. The MNK inhibitor ETC-206 also prevented HFD-induced weight gain, confirming that the activity of the MNKs facilitates weight gain due to excessive caloric consumption.

Conclusions

Disabling the MNKs in mice, either genetically or pharmacologically, strongly prevents weight gain on a calorie-rich diet. This likely results from increased energy utilisation, involving greater ATP consumption, mitochondrial oxidative metabolism and other processes.

Background

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 currently approved pharmacological treatments for NASH/NAFLD. A large number of clinical trials have been, or are being, undertaken; however, the challenge is the assessment of the clinical endpoint.

Scope of review

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. A literature search was carried out using 3 databases (PubMed, Scopus, and Google Scholar) to identify the clinical trials that included liver biopsy assessment before and after treatment. Interventional clinical trials (n=33) involving 18 different agents, alone and in combination, were identified.

Major conclusions

Pioglitazone is the only agent that has shown consistent benefit and efficacy in a number of 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. There is an urgent need for further research in this field.

Objective

Skeletal muscle mitochondrial function and energy metabolism displays day-night rhythmicity in healthy, young individuals. 24-h rhythmicity of metabolism has been implicated in the etiology of age-related metabolic disorders. Whether day-night rhythmicity in skeletal muscle mitochondrial function and energy metabolism is altered in older, metabolically comprised humans is so far unknown.

Methods

Twelve male overweight volunteers with impaired glucose tolerance and insulin sensitivity stayed in a metabolic research unit for 2 days under free living conditions with regular meals. Indirect calorimetry was performed at five time points (8AM, 1PM, 6PM, 11PM, 4AM), followed by a muscle biopsy. Mitochondrial oxidative capacity was measured in permeabilized muscle fibers using high-resolution respirometry.

Results

Mitochondrial oxidative capacity did not display rhythmicity. The expression of circadian core clock genes BMAL1 and REV-ERBA showed a clear day-night rhythm (p < 0.001), peaking at the end of the waking period. Remarkably, the repressor clock gene PER2 did not show rhythmicity, whereas PER1 and PER3 were strongly rhythmic (p < 0.001). On the whole-body level resting energy expenditure was highest in the late evening (p < 0.001). Respiratory exchange ratio did not decrease in the night, indicating metabolic inflexibility.

Conclusions

Mitochondrial oxidative capacity does not show a day-night rhythm in older, overweight participants with impaired glucose tolerance and insulin sensitivity. In addition, gene expression of PER2 in skeletal muscle indicates that rhythmicity of the negative feedback loop of the molecular clock is disturbed.

Objective

PARKIN is an E3 ubiquitin ligase that regulates mitochondrial quality control through a process called mitophagy. Recent human and rodent studies suggest that loss of hepatic mitophagy may occur during the pathogenesis of obesity-associated fatty liver and contribute to changes in mitochondrial metabolism associated with this disease. Whole-body Prkn knockout mice are paradoxically protected against diet-induced hepatic steatosis; however, liver-specific effects of Prkn deficiency cannot be discerned in this model due to pleotropic effects of germline Prkndeletion on energy balance and subsequent protection against diet-induced obesity. We therefore generated the first liver-specific Prkn knockout mouse strain (LKO) to directly address the role of hepatic Prkn.

Methods

Littermate control (WT) and LKO mice were fed regular chow (RC) or high-fat diet (HFD) and changes in body weight and composition were measured over time. Liver mitochondrial content was assessed using multiple, complementary techniques and mitochondrial respiratory capacity assessed using Oroboros O₂K platform. Liver fat was measured biochemically and assessed histologically, while global changes in hepatic gene expression were measured by RNA-seq. Whole-body and tissue-specific insulin resistance were assessed by hyperinsulinemic euglycemic clamp with isotopic tracers.

Results

Liver-specific deletion of Prkn had no effect on body weight or adiposity during RC or HFD feeding, however, hepatic steatosis was increased by 45% in HFD-fed LKO compared with WT mice (P<0.05). While there were no differences in mitochondrial content between genotypes on either diet, mitochondrial respiratory capacity and efficiency in liver were significantly reduced in LKO mice. Gene enrichment analyses from liver RNA-seq results suggested significant changes in pathways related to lipid metabolism and fibrosis in HFD-fed Prkn knockout mice. Finally, whole-body insulin sensitivity was reduced by 35% in HFD-fed LKO mice (P<0.05), which was primarily due to increased hepatic insulin resistance (60% of whole-body effect; P=0.11).

Conclusions

These data demonstrate that PARKIN contributes to mitochondrial homeostasis in liver and plays a protective role against the pathogenesis of hepatic steatosis and insulin resistance.

Objectives

Psoriasis is a chronic inflammatory skin disease that is thought to affect ∼2% of the global population. Psoriasis has been associated with ∼30% increased risk of developing type 2 diabetes (T2D), with numerous studies reporting that psoriasis is an independent risk-factor for T2D, separate from underlying obesity. Separately, studies of skin-specific transgenic mice have reported altered whole-body glucose homeostasis in these models. These studies imply a direct role for skin inflammation and dysfunction in mediating the onset of T2D in psoriasis patients, potentially via the endocrine effects of the skin secretome on key metabolic tissues. We used a combination of in vivo and ex vivo mouse models and ex vivo human imiquimod (IMQ) models to investigate the effects of psoriasis-mediated changes in the skin secretome on whole-body metabolic function.

Methods

To induce psoriatic skin inflammation, mice were topically administered 75 mg of 5% IMQ cream (or Vaseline control) to a shaved dorsal region for 4 consecutive days. On day 5, mice were fasted for glucose and insulin tolerance testing, or sacrificed in the fed state with blood and tissues collected for analysis. To determine effects of the skin secretome, mouse skin was collected at day 5 from IMQ mice and cultured for 24 h. Conditioned media (CM) was collected and used 1:1 with fresh media to treat mouse explant subcutaneous adipose tissue (sAT) and isolated pancreatic islets. For human CM experiments, human skin was exposed to 5% IMQ cream for 20 minutes, ex vivo, to induce a psoriatic phenotype, then cultured for 24h. CM was collected, combined 1:1 with fresh media and used to treat human sAT ex vivo. Markers of tissue inflammation and metabolic function were determined by qPCR. Beta cell function in isolated islets was measured by dynamic insulin secretion. Beta-cell proliferation was determined by measurement of Ki67 immunofluorescence histochemistry and BrDU uptake, whilst islet apoptosis was assessed by caspase 3/7 activity. All data is expressed as mean ± SEM.

Results

Topical treatment with IMQ induced a psoriatic-like phenotype in mouse skin, evidenced by thickening, erythema and inflammation of the skin. Topical IMQ treatment induced inflammation and signs of metabolic dysfunction in sub-cutaneous and epidydimal adipose tissue, liver, skeletal muscle and gut tissue. However, consistent with islet compensation and a pre-diabetic phenotype, IMQ mice displayed improved glucose tolerance, increased insulin and c-peptide response to glucose, and increased beta cell proliferation. Treatment of sAT with psoriatic mouse or human skin-CM replicated the in vivo phenotype, leading to increased inflammation and metabolic dysfunction in mouse and human sAT. Treatment of pancreatic islets with psoriatic mouse skin-CM induced increases in beta-proliferation and apoptosis, thus partially replicated the in vivo phenotype.

Conclusions

Psoriasis-like skin inflammation induces a pre-diabetic phenotype, characterised by tissue inflammation and markers of metabolic dysfunction, together with islet compensation in mice. The in vivo phenotype is partially replicated by exposure of sAT and pancreatic islets to psoriatic-skin conditioned media. These results support the hypothesis that psoriatic skin inflammation, potentially via the endocrine actions of the skin secretome, may constitute a novel pathophysiological pathway mediating development of T2D.

Background

Individuals with diabetes are at a greater risk of hospitalization and mortality resulting from viral, bacterial and fungal infections. The Coronavirus Disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has spread quickly to more than 213 countries across the world and has claimed 395,779 lives as of June 7, 2020. Notably, in several studies, diabetes is one of the most reported comorbidities in patients with severe COVID-19.

Scope of Review

In this review, I will summarize the clinical data on the risk for infectious diseases in individuals with diabetes, highlighting the mechanisms for altered immune regulation. A special focus will be given to coronaviruses. In the light of the new clinical data obtained from COVID-19 patients, mechanisms such as cytokine storm, pulmonary and endothelial dysfunction, hypercoagulation that may render individuals with diabetes more vulnerable to COVID-19 will be discussed in the end.

Major Conclusions

Epidemiological studies show that poorly controlled diabetes is a risk factor for various infectious diseases. Given the global burden of diabetes and pandemic nature of coronaviruses, understanding how diabetes affects COVID-19 severity is critical to design tailored treatments and clinical management of individuals affected by diabetes.